J.R. Simplot Company JRS06 Page 1 of 196

Title

Petition for Determination of Nonregulated Status for InnateTM Potatoes with 

Late Blight Resistance, Low Acrylamide Potential, Reduced Black Spot, and 

Lowered Reducing Sugars: Russet Burbank Event W8

We submit this petition under 7 CFR 340.6 to request that the Administrator make a determination that 

the articles should not be regulated under 7 CFR Part 340.

Submitted by Pete Clark Ph.D.

J.R. Simplot Company

Prepared by Pete Clark Ph.D.*, Jeff Habig Ph.D., Jingsong Ye Ph.D., and Susan Collinge Ph.D.

J.R. Simplot Company

Submitted on March 28, 2014

J.R. Simplot Company Petition JRS06

OECD Unique identifier:

SPS-W8-4

*Corresponding Author:

Pete Clark Ph.D.

J.R. Simplot Company

5369 West Irving Street

Boise, ID 83706

Tel.: (208) 780-6066

This Document (Including Appendices) Does Not Contain CBI

J.R. Simplot Company JRS06 Page 3 of 196

Release of Information

J.R. Simplot Company is submitting the information in this assessment for review by the USDA as part of 

the regulatory process. By submitting this information, J.R. Simplot Company does not authorize its 

release to any third party except to the extent it is requested and required under the Freedom of 

Information Act, 5 U.S.C., paragraph 552; USDA complies with the provisions of FOIA and USDA’s 

implementation regulations (7 CFR Part 1.4). Except in accordance with the Freedom of Information act, 

J.R. Simplot Company does not authorize the release, publication or other distribution of this 

information without J.R. Simplot Company’s prior notice and consent.
J.R. Simplot Company JRS06 Page 4 of 196

Table of Contents

Title ...............................................................................................................................................................1

Table of Contents..........................................................................................................................................4

List of Tables .................................................................................................................................................8

List of Figures................................................................................................................................................9

List of Appendices.......................................................................................................................................10

List of Abbreviations, Acronyms, Definitions, and Commonly Used Terms ...............................................11

Summary .....................................................................................................................................................14

1.0 Rationale for Potatoes with Late Blight Resistance, Low Acrylamide, Reduced Black Spot, and 

Lowered Reducing Sugars...........................................................................................................................18

1.1 Basis for Determination of Nonregulated Status .......................................................................18

1.2 Rationale and Benefits of Developing Potatoes with Late Blight Resistance.............................19

1.3 Rationale and Benefits of Developing Potatoes with Low Acrylamide Potential.......................20

1.4 Rationale and Benefits of Developing Potatoes with Reduced Black Spot ................................22

1.5 Rationale and Benefits of Developing Potatoes with Reduced Levels of Reducing Sugars........23

1.6 Conclusions: Rationale for Potatoes with Late Blight Resistance, Low Acrylamide, Reduced 

Black Spot, and Lowered Reducing Sugars .............................................................................................24

1.7 References..................................................................................................................................25

2.0 The Biology of Potato......................................................................................................................28

2.1 History of Potato.........................................................................................................................28

2.2 Use of the Potato as Food and Feed in the USA.........................................................................28

2.3 Taxonomy of the Genus Solanum...............................................................................................29

2.4 Genetics of Potato ......................................................................................................................29

2.5 Potato Growth and Life Cycle .....................................................................................................30

2.6 Seed Propagation........................................................................................................................30

2.7 Variety Development..................................................................................................................31

2.8 Recipient Potato Varieties..........................................................................................................33

2.9 Typical Agronomic Practices.......................................................................................................33

2.10 Pollination and Outcrossing........................................................................................................33

2.11 Wild potatoes in the U.S.............................................................................................................34

2.12 Weediness ..................................................................................................................................35

2.13 Characterization of the Recipient Potato Cultivar......................................................................36

2.14 Conclusions: The Biology of Potato ...........................................................................................37

2.15 References..................................................................................................................................38

3.0 Development of W8 Russet Burbank: Description of Marker-Free DNA Transformation..............40

3.1 Introduction................................................................................................................................40

3.2 Description of the Transformation System ................................................................................40

3.3 Transformation Method .............................................................................................................40

3.4 References..................................................................................................................................42

4.0 Donor Genes and Regulatory Sequences........................................................................................43

4.1 Plasmids used for Transformation..............................................................................................43

4.2 References..................................................................................................................................50

5.0 Genetic Characterization of Russet Burbank Event W8..................................................................51

5.1 Introduction................................................................................................................................51

5.2 Copy number and structure of the DNA Inserts.........................................................................51

5.2.1 Characterization of insertion number for pSIM1278 and pSIM1678 .................................54

5.2.2 Structure of the pSIM1278 DNA insert...............................................................................57
J.R. Simplot Company JRS06 Page 5 of 196

5.2.3 Structure of the pSIM1678 DNA insert...............................................................................63

5.2.4 Conclusions: Copy number and structure of the DNA Inserts...........................................79

5.3 Flanking Regions of Inserted Sequences....................................................................................79

5.4 Absence of Plasmid Backbone Sequence ...................................................................................80

5.4.1 Selection of Backbone-free Plants......................................................................................80

5.4.2 Molecular Evidence for Backbone-free Plants....................................................................81

5.4.3 Conclusions: Absence of Plasmid Backbone ......................................................................86

5.5 Genetic stability of W8 over three generations.........................................................................86

5.5.1 Molecular Evidence for Stability of the DNA Insert............................................................86

5.5.2 Phenotypic Evidence for Stability of the DNA Insert ..........................................................91

5.5.3 Summary of Genetic Stability Studies.................................................................................91

5.6 Characteristics of Transferred DNA and Gene Regulation .........................................................91

5.7 Event-Specific PCR ......................................................................................................................91

5.8 Summary of the Genetic Characterization of Event W8 ............................................................92

5.9 References..................................................................................................................................93

6.0 Characterization and Safety of the VNT1 Protein...........................................................................94

6.1 Identity and Function of the VNT1 Protein ................................................................................94

6.2 Levels of the VNT1 protein in W8 Tissues..................................................................................95

6.3 Rpi-vnt1 Gene Expression...........................................................................................................97

6.4 Assessment of VNT1 for Allergens and Toxins...........................................................................98

6.5 Conclusions on VNT1 Protein Safety ..........................................................................................98

6.6 References................................................................................................................................100

7.0 Characterization of Gene Silencing and Target Gene Expression.................................................102

7.1 Gene Silencing History and Mode of Action.............................................................................102

7.2 Gene Silencing Construct Design..............................................................................................102

7.3 Expression of Neighboring Genes.............................................................................................103

7.4 Silencing of Target Genes is Tissue Specific..............................................................................104

7.5 Summary of Gene Silencing and Target Gene Expression........................................................107

7.6 References................................................................................................................................108

8.0 Compositional Assessment...........................................................................................................109

8.1 Compositional Analysis Results ................................................................................................109

8.2 Glycoalkaloids...........................................................................................................................114

8.3 Methods for Compositional Assessment..................................................................................114

8.4 Statistical Analysis for Compositional Assessment...................................................................116

8.5 Compositional Assessment Conclusions...................................................................................120

8.6 References................................................................................................................................121

9.0 Safety of Russet Burbank W8 Potato............................................................................................123

9.1 Phenotype through Agronomic Studies ...................................................................................123

9.2 Compositional Equivalence ......................................................................................................123

9.3 A Bioinformatics Approach to Allergen and Toxin Assessment ...............................................123

9.4 Glycoalkaloid Testing................................................................................................................125

9.5 Dietary Risk Assessment of VNT1 protein ................................................................................126

9.6 Low levels of VNT1 protein.......................................................................................................127

9.7 Safety of Nucleic Acids..............................................................................................................127

9.8 Safety of Gene Silencing Methods............................................................................................127

9.9 Previous regulatory actions for plant incorporated protectants .............................................131

9.10 Conclusions: Safety of Russet Burbank W8 Potato .................................................................132

9.11 References................................................................................................................................133
J.R. Simplot Company JRS06 Page 6 of 196

10.0 Trait Efficacy..................................................................................................................................138

10.1 Composition and Field Results .................................................................................................138

10.1.1 Potato Free Amino Acids Results......................................................................................138

10.1.2 Reducing Sugars and Invertase Silencing..........................................................................139

10.1.3 Acrylamide ........................................................................................................................142

10.1.4 Black Spot..........................................................................................................................143

10.1.5 Late Blight Resistance .......................................................................................................145

10.2 Conclusions on Trait Efficacy ....................................................................................................146

10.3 Analytical Methods for Trait Efficacy and Composition ...........................................................146

10.3.1 Analytical Methods for Free Amino Acids, Sugars, Acrylamide........................................146

10.3.2 Black Spot Analysis by Catechol Assay..............................................................................148

10.3.3 Foliar Late Blight Resistance Test Methods......................................................................148

10.3.4 Tuber Late Blight Resistance Test Methods......................................................................150

10.3.5 Enzymatic Assays for PPO and Invertase ..........................................................................151

10.4 References................................................................................................................................152

11.0 Agronomic Evaluation...................................................................................................................154

11.1 Agronomic Performance...........................................................................................................154

11.1.1 Typical Agronomic Practices.............................................................................................154

11.1.2 Field Trial Locations...........................................................................................................154

11.1.3 Field Performance and Tuber Assessment Results...........................................................156

11.2 Conclusions: Agronomic Characteristics...................................................................................162

11.3 Agronomic and Phenotypic Methods.......................................................................................162

11.3.1 Agronomic Trial Experimental Design...............................................................................163

11.3.2 Phenotypic Assessments...................................................................................................163

11.3.3 Insect, Disease, and Abiotic Stressors...............................................................................165

11.3.4 Tuber Grading and Yield....................................................................................................166

11.3.5 Statistical Methods for Agronomic Studies.......................................................................167

11.3.6 BQMS Quality Management Systems...............................................................................168

11.3.7 Survival in Fields................................................................................................................169

11.4 Disease Susceptibility Assessment ..........................................................................................169

11.4.1 Disease Study Locations....................................................................................................169

11.4.2 Disease Susceptibility Assessment Results.......................................................................171

11.5 Conclusions: Disease Susceptibility ..........................................................................................171

11.6 Disease Susceptibility Methods................................................................................................173

11.6.1 Statistical Methods for Disease Studies............................................................................176

11.7 Field Test Reports.....................................................................................................................176

11.8 Conclusion on Agronomic Performance, Yield and Grading, and Disease Susceptibility.........177

11.9 References................................................................................................................................178

12.0 Environmental Safety Assessment................................................................................................179

12.1 Potential to become a weed of agriculture or to be invasive of natural habitats ...................179

12.2 Potential for gene flow to sexually compatible plants.............................................................179

12.3 Potential to become a plant pest .............................................................................................181

12.4 Potential impact on non-target species including humans......................................................181

12.5 Potential impact on biodiversity...............................................................................................181

12.6 Summary of the Environmental Safety Assessment.................................................................182

12.7 References................................................................................................................................183

13.0 Crop Production and the Potato Industry.....................................................................................184

13.1 Benefits of Potatoes with Late Blight Resistance .....................................................................184
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13.2 Benefits of Potatoes with Reduced Acrylamide Potential........................................................185

13.3 Benefits of Potatoes with Reduced Black Spot.........................................................................186

13.4 Benefits of Potatoes with Lowered Reducing Sugars...............................................................186

13.5 Potato uses and exports...........................................................................................................187

13.6 Submissions to Other Regulatory Agencies..............................................................................187

13.7 Impact on the Organic Market .................................................................................................188

13.8 History of Biotech Potatoes......................................................................................................188

13.9 Stewardship of InnateTM W8 Potatoes......................................................................................188

13.10 Conclusions: Crop Introduction and the Potato Industry........................................................190

13.11 References................................................................................................................................191

14.0 Conclusions: Determination of Nonregulated Status for Russet Burbank W8 Potatoes.............193

15.0 Acknowledgements.......................................................................................................................194

16.0 Statement of Grounds Unfavorable..............................................................................................195

17.0 Certification...................................................................................................................................196
J.R. Simplot Company JRS06 Page 8 of 196

List of Tables

Table 1. InnateTM event W8 and the OECD unique identifier.....................................................................15

Table 2-1. Example of Agronomic Inputs for Russet Varieties....................................................................33

Table 4-1. Table 4-1 Genetic elements of the pSIM1278 and pSIM1678 backbone1.................................46

Table 4-2. Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border .......47

Table 4-3 Genetic elements of the DNA Insert of pSIM1678, from Left Border site to Right Border ........49

Table 5-1. Predicted and observed bands based on Southern blots for pSIM1278 ...................................57

Table 6-1. Limit of Detection, Quantification and Expression of VNT1 in Potato.......................................96

Table 8-1: Proximates, Vitamins, and Minerals in W8 and Control..........................................................110

Table 8-2: Total Amino Acids in W8 and Control......................................................................................111

Table 8-3: Event W8 and Control Glycoalkaloids......................................................................................114

Table 8-4. Varieties in 2012 and 2013 Field Trials....................................................................................115

Table 8-5. Number of data points for each test, control and reference line............................................117

Table 8-6. Combined Literature Ranges (CLR) for Proximates, Vitamins, Minerals .................................118

Table 8-7. Combined Literature Ranges (CLR) for Total Amino Acids.......................................................119

Table 10-1. Potato Free Amino Acids at Harvest......................................................................................138

Table 10-2. Potato Sugar Levels at Harvest and After Storage at 46°F.....................................................141

Table 10-3. Potato Sugar Levels When Stored at 38°F .............................................................................141

Table 10-4. French Fry Acrylamide Levels (ppb) at Harvest and After Storage at 46°F............................142

Table 10-5. French Fry Acrylamide Levels at Harvest and After Storage at 38°F .....................................143

Table 10-6. Percent Foliar Late Blight Infection at Last Rating .................................................................145

Table 10-7. Percent Tuber Late Blight Infection .......................................................................................146

Table 10-8. Combined Literature Ranges (CLR) for Free Amino Acids......................................................148

Table 10-9. Origin of Tubers from 2013 Field Trials for PPO Assay ..........................................................148

Table 10-10. Late Blight Study Information ..............................................................................................149

Table 10-11. Plot Inoculation Information for Late Blight........................................................................149

Table 11-1. Example of Agronomic Inputs for Russet Burbank Potatoes................................................154

Table 11-2. Field Trial Locations................................................................................................................155

Table 11-3. Agronomic Characteristics.....................................................................................................157

Table 11-4. Yield and Grading Characteristics..........................................................................................158

Table 11-5. Stressor Observations in W8 and the Control .......................................................................159

Table 11-6. Beneficial Arthropod Abundance in Field Trials.....................................................................161

Table 11-7. Pest Arthropod Abundance in Field Trials .............................................................................161

Table 11-8. Varieties in Field Trials...........................................................................................................163

Table 11-9. Characteristics Evaluated.......................................................................................................164

Table 11-10. Common Potato Disease and Insect Symptoms1.................................................................166

Table 11-11. 2012 and 2013 Disease Study Details.................................................................................170

Table 11-12. Disease Susceptibility Assessment Results..........................................................................172

Table 11-13. Scab Severity Rating Scale....................................................................................................175

Table 11-14. Field release notifications for agronomic and disease susceptibility studies......................177
J.R. Simplot Company JRS06 Page 9 of 196

List of Figures

Figure 2-1. Potato Variety Development Schematic...................................................................................32

Figure 2-2. Potato Production in the United States....................................................................................35

Figure 4-1. pSIM1278 Vector......................................................................................................................44

Figure 4-2. pSIM1678 Vector......................................................................................................................45

Figure 5-1. Plasmid constructs and Southern Probes.................................................................................53

Figure 5-2. pSIM1278 DNA inserted at a single site in the genome ...........................................................54

Figure 5-3. pSIM1678 DNA inserted at a single site in the genome ...........................................................56

Figure 5-4. Overall structure of pSIM1278 DNA Insert with digestion and hybridization patterns ...........59

Figure 5-5. Structure of pSIM1678 DNA Insert Digestion and Hybridization Pattern ................................64

Figure 5-6. Russet Burbank DNA Hybridization with the AGP Probe..........................................................65

Figure 5-7. Russet Burbank DNA Hybridization with the AGP Probe.........................................................66

Figure 5-8. Russet Burbank DNA Hybridization with the ASN Probe..........................................................67

Figure 5-9. Russet Burbank DNA Hybridization with the ASN Probe..........................................................68

Figure 5-10. Russet Burbank DNA Hybridization with the ASN Probe........................................................69

Figure 5-11. Russet Burbank DNA Hybridization with the GBS1 Probe......................................................70

Figure 5-12. Russet Burbank DNA Hybridization with the GBS1 Probe......................................................71

Figure 5-13. Russet Burbank DNA Hybridization with the R1 Probe ..........................................................72

Figure 5-14. Russet Burbank DNA Hybridization with the R1 Probe ..........................................................73

Figure 5-15. Russet Burbank DNA Hybridization with the GBS2 Probe......................................................74

Figure 5-16. Russet Burbank DNA Hybridization with the VNT1 Probe......................................................75

Figure 5-17. Russet Burbank DNA Hybridization with the VNT1 Probe......................................................76

Figure 5-18. Russet Burbank DNA Hybridization with the INV Probe.........................................................77

Figure 5-19. Russet Burbank DNA Hybridization with the INV Probe.........................................................78

Figure 5-20. Predicted Chromosomal Insertion Sites for each Insert.........................................................80

Figure 5-21. Probes for the backbone of pSIM1278 and pSIM1678..........................................................81

Figure 5-22. Analysis of Plasmid Backbone DNA using Southern Blotting..................................................82

Figure 5-23. PCR Amplification of Gapped Regions in Plasmid Backbone..................................................83

Figure 5-24. PCR Primer Sets for Detection of Backbone Junction Sites....................................................84

Figure 5-25. Confirmation of the Absence of Backbone DNA Junctions ....................................................85

Figure 5-26. Southern blot probing of EcoRV digestion .............................................................................87

Figure 5-27. Southern blot probing of XbaI digestion.................................................................................89

Figure 6-1. Amino Acid Sequence of VNT1 .................................................................................................94

Figure 6-1. Limit of detection for anti-VNT1 antibody 5363.......................................................................95

Figure 6-2. Western blot analysis of VNT1 in potato tissues......................................................................96

Figure 6-3. Quantitation of VNT1 in W8 tissue samples is below LOQ.......................................................97

Figure 6-4. Rpi-vnt1 expression measured by RT-qPCR..............................................................................98

Figure 7-1. Silencing Approach Using the DNA Inserts of pSIM1278 and pSIM1678 ...............................103

Figure 7-2. Target Gene Expression in Tubers..........................................................................................104

Figure 7-3. Target Gene Expression in Leaves ..........................................................................................105

Figure 7-4. Target Gene Expression in Stems ...........................................................................................105

Figure 7-5. Target Gene Expression in Roots............................................................................................106

Figure 7-6. Target Gene Expression in Flowers.........................................................................................106

Figure 10-1. Biosynthesis of GLN and ASN in plants................................................................................139

Figure 10-2. Enzymatic Activity of Invertase in W8 and Russet Burbank Tubers.....................................140

Figure 10-3. Catechol Assay for Polyphenol Oxidase Activity...................................................................144

Figure 10-4. PPO Activity in Russet Burbank and W8 Tubers...................................................................145

Figure 13-1. Areas of late blight infection in 2012 are shaded.................................................................184
J.R. Simplot Company JRS06 Page 10 of 196

List of Appendices

Appendix A: ......................................................................... Genetic, Molecular, and Biochemical Methods

Appendix B: ............................................................................................................Characterization of VNT1

Appendix C: ..................................................................................................Allergen and Toxin Assessment

Appendix D: ...................................................................................................Dietary Exposure to Rpi-vnt1.1

Appendix E: ............................................................. Detection of Russet Burbank W8 Using Real-Time PCR
J.R. Simplot Company JRS06 Page 11 of 196

List of Abbreviations, Acronyms, Definitions, and Commonly Used Terms

Abbreviation Definition

AGP Southern blot probe used to detect Agp promoter sequence

ALA Alanine

A. tumefaciens Agrobacterium tumefacians

ARG Arginine

ASN Asparagine

ASN Southern blot probe used to detect Asn1 sequence (Chapter 5)

Asn1 Asparagine synthetase-1 gene

ASP Aspartic acid

Backbone DNA DNA associated with plasmid/vector backbone

BLAST Basic Local Alignment Search Tool

Chs Chalcone synthase gene

cwt/A Unit of measure equal to 100lbs/ acre

CYS Cysteine

DNA Deoxyribonucleic acid

DNA insert The DNA sequence from pSIM1278 located between the LB and RB intended to be 

integrated into the potato genome

dsRNA Double-stranded RNA

EB Ethidium bromide

EPA U.S. Environmental Protection Agency

EUP Experimental Use Permit (application to Environmental Protection Agency)

fASN1 Fragment of the Asn1 gene

FDA Food & Drug Administration

G0 First generation greenhouse-grown tuber seed

G1 First generation field-grown tuber seed

G2 Second generation field-grown tuber seed

G3 Third generation field-grown tuber seed

GBS1 or GBS2 Southern blot probe used to detect Gbss promoter (3 ́ and 5 ́ ends, respectively)

GLN Glutamine

GLU Glutamic acid

GLY Glycine

GM Genetically modified

gus -glucuronidase gene

HIS Histidine

IPD Inter-genebank Potato Database

ipt Isopentyltransferase gene – produces cytokinin hormones associated with plant 

growth and development

ILE Isoleucine

INV Southern blot probe used to detect VInv sequence

LB Left Border (a 25-base pair sequence) similar to A. tumefaciens T-DNA border

LEU Leucine

LYS Lysine
J.R. Simplot Company JRS06 Page 12 of 196

Abbreviations, Acronyms, and Definitions (Continued)

Abbreviation Definition

MAFF Japan Ministry of Agriculture, Forestry, and Fisheries

MET Methionine

MHLW Japan Ministry of Health, Labor and Welfare

NCBI National Center for Biotechnology Information

Non-coding DNA DNA not coding for translated RNA

ORFs Open reading frames

pAgp Promoter of the ADP glucose pyrophosphorylase gene

PCR Polymerase chain reaction

pGbss Promoter of the granule-bound starch synthase gene

PHE Phenylalanine

PhL Phosphorylase-L gene

P. infestans Phytophthora infestans, pathogen that causes late blight

Ppo5 Polyphenol oxidase-5 gene

PRO Proline

qPCR Quantitative / real-time PCR

R1 Water dikinase R1 gene

R1 Southern blot probe used to detect the R1 cassette (Chapter 5)

RB Right Border (a 25-base pair sequence) similar to A. tumefaciens T-DNA 

border

RCB Randomized complete block design

RNA Ribonucleic acid

RNAi RNA interference

Rpi-vnt1 R-gene that is resistant to Phytophtora infestans (Rpi) from Solanum 

venturii

RT-PCR Reverse transcription PCR

RT-qPCR Reverse transcription quantitative PCR

Sclerotia Compact mass of hardened mycelium containing food reserves for the 

pathogen, Rhizoctonia solani

SER Serine

siRNA Small interfering RNA from RNAi pathway

Somaclonal

variation

Genetic and/ or phenotypic variation among clonally propagated plants of a 

single donor clone; generated by tissue culture and other forms of 

vegetative propagation

sRNA All classes of small cellular RNAs

TPS True potato seed

T-DNA Transfer DNA from A. tumefaciens delineated by left and right border 

sequences

THR Threonine

Tomato Currently known as Solanum lycopersicum, previously known as 

Lycopersicon esculentum. May be referred to as Lycopseriscon esculentum 

in older papers and database searches such as BLAST.

TRP Tryptophan

TYR Tyrosine
J.R. Simplot Company JRS06 Page 13 of 196

Abbreviations, Acronyms, and Definitions (Continued)

Abbreviation Definition

VAL Valine

VNT1 Southern blot probe used to detect Rpi-vnt1 promoter sequence (Chapter 5)

VNT1 Protein expressed from Rpi-vnt1 gene
J.R. Simplot Company JRS06 Page 14 of 196

Summary

The J. R. Simplot Company has advanced plant breeding by employing Simplot’s proprietary InnateTM

branded technologies to transform plants with plant genomic DNA. In the latest innovation in potato 

breeding, we added late blight resistance, low acrylamide potential, reduced black spot, and lowered

reducing sugars into the most popular potato variety, Russet Burbank. 

To explain further, we used InnateTM branded technologies to provide resistance to the devastating 

disease, late blight, caused by the oomycete, Phytophthora infestans. Late blight was the cause of the 

Irish potato famine in the mid-1800s, resulting in mass starvation, disease, and immigration. Late blight 

is still a serious pest of potatoes and results in significant costs from fungicide applications but also 

reduces yield because of loss of vitality associated with diseased plants. Reducing fungicide application 

would save cost but has the additional benefit of releasing less fungicide into the environment. In 

addition, we address three critical potato quality issues: (1) accumulation of reducing sugars leading to 

dark spots and sugar ends, (2) large amounts of asparagine, a non-essential free amino acid that is 

rapidly oxidized to form acrylamide upon frying or baking; and (3) susceptibility to enzymatic browning 

and discoloration, which happens when polyphenol oxidase leaks out from the damaged plastids of 

bruised potatoes. In the cytoplasm, the enzyme oxidizes phenols, which then rapidly polymerize to 

produce dark pigments. Browning is also triggered non-enzymatically as a consequence of the partial 

degradation of starch into glucose and fructose. When heated, these reducing sugars react with amino 

acids, such as asparagine, through the Maillard reaction to produce a variety of desired compounds 

contributing to flavor, aroma, and browning, but also acrylamide. By silencing invertase in tubers, the 

conversion of sucrose to reducing sugars is reduced, potentially allowing for storing potatoes at colder 

temperatures. Other advantages to lower storage temperatures include reducing the use of chemicals 

to inhibit sprouting, less disease and moisture loss, all resulting in higher yield of usable potatoes and 

anticipated economic advantages.

Russet Burbank W8 potatoes provide the potato industry the opportunity to keep the highly desired 

characteristics of the leading french fry and fresh potato variety with multiple enhancements that could 

not be added through traditional breeding. Overall improvement in yield and lowered fungicide use 

with late blight resistance should benefit all members of the food value chain by reducing environmental 

impacts associated with fungicide use, and potentially reducing acreage devoted to potato production. 

The combination of low asparagine and reducing sugars results in greater than 70% reduction in 

acrylamide even after extended cold storage, addressing this potential health risk for consumers and the 

food industry. Quality improvements related to lower levels of reducing sugars and black spot provide 

benefits to processors and consumers, and invertase silencing could positively impact quality and yield 

from potato storage. There is evidence that invertase silencing reduces the incidence of high sugar 

potatoes and sugar ends, both quality attributes that result in economic loss by causing potatoes to be 

rejected by french fry and chip processors. Finally, reduced invertase could enable storage of processing 

potatoes at significantly lower temperatures, decreasing loss from disease and yield losses from higher 

respiration rates related to typical storage at 46 – 48  ̊F. 

Simplot’s InnateTM technologies allow researchers to isolate genetic elements from any plant genome, 

rearrange them, or link them together in desired permutations, and introduce them back into the 

genome. More specifically, in these events, the genomic DNA comes from potato or wild potato, a 

group of related plant species that are sexually-compatible with potato. We incorporate no viral 

markers, and no plasmid backbone sequences, into the plant genome. 
J.R. Simplot Company JRS06 Page 15 of 196

Because our InnateTM technologies effectively accelerate the process of conventional crossing, it allows 

changes in traditional varieties to occur much faster, while maintaining the desired characteristics of the 

original parent plant. For potatoes, InnateTM technologies are particularly attractive because potatoes 

are notorious for having a high degree of heterozygosity, suffering from inbreeding depression, and 

limited predominantly to clonal propagation. Historically, these inherent factors have significantly 

hindered and prevented the commercial introduction of quality, sought-after traits into this valuable 

crop. Traditional breeding techniques result in random genomic rearrangements and trait segregation, 

and do not allow for the simultaneous addition of multiple desired traits.

To illustrate this point, there currently are no varieties available that produce tubers with late blight 

resistance, low acrylamide potential, reduced black spot and reducing sugars, while displaying all other 

traits important to the food industry. Therefore, instead of attempting to develop new varieties, the J.R. 

Simplot Company used the techniques of modern biotechnology to improve the quality of the existing

Russet Burbank variety by transforming with potato genomic DNA that introduces late blight resistance 

and silences the genes related to expression of black spot, asparagine, and reducing sugars in tubers. 

The plasmids pSIM1278 and pSIM1678 were used to transform the Russet Burbank potatoes resulting in 

selection of W8. These plasmids contain DNA sequence for silencing genes through the mechanism of 

RNA interference (RNAi) resulting in lower levels of reducing sugars, asparagine, and black spot in 

potatoes. In addition, the pSIM1678 plasmid contains an expression cassette for the Rpi-vnt1 gene, 

which relies on expression of the VNT1 protein for resistance to late blight. See Table 1 for the OECD 

unique identifier for W8.

Table 1. InnateTM event W8 and the OECD unique identifier

Event number OECD Unique Identifier

W8 SPS-W8-4

A rationale is presented for the safe use of W8 potatoes. Among the supporting evidence are the 

phenotypic as well as ecological interactions data collected in a robust field trial program at 11 sites over 

two crop years. From those studies, a compositional assessment shows the W8 potatoes substantially 

equivalent to Russet Burbank controls. Open reading frames associated with the DNA inserts, including 

the late blight resistance gene itself, were screened for allergen and toxin potential, but no safety 

concerns were identified. The W8 potatoes were tested for glycoalkaloids and found no different from 

the Russet Burbank controls and well below the accepted safety limit. Similarity of the Rpi-vnt1 to other 

gene sequences in related species was assessed and greater than 90% homology discovered between 

this late blight resistance gene and the Tomato Mosaic Virus resistance gene (ToMV Tm-2) identified in 

tomatoes, which has been widely bred into tomato varieties in the fresh market and is consumed by 

humans. 

Among the risk considerations is the presence of the VNT1 protein, and although effective at controlling 

late blight, levels in potato tubers are below our detection limit of 30 ppb. Also, nucleic acids such as 

those added through plasmids pSIM1278 and pSIM1678 have long been considered safe under the 

FFDCA by EPA and FDA. An in depth review is included on the topic of the safety of using RNA 

interference for gene silencing. Numerous physiological barriers exist to uptake of RNA, confirming that 

ingested RNA is rapidly metabolized in the gut where it is converted to nutrients, and therefore unlikely 

to adversely affect human health. Regulatory actions supporting safe use of the Coat Protein Gene of 
J.R. Simplot Company JRS06 Page 16 of 196

Plum Pox Virus and Potato Leaf Roll Virus Resistance Gene provide additional evidence of the safety of 

W8. 

Accordingly, based on the results of the studies presented here, there is no reason to believe that the 

VNT1 protein will have any impact to human health through allergenicity or toxicity, or any 

environmental impacts to non-target mammals, birds, fish, or insects due to the low expression and lack 

of toxic effects of the protein. There is negligible risk of environmental contamination and no 

persistence in the environment because of the low expression of the VNT1 protein in potato tubers. The 

prevalence of similar resistance genes throughout edible crops suggests that extremely low levels of 

similar proteins are widespread in nature and unlikely to pose risk to human health, non-targets or the 

environment. 

Extensive analyses demonstrated the achievement of the desired traits, including effective resistance to 

late blight, reduced black spot, lower asparagine, and lower levels of reducing sugar. Ultimately, the 

combination of lower asparagine and reducing sugars led to greater than 70% reductions in acrylamide 

levels in cooked potatoes and, importantly, we showed that the transformed tubers were otherwise

substantially equivalent to untransformed potatoes.

The propagation of commercial potato varieties through cloning of seed potatoes mitigates concerns 

about increased weediness or plant pest potential such as seed dispersal, survival outside of cultivation, 

or outcrossing. The potatoes that were transformed, the Russet Burbank variety, produce few flowers 

and are male sterile. Other factors limiting outcrossing include the tendency for most fertile varieties to

be self-pollinated, an inability to attract honey bees because they lack nectar, and limitation of the 

pollen transfer range to about 20 meters. In addition, true seeds would be unlikely to grow into mature 

potatoes since potato seeds are not saved and propagated in the typical farming operation. If potatoes 

were grown from true potato seed, the offspring would be so diverse that they would not be useful as 

commercial potatoes. In addition, potatoes are not known to escape from commercial fields or show 

weediness potential. Wild potato varieties are rare in the United States and for the most part 

geographically isolated from commercial production areas, further reducing concerns about crosspollination with wild species.

The modifications described in this petition were intended to enhance disease resistance and quality, 

not agronomic characteristics, of potatoes. Planting, cultivation, management and harvesting 

techniques were not affected by the incorporated traits, with the exception that late blight resistant 

potatoes should require less fungicide. The InnateTM branded W8 variety is likely to be planted in areas 

that are already growing potatoes, and would not result in a significant expansion of planted acres or a 

change in the areas where potatoes would otherwise be grown. The modifications described in this 

petition are highly unlikely to increase the weediness or invasiveness of potato because the 

incorporated traits (late blight resistance, reduced free-asparagine, black spot tolerance, and lowered 

reducing sugars) would not influence the fundamental biological characteristics or ecological 

competitiveness. Field trials over multiple years with W8 did not provide any evidence for altered 

growth characteristics such as accelerated tuber sprouting, increased plant vigor, increased tuber set, or 

delayed senescence. The Potato Late Blight Resistance Gene (Rpi-vnt1) expresses a resistance protein, 

VNT1, which reacts with a pathogen-specific effector protein to halt the spread of infection. This 

resistance should partially replace fungicide treatments but would not enhance the potatoes 

survivability beyond what would be normally expected for potatoes. Thus, modified potatoes are 

unlikely to display enhanced weediness. The mechanism of action for resistance proteins like that coded 
J.R. Simplot Company JRS06 Page 17 of 196

by Rpi-vnt1 is highly specific and extremely unlikely to impact insects and other non-target organisms, 

weed or disease susceptibility, endangered species or biodiversity.

We therefore seek nonregulated status for Russet Burbank event W8 based on the weight of evidence 

demonstrating safety when compared to untransformed potatoes. The technologies presented in this 

petition and the resultant transformed plants and their tuber products satisfy the requirements for 

nonregulated status. The transformed plants and subsequent traits described in this petition should not 

be considered plant pests. In conclusion, plants displaying the incorporated traits are as safe to grow 

and as safe to eat as untransformed potatoes based on all of the data contained and referenced in this 

submission. The J.R. Simplot Company requests a determination from APHIS that event W8 described in 

this petition, and any progeny derived from this event, are granted nonregulated status and would no 

longer be considered regulated articles under 7 CFR Part 340.

The genetic services and research and development methods and associated genetic tools, disclosed in 

this petition, such as expression vectors, and the transformed plants and plant cells, and methods of 

transformation, have been trademark-branded by Simplot as InnateTM.
J.R. Simplot Company JRS06 Page 18 of 196

1.0 Rationale for Potatoes with Late Blight Resistance, Low Acrylamide, Reduced Black Spot, and 

Lowered Reducing Sugars

The Russet Burbank potatoes were transformed using InnateTM technologies in order to address the 

need of the potato industry and consumers for potatoes with late blight resistance, improve quality by 

reducing expression of the enzyme responsible for black spot and to reduce asparagine and reducing 

sugars, and thus reduce acrylamide through lowering the concentration of the reactants, asparagine and 

reducing sugars. With InnateTM branded technologies, we transformed potatoes using desirable traits 

which were simultaneously incorporated into the most popular potato processing variety, Russet 

Burbank. Such multiple trait addition would not be possible to achieve through traditional breeding 

because potato is tetraploid, highly heterozygous and sensitive to inbreeding depression. The resulting 

InnateTM branded potatoes contain highly sought after traits, none of which would contribute to 

weediness or other undesirable agronomic or environmental characteristics as verified by phenotypic

and molecular characterization. 

1.1 Basis for Determination of Nonregulated Status 

Under the authority of the Plant Protection Act and Part 340 of title 7 of the Code of Federal Regulation 

(7 CFT Part 340), USDA-APHIS regulates, among other things, the introduction of organisms and 

products altered or produced through genetic engineering that are plant pests or are believed to be 

plant pests. The event described in this petition was transformed with DNA that does not alter the pest 

characteristics of the Russet Burbank potatoes. Instead, the addition of the Potato Late Blight 

Resistance Gene (also known as Rpi-vnt1) would potentially reduce the need for pesticides and promote 

better health of the potato plants. The W8 event was confirmed to be free of Agrobacterium and free of 

backbone DNA. In addition, the scientific evidence presented here shows that the inserted genetic 

material results in a new potato event that is as safe as the untransformed potatoes.

Field evaluations demonstrated that the event displayed similar agronomic characteristics compared to 

the untransformed controls, except that the event is much more resistant to late blight. This submission 

reviews the biosafety implications of all minor differences observed. In addition to the desired 

agronomic difference of late blight resistance; the phenotypic changes in W8 potatoes include reduced 

levels of the free amino acid, asparagine, and reduced levels of reducing sugars in tubers. As predicted, 

we confirmed that changes in asparagine and reducing sugar levels were associated with a reduced 

potential to form acrylamide upon frying. The Russet Burbank event W8 also contains a silencing 

cassette to reduce the PPO enzyme in tubers, resulting in a reduced incidence of black spot.

The event contained expression cassettes that could influence reducing sugars by multiple mechanisms. 

Included in the first transformation using pSIM1278, we introduced a silencing cassette for the 

promoters of the starch associated gene (R1) and the phosphorylase-L gene (PhL) inserted between 

promoters that are predominantly active in tubers. The result of this silencing strategy was lowered 

levels of the reducing sugars glucose and fructose at harvest or when analyzed at one month after 

harvest (Collinge and Clark 2013). Another gene silencing cassette, for the acid invertase gene (Ye et al. 

2010), was introduced into event W8 using the transformation vector pSIM1678. A reduction in 

invertase through gene silencing reduces the conversion of sucrose to reducing sugars during cold 

storage, inhibits formation of sugar related defects such as sugar ends in french fries, and results in even 

lower levels of acrylamide upon frying.
J.R. Simplot Company JRS06 Page 19 of 196

The event W8 produced by transformation of the Russet Burbank variety with the specified transfer 

DNA is well characterized and safe as determined by agronomic and phenotypic evaluations, when 

compared to the untransformed controls. 

1.2 Rationale and Benefits of Developing Potatoes with Late Blight Resistance

A resistance gene, referred to as Rpi-vnt1, to the most important potato pathogen, Phytophthora

infestans, was introduced into the Russet Burbank potato variety using Agrobacterium-mediated 

transformation. The Rpi-vnt1 gene confers resistance to late blight, a major pest of potatoes in North 

America and throughout the world. Late blight was the cause of the Irish potato famine in the mid1800s, resulting in mass starvation, disease, and immigration; and remains a serious plant disease today.

Late Blight Potato Breeding History 

Resistance to late blight (Phytophthora infestans) occurs in many tuber-bearing wild Solanum species 

that belong to the highly diverse section Petota Dumort. Early potato breeding programs from the midtwentieth century used Solanum demissum, a common species in central Mexico and a great source of 

resistance against late blight. In total, 11 S. demissum resistance (R) genes designated R1-R11 are 

distinguished in a potato differential set made by Black and Mastenbroek (Black et al. 1953; Malcolmson 

and Black 1966). R1, R3, and R10, and to a lesser extent R2 and R4, have been widely used for 

introgression in European breeding programs (Colon 1994) to help control late blight. The introduction

of new cultivars containing these R genes was initially successful, but rapidly evolving populations of P. 

infestans reduced their efficacy (Fry 2008; McDonald and Linde 2002; Pink and Puddephat 1999; Wastie 

1991). However, durability in the field of a particular R gene is variable (Leach et al. 2001), and 

additional novel resistance genes against P. infestans (Rpi-genes) are being discovered from other wild 

Solanum species. To date, 68 Rpi-genes, from wild Solanum species, have been characterized (Rodewald 

and Trognitz 2013) and about 20 of them have been cloned. In addition, germplasm from those wild 

Solanum species has been integrated into numerous cultivars through breeding, for example but not 

limited to: S. bulbocastanum (Park et al. 2005; Ramanna and Hermsen 1971), S. stoloniferum (Hutten 

and van Berloo 2001, referred to as sto or CPC 2093), S. microdontum (Tan et al. 2008) and S. phureja

(Sliwka et al. 2010; 2013). 

Rpi-vnt1 History and Origin

Recently, the first Rpi-gene (Rpi-vnt1) from a South America wild Solanum species, S. venturii, was 

cloned (Pel et al. 2009; Foster et al. 2009). Three functional alleles of the gene were identified, Rpivnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3. The gene was first located on chromosome 9 (same locus as Tm-2(2) 

from S. lycopersicum which confers resistance against Tomato mosaic virus (ToMV)) of the potato wild 

species S. venturii. The gene was identified by a classical genetic and physical mapping approach 

including nucleotide binding site profiling and bulked segregant analysis, as well as a Tm-2(2) based 

allele mining strategy (Pel et al. 2009; Foster et al. 2009). Transgenic potato and tomato plants carrying 

Rpi-vnt1.1 were shown to be resistant to P. infestans. Of 11 P. infestans isolates tested, only isolate EC1 

from Ecuador was able to overcome Rpi-vnt1.1 and cause disease on the inoculated plants (Pel 2010). 

However, Rpi-vnt1.1 remains of major importance since it brings resistance against the most dominant 

late blight isolate in Europe, Blue 13, by responding to the presence of the cognate effector protein AVRVNT1 (Cooke et al. 2012). Alleles of Rpi-vnt1 (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) which differed by 

only a few nucleotides were found in other late blight resistant accessions of S. venturii. The late blight 

resistance gene Rpi-phu1 from S. phureja (Sliwka et al. 2006) has been shown to be identical to Rpivnt1.1 (Pel 2010; Sliwka et al. 2013), suggesting either that this strong resistance gene has been 

maintained since a common ancestor, due to selection pressure for blight resistance, or that genetic 
J.R. Simplot Company JRS06 Page 20 of 196

exchange between S. venturii and S. phureja has occurred at some time. In addition, a collection 

containing 200 wild Solanum species with 5 genotypes each was screened. Rpi-vnt1 alleles were found 

in S. venturii, as expected, S. mochiquense and S. weberbaueri (Pel 2010; Vleeshouwers et al. 2011; 

SOLRgene database). Moreover, Rpi-phu1 (also called Rpi-vnt1) was introgressed into S. tuberosum

(tetraploid potato varieties) from an interspecific cross between S. phureja and S. stenotomum (known 

as pinta boca in Peru and Bolivia). Both of these species are edible and research programs are underway 

in Europe to introgress Rpi-phu1/Rpi-vnt1 (identical genes) in cultivated potatoes (Sliwka et al. 2010; 

2013).

Mode of action

Expression of Rpi-vnt1 in wild and cultivated potato confers broad-spectrum resistance to late blight 

caused by P. infestans. Resistant potato parts include leaves, stems and tubers. This resistance is based 

on the recognition of a pathogen-specific protein (effector protein) by a host-specific protein (resistance 

or R protein). Upon infection, AVR-VNT1, an effector protein produced by P. infestans, is recognized by 

the R-protein VNT1 expressed in potato (Pel 2010). R-protein mediated immunity, known as Effector 

Triggered Immunity (ETI), results in a form of programmed cell death called a hypersensitive response 

(Morel and Dangl 1997). Consequently, VNT1 activates a signal transduction pathway that leads to 

localized plant cell death or hypersensitive response. Death is restricted to a few plant cells and limits 

the growth and spread of P. infestans throughout the rest of the plant.

Benefits of Potatoes with Late Blight Resistance

As one of the most important potato pathogens, resistance to late blight could have significant benefits 

with respect to reduced use of pesticides, but also could effectively improve yield compared with 

diseased potatoes. A reduction in pesticide use could reduce the cost of production through less 

chemical, fuel, and labor costs. Also, the result could be less chemical residue in food, land, and waste 

water. Even with pesticide application, some disease tends to persist in potato fields so effective 

resistance could result in healthier plants and better yield. Late blight affects foliage and tubers. There’s 

evidence to suggest that tuber damage is far more likely to occur if the plants are infected than by 

merely picking up the disease agent through soil contact. Tuber infection with late blight results in 

lesions, rot, and ultimately economic loss in storage.

1.3 Rationale and Benefits of Developing Potatoes with Low Acrylamide Potential 

The Swedish National Food Administration and Stockholm University announced in April 2002, a 

relationship between the formation of certain blood adducts and the consumption of foods later found 

to contain acrylamide (NTP 2012). Other researchers also studied the mechanism for acrylamide 

formation in food, and the J.R. Simplot Company had also earlier predicted that acrylamide would form 

as a result of the well-known but complex Maillard browning reaction. Cooking or heat treating of foods 

that are rich in reducing sugars and amino acids may result in browning, commonly referred to as the 

Maillard reaction. This reaction occurs between sugars and amino acids, affecting changes in the color, 

flavor, functional properties and nutritional value of food (Obrien and Morrissey 1989). More 

specifically, we found acrylamide formed primarily when the amino acid asparagine along with reducing 

sugars were heated at temperatures above 120°C, as would occur during frying and baking. The 

biochemical basis of acrylamide formation was later published by Stadler et al. (2002).

Various governments responded to the news about finding acrylamide in food by providing guidance 

and surveys. Through one such survey, the FDA determined that potato products contribute 35% of the 

acrylamide exposure through diet in the US. The scientific community has since gathered additional 
J.R. Simplot Company JRS06 Page 21 of 196

information, including epidemiology and toxicology studies. Along with studies to mitigate the levels of 

acrylamide in food, the scientific community has also been working to understand the health 

implications of acrylamide consumption (NTP 2012).

Based on a recent Federal Register notice (FDA 2013), FDA has proposed guidance for industry on the 

reduction of acrylamide levels in food products. In FDA’s Draft Guidance for Industry on Acrylamide in 

Foods, the FDA states that “Reducing acrylamide in foods may mitigate potential human health risks 

from exposure to acrylamide.” A rather extensive list of potential mitigation techniques were 

summarized in the guidance document (FDA 2013) focused primarily on the reducing sugar levels in 

potatoes. These include variety development and selection, focusing on varieties that are more 

resistant to cold-induced sweetening, and lower levels of reducing sugars or asparagine. Many of the 

methods in FDA’s Guidance document are consistent with those reported in the Acrylamide Toolbox 

published by Food Drink Europe (2011). 

The introduction of InnateTM potatoes with low acrylamide potential would provide potatoes that are 

largely indistinguishable from existing varieties. The reduction in asparagine and sugars using InnateTM

technologies and the resulting reduction in acrylamide upon heating will address food industry needs 

with respect to the FDA’s Draft Guidance (2013). As such, this biotechnology approach to lowering 

acrylamide was mentioned in the guidance document as a promising method to develop potato varieties 

with potentially reduced acrylamide. After deregulation, these InnateTM branded potatoes could be 

adopted readily by the food industry following completion of the FDA consultation process (57 Federal 

Register 22984, May 29, 1992). 

In addition, litigation in the state of California as a result of Proposition 65 (OEHHA 2011a; OEHHA 

2011b) resulted in legal settlements with restaurant chains and the retail french fry and potato chip 

manufacturers regarding perceived risks from acrylamide. Some settlements required signs warning 

consumers, but in addition, some manufacturers of retail french fries and potato chips would be 

required to reduce acrylamide in their products or add warning labels. As of December 1, 2011, some 

potato chip manufacturers began including a statement on packaging warning consumers of the 

presence of acrylamide and its relationship to browning in potatoes. Potato processors affected by 

these rulings in California could be motivated to adopt the low acrylamide, InnateTM potato products. 

Following the discovery of acrylamide in foods and in response to governmental concerns, many 

additives were studied and some have shown effectiveness at reducing acrylamide. Some of the more 

promising options include citric acid, enzymes such as asparaginase, and amino acids that substitute for 

asparagine in the Maillard reaction and therefore form products other than acrylamide when heated 

(FDA 2013; Food Drink Europe 2011). 

Another approach to reducing acrylamide could be based on either traditional breeding or 

biotechnology to reduce the precursors for acrylamide. The use of biotechnology allows for specific 

deactivation of genes involved in the formation of asparagine and reducing sugars with tissue specificity. 

In a crop like potatoes, which is highly heterozygous and suffers from inbreeding depression, the speed 

to develop such changes could take decades through traditional breeding. In contrast, biotechnology 

allows for specific changes in traditional varieties much faster, while maintaining the desired 

characteristics of the original parent.

The W8 event contains expression cassettes that could lower levels of reducing sugars by multiple 

mechanisms. Through the transformation with pSIM1278, we introduced a silencing cassette for the 
J.R. Simplot Company JRS06 Page 22 of 196

promoters of the starch associated gene (R1) and the phosphorylase-L gene (PhL) inserted between 

promoters that are predominantly active in tubers. Together, these traits function by slowing the 

conversion of starch to reducing sugars (glucose and fructose). The result of this silencing strategy was 

lowered levels of the reducing sugars glucose and fructose at harvest or when analyzed at one month 

after harvest (Collinge and Clark 2013). Another gene silencing cassette, for the invertase gene (Ye et al. 

2010), was introduced into event W8 using the transformation vector pSIM1678. A reduction in 

invertase from gene silencing should reduce the conversion of sucrose to reducing sugars during cold 

storage, inhibit formation of sugar related defects such as sugar ends in french fries, and result in even 

lower levels of acrylamide upon frying. Overall benefits of silencing R1, PhL, and VInv include improved 

quality, especially relating to color control, and thus contributing to the desired golden brown colors 

required by most french fry or chip customers. Also, the reducing sugars react with amino acids, such as 

asparagine, to produce Maillard products including acrylamide.

The Russet Burbank W8 produces tubers with greatly reduced potential to form acrylamide, thus 

addressing a potentially critical food safety issue for the potato industry, particularly in light of recent 

toxicology studies (NTP 2012). 

Many methods have been tested and research is ongoing to reduce acrylamide through process 

changes, reduction in dextrose, and additives such as asparaginase, citrate, and competing amino acids. 

The required capital expense to implement process changes throughout the potato industry would cost 

millions of dollars. In addition to the expense, these process changes have significant drawbacks 

including potentially negative flavors associated with additives such as asparaginase or citrate. Typically, 

fry manufacturers add dextrose during processing of french fries to develop the desired golden brown 

color, but dextrose also increases the formation of acrylamide through the Maillard reaction. Significant 

reductions in acrylamide occur by merely omitting dextrose from the process; however, the signature 

golden brown colors must then be developed some other way (such as though the addition of colors like 

annatto). The use of alternate colors, results in an absence of the typical flavors that develop through 

those browning reactions. Another challenge with the use of additives to reduce reactants like 

asparagine will be the principle of moisture migration that occurs during frozen storage resulting in a 

return of asparagine to the surface and increased acrylamide compared with the initial testing at the 

time of processing. In contrast, the InnateTM potatoes will significantly reduce acrylamide without the 

use of new additives or process changes. The resulting foods will have the same appealing color, flavors, 

and aromas as they currently have today. 

1.4 Rationale and Benefits of Developing Potatoes with Reduced Black Spot 

The blackening that occurs after potatoes are bruised affects quality and recovery in processing french 

fries and chips. Potatoes that have been damaged and show black spot must be trimmed or could be 

rejected before processing, resulting in quality challenges or economic loss or both. In many instances 

potato growers have contracts that provide incentives for delivering “bruise free” potatoes. A 

significant reduction in black spot could result in higher profit for the farmer. Also, these black spots are 

considered defects in potato chip or french fry processing, causing economic loss from trimming or 

culling potatoes with black spot. Potatoes may develop black spots from pressure bruising that results 

from the weight of potatoes in deep stacks during storage. The weight of the potatoes causes damage 

to the potato tissue resulting in dark colors that must be removed through trimming. Another possible 

advantage of silencing black spot could be the enabling of new markets for “freshly cut” potatoes, 

without pre-cooking or using sulfites or other preservatives to maintain color and flavor.
J.R. Simplot Company JRS06 Page 23 of 196

1.5 Rationale and Benefits of Developing Potatoes with Reduced Levels of Reducing Sugars

In breeding programs for processing potatoes, one of the most desired characteristics is low levels of the 

reducing sugars glucose and fructose. High levels of reducing sugars lead to undesirable dark colors and 

bitter flavors after frying of fries and chips. High sugar levels can result from any stress to the plants 

during growing or harvesting such as drought or heat stress (Bethke et al 2009), but develop rapidly with 

low temperature storage (Driskill et al. 2007). Typical storage temperatures for potatoes for the frozen 

fry market are 46 - 48 ̊F (Driskill et al. 2007). This temperature is the optimum for maintaining sugar 

levels while minimizing the chance for disease. If potatoes could be stored at a lower temperature 

without adversely affecting sugar level, the result would be less damage from disease and higher yield 

because of lower respiration rates resulting in a greater net yield of potatoes.

Multiple pathways exist for carbohydrate metabolism in potatoes and the DNA inserted into W8 targets

several of those paths through gene silencing. The vector pSIM1278 contains silencing cassettes for 

promoters of the water dikinase R1 gene and starch phosphorylase Phl gene. The R1 gene functions by 

phosphorylating the C6 position on glucosyl residues in starch (Ritte et al. 2006). Silencing of this gene 

could lead to an accumulation of starch in plants and a reduction in cold sweetening as shown by 

Lorberth et al. (1998). The phosphorylase Phl gene is also thought to be responsible for degradation of 

starch to sugar during cold storage (Sonnewald et al. 1995). Kamrani et al. (2011) showed a reduction in 

sugar accumulation in potatoes with silencing of the starch phosphorylase L gene. The vector pSIM1678 

silences the vacuolar acid invertase gene (VInv), responsible for catalyzing the reaction converting 

sucrose into glucose and fructose.

With introduction of pSIM1278 into Russet Burbank potatoes (Collinge and Clark 2013), we found 

silencing of the promoters for R1 and Phl to result in slight reductions in reducing sugars at the time of 

harvest or after 1 month of storage. In the event W8, we have silenced the VInv gene through 

introduction of a gene silencing cassette contained in the pSIM1678 DNA insert. Silencing of VInv will 

result in lower levels of reducing sugar throughout the storage period. The VInv silencing may also allow 

for lower temperature storage which will reduce yield loss from respiration and disease. There is also 

evidence that VInv silencing reduces the incidence of high sugar potatoes and sugar ends, both quality 

attributes that result in economic loss by causing potatoes to be rejected by processors, resulting in 

lower prices as they are only suitable for making dehydrated potatoes.

An early discovery of high sucrose levels in wild tomatoes led researchers to determine that some 

tomatoes contained a gene that silenced the production of acid invertase (Klann et al. 2006). 

Researchers assayed the tomatoes for several common enzymes and found that the cause of high levels 

of sucrose was associated with lack of acid invertase activity. 

More specific work with silencing of vacuolar acid invertase in potatoes has demonstrated the efficacy 

of that approach for lowering levels of reducing sugars in cold stored potatoes (Ye et al. 2010; Bhaskar

et al. 2010). Both research groups reported effective reductions in reducing sugars during cold storage 

with silencing of vacuolar acid invertase. In addition, with less reducing sugar, significantly lower levels 

of acrylamide were found after frying. 

Benefits of Potatoes with Reduced Levels of Reducing Sugars

At the time of harvest, most potatoes have low levels of the reducing sugars, fructose and glucose. For 

optimum quality, potatoes used for processing into french fries and chips, should be low in reducing 

sugars to give the processor ultimate control in creating the desired finished product color. When 
J.R. Simplot Company JRS06 Page 24 of 196

potatoes are low in reducing sugar, colors can be readily developed by adding reducing sugars, such as 

glucose before frying. If potatoes contain too much reducing sugar, they can become excessively brown 

during cooking, losing their ideal flavor and appearance. Many potatoes must be stored for as long as 3 

to 12 months before processing, and the ideal storage potato maintains a low level of reducing sugar 

throughout storage. A consistent focus of breeders for processing potatoes would be to have low 

reducing sugars at the time of harvest that remain low throughout their storage life (Driskill et al. 2007). 

High levels of reducing sugars can result in an overall excessive browning during processing or may 

result in dark ends or mottled appearance. With excessively high sugar levels, potatoes may become 

unusable for fries or chips and must be sold at a lower price into the dehydrated potato sector. 

Management of reducing sugars presents significant challenges and potential financial loss because 

processors reject loads with reducing sugar content above 2%, which is approximately 20% of potatoes 

produced. 

High levels of reducing sugars affect the overall potato color, but sugars are often concentrated into 

excessively dark areas either throughout the potato or at the end of a potato strip. If levels of sugar 

ends are extremely high, the potatoes cannot be used for premium french fries, and may result in loss of 

economic value to the processor. For the Russet Burbank event W8 presented here, there should be 

significant economic and quality benefits from lower levels of reducing sugars. In addition, less reducing 

sugar will result in lower levels of acrylamide after cooking, a significant advantage considering the 

health concerns associated with acrylamide (NTP 2012; FDA 2013).

1.6 Conclusions: Rationale for Potatoes with Late Blight Resistance, Low Acrylamide, Reduced 

Black Spot, and Lowered Reducing Sugars

In summary, there is an important need to introduce late blight resistance into the Russet Burbank 

potato variety, while simultaneously lowering reducing sugars, black spot, and the potential to form 

acrylamide in fried and baked potatoes. Considering that the most popular North American potato 

variety, the Russet Burbank, is sterile, it was not feasible to make such changes using traditional 

breeding. Therefore, we accomplished this goal by applying InnateTM technologies. The Russet Burbank 

represents 20.3% of seed acres in the US (NPC 2013), and is widely used in french fry and dehydrated 

processed potatoes, and also remains the standard of excellence for table stock. We now seek 

nonregulated status for Russet Burbank event W8 based on the weight of evidence demonstrating the 

safety of the Russet Burbank W8 potatoes. We have transformed plants with genomic DNA from potato 

and wild potato where the integrated genetic material and the transformed potatoes are as safe as the 

Russet Burbank controls.
J.R. Simplot Company JRS06 Page 25 of 196

1.7 References

Bethke, P.C., Sabba, R., Bussan, A.J. (2009). Tuber water and pressure potentials decrease and sucrose 

contents increase in response to moderate drought and heat stress. Am J Pot Res. 86: 519-532.

Bhaskar, P.B., Wu, L., Busse, J.S., Whitty, B.R., Hamernik, A.J., Jansky, S.H., Buell, R.B., Bethke, P.C., and 

Jiang, J. (2010). Suppression of the Vacular Invertase Gene Prevents Cold-Induced Sweeting in Potato. 

Plant Physiology. 154: 939-948.

Black, W., Mastenbroek, C., Mills, W.R. and Peterson, L.C. (1953). A proposal for an international 

nomenclature of races of Phytophthora infestans and of genes controlling immunity in Solanum 

demissum derivatives. Euphytica. 2: 173–178.

Collinge, S. and Clark, P. (2013). Petition for Determination of Nonregulated Status for InnateTM Potatoes 

with Low Acrylamide Potential and Reduced Black Spot Bruise: Events E12 and E24 (Russet Burbank); 

F10 and F37 (Ranger Russet); J3, J55, and J78 (Atlantic); G11 (G); H37and H50 (H). J.R. Simplot Company 

Petition JRS01 (USDA Petition 13-022-01p).

Colon, L.(1994).Resistance to Phytophthora infestans in Solanum tuberosum and wild Solanum species. Thesis 

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2.0 The Biology of Potato

This section is based, in part, on the Organization for Economic Co-Operation and Development’s (OECD) 

series on harmonization of regulatory oversight in biotechnology no. 8, Consensus document on the 

biology of Solanum tuberosum, 1997.

This section describes potato biology and how it contributes to the effective management of the 

InnateTM potatoes. All commercial potato varieties must be propagated through cloning, effectively 

mitigating many concerns about increased plant pest potential such as seed dispersal, survival outside of 

cultivation, or outcrossing. 

2.1 History of Potato

The cultivated potato traces its origin to Andean and Chilean landraces developed by pre-Colombian 

cultivators. These landraces exhibit morphological and genetic diversity, and are distributed from 

western Venezuela to northern Argentina, and in southern Chile (Spooner et al. 2005). Introduced to 

Europe by Spain in 1536, the potato was subsequently conveyed by European mariners to territories and 

ports throughout the world. Once established in Europe, the potato soon became an important food 

staple and field crop. But lack of genetic diversity, due to the fact that very few varieties were initially 

introduced, left the crop vulnerable to disease. In 1845, a plant disease known as late blight, caused by 

the fungus-like oomycete pathogen P. infestans, spread rapidly through the poorer communities of 

western Ireland, resulting in the crop failures that led to the Great Irish Famine. Late blight remains a 

serious disease of potatoes today.

Potato remains an essential crop in Europe, where per capita production is still the highest in the world, 

but the crop also made its way back over the Atlantic Ocean to North America. The industry gained 

greater importance particularly in western states after the horticulturist Luther Burbank developed the 

Russet Burbank potato in 1872. In 2012, the United States harvested 46.7 billion lbs of potatoes, enough 

to make it the world's fourth biggest producer (NPC 2013). Potatoes in the United States are grown in 

many states, although about half of the crop comes from Idaho and Washington. The top ten potato 

producing states of Idaho, Washington, Wisconsin, Oregon, Colorado, North Dakota, Minnesota,

California, Michigan, and Maine account for almost 88% of the United States potato crop (NPC 2013). 

Most potatoes are harvested in July through October. Americans eat, on average, approximately 50 kg 

of potatoes per person per year (NPC 2013). The most rapid expansion in production and consumption 

of potatoes has occurred in southern and eastern Asia. China is now the world's largest potatoproducing country, and nearly a third of the world's potatoes are harvested in China and India.

2.2 Use of the Potato as Food and Feed in the USA

Most harvested potatoes are used for food but some are grown for seed for planting. According to the

National Potato Council, the commercial uses include 36% as frozen, 26% sold fresh, 15% chips and 

shoestrings (julienne cut crispy snacks), 11% dehydrated, 6% seed potatoes, 4% other frozen products,

and 1% canned (NPC 2013).

Raw potato waste products (peels, out of specification raw potatoes, or other non-processed raw potato 

products) and processed discards (french fry, hash brown, etc.) are routinely incorporated into feed 

rations at livestock feedlot operations including those owned by the J.R. Simplot Company. 

Approximately 15,000 tons of processed potato waste and 60,000 tons of raw potato waste are 
J.R. Simplot Company JRS06 Page 29 of 196

integrated into livestock feed at the J.R. Simplot Company’s Grandview, ID feedlot on an annual basis. 

The typical feed ration used for cattle finishing at J.R. Simplot Company feedlots includes 8-14% potato 

waste. One of the major benefits of using the potato waste is that it is not discarded into landfills, but 

used in a sustainable manner as part of a long-term approach to reducing waste and integrating

sustainability with respect to the Earth’s valuable natural resources.

2.3 Taxonomy of the Genus Solanum

The Solanaceae family contains several well-known cultivated crops such as tomato (S. lycopersicum also 

referred to as Lycopersicon esculentum), eggplant (S. melogena), tobacco (Nicotiana tabacum), pepper 

(Capsicum annuum) and potato (S. tuberosum). Within the genus Solanum, over a thousand species 

have been recognized. Potatoes will not hybridize with non-tuber bearing species (tomato, eggplant, 

etc.) including weeds commonly found in and around commercial potato fields (Love 1994). 

The genus Solanum is divided into several subsections, of which the subsection potatoe contains all 

tuber-bearing potatoes. The subsection potatoe is divided into series, of which tuberosa is relevant to 

this document. Within the series tuberosa approximately 54 species of wild and cultivated potatoes are 

found. One of these is S. tuberosum.

S. tuberosum is divided into two subspecies: tuberosum and andigena. The subspecies tuberosum is the 

cultivated potato widely in use as a crop plant in, for example, North America and Europe. The 

subspecies andigena is also a cultivated species, but cultivation is restricted to Central and South 

America (Hanneman 1994).

2.4 Genetics of Potato

The basic chromosome number in the genus Solanum is twelve. S. tuberosum subsp. tuberosum can be 

diploids (2n=2x=24) or tetraploids (2n=4x=48). The diploids have a limited range in parts of South 

America, while the tetraploids are the most commonly cultivated all over the world. How tetraploidy 

originated in potato is unclear. The cultivated S. tuberosum subsp. tuberosum can be either an 

autotetraploid (doubling of the chromosomes of a diploid species) or an allotetraploid (doubling of the 

chromosomes of a diploid hybrid between two related species).

While nearly all diploid species are self-incompatible, the cultivated tetraploid S. tuberosum subsp. 

tuberosum is capable of self-pollination (selfing). Plaisted (1980) has shown that under field conditions 

selfing is most likely for tetraploid S. tuberosum, with 80-100 percent of the seeds formed due to selfing. 

Conner and Dale (1996) collected outcrossing data from several field experiments with genetically 

modified potatoes, performed in New Zealand, the United Kingdom and Sweden. In each study, the 

outcrossing rate was zero when receiving plants were separated by more than 20 meters from the 

genetically modified ones. Although many Solanum species are fertile, it appears that a large number of 

the tetraploid cultivated S. tuberosum subsp. tuberosum cultivars have reduced fertility. The Russet 

Burbank variety is sterile and has no outcrossing potential, and the same is true with respect to the W8 

Russet Burbank.
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2.5 Potato Growth and Life Cycle

Potatoes grown in both commercial and organic farming operations go through the following five

general growth stages of development (Dwelle 2003):

1) Sprouts develop from eyes on seed tubers, grow upwards, and emerge from the soil. Roots 

initiate development at the base of the emerging sprouts.

2) Vegetative growth begins. Leaves and branch stems develop from aboveground nodes. Roots 

and stolons develop at belowground nodes.

3) Tubers begin to form at stolon tips, but do not enlarge. Flowering is initiated at the end of this 

stage.

4) Tuber cells expand with the accumulation of water, nutrients, and carbohydrates.

5) Vines turn yellow and lose leaves, tuber growth slows and vines die. Tuber maturation is 

completed as tuber skins set.

In greater detail, the potato life cycle begins with certified seed potatoes, which are cut into pieces 

containing at least one eye and are typically planted in the field from February through May. Plants will 

establish by forming vegetative tissues, including roots, leaves, stems, and stolons. Tuber formation 

begins approximately 30-60 days after planting. Tubers are derived from lateral underground buds that 

develop at the base of the main stem, when kept underground. These develop into stolons due to 

diagravitropical growth. When conditions are favorable for tuber initiation, the elongation of the stolon 

stops, and cells located in the pith and the cortex of the apical region of the stolon first enlarge and then 

divide longitudinally. The combination of these processes results in the swelling of the subapical part of 

the stolon. During enlargement, tubers become the largest nutrient sink of the potato plant storing large

amounts of carbohydrates (mainly starch) and also significant amounts of protein. Furthermore, potato

tubers decrease their general metabolic activity and as such behave as typical storage sinks. 

Potato tubers of the Russet Burbank variety are harvested from 140 to 150 days after planting (PAA 

2013), which may vary with production area and marketing conditions. Typical potatoes are about 20-

24% dry matter, of which approximately 60-80% consists of starch. After potato vines die back, the 

tuber skins thicken and harden which provides greater protection to tubers during harvest and blocks 

entry of pathogens. Although dormancy is defined as the absence of visible growth, dormant tuber 

meristems are metabolically active. In general, rates of many cellular processes such as respiration, 

transcription, and translation are suppressed during dormancy and non-dividing, dormant tuber 

meristems are arrested in the G-1 phase (a period prior to the synthesis of DNA in mitosis). Activation of 

cell metabolism during early spring triggers the development of sprouts from the eyes of the primary 

tuber.

2.6 Seed Propagation 

Potato is clonally propagated, which means that tubers rather than seeds are used for planting. A single 

potato plant produces approximately 12 tubers, therefore it takes multiple years to propagate a new 

potato variety and generate sufficient amounts of tuber seed needed for commercialization. A typical 

seed bulk-up program would progress as follows. In the initial year, propagation in tissue culture would

produce about 100 plants, and then these plants are grown in a greenhouse to produce thousands of 

mini-tubers, also called “nuclear seed”. In the second year, mini-tubers are planted at field sites with 

the lowest possible incidence of diseases and pests, producing generation-1 (G1) seed. Those G1 tubers 

are grown in the third year to increase the amount of tubers, producing G2 seed. In the fourth, fifth, and 
J.R. Simplot Company JRS06 Page 31 of 196

sixth years, bulk-up continues with commercial seed often representing G3, G4, and G5 tubers 

respectively.

2.7 Variety Development

Potato varieties take many years to develop (see Figure 2-1). The decision to establish a new variety is 

based on many factors such as need in the market place, potential consumer acceptance, and pest 

tolerance or resistance. Potato varieties do not have a high frequency of introduction and 

discontinuation compared to some other crops such as field corn or soybeans. Since potatoes are 

clonally propagated, there is a reduced risk of varietal dilution due to cross pollination.
J.R. Simplot Company JRS06 Page 32 of 196

Figure 2-1. Potato Variety Development Schematic
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2.8 Recipient Potato Varieties

The potato variety chosen for modification, Russet Burbank, represents significant value to the potato 

industry and a relatively large percentage of the overall acreage. Used for fries and table stock, in 2012 it 

comprised 20.3% of seed acreage in the US (NPC 2013).

2.9 Typical Agronomic Practices

Examples of typical agronomic practices for a successful potato crop are described in Table 2-1.

Table 2-1. Example of Agronomic Inputs for Russet Varieties

Russet Varieties

Planting Date April 1 to May 10

Planting Rate 15,000 - 18,000 seed pc or 17 – 23 cwt/A

Row Spacing 34-36” between rows 

Seed Spacing 10-12” within row

Fertilizer For 600 cwt/A yields and optimum soil 

test levels:

250 lb N – 100 lb P2O5 – 330 lb K2O per 

acre

Yield/Plant 2-4 lb

Yield/Acre 400-700 cwt/A

Harvest Date September 1 to October 15

2.10 Pollination and Outcrossing

Potato is clonally propagated, which means that tubers rather than seeds are used for planting. 

Therefore, pollination to produce seed is not a factor in major commercial potato production. For 

example, if InnateTM potatoes were planted in close proximity to organic potatoes, any cross pollination 

would not impact the organic tubers.

Some of the most important potato varieties, including Russet Burbank, produce few flowers and are 

male sterile. Many cultivars flower less than wild material, and flowers that do form often drop after 

pollination resulting in no berry formation. Flowering has no impact on tuber development.

Approximately 80% of seed produced by fertile varieties is derived from self-pollination (Plaisted 1980). 

Cross-pollination is typically mediated by certain insects including bumblebees (Bombus spp.) that 

typically do not travel much more than 3 kilometers, rather than wind pollination (OECD 1997). 

Additionally, other bee species such as honey bees (Apis mellifera) are not pollinators of potatoes since 

potato flowers do not contain ample nectar (OECD 1997). Field evaluations in New Zealand, the United 

Kingdom and Sweden demonstrated through genetic testing that outcrossing rates were zero when

receiving plants were separated by more than 20 meters from genetically modified plants (Conner and 

Dale 1996). Additionally, potato pollen grains are extremely temperature sensitive. Pallais et al. (1988) 

showed that pollen viability decreased by 30 to 70% when exposed to temperatures of 30°C for up to 30 

minutes.
J.R. Simplot Company JRS06 Page 34 of 196

2.11 Wild potatoes in the U.S. 

This discussion is based on data in the U.S. Potato Genebank and on a monogram published by the 

American Society of Plant Taxonomists entitled Wild Potatoes (Solanum section Petota; Solanaceae) of 

North and Central America (Spooner et al. 2004). The USDA maintains the US Potato Genebank and 

participates in the Association of Potato Inter-genebank Collaborators (APIC), which has produced a 

global inventory of wild potato genetic resources available on the Internet, the Inter-genebank Potato 

Database (IPD 2011), which includes the U.S. Potato Genebank records. Many of the IPD records from 

the U.S. were obtained by John Bamberg and associates (including Spooner) during a decade of 

collecting wild potatoes in the Southwest U.S. (Bamberg et al. 2003). 

The only two wild potato species that grow within the borders of the USA, and for which specimens exist 

in gene banks, include the tetraploid species S. fendleri (recently reclassified as S. stoloniferum; 

however, some sources, including the IPD, still use the S. fendleri designation) and the diploid species S.

jamesii (Bamberg et al. 2003; IPD 2011; Bamberg and del Rio 2011a; Bamberg and del Rio 2011b; 

Spooner et al. 2004). Love (1994) reported that a third species, S. pinnatisectum, is also a native species 

in the USA. However, Spooner et al. (2004) determined that what was previously thought to be S. 

pinnatisectum was in fact S. jamesii. Through more than 10 years of field work and assessments of 

existing records, Bamberg et al. (2003) and Spooner et al. (2004) established the presence of only these 

two species, S. fendleri and S. jamesii, in the U.S. These researchers also attempted to verify previously 

recorded locations, and through this process, updated the maps of current known locations of these 

species, providing latitude and longitude locations for each documented population (Bamberg et al.

2003) and distribution maps (Spooner et al. 2004). These species mostly reside in dry forests, scrub 

desert, and sandy areas at altitudes of 5,000 to 10,000 feet, well isolated from most commercial 

production areas (Bamberg and del Rio 2011a).

While there is some overlap between the acreage used for commercial production and occurrence of 

wild species on a county level, the majority of the potato production in the United States is not in wild 

potato zones (Figure 2-2). However, there is a possibility that a few wild potato plants may be growing 

near potato fields (Love 1994). Spooner et al. (2004) describe S. jamesii habitat in the U.S. as among 

boulders on hillsides, sandy alluvial stream bottoms, in gravel along trails or roadways, rich organic soil 

of alluvial valleys, sandy fallow fields, grasslands, juniper-pinyon scrub deserts, oak thicket, coniferous 

and deciduous forests at elevations between 4,500 to 9,400 feet. They describe S. fendleri habitat 

similarly, and at elevations between 4700 to 11,200 feet. The risk of hybridization between commercial 

varieties and wild species is low because of the lack of geographical overlap and the fact that potatoes 

are grown from tuber seed. Furthermore, studies discussed in Conner and Dale (1996) indicated that a 

separation of 20 meters will be sufficient to prevent outcrossing between wild and commercial 

potatoes. 

Love (1994) evaluated the risk associated with growing transgenic potatoes in Canada and the USA and 

concluded that, given the number and potency of barriers to hybridization and more specifically to 

introgression and stabilization, the only sound conclusion is that gene introgression into wild Solanum

species will not occur under natural conditions in these geographies. Therefore, according to Love 

(1994), potato gene movement from commercial fields to wild potato species would not occur.
J.R. Simplot Company JRS06 Page 35 of 196

Figure 2-2. Potato Production in the United States

(County Information is for states with wild potato populations (Hijmans and Spooner 2001))

Based on conversations with Bamberg, Love (1994) reported that no one has ever reported finding 

hybrids between native and cultivated potatoes in the U.S., although gene transfer has been 

accomplished using special laboratory techniques (Love 1994). Love concluded that, based on the 

barriers that exist (including geographic isolation as described previously), endosperm imbalances, and 

multiple ploidy levels, natural hybridization is highly unlikely, and gene introgression (cross hybridization 

over multiple generations) is impossible or at least highly improbable (Love 1994). More recently, the 

US EPA has concluded that, based on its review of the scientific literature, successful gene introgression 

between native and cultivated potatoes in the U.S. is virtually excluded due to constraints of 

geographical isolation and other barriers to natural hybridization. These barriers include incompatible 

(unequal) endosperm balance numbers that lead to endosperm failure and embryo abortion, multiple 

ploidy levels and incompatible mechanisms that do not express reciprocal genes to allow fertilization to 

proceed. No natural hybrids have been observed between these species and cultivated potatoes in the 

U.S. (US EPA 2011). Based upon these biological barriers to hybridization, it is unlikely that inter-species 

pollination would occur with the InnateTM potatoes that are the subject of this petition.

2.12 Weediness

Standard growing practices for potatoes make it highly unlikely that potatoes would persist in a field 

from one crop cycle to the next. Where potatoes are a rotation crop, as is often the case, other crops 

such as alfalfa, corn or wheat would be grown following potatoes. In that situation, any potatoes left in 
J.R. Simplot Company JRS06 Page 36 of 196

the field would be eliminated by tilling, field preparations with herbicides, and harsh winters. In the rare 

event that potatoes survived mixed in with another crop, they would have to face the same challenges 

during the next year. 

Potatoes are typically grown on a 3 year minimum rotation to minimize soil-borne disease buildup such 

as white mold, pink rot, Pythium leak, Verticillium wilt, and powdery scab (Hopkins et al. 2003). 

Potatoes left to grow in the field are not desired as they can harbor disease, have no protection with 

fungicide, and could compete with the desired rotation crop the following year. For these reasons, it is 

standard practice for potato growers to monitor and control such growth, either with herbicide or 

tillage. Soil is disturbed greatly during potato growth and harvest operations, therefore requiring tillage 

prior to planting the rotation crop. Typically a broad spectrum pre-emergence herbicide is applied in the 

rotation crop to control a wide variety of weeds. Potato growers rarely leave the ground fallow 

following harvest to maximize economic return. If potatoes grew inadvertently in the field left fallow, 

they would not be protected with insecticide or fungicide and would be susceptible to insects such as 

Colorado potato beetle and diseases such as early blight, further decreasing the chance of survival and 

reproduction.

Outside of cultivation, potato seedlings from tubers have difficulty establishing themselves as they 

cannot compete effectively with other plants (Love 1994). Potato is not known as a colonizer of 

unmanaged ecosystems. In climax vegetation, potatoes are not able to compete with other species such 

as grasses, trees and shrubs (CFIA 1996), virtually eliminating any chance for them to become feral. 

Furthermore, potatoes generally do not cause toxic effects to other plants or animals including nontarget organisms.

Although almost all growers use tubers for planting, there is a small market for “true potato seed” (TPS). 

TPS is seed that is produced as a result of pollination. The seed is produced inside fruits that resemble 

small tomatoes on the potato vine. The major disadvantage of TPS is that it segregates for numerous 

traits because potato is highly heterozygous and each seed produced is potentially a new variety. When 

TPS is used for planting, harvested tubers will be variable in quality. TPS plants take longer to establish 

themselves and set tubers, resulting in lower yield compared to plants grown from tuber seed. Some 

growers in tropical areas use TPS because it is easy to ship and free of pathogens (Dwelle 2003). Love 

(1994) reported that botanical seed (TPS, not tubers or “seed potatoes”) can survive and germinate for 

periods of time in excess of seven years however, it is unlikely that plants from TPS would persist for the 

reasons stated above.

2.13 Characterization of the Recipient Potato Cultivar

Russet Burbank. Luther Burbank developed this variety in the early 1870s. Plants are vigorous and 

continue vine growth throughout the season. Stems are thick, prominently angled and finely mottled. 

Leaflets are long to medium in width and light to medium green in color. The blossoms are few, white 

and not fertile. The cultivar is tolerant to common scab but is susceptible to Fusarium and Verticillium

wilts, leafroll and net necrosis, potato virus Y, and late blight. Plants require conditions of high and 

uniform soil moisture and controlled nitrogen fertility to produce tubers free from knobs, pointed ends 

and dumbbells. Jelly-end and sugar-end develop in tubers when plants are subjected to stress. The 

tubers produced are large brown-skinned and white-fleshed, display good long-term storage 

characteristics, and represent the standard for excellent baking and processing quality. The variety is 

sterile and widely grown in the Northwest and Midwest, especially for the production of french fries.
J.R. Simplot Company JRS06 Page 37 of 196

2.14 Conclusions: The Biology of Potato

The propagation of commercial potato varieties through cloning mitigates concerns about increased 

plant pest potential such as seed dispersal, survival outside of cultivation, or outcrossing. Potatoes are 

rarely grown from true potato seed because the offspring would show such extreme diversity they 

would not be useful as commercial potatoes. In addition, potatoes are not known to escape from 

commercial fields or show weediness potential. Wild potato varieties are rare in the United States and 

geographically isolated from commercial production areas, further reducing concerns about crosspollination with wild species.

An understanding of the biology of potatoes shows the extremely difficult challenges associated with 

simultaneously incorporating multiple traits through traditional breeding. Thus, potatoes benefit greatly 

by the use of InnateTM technologies which provide a superior alternative to breeding, through the 

addition of non-coding DNA, and result in new potato varieties that are not plant pests.
J.R. Simplot Company JRS06 Page 38 of 196

2.15 References

Bamberg, J.B., del Rio, A., Huaman, Z., Vega, S., Martin, M., Salas, A., Pavek, J., Kiru, S., Fernandez, C., 

Spooner, D. (2003) A decade of collecting and research on wild potatoes of the southwest. Am J of 

Potato Res, 80: 159-172.

CFIA. (1996). Biology Document BIO1996-09: The Biology of Solanum tuberosum (L.) (Potatoes). 

Canadian Food Inspection Agency. http://www.inspection.gc.ca/plants/plants-with-noveltraits/applicants/directive-94-08/biology-documents/solanum-tuberosum-l-

/eng/1330982063974/1330982145930 (Accessed 23 August 2012).

Conner, A.J., Dale, P.J., (1996). Reconsideration of pollen dispersal data from field trials of transgenic 

potatoes. Theor Appl Genet. 92: 505-508.

Dwelle, R.B. (2003). Potato growth and development. In: Potato Production Systems (Eds. Stark JC and 

Love S) University of Idaho Agricultural Communications, Moscow. 9-19.

Hanneman, R.E. (1994). The testing and release of transgenic potatoes in the North American Center of 

diversity. In: Biosafety of Sustainable Agriculture: Sharing Biotechnology Regulatory Experiences of the 

Western Hemisphere (Eds. Krattiger AF and Rosemarin A). ISAAA, Ithaca and SEI, Stockholm, pp. 47-67.

Hijmans, R.J., Spooner, D.M. (2001). Geographic distribution of wild potato species. Am J of Botany. 88: 

2101-2112.

Hopkins, B.G., Stark, J., Hafez, S.L., Hutchinson, P.J.S., Patterson, P., Miller, J., Thornton, M., and Alvarez, 

J. (2003). Field Selection, Crop Rotations, and Soil Management. In: Potato Production Systems (Eds. 

Stark JC and Love S) University of Idaho Agriculutral Communications, Moscow, pp. 1-7.

IPD. (2011). Inter-genebank Potato Database. Wild Potato Collection. http://research.cip.cgiar.org/IPD/ 

(Accessed 10 November 2011).

Love, S. (1994). Ecological risk of growing transgenic potatoes in the United States and Canada. Am 

Potato J. 71: 647-658.

NPC. (2013). National Potato Council, 2013 Potato Statistical Yearbook.

OECD. (1997). Organization for Economic Co-Operation and Development, Consensus document on the 

biology of Solanum tuberosum subsp. tuberosum (potato). 38 pages.

Pallais, N., Mulcahy, D., Fong, N., Falcon, R., and Schmiediche, P. (1988). The relationship between 

potato pollen and true seed: effects of high temperature and pollen size. In Sexual Reproduction in 

Higher Plants. Spinger-Verlag, Berlin Heidelberg.

Plaisted, R.L. (1980). Potato. In: Hybridization of crop plants (Eds. Fehr, W.R. and Hadley, H.H.). American 

Society of Agronomy and Crop Science Society of America, publishers, Madison, Wisconsin. 483-494.
J.R. Simplot Company JRS06 Page 39 of 196

PAA. (2013). Potato Association of America Website. 

http://potatoassociation.org/Industry%20Outreach/varieties/Russets/russet_burbank.html. (Accessed 

Dec 18, 2013)

Spooner, D.M., van den Berg, R.G., Rodriguez, A., Bamberg, J.B., Hijmans, R.J., and Cabrera, S.I.L. (2004).

Wild potatoes (Solanum section Petota: Solanaceae) of North and Central America. Syst Bot Monogr. 68: 

1-209.

Spooner, D.M., McLean, K., Ramsay, G., Waugh, R., and Bryan, G.J. (2005). A single domestication for 

potato based on multilocus amplified fragment length polymorphism genotyping. P Natl Acad Sci. 102: 

14694-14699.

US EPA. (2011). United States Environmental Protection Agency. Introduction to Biotechnology 

Regulation for Pesticides. http://epa.gov/oppbppd1/biopesticides/regtools/biotech-reg-prod.htm 

(Accessed 27 January 2012).
J.R. Simplot Company JRS06 Page 40 of 196

3.0 Development of W8 Russet Burbank: Description of Marker-Free DNA Transformation

3.1 Introduction

Simplot’s InnateTM technologies comprise many aspects of plant biology all working together to produce 

traits of interest in transformed plants. Specifically, the potatoes were modified to introduce late blight 

resistance while decreasing acrylamide potential, reducing sugars, and black spot in the tuber. The 

expression cassettes, including promoters and their silencing or expression cassettes, used to confer 

these traits are all derived from the genomes of potatoes or sexually compatible species. The trait 

specific genes were combined with backbone elements to create the vectors for Agrobacteriummediated transformation resulting in potatoes that specifically incorporate the expression cassette and 

not backbone elements. We chose the most prominent variety for table stock and french fries, Russet 

Burbank, as the target for transformation.

The W8 event was developed by first transforming Russet Burbank with the plasmid pSIM1278 to 

reduce expression of asparagine synthetase (ASN1), polyphenol oxidase (PPO5), and reducing sugars. In 

a second transformation, another construct, pSIM1678, was introduced which includes the late blight 

resistance gene and an invertase silencing cassette. These constructs are described in detail in Chapter 

4: Donor Genes and Regulatory Sequences.

3.2 Description of the Transformation System

The C58-derived Agrobacterium strain AGL1 was developed by precisely deleting the transfer DNA of the 

hyper-virulent plasmid, pTiBo542 (Lazo et al. 1991). Transformed plants were grown on media 

containing the antibiotic, timentin, which prevents survival of Agrobacterium, and thus selects for plants 

free of Agrobacterium. Following selection, plants are both antibiotic and Agrobacterium free, with the 

potato-derived expression cassettes inserted into the plant’s genome. 

3.3 Transformation Method

Stock plants were maintained in magenta boxes with 40 ml half-strength M516 (Phytotechnology)

medium containing 3% sucrose and 2 g/l gelzan (propagation medium). Potato internode segments of 

four to six mm were cut from four-week old plants, infected with the Agrobacterium AGL1 strain 

carrying pSIM1278, and transferred to tissue culture media containing 3% sucrose and 2 g/l gelzan (cocultivation medium). Infected explants were transferred, after two days, to M404 (Phytotechnology) 

medium containing 3% sucrose, 2 g/l gelzan, 300 mg/l timentin and 1.2 ml plant protection medium 

(Phytotechnology) to eliminate Agrobacterium (hormone-free medium). Evidence that the plants were 

Agrobacterium-free was obtained by incubating stem and/or leaf fragments of transformed events on 

nutrient broth-yeast extract (NBY medium) for 2 weeks at 28OC (repeated twice) with no outgrowth. In 

accordance with 7 CFR Part 340, transformed plants were transported and planted in the field only 

when free of live Agrobacterium. Details of the methods are described elsewhere (Richael et al. 2008).

Although Agrobacterium is effective in cleaving at the Right Border (RB) site, it often fails to fully release 

the DNA insert from its plasmid vector by also cutting at the Left Border (LB) site (Gelvin 2003). 

Consequently, some infected plant cells received the DNA insert itself as well as additional plasmid

backbone sequences containing the backbone marker gene, isopentenyltyransferase (ipt), for a plant 

hormone cytokinin, which commonly regulates growth and development processes in plants. 

Overexpression results in stunted phenotypes, abnormal leaves, or the inability to root due to the 
J.R. Simplot Company JRS06 Page 41 of 196

cytokinin overproduction, which were used to select against plants containing backbone DNA (Richael et 

al. 2008). Every two weeks, the infected explants were transferred to fresh medium lacking any 

synthetic hormones and incubated in a Percival growth chamber under a 16-hr photoperiod at 24oC 

where they started to form shoots. Many shoots expressed the ipt gene and displayed the cytokininoverproduction phenotype; these shoots were discarded and not considered for further analyses. PCR 

genotyping demonstrated that about 0.3 to 1.5% of the remaining shoots contained at least part of the 

DNA insert while lacking the ipt gene.

Following development and isolation of events containing an insert associated with the pSIM1278 

vector, those plants were transformed using the methods described for a second plasmid, pSIM1678, to 

produce stacked events containing two independent inserts. 

The following methods were used to establish that backbone portions of the plasmids pSIM1278 and

pSIM1678 were not present in events developed for commercial purposes: 1) If plants had phenotypes 

associated with the negative selectable isopentenyl isomerase (ipt) marker gene in the plasmid

backbone, they were discarded; 2) Absence of the backbone DNA was confirmed with Southern blot

hybridization; 3) PCR was used to confirm no fragments of the backbone DNA were present. Evidence 

showing that event W8 does not contain backbone DNA is provided in Chapter 5: Genetic 

Characterization.
J.R. Simplot Company JRS06 Page 42 of 196

3.4 References

Gelvin, S.B. (2003). Agrobacterium-mediated plant transformation: the biology behind the "genejockeying" tool. Microbiol. Mol Biol R. 67: 16-37.

Lazo, G.R., Stein, P.A., and Ludwig, R.A. (1991). A DNA transformation-competent Arabidopsis genomic 

library in Agrobacterium. Biotechnology. 9: 963-967.

Richael, C.M., Kalyaeva, M., Chretien, R.C., Yan, H., Adimulam, S., Stivison, A., Weeks, J.T., and 

Rommens, C.M. (2008). Cytokinin vectors mediate marker-free and backbone-free plant transformation. 

Transgenic Res. 17: 905-917. 
J.R. Simplot Company JRS06 Page 43 of 196

4.0 Donor Genes and Regulatory Sequences

4.1 Plasmids used for Transformation

W8 was developed by first transforming Russet Burbank with plasmid, pSIM1278, (Figure 4-1) followed 

by stacking with a second plasmid, pSIM1678 (Figure 4-2). The pSIM1278 insert contains cassettes to 

reduce expression of asparagine synthetase (ASN1), polyphenol oxidase (PPO5), and reducing sugars by 

silencing the R1 and PhL genes, whereas the pSIM1678 construct provides the late blight resistance gene 

(Rpi-vnt1) and a vacuolar invertase (VInv) silencing cassette. The plasmids, pSIM1278 and pSIM1678, are

binary transformation vectors that consist of two parts: plasmid backbone and the DNA insert. 

The plasmids were developed using the same parental plasmid and thus share identical backbone 

sequences (Table 4-1), containing well-characterized bacterial origins of replication from plasmids pVS1 

and pBR322, and the nptIII gene for bacterial resistance to kanamycin. In addition, the backbone 

contains an expression cassette comprising the Agrobacterium ipt gene flanked by the plant’s 

polyubiquitin (Ubi7) promoter and polyubiquitin (Ubi3) terminator, which was introduced as a 2.6-kb 

SacII fragment into the vector backbone (Garbarino and Belknap 1994).

Maps of the transformation plasmids, pSIM1278 and pSIM1268, are provided in Figures 4-1 and 4-2 with 

descriptions of the genetic elements for the DNA inserts provided in Tables 4-2 and 4-3. Briefly, the 

pSIM1278 insert consists of two independent inverted repeats and their associated spacer elements 

flanked by opposing plant promoter elements, pGbss and pAgp. One inverted repeat consists of 

sequence from the potato Asn-1 gene adjacent to sequence from the potato gene, Ppo5. The other 

inverted repeat consists of adjacent sequences derived from the promoters of two potato genes, PhL

and R1. The pSIM1678 insert consists of the Rpi-vnt1 expression cassette and a silencing cassette for the 

plant vacuolar invertase gene, VInv. The Rpi-vnt1 gene cassette consists of the VNT1 protein coding 

region regulated by its native promoter and terminator sequences, whereas the silencing cassette 

consists of an inverted repeat of sequence from the potato VInv gene flanked by opposing plant 

promoters, pGbss and pAgp.
J.R. Simplot Company JRS06 Page 44 of 196

Figure 4-1. pSIM1278 Vector

The vector backbone region, indicated in grey, starts at position 10,149-bp and ends at 19,660-bp 

(9,512-bp total). The backbone DNA consists mainly of bacterial DNA and is intended only to support 

maintenance of the DNA insert prior to plant transformation. The DNA insert region, is on the right from 

1-bp to 10,148-bp, for a total of 10,148-bp. The DNA insert consists of T-DNA border-like sequences and 

potato genomic DNA which are intended for stable integration into the plant genome.

Spacer 2

Asn1

tPpo5

pR1

pPhL

pR1

Spacer 1

pAgp

pGbss

Kpn l

pBR322 bom

PVS1 Sta

overdrive

nptIII

pVS1Rep

pUbi7

pBR322 ori

tUbi3

RB-like

ipt

Sac ll

Sac ll

Sac 1

LB-like

pPhL

pGbss

pAgp

Asn1

tPpo5

pSIM1278

19,660 bp

Vector Backbone

Potato-Derived Transfer DNA

Start DNA insert at 1

End DNA insert at 10,148
J.R. Simplot Company JRS06 Page 45 of 196

Figure 4-2. pSIM1678 Vector

The vector backbone region, indicated in grey, starts at position 9,091-bp and ends at 18,602-bp (9,512-

bp total). The backbone DNA consists mainly of bacterial DNA and is intended only to support 

maintenance of the DNA insert prior to plant transformation. The DNA insert region, including flanking 

Border sequences, is on the right from 1-bp to 9,090-bp, which is a total of 9090-bp. The DNA insert 

consists of T-DNA border-like sequences and potato genomic DNA which are intended for stable 

integration into the plant genome.

pSIM1678

18,602 bp

Vnt1

pBR322 bom

PVS1 Sta Inv

Inv

nptIII

overdrive

pVS1Rep

pVnt1

pGbss

pAgp

pUbi7

pBR322 ori

tVnt1

Ubi3T

RB-like

LB-like

ipt

KpnI

Sac1

SacII

Potato-Derived Transfer DNA

Start DNA insert at 1 

End DNA insert at 9,090 

Vector Backbone

SacII
J.R. Simplot Company JRS06 Page 46 of 196

Table 4-1. Table 4-1 Genetic elements of the pSIM1278 and pSIM1678 backbone1

Genetic Element Origin Accession 

Number

Position

(pSIM1278) 1

Function

SacII restriction site S. tuberosum AJ272136.1 19,411-

19,416

Restriction site used to connect Ubi7 promoter with 

LB flanking sequence.

Polyubiquitin promoter 

(Ubi7) including the 

coding sequence for a 

76- amino-acid potato 

ubiquitin monomer 

(UBQmon)

S. tuberosum var. 

Ranger Russet

U26831.1 17,671-

19,410

Promoter to drive expression of the ipt backbone 

marker gene

Isopentenyl transferase

(ipt) gene

Agrobacterium

tumefaciens

NC_002377.

1

16,936-

17,658

Condensation of AMP and 

isopentenylpyrophosphate to form isopentenylAMP, a cytokinin in the plant. Results in abnormal 

growth phenotypes in plant (Smigocki and Owens 

1988)

Terminator of the 

ubiquitin-3 gene (tUbi3)

S. tuberosum GP755544.1 16,230-

16,584

Terminator for ipt gene transcription (Garbarino and 

Belknap 1994)

Neomycin 

phosphotransferase III 

(nptIII) gene

E. coli FJ362602.1 15,240-

16,034

Aminoglycoside phosphotransferase (Courvalin et 

al. 1977)

Origin of replication for 

pBR322 (pBR322 ori)

E. coli J01784.1 14,669-

14,949

Bacterial origin of replication 2

(pBR322 bom) E. coli J01749.1 14,269-

14,529

pBR322 region for replication in E. coli 2

pVS1 replicon 

( pVS1Rep)

Pseudomonas 

fluorescens 

plasmid pVS1

AJ537514.1 

(4,501-

5,501)

12,859-

13,859

pVS1 region for replication in Agrobacterium2

pVS1 partitioning 

protein StaA (PVS1 Sta)

Pseudomonas 

fluorescens 

plasmid pVS1

AJ537514.1 

(6,095-

7,095)

11,266-

12,266

pVS1 stability 2

Overdrive Agrobacterium

tumefaciens

K00549.1 

(103-132)

10,155-

10,184

Enhances cleavage at the Right Border site 2

1

Numbering system based on pSIM1278, but backbone sequences identical for pSIM1678.

2

http://www.cambia.org/daisy/cambia/585.html - (General structure map of pCAMBIA vectors)
J.R. Simplot Company JRS06 Page 47 of 196

Table 4-2. Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border

Genetic Element Origin Accession 

Number

Position

(pSIM1278)

Intended Function

1. Left Border (LB) site1Synthetic AY5665552

(bases 1-25)

1 – 25 Site for secondary cleavage to release single-stranded 

DNA insert from pSIM1278 (van Haaren et al. 1989)

2. Left Border region sequence

including LB

S. tuberosum var. 

Ranger Russet.

AY5665552

(bases1-187)

1 – 187 Supports secondary cleavage at LB

3. KpnI restriction site S. tuberosum AF393847.1 188 –193 Site for connection of DNA insert with LB flanking 

sequence.

4. Promoter for the ADP glucose 

pyrophosphorylase gene (pAgp), 

1st copy

S. tuberosum var. 

Ranger Russet

HM363752 194-2,453 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of Asn1 and Ppo5, especially in tubers

5. Fragment of the asparagine 

synthetase-1 (Asn1) gene (1st

copy antisense orientation)

S. tuberosum var. 

Ranger Russet

HM363759 2,454-2,858 Generates with (10) double stranded RNA that 

triggers the degradation of Asn1 transcripts to impair 

asparagine formation (Chawla et al. 20123)

6. 3’-untranslated sequence of 

the polyphenol oxidase-5 gene 

(Ppo5) (1st copy, in antisense 

orientation)

S. verrucosum HM363754 2,859-3,002 Generates with (9) double stranded RNA that triggers 

the degradation of Ppo5 transcripts to block black 

spot development

7. XbaI restriction site S. tuberosum DQ478950.1 3,003-3,008 Site for connection of the first Ppo5 copy to spacer-1.

8. Spacer-1 S. tuberosum var. 

Ranger Russet

HM363753 3,009-3,166 Sequence between the 1st inverted repeats

9. 3’-untranslated sequence of 

the polyphenol oxidase-5 gene 

(Ppo5) (2nd copy, in sense 

orientation)

S. verrucosum HM363754 3,167-3,310 Generates with (6) double stranded RNA that triggers 

the degradation of Ppo5 transcripts to block black 

spot development

10. Fragment of the asparagine 

synthetase-1 (Asn1) gene (2nd

copy, in sense orientation)

S. tuberosum var. 

Ranger Russet

HM363759 3,311-3,715 Generates with (5) double stranded RNA that triggers 

the degradation of Asn1 transcripts to impair 

asparagine formation (Chawla et al. 20123)

11. EcoRI restriction site S. tuberosum var. 

Ranger Russet

X73477 3,716-3,721 Site for connection of the second Asn1 copy to Gbss 

promoter.

12. Promoter for the granulebound starch synthase (pGbss) 

gene (1st copy, convergent 

orientation relative to the 1st

copy of pAgp)

S. tuberosum var. 

Ranger Russet

HM363755 3,722-4,407 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of Asn1 and Ppo5, especially in tubers

13. Spe1 / KpnI restriction sites S. tuberosum var. 

Ranger Russet

X95996 / 

AF393847.1

4,408-4,423 Polylinker site for connection of Gbss promoter to the 

second Agp promoter.

14. pAgp, 2nd copy S. tuberosum var. 

Ranger Russet

HM363752 4,424-6,683 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of the promoters of PhL and R1, especially 

in tubers

15. Fragment of promoter for 

the potato phosphorylase-L 

(pPhL) gene (1st copy, in 

antisense orientation)

S. tuberosum var. 

Ranger Russet

HM363758 6,684-7,192 Generates with (20) double stranded RNA that 

triggers the degradation of PhL transcripts to limit the 

formation of reducing sugars through starch 

degradation

16. Fragment of promoter for 

the potato R1 gene (pR1) (1st

copy, in antisense orientation)

S. tuberosum var. 

Ranger Russet

HM363757 7,193-7,724 Generates with (19) double stranded RNA that 

triggers the degradation of R1 transcripts to limit the 

formation of reducing sugars through starch 

degradation

17. Pst1 restriction site S. tuberosum var. 

Ranger Russet

DQ478950.1 7,725-7,730 Site for connection of the first R1 promoter fragment 

to the spacer2

18. Spacer-2 S. tuberosum var. 

Ranger Russet

HM363756 7,731-7,988 Sequence between the 2nd inverted repeat

19. Fragment of promoter for 

the potato R1 gene (pR1) (2nd 

copy, in sense orientation)

S. tuberosum var. 

Ranger Russet

HM363757 7,989-8,520 Generates with (16) double stranded RNA that 

triggers the degradation of R1 transcripts to limit the 

formation of reducing sugars through starch 

degradation
J.R. Simplot Company JRS06 Page 48 of 196

Table 4-2 (Continued). Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border

Genetic Element Origin Accession 

Number

Position

(pSIM1278)

Intended Function

20. Fragment of promoter for 

the potato phosphorylase-L 

(pPhL) gene (2nd copy, in sense 

orientation)

S. tuberosum var. 

Ranger Russet

HM363758 8,521-9,029 Generates with (15) double stranded RNA that

triggers the degradation of PhL transcript to limit the 

formation of reducing sugars through starch 

degradation

21. pGbss (2nd copy, 

convergent orientation relative 

to the 2nd copy of pAgp)

S. tuberosum var. 

Ranger Russet

HM363755 9,030-9,953 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of the promoters of PhL and R1, especially 

in tubers

22. SacI restriction site S. tuberosum AF143202 9,954 – 9,962 Site for connection of DNA insert with RB flanking 

sequence.

23. Right Border region

sequence including RB

S. tuberosum var. 

Ranger Russet 

AY5665552

(bases 231-

416)

9,963 –

10,148

Supports primary cleavage at RB-Like site

24. Right Border (RB) sequence1Synthetic AY5665552

(bases 392-

416)

10,124 –

10,148

Site for primary cleavage to release single stranded 

DNA insert from pSIM1278 (van Haaren et al. 1989)

1

The LB and RB sequences (25-bp each) were synthetically designed to be similar to and function like T-DNA borders from Agrobacterium 

tumefaciens.

2

GenBank Accession AY566555 was revised to clarify the sources of DNA for the Border regions.

3

ASN1 described as genetic elements 5 and 10 is referred to as StAst1 in Chawla et al. 2012.
J.R. Simplot Company JRS06 Page 49 of 196

Table 4-3 Genetic elements of the DNA Insert of pSIM1678, from Left Border site to Right Border

Genetic Element Origin Accession 

Number

Position

(pSIM1678)

Intended Function

1. Left Border (LB) site1Synthetic AY5665552

(bases 1-25)

1 – 25 Site for secondary cleavage to release singlestranded DNA insert from pSIM1678

2. Left Border region sequence

including LB

S. tuberosum

var. Ranger 

Russet.

AY5665552

(bases 1-187)

1 - 187 Supports secondary cleavage at LB

3. KpnI restriction site S. tuberosum AF393847.1 188 - 193 Site for connection of DNA insert with LB flanking 

sequence.

4. Native promoter for the late 

blight resistance gene (Rpivnt1)

S. venturii FJ423044.1 194 -902 Drives expression of late blight resistance gene 

vnt1

5. Late blight resistance gene 

(Rpi-vnt1)

S. venturii FJ423044.1 903 -3,578 Solanum venturii late blight resistance protein 

gene

6. Native terminator for the 

Rpi-vnt1 gene

S. venturii FJ423044.1 3,579 -4,503 Ends transcription of late blight resistance gene 

vnt1

7. Apa1 S. tuberosumt HM363755 4,504 -4,509 Site for connection of vnt1 terminator with Agp 

promoter

8. Promoter for the ADP 

glucose pyrophosphorylase 

gene (pAgp)

S. tuberosum

var. Ranger 

Russet

HM363752 4,510 - 6,770 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of acid invertase gene.

9. BamH1 S. tuberosum

var. Ranger 

Russet

DQ206630 6,771 - 6,776 Site for connection of Agp promoter with 

invertase 

10. Fragment of the acid 

invertase (sense orientation)

S. tuberosum

var. Ranger 

Russet

DQ478950.1 6,777 - 7,455 Generates with (12) double stranded RNA that 

triggers the degradation of invertase transcripts

11. EcoRI S. tuberosum

var. Ranger 

Russet

X73477 7,456 - 7,461 Site for connection of an invertase fragment 

(sense) with an invertase fragment (anti-sense)

12. Fragment of the acid 

invertase (anti-sense 

orientation)

S. tuberosum

var. Ranger 

Russet

DQ478950.1 7,462 - 7,965 Generates with (10) double stranded RNA that 

triggers the degradation of invertase transcripts

13. Spe1 S. tuberosum

var. Ranger 

Russet

X95996 7,966 - 7,971 Site for connection of an invertase fragment 

(anti-sense) with GBSS promoter

14. Promoter for the granulebound starch synthase (pGbss) 

gene ( convergent orientation 

relative to the pAgp)

S. tuberosum

var. Ranger 

Russet

HM363755 7,972 - 8,895 One of the two convergent promoters that drives 

expression of an inverted repeat containing 

fragments of invertase gene, especially in tubers

15. SacI restriction site S. tuberosum AF143202 8,896 – 8,901 Site for connection of DNA insert with RB flanking 

sequence.

16. Right Border region

sequence including RB

S. tuberosum

var. Ranger 

Russet 

AY5665552

(bases 231-416)

8,902 – 9,090 Supports primary cleavage at RB-Like site

17. Right Border (RB) 

sequence

1

Synthetic AY5665552

(bases 392-416)

9,066 – 9,090 Site for primary cleavage to release single 

stranded DNA insert from pSIM1278 (van Haaren 

et al. 1989)

1

The LB and RB sequences (25-bp each) were synthetically designed to be similar to and function like T-DNA borders from Agrobacterium 

tumefaciens.

2

GenBank Accession AY566555 was revised to clarify the sources of DNA for the Border regions.
J.R. Simplot Company JRS06 Page 50 of 196

4.2 References

Chawla, R., Shakya, R., and Rommens, C.M. (2012). Tuber specific silencing of asparagine synthetase-1 

reduces the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape 

and yield. Plant Biotechnol J. 10: 913-924.

Courvalin, P., Weisblum, B., and Davies, J. (1977). Aminoglycoside-modifying enzyme of an antibioticproducing bacterium acts as a determinant of antibiotic resistance in Escherichia coli. P Natl Acad Sci.

74: 999-1003.

Garbarino, J.E., and Belknap, W.R. (1994). Isolation of a ubiquitin-ribosomal protein gene (ubi3) from 

potato and expression of its promoter in transgenic plants. Plant Mol Biol. 24: 119-127.

Smigocki, A.C., and Owens, L.D. (1988). Cytokinin gene fused with a strong promoter enhances shoot 

organogenesis and zeatin levels in transformed plant cells. P Natl Acad Sci. 85: 5131-5135.

Van Haaren, M.J.J., Sedee, N.J.A., de Boer, H.A., Schilperoort, R.A., and Hooykaas, P.J.J. (1989).

Mutational analysis of the conserved domains of a T-region border repeat of Agrobacterium 

tumefaciens. Plant Mol Bio. 13: 523-531.
J.R. Simplot Company JRS06 Page 51 of 196

5.0 Genetic Characterization of Russet Burbank Event W8

5.1 Introduction

The Russet Burbank W8 event contains inserts derived from two separate transformations with different 

plasmids. The first insert, plasmid pSIM1278, contains two cassettes consisting of inverted repeats 

designed to silence up to four potato genes, Asn1, Ppo5, R1, and PhL, in tubers. Similarly, the second 

plasmid, pSIM1678, contains a cassette consisting of an inverted repeat to silence the VInv gene in 

tubers, while also containing a copy of the Rpi-vnt1 gene under its native potato promoter. The

following studies were carried out as part of the characterization and biosafety assessment of the Russet 

Burbank W8 event.

In the following section, we provide a detailed characterization of the genetic inserts associated with 

transformation of the Russet Burbank variety to produce event W8. The characterization includes a 

combination of Southern blot analyses, polymerase chain reactions (PCR), and DNA sequencing to assess 

the structure, stability, and flanking regions of each insert. After determining the DNA insert and 

flanking region structures, bioinformatic methods were used to evaluate putative open reading frames 

to show that modifications to the genome did not generate new or modify existing open reading frames 

to produce potential allergens or toxins (see Chapter 9: Safety of the W8 Potato for the detailed 

analysis). The methods used in the following studies are described in Appendix A: Genetic, Molecular, 

and Biochemical Methods.

5.2 Copy number and structure of the DNA Inserts

An ideal transformation event consists of the plasmid insert being introduced into a single locus within 

the plant genome including intact copies of all expression cassettes. In some cases, the structure of the 

insert within the plant genome matches the structure from the original plasmid. However, in other 

cases, recombination events can lead to changes in the structure and/or duplications of cassettes within 

the original plasmid. Although one result is not better than the other, the presence of rearrangements 

and duplications can make structural determination more challenging.

Our characterization of copy number and structure of the Russet Burbank W8 Event (W8) included: (1) 

Southern blot verification that pSIM1278 and pSIM1678 each integrated into a single genomic locus, (2) 

Southern blot determination that the structure of the pSIM1678 insert was similar to the original 

construct, whereas the pSIM1278 insert was significantly more complex, and (3) structural 

determination of both inserts using numerous Southern blot analyses combined with PCR and 

sequencing analysis. 

The Southern blot analyses presented in this section use a set of probes that specifically hybridize to 

elements contained within our inserts, but also recognize endogenous sequence within the plant 

genome. The AGP probe hybridizes to the 5 ́ end of the Agp promoter, which exists at two locations in 

the original pSIM1278 construct. The ASN probe hybridizes to both sides of the Asn1/Ppo5 silencing 

cassette, whereas the R1 probe hybridizes to both sides of the pR1/pPHL silencing cassette. The Gbss

promoter associated with the Asn1/Ppo5 silencing cassette is a truncated form of the Gbss promoter 

associated with the pR1/pPHL silencing cassette. Whereas the GBS1 probe detects both promoters, the 

GBS2 probe can distinguish between these two promoters as it specifically detects the longer one 

associated with the pR1/pPHL silencing cassette.
J.R. Simplot Company JRS06 Page 52 of 196

As shown in Figure 5-1A, the pSIM1278 insert consists of an Asn1/Ppo5 silencing cassette flanked by 

converging Agp and Gbss promoters and a second pPHL/pR1 silencing cassette flanked by the same set 

of converging promoters. Probes that hybridize to the pAgp, Asn1, pGbss, pR1 elements were used to 

analyze the copy number and structure using Southern blot analysis following digestion with restriction 

enzymes. A number of informative restriction enzyme sites are highlighted, including EcoRV, HindIII, 

EcoRI, and ScaI, with a map showing the expected fragment sizes provided below the insert map for 

convenience. 

Similarly, Figure 5-1B shows the structure of the original pSIM1678 insert along with Southern probes 

and a map of commonly used restriction digests. Note there are two promoter elements, pAgp and the 

longer pGbss, in common between the pSIM1278 and pSIM1678 inserts. Therefore, the AGP, GBS1, and 

GBS2 probes will detect bands from both inserts on all Southern blots. However, the VNT1 and INV 

probes are specific to the Rpi-vnt1 promoter and VInv silencing cassette of the pSIM1678 insert, 

respectively.
J.R. Simplot Company JRS06 Page 53 of 196

Figure 5-1. Plasmid constructs and Southern Probes

A. pSIM1278

B. pSIM1678 

Restriction enzyme abbreviations: RV=EcoRV, RI=EcoRI, Sc=ScaI, Hd=HindIII, Xb=XbaI, Sp=SphI

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.

AGP

RB

RI (3718)Sc(8247)Hd(2444)Hd(6674)Hd(7987)RV (1128)RV (2353)RV (3065)RV(5358)RV(6583)

pGbss pGbss

Sc(7469)

LB

ASN ASN GBS1 AGP R1 R1 GBS1

pAgp (1) (194) (10148)

GBS2

Asn1 Asn1 pPHL pR1 pR1 pPHL

EcoRV

HindIII

>1.1 kb 0.7 kb 2.3 kb >3.6 kb

>2.4 kb 4.2 kb 1.3 kb >2.1 kb

>3.7 kb 3.8 kb 0.8 kb >1.9 kb

Probes

EcoRI/ScaI

pAgp Sc (834) Hd(1947) Sc (2132) Hd(2608) Xb(2832) Hd(3265) Hd(4106)

RV(5444)RV(6669) Hd(6760) Xb(7383) RI(7457) (1) (9090)

Rpi-vnt1 tVnt1 pAgp fInv fInv RB

VNT1 AGP INV INV GBS1

EcoRV

HindIII

EcoRI/ScaI

XbaI >2.8 kb 4.6 kb >1.7 kb

>0.8 kb 5.3 kb >1.6 kb

>2.3 kb 2.7 kb >1.9 kb

>5.4 kb >2.4 kb

GBS2 Probes

LB pVnt1 pGbss Sp (1558)

SphI >1.6 kb >7.5 kb
J.R. Simplot Company JRS06 Page 54 of 196

5.2.1 Characterization of insertion number for pSIM1278 and pSIM1678

Southern blots were performed using genomic DNA isolated from Russet Burbank W8 Event and Russet 

Burbank controls (WT) to identify the number of insertion events associated with each plasmid. These 

analyses were performed using restriction enzymes that frequently cut within the potato genome, but 

do not cut within our inserts, resulting in individual intact loci on Southern blots. 

Single insertion site associated with pSIM1278

To assess the number of insertion events for the pSIM1278 plasmid, we digested genomic DNA with the 

restriction enzymes, BciVI and NdeI, which do not cut within the insert itself. Since the restriction 

enzyme recognition site is common within the potato genome, it is unlikely that more than one insertion 

event will contain the same size following digestion and that two insertion events will fall within the 

same fragment.

Figure 5-2. pSIM1278 DNA inserted at a single site in the genome

DNA digests were separated on agarose gels (TAE) using extended electrophoresis to ensure good 

separation and resolution of high-molecular weight bands. Southern blots were hybridized with the 

GBS1, ASN, and R1 probes depicted in Figure 5-1 to verify that a single band is detected with each probe 

(Figure 5-2). In addition to the two endogenous bands common to both samples, two higher molecular 

GBS1 Probe

23.1

kb

2.8

2.3

1.52

1.48

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

2.0

1.95

1.88

DIGII DIGVII WT W8 WT W8 DIGII DIGVII WT W8 WT W8 DIGII DIGVII WT W8 WT W8 

Ndel BciVI Ndel BciVI Ndel BciVI

ASN Probe R1 Probe

Red arrows indicate bands associated with pSIM1278 DNA and blue arrows indicate bands associated 

with pSIM1678 DNA.

pSIM1278 

insert

pSIM1278 

insert

pSIM1278 

insert

pSIM1678 

insert
J.R. Simplot Company JRS06 Page 55 of 196

weight species were identified in the NdeI digested samples. The lower band corresponds to the 

pSIM1678 insert, whereas the larger band contains the pSIM1278 insert. A similar digestion pattern was 

observed following digestions with BciVI, a single pSIM1278 band and a smaller pSIM1678 band. The 

identity of the smaller bands was consistent with known NdeI and BciVI restriction sites within the 

pSIM1678 construct and was confirmed by INV and VNT1 probes specific to this insert. The identity of 

the high molecular weight pSIM1278 bands was confirmed by Southern blots probed with ASN and R1, 

which are specific to that insert (Figure 5-2, gels on right). Collectively, these data show the pSIM1278 

insert was integrated at a single locus within the Russet Burbank genome.

Single insertion site associated with pSIM1678

To assess the number of integration sites for the pSIM1678 insert, genomic DNA was digested with the 

restriction enzymes, BglII and SphI. The BglII restriction enzyme does not cut within the pSIM1678 DNA 

insert, and thus should generate a single band containing the entire construct. As shown in Figure 5-3, a 

single band of the same size was detected when BglII digested DNA was probed with either the VNT1 or 

INV probe. As shown in Figure 5-1(B), the regions detected by these probes are located on opposite 

ends of the insert and are not found in pSIM1278. To corroborate these findings, we also digested with 

the restriction enzyme, SphI, which cleaves the pSIM1678 insert within the Rpi-vnt1 gene (Figure 5-1b), 

which resulted in a single band when probed with either VNT1 or INV, as expected. Since the SphI 

restriction site is located between the VNT1 and INV probe binding sites, this digest was expected to 

yield the two different sized species that was observed (Figure 5-3). 
J.R. Simplot Company JRS06 Page 56 of 196

Figure 5-3. pSIM1678 DNA inserted at a single site in the genome

Summary of Integration Analysis

Our Southern blot analyses indicated a single insertion site associated with each plasmid transformation. 

As discussed in Section 5-3, the flanking sequence for the pSIM1278 insert indicated the presence of 

BciVI restriction sites within 1kb of the insert junctions. Given the close proximity of these restriction 

sites to our inserted DNA, a much smaller band was expected in our Southern blots (Figure 5-2) if the 

insertion represented the structure shown in Figure 5-1. Since the observed band was roughly 2-fold 

larger than expected, it suggested the structure of the insert was more complex than the original 

construct of pSIM1278 (Figure 5-1). A similar analysis could not be performed for pSIM1678 as there 

were no SphI or BglII restriction sites identified in the flanking sequence, but as described later the 

structure of this insert was very similar to the input sequence.

Blue arrows indicate bands associated with pSIM1678 DNA.

VNT1 Probe

23.1

kb

2.8

2.3

1.52

1.48

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

2.0

1.95

1.88

DIGII DIGVII WT W8 WT W8

BglII

INV Probe

pSIM1678 

insert

BglII SphI SphI

DIGII DIGVII WT W8 WT W8

pSIM1678 

insert
J.R. Simplot Company JRS06 Page 57 of 196

5.2.2 Structure of the pSIM1278 DNA insert

We used Southern blot analyses to test our hypothesis that the pSIM1278 construct was more complex 

than a simple, single copy integration of our plasmid DNA insert. Table 5-1 presents a summary of a set 

of Southern blots highlighting differences between the expected and observed band numbers, sizes, and 

intensities. The expected values are based upon integration of a single copy of the pSIM1278 DNA insert 

described in Figure 5-1A.

Table 5-1. Predicted and observed bands based on Southern blots for pSIM1278 

Enzyme Probe Expected sizes (kb) Observed sizes (kb)* References

EcoRV AGP

ASN

GBS1

R1

>1.1, 2.3

 0.7, 2.3

 2.3, >3.6

>3.6 

10, 2.3

0.7, 2.3

2.3, 14.0 

14.0

Figure 5-6

Figure 5-8

Figure 5-11

Figure 5-13

HindIII AGP

ASN

GBS1

R1

>2.4, 4.2

 4.2

 4.2, >2.1

 1.3, >2.1

4.2

4.2

4.2, 6.0

1.3, 4.3, 6.0

Figure 5-6

Figure 5-8

Figure 5-11

Figure 5-13

EcoRI/ScaI AGP

ASN

GBS1

R1

>3.7, 3.8

>3.7

 3.8, >1.9

>3.7, 3.8, 0.8

3.8, 4.2

4.2

3.7, 3.8, 4.2, 9.0

0.8, 3.8, 9.0

Figure 5-6

Figure 5-8

Figure 5-11

Figure 5-13

*Does not include endogenous or pSIM1678 bands.

- Sizes marked by bold print indicate bands had higher than normal intensity, suggesting >1 copy.

The presence of unexpected bands and signal intensity confirmed our expectation that the actual insert 

consisted of a complex structure. As we were unable to reconcile the Southern data presented in Table 

5-1 with a tandem or inverted duplication of the pSIM1278 insert DNA, a number of additional Southern 

blots were performed in combination with PCR and sequencing studies to elucidate the structure of the 

insert at this locus. A detailed analysis of these studies is upcoming, but a summary of the final structure 

is provided in Figure 5-4 (top) and is broken into sections (Left, Middle, and Right) to facilitate discussion 

as shown in Figure 5-4a and 5-4b. Briefly, the Middle section consists of a full-length copy of the original 

pSIM1278 insert with a deletion of the T-DNA left border. The Middle section is flanked on the left (Left 

Section) by a tandem repeat of the Asn1/Ppo5 silencing cassette, both of which are in their original 

orientation, but also include the T-DNA left border deletion. The Right Section consists of an inverted 

repeat containing the pR1/pPHL silencing cassette flanked by converging Gbss promoters, where the 

right edge of the inverted repeat is juxtaposed to a tandem copy of the longer GBSS promoter separated 

by right border and a small amount of pPHL sequence (Figure 5-4).

Although the structure associated with pSIM1278 is more complicated than anticipated, the duplicated 

silencing cassettes are still under the control of the tissue-specific promoters and the current structure 

does not negatively impact safety or trait efficacy of the product (See Chapter 9: Safety of Russet 

Burbank W8 Potato and Chapter 10: Trait Efficacy). The data supporting the proposed structure will be 

presented next.

Southern blot presentation notes

Each Southern blot has been presented with a table that indicates the estimated sizes of the observed 

bands and whether they are interpreted as either endogenous original bands (OB), internal bands (IB) or 
J.R. Simplot Company JRS06 Page 58 of 196

junction bands (JB). The endogenous original bands represent sequences found in the Russet Burbank 

control and are not related to the DNA inserts. The internal bands are those bands that reside 

completely within the junctions of the inserted DNA and thus have predicted sizes. The junction bands 

correspond to digestion products that contain sequence from the DNA insert as well as the plant 

genome. These bands have unique sizes that are not always predictable due to uncertainty in the plant 

genome sequence. Nonetheless, the minimal size of these bands can be predicted based upon the 

distance between the location of the restriction site within the DNA insert and the junction between the 

DNA insert and plant genome sequence. The junction bands are valuable for confirming the number of 

integration sites in the genome as there should never be more than two junction bands for a single 

integration event.

We have observed two phenomena that make it challenging to accurately determine the size of 

digestion products from potato genomic DNA. First, we have observed a tendency for bands above ~3kb 

to migrate slightly faster than similarly-sized molecular weight markers in the same gel. The anomalous 

migration appears to be related to residual polysaccharides in the genomic DNA samples as molecular 

weight markers also run faster when they are mixed with potato DNA samples. That said, the markers 

were not generally spiked with genomic DNA as the presence of genomic DNA has a tendency to make 

the ladder difficult to interpret due to hybridization of probes to endogenous DNA. Instead, we validated 

the size of IBs, such as the numerous 4.2 kb IBs and the 7.0 kb IB, by comparing their migration with 

digested plasmid DNA that had been mixed with Russet Burbank genomic DNA. Under these conditions, 

the plasmid and genomic bands co-migrated, although their migration was consistently faster than the 

unspiked molecular weight markers. In addition, we confirmed the identity of selected, faster migrating 

species by PCR and sequencing as described in Appendix A: Genetic, Molecular, and Biochemical 

Methods.

Additionally, many of the higher molecular weight bands in these samples have a tendency to produce 

smiles during electrophoresis. Size measurements taken from the top of these bands have proven to be 

the most reliable and reproducible so all sizes reported here used this technique (see line marking top of 

band in following example). 
J.R. Simplot Company JRS06 Page 59 of 196

Figure 5-4. Overall structure of pSIM1278 DNA Insert with digestion and hybridization patterns

2.0 kb HindIII/SacI

>2.8 kb XbaI (3.3 kb) 4.2 kb XbaI 7.0 kb Xba1/SacI

>2.2 kb HindIII (4.2 kb) 4.2 kb HindIII 4.2 kb HindIII 1.3 kb HindII

4.2 kb EcoRI 3.7 kb EcoRI/Scal 0.8 kb

4.2 kb PacI 5.3 kb PacI

>0.9 kb (10 kb) 0.7 kb 2.3 kb EcoRV 0.7 kb 2.3 kb EcoRV

EcoRV

>4.1 kb PacI (15 kb)

AGP ASN ASN GBS1 AGP ASN ASN GBS1 AGP R1 R1 GBS1 GBS2GBS2 XbaI (7005) XbaI (11235) PacI (8299) PacI (12529) PacI (17838) SacI (18186) EcoRV (13586) EcoRV (9356) EcoRV (5126) EcoRV (6351) EcoRV (10581) EcoRV (14811) HindIII (6444) EcoRI (7718) HindIII (10674) EcoRI (11948) HindIII (14904) ScaI (15697) HindIII (16217) ScaI (16475) EcoRV (7063) EcoRV (11293)

Middle

pAgp Asn Ppo Ppo Asn pGbss pAgp Asn Ppo Ppo Asn pGbss pAgp PHL R1 R1 PHL pGbss

Left

AGP ASN ASN GBS1 XbaI (2775) PacI (4069) EcoRV (896) EcoRV (2121) HindIII (2214) EcoRI (3488) EcoRV (2833)

pAgp Asn Ppo Ppo Asn pGbss ScaI (-603) Xba1 (-510)

0.7 kb 2.3 kb EcoRV

>3.5 kb Scal/EcoRI (4.1 kb)

4.2 kb HindIII

4.2 kb XbaI

4.2 kb PacI

4.2 kb EcoRI

AGP ASN ASN GBS1AGP ASN ASN GBS1AGP R1 R1 GBS1

GBS2

GBS1

GBS2

GBS1

GBS1 GBS2

GBS2 R1 R1

Left Middle Right

AGP ASN ASN GBS1

(a)
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2.0 kb HindIII/SacI 2.0 kb HindIII/SacI 2.2 kb HindIII/SacI 1.2 kb SacI

7.0 kb XbaI/SacI 4.2 kb SacI 1.2 kb SacI

4.2 kb HindIII 1.3 kb 4.3 kb HindIII >3.6 HindIII kb (5.9 kb)

3.7 kb EcoR1/Scal 0.8 kb 3.8 kb Scal 0.8 kb >3.1 kb Scal (8.0 kb)

5.3 kb PacI 1.1 kb 3.5 kb PacI 1.2 kb

Pacl Pacl

1.3 kb

Pacl

0.7 kb 2.3 kb EcoRV >9.2 kb EcoRV (14 kb)

AGP ASN ASN GBS1 AGP R1 R1 GBS1 GBS2 GBS2GBS1 GBS1 GBS2 GBS1GBS2 XbaI (11235) PacI (12529) PacI (17838) PacI (18867) PacI (22372) PacI (23600) SacI (22720) SacI (23948) SacI (18521) SacI (18186) EcoRV (13586) EcoRV (9356) EcoRV (10581) EcoRV (14811) HindIII (10674) EcoRI (11948) HindIII (14904) ScaI (15697) HindIII (16217) ScaI (16475) ScaI (20232) HindIII (20490) ScaI (21010) EcoRV (11293)

R1 R1

Middle Right

pAgp Asn Ppo Ppo Asn pGbss pAgp PHL R1 R1 PHL pGbss pGbss PHL R1 R1 PHL pGbss pGbss PacI (+24986)

AGP ASN ASN GBS1AGP ASN ASN GBS1AGP R1 R1 GBS1

GBS2

GBS1

GBS2

GBS1

GBS1 GBS2

GBS2 R1 R1

Left Middle Right

AGP ASN ASN GBS1

(b)
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Figure 5-4 legend. The upper figure in (a) and (b) represents the entire pSIM1278 insert containing the left, middle, and right 

sections. Underneath is an enlarged image of (a) the left and middle sections or (b) the middle and right sections, including 

designated restriction sites. The digestion pattern for selected enzymes is shown as colored boxes with the digest and fragment 

size indicated. The probes that are expected to detect each digestion product are indicated below the fragment with a colored

line. All expected probe binding sites are indicated by bands, but only the digest/probe combinations necessary to support the 

model are provided in this petition. Red boxes denote internal bands (IB) associated with the original pSIM1278 DNA construct. 

Blue closed boxes indicate internal bands or bands of known sizes due to identification of restriction sites within flanking DNA. 

Open-ended blue boxes indicate junction bands where the second restriction site is unknown. The estimated size of junction 

bands identified on Southern blots is indicated in parenthesis for all junction bands.

Southern Blot structure discussion 

Our molecular studies led to the elucidation of the model for the pSIM1278 insert presented in Figure 5-

4. As mentioned earlier, we have separated the structure into three sections (Left, Middle, and Right) 

and will describe the molecular data supporting each of those sections independently for simplicity and 

clarity. Although not all bands will be discussed, all of the digests and probes depicted in Figure 5-4 are 

represented in the Southern blots shown in Figures 5-6 through 5-15. In addition, the size of each 

digestion product shown in Figure 5-4 can be cross-referenced against the tables adjacent to the 

appropriate Southern blot.

Structure of the Left Section and Junctions 

Based upon sequence information (Section 5.3 Flanking Regions of Inserted Sequences) we determined 

that the left side of the insert consists of a slightly truncated (35 bp) Agp promoter fused to Russet 

Burbank DNA, which contained useful restriction sites as described in Figure 5-4. These restriction sites 

predicted precise junction band sizes for the ScaI/EcoRI (4.1 kb) and XbaI (3.3 kb) digests, which were 

observed in gels hybridized with AGP and ASN probes (Figures 5-6 and 5-7 for AGP; Figures 5-8 and 5-9 

for ASN). The expected sizes of the junction bands associated with the other three digests were not 

known; however, after extending the flanking sequence using the reference genome available from 

Michigan State University’s repository, a HindIII site was predicted to exist ~2 kb upstream of the insert 

and result in a 4.2 kb junction band, which is what was observed (Figure 5-6).

Initial evidence that the left end of the insert consisted of a duplicated Asn1/Ppo5 silencing cassette 

came from the number and intensity of 4.2 kb bands in our Southern blots when W8 DNA was digested 

with the enzymes, HindIII, XbaI, PacI, and EcoRI/ScaI, and probed with AGP, ASN, and GBS1, which all 

hybridize to sequence within the 4.2 kb Asn1/Ppo5 silencing cassette. The 4.2 kb bands present in the 

EcoRI, HindIII, and XbaI digests were all consistent with tandem duplications of the silencing cassette 

and spanned the border between the two sections (Figure 5-4a). This arrangement and duplication was 

further corroborated by higher intensity 2.3 and 0.7 kb bands on EcoRV blots hybridized with the same 

set of probes (Figures 5-6, 5-8, and 5-11). The second copy of the 2.3 kb band covers the border 

between the left and middle sections to further establish the tandem arrangement. Lastly, the 4.2 kb 

band observed when the PacI digests were probed with the AGP, ASN and GBS1 probes (Figures 5-7, 5-9 

and 5-12) strongly supports the tandem arrangement of two Asn1/Ppo5 silencing cassettes. Not only 

does it cross the junction between the left and middle sections, but it also supports the existence of a 

Gbss promoter at the junction between the two sections. The Gbss promoter is the only element in the 

pSIM1278 insert that contains a PacI restriction site, which is necessary to generate this 4.2 kb band 

containing ASN, AGP, and GBS1 hybridization sites (consider PacI digestion pattern in Figure 5-4).

The intensity of the 4.2, 2.3, and 0.7 kb bands described above appeared greater than expected for a 

structure including a simple tandem duplication of the Asn1/Ppo5 silencing cassette. We performed a 
J.R. Simplot Company JRS06 Page 62 of 196

set of digests to determine the size of the left side of the insert and determine whether there were two 

or three copies of the Asn1/Ppo5 cassette on that side. The restriction enzymes, ScaI and BsrGI, both cut 

the insert exclusively in or adjacent to R1, leaving the entire left side of the insert intact. A known 

restriction site was identified in the flanking region for ScaI and another was predicted for MfeI by the 

reference genome at the insertion site. Thus, these two digests were capable of determining the size of 

the insert to the left of the internal R1 cassette. Southern blots probed with an ASN probe identified a 

single band, as expected. The sizes of the MfeI/BsrGI and ScaI digests were 17.6 and 16.3 kb, 

respectively (Figure 5-10). These sizes were both 4.2 kb larger than expected for an insert containing a 

single Asn1/Ppo5 cassette in the left section. This additional cassette does not alter the digestion 

patterns of any of our other digests due to the repetitive nature of the 4.2 kb duplication, but the third 

cassette identified by these digests does account for the higher than expected intensity of the bands 

corresponding to this region. Thus, these data support the tandem duplication of the Asn1/Ppo5 

cassette represented in the left section of the model (Figure 5-4). We did not uncover any sequence 

differences among the pGbss-pAgp junctions within these repeats using PCR and Sanger sequencing.

Structure of the Middle Section: The middle section of the structure represents a complete pSIM1278 

DNA insert except for a deletion of the T-DNA left border, originally adjacent to the Agp promoter 

(compare middle section with Figure 5-1). All of the expected internal bands shown as red boxes in 

Figure 5-4 were observed on the appropriate Southern blot (Figures 5-6 through 5-9 and Figures 5-11 

through 5-15). These digests confirm the existence of a full copy of the pSIM1278 DNA insert in the 

middle section of our structure. 

The EcoRV digests produced the high intensity 2.3 kb band already mentioned that exists in the left and 

middle sections and is detected by AGP, ASN, and GBS1 probes (Figures 5-6, 5-8, and 5-11). The 4.2 kb 

internal band generated by the HindIII digest was detected by the AGP, ASN and GBS1 probes, but 

appears as a high intensity band due to the presence of other 4.2 kb bands in those same digests as 

described in Figure 5-4. Similarly, the 0.8 kb and 1.3 kb internal bands generated by digestion with Scal 

and HindIII, respectively, were detected by the R1 probe (Figure 5-13). Additionally, the 5.3 kb and 3.7 

kb internal bands resulting from PacI and EcoRI/Scal digests were detected by AGP, R1, and GBS1 

probes. Lastly, the 7.0 kb internal band generated by a combined XbaI/Sac1 digest was detected by R1 

probe (Figure 5-14).

Right Section and Junction: The right section of the structure is essentially an inverted repeat of the 

pPhl/pR1 silencing cassette adjacent to a pGbss-RB tandem repeat (Figure 5-4, right). The pGbss-RB 

tandem repeat was initially determined by identification and sequencing of the right junction (Section 

5.3). In addition to the repeat and the right flanking sequence, these data revealed a small intervening 

region of sequence derived from the Phl promoter as shown by the small green arrow at the right side of 

Figure 5-4b. The flanking sequence also identified a nearby PacI restriction site as indicated in the 

structure.

The SacI and SacI/XbaI digests probed with the R1 probe were central to the elucidation of a second 

copy of the pPhl/R1 silencing cassette (Figure 5-14). The XbaI enzyme only cuts pSIM1278 DNA within 

the Asn1/Ppo5 inverted repeat, which results in a single, large right junction band (~18kb) when probed 

with R1. However, when XbaI is combined with SacI, we observe a 4.2 kb band, in addition to the 7.0 kb 

internal band, indicating the presence of a second copy of R1. This conclusion was corroborated by the 

presence of two appropriately sized R1 bands in the PacI digests (Figure 5-14). From the same set of 

gels, the 5.3 kb band corresponded to the internal band described in the previous section, whereas the 

3.5 kb band corresponds to the second copy. Lastly, the numerous small, but distinct internal bands 
J.R. Simplot Company JRS06 Page 63 of 196

ranging from 1.0 to 2.2 kb shown in Figure 5-4b were detected on the GBS1 and GBS2 blots (Figures 5-12 

and 5-15).

Summary of pSIM1278 structure

The structural data presented support a single integration event for pSIM1278 where the structure 

contains multiple copies of the two silencing cassettes as depicted in Figure 5-4. The flanking sequences 

were identified and mapped to chromosome 2 as described in Section 5.3.

5.2.3 Structure of the pSIM1678 DNA insert

As established earlier, the pSIM1678 DNA insert integrated at a single location in the Russet Burbank 

genome. Identification of the flanking regions (Section 5.3) revealed a number of nearby restriction sites 

that were valuable in our Southern blot analyses and are depicted in Figure 5-5. Many of the same 

Southern blots already discussed for pSIM1278 were instrumental in elucidating the structure of the 

insert derived from the pSIM1678 plasmid as both DNA inserts contain Agp and Gbss promoter 

elements. The data did not suggest the inserted structure deviated from the original pSIM1678 DNA 

insertion except for a deletion of the T-DNA left border and a small piece (137 bp) of the Rpi-vnt1

promoter (Figure 5-5). 

The structure of the left junction, including the promoter truncation, was confirmed by the presence of 

the 0.5 kb ScaI and 3.1 kb HindIII (Figure 5-16) bands and the 3.6 kb PacI band (Figure 5-17) detected 

using the VNT1 probe. All three junction bands involved restriction sites predicted by the left flanking 

sequence. As expected, only a single junction band was identified for each of the digests probed by 

VNT1. The internal bands depicted in Figure 5-5 by red boxes were all identified by the appropriate 

probes suggesting integrity of the internal region (Figures 5-6, 5-7, 5-18, and 5-19). 

Consistent with a single integration event, we observed a single right junction band for each of the 

digests described in Figure 5-5, including 1.2 kb PacI band (GBS2 probe, Figure 5-15) and a 3.6 kb EcoRV 

band (GBS1 and INV probes; Figures 5-11 and 5-19) bands as predicted by the flanking sequence at the 

right junction. The 2.1 kb internal HindIII/SacI band detected by the GBS2 probe confirmed the integrity 

of the VInv inverted repeat and adjacent Gbss promoter (Figure 5-15). 

Collectively, our data are consistent with a single copy and insertion of the pSIM1678 DNA insert with a 

deletion of the left border and a small region of adjoining promoter sequence. 
J.R. Simplot Company JRS06 Page 64 of 196

Figure 5-5. Structure of pSIM1678 DNA Insert Digestion and Hybridization Pattern

The upper figure represents the structure of the insert associated with the pSIM1678 plasmid, including designated restriction sites. The digestion pattern for selected enzymes 

is shown as colored boxes with the digest and fragment size indicated. The probes that are expected to detect each digestion product are indicated below the fragment with a 

colored line. All expected probe binding sites are indicated by bands, but only the digest/probe combinations necessary to support the model are provided in this petition. Red 

boxes denote internal bands (IB) associated with the original pSIM1678 DNA construct. Blue closed boxes indicate internal bands or bands of known sizes due to identification of 

restriction sites within flanking DNA. Open-ended blue boxes indicate junction bands where the second restriction site is unknown. The estimated size of junction bands 

identified on Southern blots is indicated in parenthesis for all junction bands.

pVnt1 Vnt1 tVnt1 pAgp Inv pGbss RB EcoRI (7127) EcoRV (51114) EcoRV (6339) Scal (504) Scal (1802) XbaI (2502) XbaI (7053) HindIII (1617) HindIII (2788) HindIII (2935) HindIII (3772) HindIII (6430) Inv

VNT1 AGP INV INV GBS1 GBS2 Pacl (8223) Pacl (3511) SacI (8567) PacI (- 87) HindIII (- 1527) ScaI(- 21) Pacl (+ 748) EcoRV (+ 1238)

>2.5 kb XbaI (6.1 kb) 4.6 kb XbaI >1.6 kb XbaI (5.5 kb)

>1.6 kb HindIII (3.1 kb) 2.7 kb HindIII >2.2 kb HindIII (4.2 kb) 

5.3 kb Scal/EcoRI 0.5 kb ScaI >1.5 kb EcoRI/Scal (7.0 kb)

>3.5 kb Pacl (3.6 kb) 4.7 kb Pacl 1.2 kb Pacl

>5.1 kb EcoRV (7 kb) >2.3 kb EcoRV (3.6 kb)

2.1 kb HindIII/SacI

(1) (8656)
J.R. Simplot Company JRS06 Page 65 of 196

Figure 5-6. Russet Burbank DNA Hybridization with the AGP Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with EcoRV, HindIII, and 

EcoRI/Scal and hybridized with the AGP probe. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image. The estimated sizes of bands are summarized in the table and 

classified into three groups based on the structure of the DNA insert: original endogenous bands (OBs,

in green), internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are 

in red. The DNA fragments of pSIM1678 are in blue. All molecular weights are presented in kilobases.

EcoRV HindIII EcoRI / 

Scal

JB 10

JB 7

OB 5.3

OB 4.4

IB 2.3

IB 2.3

IB 2.3 

OB 1.4

OB 9

OB 7.5

OB 7.0

OB 6.0

IB 4.2

IB 4.2

IB 4.2

JB 4.2

IB 2.7

IB 5.3

OB 5.1

IB 4.2

IB 4.2

JB 4.2

IB 3.8

OB 2.2

OB 1.1

DIGII DIGVII WT W8 WT W8 WT W8

EcoRV HindIII EcoR1/Scal

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0
J.R. Simplot Company JRS06 Page 66 of 196

Figure 5-7. Russet Burbank DNA Hybridization with the AGP Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with XbaI, Sac1, and PacI

and hybridized with the AGP probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are in red. The 

DNA fragments of pSIM1678 are in blue. All molecular weights are presented in kilobases.

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb DIGII DIGVII WT W8 WT W8 WT W8 

XbaI SacI PacI

XbaI SacI PacI

OB 20

JB 18

OB 8.2

OB 7.4

OB 6.7

IB 4.6

IB 4.2

JB 3.3

JB 25 

JB 18

OB 23

JB 15

OB 14

OB 12

IB 5.3

IB 4.7

IB 4.2

IB 4.2
J.R. Simplot Company JRS06 Page 67 of 196

Figure 5-8. Russet Burbank DNA Hybridization with the ASN Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with EcoRV, HindIII, and 

EcoRI/Scal and hybridized with the ASN probe. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image. The estimated sizes of bands are summarized in the table and 

classified into three groups based on the structure of the DNA insert: original endogenous bands (OBs,

in green), internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are 

in red. All molecular weights are presented in kilobases.

EcoRV HindIII EcoRI / 

Scal

OB 8

OB 4.3

IB 2.3

IB 2.3

IB 2.3

OB 2.2

OB 1.4

IB 0.7

IB 0.7

IB 0.7

OB 10

OB 4.5

IB 4.2

IB 4.2

IB 4.2

OB 3.1

OB 2.8

OB 1.9

OB 8

OB 7.5

OB 6.5

OB 4.8

OB 4.2

IB 4.2

IB 4.2

JB 4.2

OB 3.5

OB 2.5

DIGII DIGVII WT W8 WT W8 WT W8

EcoRV HindIII EcoRI/Scal

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7
J.R. Simplot Company JRS06 Page 68 of 196

Figure 5-9. Russet Burbank DNA Hybridization with the ASN Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with XbaI and PacI and 

hybridized with the ASN probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are in red. All 

molecular weights are presented in kilobases.

XbaI PacI

JB 18 

OB 12

OB 9.0

OB 5.3

IB 4.2

IB 4.2

OB 4.0

OB 3.6

JB 3.3

JB 15

OB 11

OB 8.0

IB 4.2

IB 4.2

23.1

kb

2.8

2.3

1.52

1.48

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

2.0

1.95

1.88

DIGII DIGVII WT W8 WT W8 

XbaI PacI
J.R. Simplot Company JRS06 Page 69 of 196

Figure 5-10. Russet Burbank DNA Hybridization with the ASN Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with MfeI/BsrGI and ScaI 

and hybridized with the ASN probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image on the left. pSIM1278 digests were used as additional size markers and are 

shown at the right with their sizes indicated in kb. The sizes of the two digests are indicated with the 

observed sizes 4.2kb greater than expected for a single Asn1/Ppo5 cassette in the left section. All 

molecular weights are presented in kilobases.

7.4

4.9

4.4

9.4

8.6

6.1

6.6

WT W8

MfeI/BsrGI

WT W8

ScaI

MW markers

23.1

kb

2.8

2.3

1.52

1.48

3.6

2.0

1.95

1.88

1.2

1.0

19.7

15.6

14.4

kb

17.6 kb

16.3 kb
J.R. Simplot Company JRS06 Page 70 of 196

Figure 5-11. Russet Burbank DNA Hybridization with the GBS1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with EcoRV, HindIII, and 

EcoRI/Scal and hybridized with the GBS1 probe. Size of the DigII and DigVII molecular weight markers 

are indicated adjacent to the blot image. The estimated sizes of bands are summarized in the table and 

classified into three groups based on the structure of the DNA insert: original endogenous bands (OBs,

in green), internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are 

in red. The DNA fragments of pSIM1678 are in blue. All molecular weights are presented in kilobases.

EcoRV HindIII EcoRI / 

Scal

JB 14 

OB 9.5

OB 8.0

JB 3.6

IB 2.3

IB 2.3

IB 2.3

JB 6.0

IB 4.3

IB 4.2

IB 4.2

IB 4.2

JB 4.2

OB 1.3

JB 9.0

JB 7.0

OB 4.2

IB 4.2

IB 4.2

IB 3.8

IB 3.7

OB 3.5

DIGII DIGVII WT W8 WT W8 WT W8

EcoRV HindIII EcoRI/Scal

kb

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

1.0

0.7
J.R. Simplot Company JRS06 Page 71 of 196

Figure 5-12. Russet Burbank DNA Hybridization with the GBS1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with XbaI and PacI and 

hybridized with the GBS1 probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are in red. The 

DNA fragments of pSIM1678 are in blue. All molecular weights are presented in kilobases.

23.1

kb

2.8

2.3

1.52

1.48

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

2.0

1.95

1.88

1.2

DIGII DIGVII WT W8 WT W8 

XbaI PacI

XbaI PacI

OB 20

JB 18 

JB 5.5

IB 4.2

IB 4.2

OB 16

OB 15

JB 15

IB 5.3

IB 4.7

IB 4.2

IB 4.2

IB 3.5 

IB 1.3
J.R. Simplot Company JRS06 Page 72 of 196

Figure 5-13. Russet Burbank DNA Hybridization with the R1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with EcoRV, HindIII, and 

EcoRI/Scal and hybridized with the R1 probe. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image. The estimated sizes of bands are summarized in the table and 

classified into three groups based on the structure of the DNA insert: original endogenous bands (OBs,

in green), internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are 

in red. All molecular weights are presented in kilobases.

EcoRV HindIII EcoRI / 

Scal

JB 14

OB 8

OB 6.3

OB 2.2

OB 10

JB 6.0

IB 4.3

OB 3.2

OB 2.3

IB 1.3

JB 9.0

IB 3.8

IB 3.8

OB 1.0

IB 0.8 

IB 0.8

OB 0.5

DIGII DIGVII WT W8 WT W8 WT W8

EcoRV HindIII EcoRI/Scal

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7
J.R. Simplot Company JRS06 Page 73 of 196

Figure 5-14. Russet Burbank DNA Hybridization with the R1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with XbaI, PacI, and 

XbaI/Sac1 and hybridized with the R1 probe. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image. The estimated sizes of bands are summarized in the table and 

classified into three groups based on the structure of the DNA insert: original endogenous bands (OBs,

in green), internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are 

in red. All molecular weights are presented in kilobases.

XbaI PacI

JB 18 

OB 1.5

OB 1.4

OB 21

OB 20

IB 5.3

IB 3.5

23.1

kb

2.8

2.3

1.52

1.48

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

2.0

1.95

1.88

DIGII DIGVII WT W8 WT W8 

XbaI PacI

1.2

XbaI/SacI

IB 7.0

IB 4.2

OB 1.4

OB 1.3

DIGII DIGVII WT W8 

XbaI/SacI

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7
J.R. Simplot Company JRS06 Page 74 of 196

Figure 5-15. Russet Burbank DNA Hybridization with the GBS2 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with HindIII/Sac1 and PacI

and hybridized with the GBS2 probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1278 insert are in red. The 

DNA fragments of pSIM1678 are in blue. All molecular weights are presented in kilobases.

HindIII/SacI Pacl

IB 2.2 kb

IB 2.1 kb

IB 2.0 kb

IB 2.0 kb

IB 1.2 kb

OB 1.2 kb

OB 10

OB 5.5

JB 1.3 kb

IB 1.2 kb

JB 1.2 kb

IB 1.1 kb

DIGII DIGVII WT W8 WT W8 

HindIII/SacI Pacl

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0
J.R. Simplot Company JRS06 Page 75 of 196

Figure 5-16. Russet Burbank DNA Hybridization with the VNT1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with HindIII and EcoRI/Scal

and hybridized with the VNT probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1678 are in blue. All 

molecular weights are presented in kilobases.

HindIII EcoRI / 

Scal

OB 6.6

0B 3.6

JB 3.1

OB 3.5

OB 3.2

JB 0.5

DIGII DIGVII WT W8 WT W8 

HindIII EcoRI/Scal

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7

0.56

0.49
J.R. Simplot Company JRS06 Page 76 of 196

Figure 5-17. Russet Burbank DNA Hybridization with the VNT1 Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with HindIII/Sac1 and PacI

and hybridized with the VNT probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1678 are in blue. All 

molecular weights are presented in kilobases.

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb DIGII DIGVII WT W8 WT W8 WT W8 

XbaI PacI EcoRV

XbaI PacI EcoRV

OB 6.7

JB 6.1

OB 5.5

OB 4.8

OB 4.0

OB 15

OB 13

OB 5.2

JB 3.6

OB 15

JB 7

OB 4.9
J.R. Simplot Company JRS06 Page 77 of 196

Figure 5-18. Russet Burbank DNA Hybridization with the INV Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with HindIII and EcoRI/Scal

and hybridized with the INV probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1678 are in blue. All 

molecular weights are presented in kilobases.

HindIII EcoRI / 

Scal

OB 6.6

OB 6.1

JB 4.2

JB 7.0

IB 5.3

OB 1.8

OB 1.6

OB 1.1

DIGII DIGVII WT W8 WT W8 

HindIII EcoRI/Scal

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7
J.R. Simplot Company JRS06 Page 78 of 196

Figure 5-19. Russet Burbank DNA Hybridization with the INV Probe

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with XbaI, PacI and EcoRV

and hybridized with the INV probe. Size of the DigII and DigVII molecular weight markers are indicated 

adjacent to the blot image. The estimated sizes of bands are summarized in the table and classified into 

three groups based on the structure of the DNA insert: original endogenous bands (OBs, in green),

internal bands (IBs) and the junction bands (JBs). The DNA fragments of pSIM1678 are in blue. All 

molecular weights are presented in kilobases.

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb DIGII DIGVII WT W8 WT W8 WT W8 

XbaI PacI EcoRV

XbaI PacI EcoRV

OB 13

OB 6.7

OB 6.2

JB 5.5

IB 4.6

OB 11

OB 10

OB 8.5

OB 6.3

IB 4.7

OB 3.1

OB 14

OB 13

OB 12

JB 3.6
J.R. Simplot Company JRS06 Page 79 of 196

5.2.4 Conclusions: Copy number and structure of the DNA Inserts 

Genetic and structural characterization of the inserts associated with transformation of Russet Burbank 

by pSIM1278 and pSIM1678 to produce event W8 showed that both transformations resulted in a single 

integration site for each plasmid. The structure of the DNA derived from transformation of pSIM1278 

was complex (Figure 5-4) relative to the structure of the original insert. The inserted DNA appears to 

have undergone rearrangement during transformation resulting in a structure consisting of a tandem 

repeat of the Asn1/Ppo5 silencing cassette, followed by a nearly complete pSIM1278 construct, and an 

inverted repeat containing a duplication of the pR1/pPhl silencing cassette and a tandem duplication of 

the Gbss promoter with intervening Phl sequence (Figure 5-4). 

Although this structure is more complicated than anticipated, the duplicated silencing cassettes are 

intact and remain under the control of the tissue-specific promoters. The structure does not negatively 

impact safety or trait efficacy of the product (See Chapter 9: Safety of Russet Burbank W8 Potato and 

Chapter 10: Trait Efficacy). 

W8 also contains a single copy of the DNA from pSIM1678 that resides at a single locus of integration 

(Figure 5-5). The DNA insert of pSIM1678 contains a nearly intact DNA insert with a 330-bp deletion, 

which removes the entire T-DNA left border and 137-bp of the Rpi-vnt1 promoter. This small deletion in 

the promoter does not affect the gene’s ability to confer late blight resistance (Chapter 10: Trait 

Efficacy). Also, RNA expression associated with the Rpi-vnt1 gene has been demonstrated using RT-PCR, 

which is included in Chapter 6, Section 6.3: Rpi-vnt1 Gene Expression.

5.3 Flanking Regions of Inserted Sequences

As described in the previous section, each construct was integrated at a single locus in the Russet 

Burbank genome. We identified the flanking sequence for each construct using standard molecular 

approaches (see Appendix A: Genetic, Molecular, and Biochemical Methods for details) and aligned 

them to the reference genome sequence using the genomic resources available through Michigan State 

University’s Plant Biology Group. 

The reference genome is based upon sequencing a unique homozygous, doubled monoploid, form of 

the potato developed using tissue culture techniques (Potato Genome Sequencing Consortium, 2011)

and was later integrated with sequences from a heterozygous diploid line. Together, these efforts have 

provided a reference genome that is valuable for investigating the evolution and genome organization 

of potatoes, but is still lacking for detailed analysis of individual loci in commercial cultivars, which are 

mostly tetraploid. Although this reference genome can be valuable, many differences between it and 

Russet Burbank have been identified through the course of our work, and reliance upon it should be 

undertaken with caution. 

Based upon the reference genome assembly, the pSIM1278 construct appears to have been inserted on 

chromosome 2, whereas the pSIM1678 construct was mapped to a location on chromosome 10 (Figure 

5-20).
J.R. Simplot Company JRS06 Page 80 of 196

Figure 5-20. Predicted Chromosomal Insertion Sites for each Insert

Among the evidence supporting safety relative to the insertion site are the following: (1) most insertions 

do not disrupt gene expression because DNA integrates at random when no selection is used during 

transformation (Kim et al. 2007) and only approximately 5.5% of the potato genome is occupied by 

genes (Zhu et al. 2008); (2) insertions that do disrupt native potato gene functions are almost always 

recessive (Hagio et al. 2002; Chiou et al. 2006; Daxinger et al. 2008), which means that disruption of a 

single copy of the native gene would be unlikely to result in an observable phenotype; and (3) if an 

unusual and undesirable phenotype occurred, that event would be selected only if equivalent or 

superior to the untransformed potato variety. As discussed in Chapter 9: Safety of the W8 Potato, there 

is no evidence suggesting these insertions have disrupted any native Russet Burbank genes or introduce 

any safety concerns related to generation of allergens or toxins.

5.4 Absence of Plasmid Backbone Sequence

The W8 event was developed by transforming Russet Burbank with two constructs in series, pSIM1278 

followed by pSIM1678. Only the potato-derived transfer DNA sequence located between the left and 

right border sequences is designed to be inserted into the potato genome, but not the plasmid 

backbone sequences (Chapter 4: Donor Genes, Figures 4-1 and 4-2; grey highlighting). As the backbone 

sequence of the two plasmids is identical, molecular evidence showing the absence of backbone 

sequence simultaneously confirms its absence from both transformations. 

The following methods were used to establish that backbone portions of the plasmid were not present 

in events developed for commercial purposes: 1) If plants had phenotypes associated with the negative 

selectable isopentenyl isomerase (ipt) marker gene in the plasmid backbone, they were discarded; 2) 

Absence of the backbone DNA was confirmed with Southern blot hybridization; 3) PCR was used to 

confirm no fragments of the backbone DNA were present. Our results indicate that the W8 event does 

not contain backbone sequence as described below.

5.4.1 Selection of Backbone-free Plants

Agrobacterium-mediated transformation often results in transfer of plasmid backbone DNA, in addition 

to the intended DNA positioned between the left and right borders (LB and RB) of the plant-derived DNA

insert. The frequency of transfer of backbone DNA has been estimated at 75% for tobacco (Kononov et 

al. 1997) and 47% - 67% for Arabidopsis (Oltmanns et al. 2010). To reduce the number of transformants 

that must be characterized molecularly, we employed a phenotypic screen for plants that contain the 

Agrobacterium ipt gene, which is present in the plasmid backbone (Richael et al. 2008). When this gene 

is introduced into potatoes, its overexpression results in stunted growth, abnormal leaves, or the 

inability to root due to overproduction of cytokinin. Thus, these phenotypes were used to select against 

plants containing backbone DNA. The Russet Burbank W8 event did not present abnormal growth 

phenotypes and was further characterized using molecular methods to show that it did not contain any 
J.R. Simplot Company JRS06 Page 81 of 196

backbone sequences integrated into the plant genome. Additional details regarding the transformation 

process and selection method can be found in Chapter 3: Transformation Method.

5.4.2 Molecular Evidence for Backbone-free Plants

A detailed molecular characterization of W8 genomic DNA was undertaken to show the absence of 

backbone DNA using a combination of Southern blot and PCR-based analyses. 

Absence of backbone DNA in W8 using Southern blotting methods 

A series of Southern probes were designed to span the length of the plasmid backbone for detection of 

any backbone DNA residing in the genome of the transformed potatoes (Figure 5-21). There were two 

gaps, Gap A and Gap B, not detected by the probe set, but were instead analyzed by PCR. Since the 

absence of DNA is based upon a lack of detection or amplification, a positive control (T130) 

transformant was developed, which contains an integrated copy of the entire plasmid backbone. 

Genomic DNA isolated from W8, T130, and Russet Burbank controls (WT) were digested with EcoRI and 

probed with each of the six probes. As shown in Figure 5-22, bands were limited to the positive control 

(T130) samples in all Southern blots, except for probe 6, which contained a pair of bands common 

between the Russet Burbank control and W8. Backbone would only be confirmed in an event if there 

were unique bands associated with the positive controls and not found in the untransformed controls. 

Thus, these bands are not associated with backbone DNA, and all Southern blots are consistent with a 

lack of backbone DNA in the W8 genome.

Figure 5-21. Probes for the backbone of pSIM1278 and pSIM1678

1

Numbering system based on pSIM1278 (Chapter 4: Donor Genes, Table 4-1 and Figure 4-1), but backbone 

sequences are identical to pSIM1678. The backbone DNA is spanned by the probes shown as blue rectangles with 

two small gaps labeled as Gap A and Gap B detected by PCR using the indicated primer sets. The gapped regions 

exist in the potato genome and can be amplified using the internal primer sets, while the flanking set of primers is 

unique to the backbone DNA. 

1474 bp

Probe 1

Probe 4 Probe 5 Probe 6 Probe 2

1515 bp Probe 3

1588 bp

1660 bp 1074 bp 834 bp

Pat P pBR322 bom

PVS1 Sta

nptIII

pVS1Rep

pBR322 ori

Ubi3T RB LB

UBQmon

ipt

EcoRI (16227)

Ubi7

EcoRI (17793)

Y277 - Y278

Y279 - Y280

Y281 - Y282

Y283 - Y284

• Amplification from Potato

• No Amplification from Potato

Gap A Gap B
J.R. Simplot Company JRS06 Page 82 of 196

Figure 5-22. Analysis of Plasmid Backbone DNA using Southern Blotting 

Genomic DNA was digested with EcoRI and analyzed by Southern blot using probes 1-6. WT = Russet Burbank control, W3 = 

additional event that was not submitted, W8 = Event W8, T130 = positive control containing backbone DNA. Lanes 1 and 2 are 

molecular weight markers (DIG II, and DIGVII, respectively) with sizes indicated next to gel.

Probe 1 Probe 2 Probe 3

Probe 4 Probe 5 Probe 6
J.R. Simplot Company JRS06 Page 83 of 196

Absence of backbone DNA in W8 using PCR-based methods

The two gapped regions not covered by Southern probes were independently characterized using a set 

of PCR assays. First, PCR was performed using a set of primers that hybridized fully within the Gap A 

(Y277-Y278) and Gap B (Y281-Y282) regions (Figure 5-21). Each of these primer sets successfully 

amplified sequence corresponding to the gapped regions (Figure 5-23, left panel of gels), but the 

sequence corresponded to the Russet Burbank genome, not to the plasmid backbone. Sequence 

comparisons showed that the sequence of the W8 and Russet Burbank PCR products were identical, but 

nucleotide polymorphisms distinguished these sequences from the backbone DNA and T130. Further 

evidence for the absence of these gapped regions in W8 was obtained by a second set of PCR using 

primers specific to the flanking regions, just outside of the gaps (Y279-Y280, Gap A; Y283-Y284, Gap B; 

Figure 5-21). Unlike the internal gap-specific primers, these primers do not amplify Russet Burbank 

sequence (WT) as shown in Figure 5-23 (right panel of gels), but do amplify the gapped regions from the 

plasmid backbone as shown in the positive control (PC) sample. Importantly, these primers did not 

detect backbone in the W8 samples.

Figure 5-23. PCR Amplification of Gapped Regions in Plasmid Backbone

Ethidium bromide stained agarose gels for the PCR reactions using the primer sets indicated. PC = positive control, Lane 1 = 

100-bp DNA marker (Invitrogen), WT = Control Russet Burbank, W3 = Additional event that was not submitted, W8 = Submitted 

Event, PC = Positive control, NC = Negative control.

Y277/Y278

Y281/Y282

WT W3 W8 PC NC

Gap A

Gap B

Y283/Y284

WT W3 W8 PC NC

Y281/Y282

WT W3 W8 PC NC

Y279/Y280

WT W3 W8 PC NC

Y277/Y278
J.R. Simplot Company JRS06 Page 84 of 196

Another PCR assay was used to verify the absence of sequence containing the junction between 

backbone and the DNA insert from either pSIM1278 or pSIM1678. As diagrammed in Figure 5-24, each 

junction was tested using two sets of primers. Since the plasmids have identical right junctions, its 

presence was tested using a shared set of PCR primers (JY725-JY726, JY915-JY749). The left junction was 

tested using two sets of primers that were specific to either pSIM1278 or pSIM1678. All of the PCR 

reactions failed to identify junction regions in either W8 or WT samples, whereas in each case positive 

controls amplified as expected (Figure 5-25). Thus, the backbone DNA adjacent to the left and right 

border regions was not introduced into W8 for either transformation. These findings are further 

supported by the lack of backbone DNA within the flanking sequences for each insert (Section 5.3).

Figure 5-24. PCR Primer Sets for Detection of Backbone Junction Sites
J.R. Simplot Company JRS06 Page 85 of 196

Figure 5-25. Confirmation of the Absence of Backbone DNA Junctions

Detection of Left Border of the DNA insert and flanking backbone of pSIM1278

Detection of Left Border of the DNA insert and flanking backbone of pSIM1678

Detection of Right Border of the DNA insert and flanking backbone of pSIM1278 and pSIM1678

Ethidium bromide stained agarose gels for the PCR reactions using the primer sets indicated. PC = positive control, M = 100-bp 

DNA marker (Invitrogen), W3 = Additional event that was not submitted, W8 = Submitted Event, WT = Control Russet Burbank, 

NC = Negative control.

DNA Ladders PC M W3 W8 WC NC

(bp)

1500

1000

600

300

100

JY719-JY718

DNA Ladders

(bp)

1500 

1000

600

300

100

PC M W3 W8 WC NC

JY1029-JY1030

DNA Ladders PC M W3 W8 WC NC

(bp)

1500

1000

600

300

100

JY719-JY718

DNA Ladders

(bp)

1500

1000

600

300

100

PC M W3 W8 WC NC

JY719-Y180 JY1029 Y179--JY1030 Y180

DNA Ladders PC M W3 W8 WC NC

(bp)

JY719-JY718

1500

1000

600

300

100

DNA Ladders

(bp)

1500

1000

600

300

100

JY1029-JY1030

PC M W3 W8 WC NC

JY725-JY726 JY915-JY749
J.R. Simplot Company JRS06 Page 86 of 196

5.4.3 Conclusions: Absence of Plasmid Backbone

Collectively, our Southern blot and PCR analyses have shown that the Russet Burbank W8 event does 

not contain backbone from either plasmid used in the transformations.

5.5 Genetic stability of W8 over three generations

Bacterial T-DNAs are not always stable after insertion into a plant. The estimated instability rate (0.5-

5.9×10-4) is associated with meiosis (Müller et al. 1987; Conner et al. 1998), which is not relevant to 

potatoes as they reproduce vegetatively. Thus, DNA insertions are expected to be stable. Tubers rather 

than seeds were used to define subsequent generations since tubers are what are commercially planted.

Genetic stability was assessed using both molecular and phenotypic assays. The structure of the insert 

was shown to be stable using Southern blot analysis of genomic DNA isolated over three generations of 

W8 potatoes (G0 - G3), whereas the phenotypic stability was assessed by measuring polyphenol oxidase 

activity, in the second generation of field-grown tubers. This method shows visual evidence of PPO 

silencing after applying catechol to the cut surface of potatoes as shown in Chapter 10, Trait Efficacy. 

These studies were carried out to ensure that the desired genetic changes in W8 remained stable over 

multiple clonal cycles while maintaining the traits.

5.5.1 Molecular Evidence for Stability of the DNA Insert 

The stability of the DNA inserts was evaluated by comparing three successive clonal generations (G1, G2, 

and G3) to the original transformant (G0) using Southern blots. Stable DNA inserts are expected to 

maintain the same structure and thus produce the same digestion patterns over multiple generations of 

the plant. To test stability of the inserts in the W8 event, we compared its digestion pattern using two 

probes (GBS1 and AGP) that hybridize to regions of the inserts from both pSIM1278 and pSIM1678, and 

two probes (INV and VNT1) that are specific to the pSIM1678 insert. Since the DNA sequences these 

probes hybridize with are contained in the potato genome as well as within the DNA insert(s), both 

endogenous and insert-specific bands are expected in the Southern blots.

All genomic DNA samples were digested with the restriction enzyme, EcoRV, and hybridized with a 

probe specific to either AGP or GBS1. EcoRV was chosen for these studies as it digests within both 

inserts to provide a unique banding pattern with internal bands of predicted size in the pSIM1278 insert 

(e.g. 2.3 kb). The banding patterns between all samples of W8 were identical to each other for both 

probes (Figure 5-26). The multiple bands present in the Russet Burbank control are also found in W8, 

but W8 also contains bands corresponding to the pSIM1278 and pSIM1678 inserts. These bands are 

similarly consistent between all generations of W8 analyzed indicating genetic stability of both inserts.

A second analysis was performed using two probes specific to the pSIM1678 insert. For this analysis, 

genomic DNA samples were digested with the restriction enzyme, XbaI, and hybridized with VNT1 and 

INV probes. XbaI was chosen as the restriction enzyme for these studies as it digests the pSIM1678 

internally and produces a band of known size (e.g. 4.6 kb for the INV probe). Again, both endogenous 

and insert-specific bands were detected with consistent banding patterns between the three 

generations analyzed (Figure 5-27).
J.R. Simplot Company JRS06 Page 87 of 196

Figure 5-26. Southern blot probing of EcoRV digestion 

Southern blots of genomic DNA (3 μg) following digestion with EcoRV and probed for AGP sequence. Each blot compares the indicated generation of DNA from the initial 

transformant (G0). Russet Burbank untransformed controls (WT), W3 = additional event that was not submitted, W8 = submitted event. Genetic stability is established by the 

consistent digestion pattern for each line between the first generation (G0) and each subsequent generation (G1 - G3) within a set of gels.

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

0.7

WT WT WT W3 W3 W3 W8 W8 W8

MII MVII G0 G1 G1 G0 G1 G1 G0 G1 G1

kb

WT WT WT W3 W3 W3 W8 W8 W8

MII MVII G0 G2 G2 G0 G2 G2 G0 G2 G2

2.8

2.3

2.0

1.52

1.48

1.2

23.1

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8.6

7.4

6.6

6.1

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1.95

1.88

1.0

0.7

kb

WT WT WT W3 W3 W3 W8 W8 W8

MII MVII G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

1.0

0.7

(A) AGP Probe

Generation 1 Generation 2 Generation 3
J.R. Simplot Company JRS06 Page 88 of 196

Figure 5-26. Southern blot probing of EcoRV digestion (cont.)

Southern blots of genomic DNA (3 μg) following digestion with EcoRV and probed for GBS1 sequence. Each blot compares the indicated generation of DNA from the initial 

transformant (G0). Russet Burbank untransformed controls (WT), W3 = additional event that was not submitted, W8 = submitted event. Genetic stability is established by the 

consistent digestion pattern for each line between the first generation (G0) and each subsequent generation (G1 - G3) within a set of gels.

(B) GBS1 Probe

Generation 1 Generation 2 Generation 3

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

WT WT WT W3 W3 W3 W8 W8 W8

MII MVII G0 G1 G1 G0 G1 G1 G0 G1 G1

WT WT WT W3 W3 W3 W8 W8 W8

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb MII MVII G0 G2 G2 G0 G2 G2 G0 G2 G2

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb WT WT WT W3 W3 W3 W8 W8 W8

MII MVII G0 G3 G3 G0 G3 G3 G0 G3 G3
J.R. Simplot Company JRS06 Page 89 of 196

Figure 5-27. Southern blot probing of XbaI digestion 

Southern blots of genomic DNA (3 μg) following digestion with XbaI and probed for Rpi-vnt1 sequence. Each blot compares the indicated generation of DNA from the initial 

transformant (G0). Russet Burbank untransformed controls (WT), W3 = additional event that was not submitted, W8 = submitted event. Genetic stability is established by the 

consistent digestion pattern for each line between the first generation (G0) and each subsequent generation (G1 - G3) within a set of gels.

(A) VNT1 Probe

Generation 1 Generation 2 Generation 3

kb

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

1.0

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G1 G1 G0 G1 G1 G0 G1 G1

2.8

2.3

2.0

1.95

1.88

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

kb

1.0

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G2 G2 G0 G2 G2 G0 G2 G2

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

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3.6

1.95

1.88

kb

1.0

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G3 G3 G0 G3 G3 G0 G3 G3
J.R. Simplot Company JRS06 Page 90 of 196

Figure 5-27. Southern blot probing of XbaI digestion (cont.) 

Southern blots of genomic DNA (3 μg) following digestion with XbaI and probed for VInv sequence. Each blot compares the indicated generation of DNA from the initial 

transformant (G0). Russet Burbank untransformed controls (WT), W3 = additional event that was not submitted, W8 = submitted event. Genetic stability is established by the 

consistent digestion pattern for each line between the first generation (G0) and each subsequent generation (G1 - G3) within a set of gels. 

(B) INV Probe

Generation 1 Generation 2 Generation 3

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G1 G1 G0 G1 G1 G0 G1 G1

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G2 G2 G0 G2 G2 G0 G2 G2

2.8

2.3

2.0

1.52

1.48

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

2.8

2.3

2.0

1.52

1.48

1.2

23.1

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8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.95

1.88

kb

1.0

WT WT WT W3 W3 W3 W8 W8 W8 MII MVII G0 G3 G3 G0 G3 G3 G0 G3 G3

0.7
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5.5.2 Phenotypic Evidence for Stability of the DNA Insert

Additional evidence for insert stability was obtained by measuring resistance to black spot in W8 

potatoes using a catechol assay that measures PPO activity (see Chapter 10, Section 10.1.4 Black Spot

for additional data and details of the assay). Black spot resistance is conferred by silencing of the Ppo5

gene in tubers, which is being suppressed by a silencing cassette within the pSIM1278 insert. This assay 

provided phenotypic evidence for maintenance of a functional Ppo5 silencing cassette in the Russet 

Burbank W8 event. 

5.5.3 Summary of Genetic Stability Studies

The genetic and phenotypic analyses indicated the insertions arising from transformation of both 

pSIM1278 and pSIM1678 are stable over three generations. Given the demonstrated stability over three 

generations, it is likely that stability will be maintained during subsequent cycles of vegetative 

propagation. The catechol assay provides a convenient assay for monitoring stability as it is indicative of 

black spot tolerance, but also low asparagine formation because Ppo5 and Asn1 gene silencing are 

mediated by the same silencing cassette in the pSIM1278 insert. Although instability is unlikely and 

undesired, the result would be a loss of activity and reversion to wild-type, which would not trigger any 

biosafety issues as a result.

5.6 Characteristics of Transferred DNA and Gene Regulation

The DNA inserts introduced into W8 include three expression cassettes designed to partially silence up 

to five endogenous potato genes in a tissue-specific manner. Transcription of each cassette leads to the 

production of double-stranded RNA containing sequence derived from and specific to target potato 

genes, which are silenced using the cellular RNA interference (RNAi) pathway. Silencing of target genes 

leads to a reduction in acrylamide, black spot bruising, and accumulation of reducing sugars in W8 

potatoes. A detailed description of the gene silencing cassettes, mode of action, and efficacy is 

described in Chapter 7: Characterization of Gene Silencing and Target Gene Expression.

The DNA insert derived from pSIM1678 contains an additional expression cassette consisting of the Rpivnt1 gene. This gene is expressed under its native potato promoter and provides late blight resistance 

against P. infestans. A detailed description and characterization of Rpi-vnt1 gene expression, VNT1 

protein accumulation, safety and mode of action is described in Chapter 6: Characterization and Safety 

of the VNT1 Protein.

5.7 Event-Specific PCR 

As part of our stewardship and identity preservation plan a method was developed to allow low-level 

detection of Russet Burbank W8 potatoes in plants and food products. The quantitative PCR (qPCR) 

method reliably detects the presence of W8 at concentrations of 0.2% with a 95% confidence interval 

(Appendix E: Detection of Russet Burbank W8 Using Real-Time PCR). The method has high specificity as 

it distinguishes between W8 and other events established through transformation with the same 

plasmids. The methods developed will be used to monitor plants and tubers in field and storage as part 

of quality management programs.
J.R. Simplot Company JRS06 Page 92 of 196

5.8 Summary of the Genetic Characterization of Event W8

The Russet Burbank W8 Event was transformed with two plasmids, pSIM1278 and pSIM1678. The DNA 

inserted from each transformation consisted solely of sequence targeted for insertion and did not 

contain any detectable plasmid backbone in the genome of W8. Transformation of each plasmid led to a 

single integration event in the genome. The sequence and structure of the DNA inserts and the genomic 

flanking regions were determined for both plasmids. The structure of the insert associated with the 

pSIM1278 plasmid was more complex than the original plasmid, but the duplicated silencing cassettes 

remained intact and under the control of tissue-specific promoters (Figure 5-4). The insert associated 

with the pSIM1678 plasmid was very similar to the input sequence, but contained a small deletion 

consisting of the left border and a small region of the Rpi-vnt1 promoter (Figure 5-5). This small deletion 

in the promoter does not affect the gene’s ability to confer late blight resistance (Chapter 10: Trait 

Efficacy). The structures of the DNA inserts were shown to be stable over three generations and a 

method of detecting low levels of W8 was developed. 

No biosafety concerns associated with the DNA inserts or the integration site were identified (See 

Chapter 9: Safety of Russet Burbank W8 Potato and Chapter 10: Trait Efficacy). 
J.R. Simplot Company JRS06 Page 93 of 196

5.9 References

Chiou, T.J., Aung, K., Lin, S.I., Wu, C.C., Chiang, S.F., and Su, C.I. (2006). Regulation of phosphate 

homeostasis by microRNA in Arabidopsis. Plant Cell. 18: 412-421.

Conner, A.J., Mlynárová, L., Stiekema, W.J., and Nap, J.P. (1998). Meiotic stability of transgene 

expression is unaffected by flanking matrix-associated regions. Mol Breeding. 4: 47-58.

Daxinger, L., Hunter, B., Sheikh, M., Jauvion, V., Gasciolli, V., Vaucheret, H., Matzke, M., and Furner,I.

(2008). Unexpected silencing effects from T_DNA tags in Arabidopsis. Trends Plant Sci. 13: 4-6.

Hagio, M., Sakurai, I., Sato, S., Kato, T., Tabata, S., and Wada, H. (2002). Phosphatidyglycerol is essential 

for the development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol. 43: 1456-1464.

Kim, S.I., Veena, and Gelvin, S.B. (2007). Genome-wide analysis of Agrobacterium T-DNA integration 

sites in the Arabidopsis genome generated under non-selective conditions. Plant J. 51: 779-791.

Kononov, M.E., Bassuner, B., and Gelvin, S.B. (1997). Integration of T-DNA binary vector ‘backbone’ 

sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J. 11: 

945-957.

Müller, A.J., Mendel, R.R., Schiemann, J., Simoens, C., and Inzé, D. (1987). High meiotic stability of a 

foreign gene introduced into tobacco by Agrobacterium-mediated transformation. Mol Gen Genet. 207: 

171-175.

Oltmanns, H., Frame, B., Lee, L.Y., Johnson, S., Li, B., Wang, K., and Gelvin, S.B. (2010). Generation of 

backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. 

Plant Physiol. 152: 1158-1166.

The Potato Genome Sequencing Consortium, “Genome sequence and analysis of the tuber crop potato,” 

Nature. 2011. 475: 189-197.

Richael, C.M., Kalyaeva, M., Chretien, R.C., Yan, H., Adimulam, S., Stivison, A., Weeks, J.T., and 

Rommens, C.M. (2008). Cytokinin vectors mediate marker-free and backbone-free plant transformation. 

Transgenic Res. 17: 905-917. 

Zhu, W., Ouyang, S., Iovene, M., O'Brien, K., Vuong, H., Jiang, J., and Buell, C.R. (2008). Analysis of 90 Mb 

of the potato genome reveals conservation of gene structures and order with tomato but divergence in 

repetitive sequence composition. BMC Genomics. 9: 286.
J.R. Simplot Company JRS06 Page 94 of 196

6.0 Characterization and Safety of the VNT1 Protein

The VNT1 protein produced through introduction of the Rpi-vnt1 gene into the Russet Burbank Event 

W8 has been characterized and assessed for safety. A description of VNT1’s origin, function, mode of 

action, and an assessment for toxins and allergens are presented along with its pattern of expression 

and protein concentration. The details of the materials and methods used in these studies are provided

in Appendix A: Genetic, Molecular, and Biochemical Methods and Appendix B: Characterization of VNT1.

6.1 Identity and Function of the VNT1 Protein

Identity

Resistance proteins (R-proteins) such as VNT1 are signal transduction ATPases with homologs found in 

all domains of life (Leipe et al. 2004). They have a conserved domain structure consisting of a central 

nucleotide-binding domain flanked by either an N-terminal toll-like/interleukin-1 (TIR) or coiled-coil (CC) 

domain and a C-terminal leucine-rich repeat (LRR) (Takken and Goverse 2012). The LRR is the least 

conserved and has been hypothesized to function in effector recognition. 

Using Simplot InnateTM Technologies, an Rpi-vnt1 gene (accession: FJ423044.1) was transformed into the

Russet Burbank potato variety after insertion into the pSIM1678 plasmid, resulting in late blight 

resistance. This allele is one of three identified in the wild potato species, Solanum venturii, and is 

identical to the Rpi-phu1 gene from the related species, S. phureja (Sliwka et al. 2013). The VNT1 protein 

confers resistance to late blight with a relatively broad resistance spectrum against the plant pathogen 

P. infestans, a member of the oomycete class of fungus-like microorganisms. It is an 891 amino acid 

(Figure 6-1) CC-NB-LRR R-protein and a homolog of the Tm-22 tomato mosaic virus disease resistance 

protein (Foster et al. 2009). 

Figure 6-1. Amino Acid Sequence of VNT1

001 MNYCVYKTWA VDSYFPFLIL TFRKKKFNEK LKEMAEILLT AVINKSIEIA 

051 GNVLFQEGTR LYWLKEDIDW LQREMRHIRS YVDNAKAKEV GGDSRVKNLL

101 KDIQQLAGDV EDLLDEFLPK IQQSNKFICC LKTVSFADEF AMEIEKIKRR

151 VADIDRVRTT YSITDTSNNN DDCIPLDRRR LFLHADETEV IGLEDDFNTL

201 QAKLLDHDLP YGVVSIVGMP GLGKTTLAKK LYRHVCHQFE CSGLVYVSQQ

251 PRAGEILHDI AKQVGLTEEE RKENLENNLR SLLKIKRYVI LLDDIWDVEI

301 WDDLKLVLPE CDSKIGSRII ITSRNSNVGR YIGGDFSIHV LQPLDSEKSF

351 ELFTKKIFNF VNDNWANASP DLVNIGRCIV ERCGGIPLAI VVTAGMLRAR

401 GRTEHAWNRV LESMAHKIQD GCGKVLALSY NDLPIALRPC FLYFGLYPED

451 HEIRAFDLTN MWIAEKLIVV NTGNGREAES LADDVLNDLV SRNLIQVAKR

501 TYDGRISSCR IHDLLHSLCV DLAKESNFFH TEHNAFGDPS NVARVRRITF

551 YSDDNAMNEF FHLNPKPMKL RSLFCFTKDR CIFSQMAHLN FKLLQVLVVV

601 MSQKGYQHVT FPKKIGNMSC LRYVRLEGAI RVKLPNSIVK LKCLETLDIF

651 HSSSKLPFGV WESKILRHLC YTEECYCVSF ASPFCRIMPP NNLQTLMWVD

701 DKFCEPRLLH RLINLRTLCI MDVSGSTIKI LSALSPVPRA LEVLKLRFFK

751 NTSEQINLSS HPNIVELGLV GFSAMLLNIE AFPPNLVKLN LVGLMVDGHL

801 LAVLKKLPKL RILILLWCRH DAEKMDLSGD SFPQLEVLYI EDAQGLSEVT

851 CMDDMSMPKL KKLFLVQGPN ISPISLRVSE RLAKLRISQV L
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Mode of Action

Expression of Rpi-vnt1 in wild and cultivated potato confers broad-spectrum resistance to late blight 

caused by P. infestans. A critical layer of the plant defense system against pathogens relies upon 

recognition of pathogen-secreted effector proteins by resistance protein receptors (R-proteins). Rprotein mediated immunity is part of a hypersensitive response that leads to destruction of infected 

plant tissue through programmed cell death (Moffett et al. 2002; Qi et al. 2012; Rairdan et al. 2008;

Ayliffe et al. 1999; Tan et al. 2007; Morel and Dangl 1997). These R-proteins are tightly regulated and are 

maintained in an inactive state at low concentrations in the cell and are activated by their specific 

cognate ligands from the pathogen (Spoel and Dong 2012).

Specifically, following infection, P. infestans secretes an effector protein (AVR-VNT1), which is 

recognized by the R-protein, VNT1, expressed in potato. Consequently, VNT1 activates a signal 

transduction cascade that leads to localized plant cell death, which restricts growth and spread of the 

pathogen to the rest of the plant (Pel Dissertation 2010). Importantly, unlike Bt proteins, R-proteins do 

not confer pest resistance by directly targeting the pest or acting as toxins, but instead activate an 

immune response within the host plant. 

6.2 Levels of the VNT1 protein in W8 Tissues

A number of VNT1 antibodies were generated and tested for sensitivity and specificity, and the antibody 

(5363) recognizing the peptide, FHSSKLPFGVWESKIL, of the LRR domain was chosen for detection and 

quantitative analyses. Recombinant VNT1 protein (VNT1-LRR domain) was purified from E. coli and used 

to determine the limit of detection and quantitation. As shown in Figure 6-1, antibody 5363 was 

sensitive enough to detect as little as 9 pg of purified VNT1 protein. For methods and supporting data 

refer to Appendix B: Characterization of VNT1. 

Figure 6-1. Limit of detection for anti-VNT1 antibody 5363

Limit of detection for anti-VNT1 antibody 5363. (A) Western blot analysis of a serial dilution of recombinant VNT1-LRR. (B) 

Quantitative curve showing linearity of western blot data.

A B
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Western blots using the sensitive antibody, 5363, were used to investigate the expression levels of VNT1 

in various plant tissues. The VNT1 protein was undetectable in all tissues analyzed, indicating it was 

expressed at very low levels as expected (Figure 6-2).

Figure 6-2. Western blot analysis of VNT1 in potato tissues

Detection of VNT1 in Russet Burbank, S. venturii, W3, and W8 tissues. Proteins were extracted from each tissue and quantified 

by BCA assay. 320 ± 75 ng of total protein extracted from tissues from Russet Burbank (RB), S. venturii (S.vtri), W3, and W8 

plants were loaded into each well of an SDS-PAGE gel. Western Blot analysis was performed using anti-VNT1 (5363) antibody. 

MW, molecular weight marker; NB, N. benthamiana expressed VNT1 (2.5 ng); LRR, E. coli expressed VNT1-LRR domain (2.5 ng). 

W3 = Additional event that was not submitted.

Given the low levels of VNT1 in the W8 tissue samples, we measured the limit of quantitation (LOQ) in 

samples isolated from those tissues to identify an upper bound on VNT1 expression levels. The results of 

those experiments are presented in Appendix B: Characterization of VNT1 and summarized here in Table 

6-1.

Table 6-1. Limit of Detection, Quantification and Expression of VNT1 in Potato.

Tissue Type LOD

(pg)

LOQ

(ng g-1or ppb)

Russet Burbank

(ng g-1or ppb)

W8

(ng g-1or ppb)

Leaf 9 60 < 60 < 60

Tuber 9 30 < 30 < 30

Stem 9 20 < 20 < 20

Root 9 15 < 15 < 15

Flower 9 60 < 60 < 60

VNT1 concentrations were calculated using ng g-1dry weight basis (equivalent to parts per billion, ppb).
J.R. Simplot Company JRS06 Page 97 of 196

We found that the LOQ ranged between 15 and 60 ppb depending on the tissue sample. Western blot 

analysis was used to measure VNT1 concentration in protein extracts isolated from tissue where the 

amount of protein loaded was sufficient to allow detection of VNT1 at or above our measured LOQ 

(Figure 6-3). As shown, we were unable to detect VNT1 protein expressed in any tissues isolated from 

the Russet Burbank W8 event, but were able to detect VNT1 spiked in at concentrations similar to our 

reported LOQ.

Importantly, VNT1 was undetectable using an assay able to measure concentrations as low as 30 ppb in 

tubers, yet VNT1 expression was still able to provide resistance to late blight as described in Chapter 10:

Trait Efficacy. In general, R-proteins are naturally low abundance and are estimated to be at 

concentrations as low as 18 ppt with low exposure levels in humans (3.6 ng protein/year) (Bushey, 

2014). The concentration of VNT1 in tubers was minimally multiple orders of magnitude lower than has 

been determined for Bt protein in corn, soybean, cotton, and other crops deregulated by APHIS. In one 

example, the Cry1Ac protein in Bt soybean samples was expressed at mean levels of 25.4 ppm in leaves 

and 1.04 ppm in grain (Han 2012).

Figure 6-3. Quantitation of VNT1 in W8 tissue samples is below LOQ. 

Spiked samples labeled as +VNT1.

Western Blot analysis using antibody 5363 demonstrates that VNT1 is below detectable limits in all W8 tissues. Protein was 

extracted from 0.5 mg of leaf, 1 mg of tuber, 1mg of root, 1.5 mg of stem, and 0.5 mg flower. 30 pg of VNT1 were spiked into 

leaf, tuber, stem, and flower extracts and 15 pg of VNT1 were spiked into root extracts. LOQ values are shown in ppb.

6.3 Rpi-vnt1 Gene Expression

Since the concentration of VNT1 protein was too low to detect using a highly sensitive antibody, 

quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) was used to verify expression of 

the Rpi-vnt1 transcript in W8, which was not readily detectable by northern blot (data not shown). Total 

RNA was isolated from tissues of both Russet Burbank and the W8 event and subjected to quantitative 

RT-PCR using Rpi-vnt1 specific primers (see Appendix A: Genetic, Molecular, and Biochemical Methods

for details). Expression of Rpi-vnt1 was normalized to a set of endogenous housekeeping genes, αtubulin and elongation factor 1α, within each sample. Although expression was low in W8 tissue 

samples, it was considerably higher than in the Russet Burbank controls (Figure 6-4).

These data indicate that the Rpi-vnt1 gene is transcribed in all W8 tissues analyzed, but are consistent 

with low levels of expression and protein accumulation in these potatoes.
J.R. Simplot Company JRS06 Page 98 of 196

Figure 6-4. Rpi-vnt1 expression measured by RT-qPCR

Rpi-vnt1 expression measured by RT-qPCR. Each set of samples were analyzed in triplicate and normalized to a pair of 

endogenous genes (α-tubulin and elongation factor 1α). All analyses were performed using SYBR green dye with an associated 

melting curve analysis. RB (Russet Burbank); W8 (Event W8).

6.4 Assessment of VNT1 for Allergens and Toxins

Using a number of well-established bioinformatics tools, we performed a comprehensive analysis of 

possible toxins and allergens associated with the Rpi-vnt1 gene (accession FJ423044.1) and the 

associated VNT1 protein sequence (Chapter 9: Safety of Russet Burbank W8 Potato). There was no 

significant sequence identity between ORFs within the Rpi-vnt1 gene and known allergens or toxins, 

indicating VNT1 is not homologous with any known allergens or toxins. Thus, it is highly unlikely that 

introduction of this gene into Russet Burbank potatoes would represent a safety risk related to allergens

or toxins.

6.5 Conclusions on VNT1 Protein Safety

Biochemical characterization and safety assessment of the VNT1 protein suggested a negligible risk 

associated with using VNT1 to confer late blight resistance to potato cultivars. The encoded VNT1 

protein is native to Solanum species and provides protection against P. infestans, the causal agent of 

late-blight disease. In fact, the Rpi-vnt1 (also called Rpi-phu1) gene has been introgressed into S. 

tuberosum (tetraploid potato varieties) from an interspecific cross between Solanum phureja and 

Solanum stenotomum (known as pinta boca in Peru and Bolivia). Both of these species are edible and 

research programs are underway in Europe to introgress Rpi-phu1/Rpi-vnt1 in cultivated potatoes 

(Sliwka et al. 2010; 2013). Currently VNT1 represents only one of 68 distinct R-genes that have been 

characterized from wild Solanum species, which shows the breadth and importance of these genes to 

potato biology (Rodewald and Trognitz 2013). 

Although we were able to confirm expression of the Rpi-vnt1 gene in W8 plants, the protein 

concentration was below our limit of quantitation in all tissues tested, yet Rpi-vnt1 conferred late blight 

resistance (Chapter 10: Trait Efficacy). A bioinformatics assessment of possible allergens and toxins 

associated with the VNT1 protein did not identify any safety concerns.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

RB root W8 root RB Leaf W8 Leaf RB stem W8 stem RB flower W8 flower RB tuber W8 tuber

Relative quantity of Rpi-vnt1 transcripts
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Collectively, the ubiquitous nature of R-genes and their encoded R-proteins in Solanum species, 

including tomato and edible potatoes, lack of sequence identity to known toxins or allergens, mode of 

action not based upon inducing pest toxicity, and extremely low expression levels provides confidence in 

the safe use of potato varieties created to express VNT1.
J.R. Simplot Company JRS06 Page 100 of 196

6.6 References

Ayliffe, M.A., Frost, D.V., Finnegan, E.J., Lawrence, G.J., Anderson, P.A., and Ellis, J.G. (1999). Analysis of 

Alternative Transcripts of the Flax L6 Rust Resistance Gene. The Plant Journal. 17: 287–292.

Bushey, D.F., Bannon, G.A., Delaney, B.F., Graser, G., Hefford, M., Jiang, X., Lee, T.C., Madduri, K.M., 

Pariza, M., Privalle, L.S., et al. (2014). Characteristics and Safety Assessment of Intractable Proteins in 

Genetically Modified Crops. Regulatory Toxicology and Pharmacology doi: 

http://dx.doi.org/10.1016/j.yrtph.2014.03.003.

Foster, S.J., Park, T.H., Pel, M., Brigneti, G., Sliwka, J., Jagger, L., van der Vossen, E., and Jones, J.D.G. 

(2009). Rpi-vnt1. 1, a Tm-22 Homolog from Solanum Venturii, Confers Resistance to Potato Late Blight. 

Molecular Plant-Microbe Interactions 22: 589–600.

Han, L. (2012). Petition for Determination of Nonregulated Status for Insect-Resistant DAS-81419-2 

Soybean. http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml.

Leipe, D.D., Koonin, E.V., and Aravind, L. (2004). STAND, a class of P-Loop NTPases including animal and 

plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic 

patters, and evolution by horizontal gene transfer. J Mol Bio. 343: 2-18.

Moffett, P., Farnham, G., Peart, J., and Baulcombe, D.C. (2002). Interaction Between Domains of a Plant 

NBS-LRR Protein in Disease Resistance-Related Cell Death. The EMBO Journal. 21: 4511–4519.

Morel, J.B., and Dangl, J.L. (1997). The Hypersensitive Response and the Induction of Cell Death in 

Plants. Cell Death and Differentiation. 4: 671–683.

Pel, MA. (2010). Mapping, isolation and characterisation of genes responsible for late blight 

resistanceIn: mapping, isolation and characterisation of genes responsible for late blight resistance. 

Thesis.

Qi, D., DeYoung, B.J., and Innes, R.W. (2012). Structure-function analysis of the coiled-coil and leucinerich repeat domains of the RPS5 disease resistance protein. Plant Physiology. 158: 1819–1832.

Rairdan, G.J., Collier, S.M., Sacco, M.A., Baldwin, T.T., Boettrich, T., and Moffett, P. (2008). The CoiledCoil and Nucleotide Binding Domains of the Potato Rx Disease Resistance Protein Function in Pathogen 

Recognition and Signaling. The Plant Cell. 20: 739–751.

Rodewald, J., and Trognitz, B. (2013). Solanum Resistance Genes Against Phytophthora Infestans and 

their Corresponding Avirulence Genes. Molecular Plant Pathology. 14: 740–757.

Sliwka, J., Jakuczun, H., Kamiñski, P., and Zimnoch-Guzowska, E. (2010). Marker-assisted selection of 

diploid and tetraploid potatoes carrying Rpi-phu1, a major gene for resistance to Phytophthora 

infestans. J Appl Genet. 51: 133–140.

Sliwka, J., Swiatek, M., Tomczynska, I., Stefanczyk, E., Chmielarz, M., and Zimnoch-Guzowska, E. (2013).

Influence of genetic background and plant age on expression of the potato late blight resistance gene 
J.R. Simplot Company JRS06 Page 101 of 196

Rpi-phu1 during incompatible interactions with Phytophthora infestans. Plant Pathology 62: 1072–1080.

Spoel, S.H., and Dong, X. (2012). How do Plants Achieve Immunity? Defence without Specialized Immune 

Cells. Nature Reviews Immunology. 12: 89–100.

Takken, F.L.W., and Goverse, A. (2012). How to Build a Pathogen Detector: Structural Basis of NB-LRR 

Function. Current Opinion in Plant Biology. 15: 375–384.

Tan, X., Meyers, B.C., Kozik, A., West, M.A.L., Morgante, M., St Clair, D.A., Bent, A.F., and Michelmore, 

R.W. (2007). Global Expression Analysis of Nucleotide Binding Site-Leucine Rich Repeat-Encoding and 

Related Genes in Arabidopsis. BMC Plant Biology. 7: 56.
J.R. Simplot Company JRS06 Page 102 of 196

7.0 Characterization of Gene Silencing and Target Gene Expression

The DNA inserts introduced into W8 include three expression cassettes designed to partially silence as 

many as five endogenous potato genes in a tissue-specific manner. Transcription of each cassette leads 

to the production of double-stranded RNA containing sequence derived from and specific to the target 

potato genes, which are then silenced through the RNA interference (RNAi) pathway.

7.1 Gene Silencing History and Mode of Action

RNA interference (RNAi) refers to a cellular pathway used by plant and animal cells to regulate gene 

expression through destruction of selected or target RNAs within the cell. Mechanistically, enzymes 

within cells detect a unique, double-stranded form of RNA (dsRNA) and process it into small silencing

RNAs (siRNA) that are then used to identify and specifically destroy RNAs containing complementary 

sequence (Chau and Lee 2007; Fire et al. 1998). This mechanism is particularly useful as part of the 

immune defense against invading organisms, such as viruses, many of which produce dsRNA as part of 

their normal replication cycle (Fusaro et al. 2006; Pumplin and Voinnet 2013). The utility of this 

mechanism is far reaching as it can be harnessed to selectively silence any gene within a cell without 

introduction of foreign sequence.

Traditional breeding practices have produced a number of cultivars that use RNAi to silence their own 

genes to produce desired traits (Petrick et al. 2013; Tuteja et al. 2004; Tuteja et al. 2009). Similarly, our 

InnateTM technologies have allowed us to create commercially desirable potatoes by using RNAi to 

specifically silence a set of target genes in the potato and reduce levels of enzymes responsible for 

quality related traits in tubers. These technologies allowed us to specifically silence the target genes in 

the desired plant tissue with minimal impact on the overall plant biology.

The expression of an inverted repeat is a regulatory system that has evolved naturally as a consequence 

of the duplication of genes and regulatory elements. One example relates to a 27-kb region of the 

soybean genome containing two perfectly repeated and inverted clusters of three chalcone synthase 

(Chs) genes (Tuteja et al. 2009). This results in the silencing of all Chs gene family members through the 

formation of double-stranded RNA. In this example, the double-stranded RNA initiates gene silencing, 

and is an example of naturally occurring inhibition of a metabolic pathway, which functions specifically 

in one tissue while allowing expression in other parts of the plant.

In order to create effective siRNA populations in plants, it is common to transcribe an extended inverted 

repeat sequence consisting of 100-500 nucleotides into a dsRNA precursor. The siRNAs generated 

generally consist of a mixture of sequences and sizes between 21 and 24-nts in length, where the 21-nt 

siRNA pool degrades mRNA and the 24-nt pool silences transcription in plants (Fusaro et al. 2006; 

Pumplin and Voinnet 2013).

7.2 Gene Silencing Construct Design

Silencing was achieved by introducing inverted repeats containing sequences derived from the genes 

and promoters targeted for silencing. Although there are a number of parallel pathways involved in 

double-stranded RNA mediated silencing, transcription of these inverted repeats is thought to be 

processed by the cellular machinery involved in the viral defense (Fusaro et al. 2006). W8 potatoes

contain three unique cassettes, which contain sequence from a total of five different potato genes. The 

pSIM1278 construct consists of two gene silencing cassettes (Figure 7-1, upper construct). One cassette 
J.R. Simplot Company JRS06 Page 103 of 196

contains an inverted repeat of sequence from two genes, asparagine synthetase-1 (Asn1) and

polyphenol oxidase-5 (Ppo5). The second cassette includes sequence from the promoters of the starch 

associated genes, R1 (531-bp) and phosphorylase-L (PhL) (508-bp). The final cassette was introduced 

through the pSIM1678 construct, which includes an inverted repeat containing sequence from the 

vacuolar invertase (VInv) gene (Figure 7-1, lower construct). 

All three silencing cassettes are regulated by the same set of well-characterized and tissue-specific 

promoters from the Agp and Gbss genes of potato, which are highly active in tubers compared with 

photosynthetically-active tissues and roots (Nakata et al. 1994; Visser et al. 1991). Therefore, expression 

and gene silencing was expected to be most effective in and largely limited to tubers.

Figure 7-1. Silencing Approach Using the DNA Inserts of pSIM1278 and pSIM1678

7.3 Expression of Neighboring Genes

Previous studies on convergent transcription have shown that transcripts are of variable size but smaller 

than the distance between the two promoters, both in yeast (Prescott and Proudfoot 2002) and plants 

(Yan et al. 2006). In a study on convergent transcription-based silencing of the -glucuronidase (gus)

gene, no expression of neighboring genes was found (Yan et al. 2006). 

Although the possibility of rare read-through transcripts cannot be excluded, it is unlikely that such 

transcripts would interfere with the expression of neighboring genes because single stranded transcripts 

are ineffective in triggering gene silencing (Yan et al. 2006). Furthermore, studies on the expression of 

an antibiotic selectable marker gene next to a convergent transcription-based silencing construct 

demonstrated that this antibiotic gene was not affected by expression of the silencing construct (Yan et 

al. 2006). A complete bioinformatics analysis of open reading frames associated with the DNA inserts 

and flanking regions is discussed in Chapter 9: Safety of Russet Burbank W8 Potato, with additional 

details of the methods in Appendix C: Allergen and Toxin Assessment for Russet Burbank W8. 

(Pro = functionally-active promoter, F =transcript fragment, S1 = spacer 1, 

P = promoter fragment, S2 = spacer 2, S3 = spacer 3)

F

Asn1

F

Ppo5

F

Ppo5

F Pro Asn1 Pro Pro P

PhL

P

R1

P

R1

P

PhL

S

1

S

2

Transcription Transcription

Transcription Transcription

Pro

Pro Pro

Transcription

Transcription

pSIM1278

pSIM1678 S

3

F

Inv

F

Inv
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7.4 Silencing of Target Genes is Tissue Specific 

The expression of all five target genes was characterized by northern blot analysis to determine the 

effectiveness of gene silencing from each cassette. Note that data for two events is presented in each of 

the figures in this chapter, but only event W8 is under consideration in this submission. Robust silencing 

of Asn1, Ppo5, and VInv was observed in tubers while silencing of R1 was less effective (Figure 7-2).

Silencing of PhL was considered ineffective as no measurable differences between control and W8 

samples were observed. In other events with the same pSIM1278 construct, partial silencing of the 

promoters for PhL and R1 in tubers was observed (Collinge and Clark 2013).

Previous studies have shown that Ppo gene silencing reduces the amount of associated protein to levels 

undetectable by western blot analysis (Llorente et al. 2011). Similarly, silencing of the R1 gene 

diminished accumulation of a ~160kDa protein that is at least partially bound to starch granules 

(Lorberth et al. 1998). 

Figure 7-2. Target Gene Expression in Tubers

Northern blot analysis of total RNA (20 μg) isolated from tubers of field-grown plants for two events (W3 and W8) along with 

the Russet Burbank control (WT). Blots were hybridized with probes specific to the Asn1, Ppo5, PhL, R1, and VInv transcripts 

(upper panels). A probe specific to the internal control 18s rRNA (middle panels) and ethidium bromide stained total RNA 

(lower panels) were used as internal and loading controls. Each blot includes two independent biological replicates for each 

sample. W3 = Additional event that was not submitted.

We evaluated target gene expression in other plant tissues to determine the specificity of our gene 

silencing. Northern blot analysis was similarly performed on RNA isolated from leaves, stems, roots, and 

flowers from W8 and the Russet Burbank control. As shown in Figures 7-3, 7-4, and 7-5 there was no 

silencing of target genes in leaves, stems, or roots relative to the Russet Burbank controls. All transcripts 

were readily detectable, except those corresponding to the VInv gene, which was weakly expressed in all 

leaf and stem samples, including controls.

Asn1 Ppo5

PhL R1 VInv
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The only tissue other than tubers where some target silencing was observed was in the flower samples. 

These samples indicated some silencing of the Asn1 transcript in W8 relative to the Russet Burbank 

controls, which may be due to some leaky expression in that tissue (Figure 7-6).

Figure 7-3. Target Gene Expression in Leaves

Northern blot analysis of total RNA (20 μg) isolated from leaves of field-grown plants for two events (W3 and W8) along with 

the Russet Burbank control (WT). Blots were hybridized with probes specific to the Asn1, Ppo5, PhL, R1, and VInv transcripts 

(upper panels). A probe specific to the internal control 18s rRNA (middle panels) and ethidium bromide stained total RNA 

(lower panels) were used as internal and loading controls. Each blot includes two independent biological replicates for each 

sample. W3 = Additional event that was not submitted.

Figure 7-4. Target Gene Expression in Stems

Northern blot analysis of total RNA (20 μg) isolated from stems of field-grown plants for two events (W3 and W8) along with 

the Russet Burbank control (WT). Blots were hybridized with probes specific to the Asn1, Ppo5, PhL, R1, and VInv transcripts 

(upper panels). A probe specific to the internal control 18s rRNA (middle panels) and ethidium bromide stained total RNA 

(lower panels) were used as internal and loading controls. Each blot includes two independent biological replicates for each 

sample. W3 = Additional event that was not submitted.

Asn1 Ppo5

PhL R1 VInv

Asn1 Ppo5

PhL R1 VInv
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Figure 7-5. Target Gene Expression in Roots

Northern blot analysis of total RNA (20 μg) isolated from roots of field-grown plants for two events (W3 and W8) along with the 

Russet Burbank control (WT). Blots were hybridized with probes specific to the Asn1, Ppo5, PhL, R1, and VInv transcripts (upper 

panels). A probe specific to the internal control 18s rRNA (middle panels) and ethidium bromide stained total RNA (lower 

panels) were used as internal and loading controls. Each blot includes two independent biological replicates for each sample.

W3 = Additional event that was not submitted.

Figure 7-6. Target Gene Expression in Flowers

Northern blot analysis of total RNA (20 μg) isolated from flowers of field-grown plants for two events (W3 and W8) along with 

the Russet Burbank control (WT). Blots were hybridized with probes specific to the Asn1, Ppo5, PhL, R1, and VInv transcripts 

(upper panels). A probe specific to the internal control 18s rRNA (middle panels) and ethidium bromide stained total RNA 

(lower panels) were used as internal and loading controls. Each blot includes two independent biological replicates for each 

sample. W3 = Additional event that was not submitted.

Asn1 Ppo5

PhL R1 VInv
J.R. Simplot Company JRS06 Page 107 of 196

7.5 Summary of Gene Silencing and Target Gene Expression

Two of the three gene silencing cassettes introduced into Russet Burbank to generate the W8 event 

were very effective at silencing their target transcripts for RNAi-mediated silencing. These two 

constructs effectively silenced Asn1, Ppo5, and VInv in the tubers of W8. The specificity of silencing to 

the tubers indicates that few, if any, of the siRNA generated by the RNAi machinery spread to other 

tissues or that their levels were insufficient to invoke an RNAi response in those tissues. The only 

evidence for silencing outside of tubers was in flowers where lower levels of Asn1 were observed, yet 

the magnitude of change was much lower than in tubers. The promoter silencing strategy with PhL and 

R1 had minimal effect, which was consistent with other events containing the same pSIM1278 construct 

(Collinge and Clark 2013). 

As expected, the reduced expression of RNA transcripts associated with Asn1, Ppo5, and VInv were 

further corroborated as described in Chapter 10: Trait Efficacy. In addition, the compositional and 

agronomic data presented in Chapters 8: Compositional Assessment and 11: Agronomic Evaluation did 

not reveal any unexpected phenotypes that would be associated with significant off-target effects or 

unintended silencing. For instance, strong silencing of Asn1 in tubers limits the accumulation of the 

amino acid asparagine as desired, whereas silencing of Asn1 in leaves or stems might adversely affect 

growth and development, which was not the case. Thus, the RNAi response was both effective and 

specific and there is no indication that silencing these potato genes would affect weediness or other 

plant-pest characteristics (See Chapter 12: Environmental Safety Assessment for additional information). 
J.R. Simplot Company JRS06 Page 108 of 196

7.6 References

Chau, B.L., and Lee, K.A. (2007). Function and anatomy of plant siRNA pools derived from hairpin 

transgenes. Plant Methods. 3: 13. 

Collinge, S., and Clark, P., 2013. Petition for Determination of Nonregulated Status for InnateTM Potatoes 

with Low Acrylamide Potential and Reduced Black Spot Bruise: Events E12 and E24 (Russet Burbank); 

F10 and F37 (Ranger Russet); J3, J55, and J78 (Atlantic); G11 (G); H37and H50 (H). J.R. Simplot Company 

Petition JRS01 (USDA Petition 13-022-01p).

Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific 

genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391: 806–811.

Fusaro, A.F., Matthew, L., Smith, N.A., Curtin, S.J., Dedic-Hagan, J., Ellacott, G.A., and Waterhouse, P.M. 

(2006). RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO 

Reports. 7: 1168–1175. 

Llorente, B., Alonso, G.D., Bravo-Almonacid, F., Rodríguez, V., López, M.G., Carrari, F., and Flawiá, M.M. 

(2011). Safety assessment of nonbrowning potatoes: opening the discussion about the relevance of 

substantial equivalence on next generation biotech crops. Plant Biotechnology Journal. 9: 136–50. 

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protein leads to modified starch and repression of cold sweetening. Nature Biotechnology. 16: 473–477. 

Nakata, P.A., Anderson, J.M., and Okita, T.W. (1994). Structure and expression of the potato ADPglucose pyrophosphorylase small subunit. Journal of Biological Chemistry. 269: 30798–30807. 

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from biotechnology-derived crops employing RNA-mediated gene regulation to achieve desired traits: a 

scientific review. Regulatory Toxicology and Pharmacology: RTP., 66: 167–176. 

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Tuteja, J.H., Clough, S.J., Chan, W.C., and Vodkin, L.O. (2004). Tissue-specific gene silencing mediated by 

a naturally occurring chalcone synthase gene cluster in Glycine max. Plant Cell 16: 819–835.

Tuteja, J.H., Zabala, G., Varala, K., Hudson, M., and Vodkin, L.O. (2009). Endogenous, Tissue-Specific 

Short Interfering RNAs Silence the Chalcone Synthase Gene Family in Glycine max Seed Coats. Plant Cell . 

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synthase-GUS gene in transgenic potato plants. Plant Molecular Biology. 17: 691–699. 

Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in 

plants. Plant Physiol, 141: 1508-1518.
J.R. Simplot Company JRS06 Page 109 of 196

8.0 Compositional Assessment

The compositional assessment study evaluating proximates, vitamins, amino acids, minerals, and 

glycoalkaloids, was conducted on Russet Burbank Event W8 to 1) show equivalence to the 

untransformed control, 2) compare W8 to literature ranges, where applicable; and 3) show that there 

were no changes in potential toxins. Ultimately, results of the composition studies showed food safety 

equivalence between W8 and the control Russet Burbank.

8.1 Compositional Analysis Results

These analyses were conducted to confirm that composition of Event W8 remained within the normal 

levels for potato and would have equivalent food quality, feed quality, and safety when compared to 

untransformed potatoes. The compositional assessments determined the amounts of 1) proximates: 

protein, fat, ash, crude fiber, carbohydrate, calories, and moisture (Table 8-1); 2) vitamins: B3, B6, and C 

(Table 8-1); 3) minerals: copper, magnesium, and potassium (Table 8-1); and 4) total amino acids (Table 

8-2). These assessments were conducted in W8, the control, and conventional reference varieties grown 

in potato-growing areas of the United States. A detailed list of reference varieties is shown in Table 8-5. 

For all the nutrients listed above, the goal was to determine if W8 contained equivalent amounts 

compared with the untransformed control at the time of harvest.
J.R. Simplot Company JRS06 Page 110 of 196

Table 8-1: Proximates, Vitamins, and Minerals in W8 and Control

Compound Variety Mean P-value N

Range 

Min

Range 

Max TI Low TI High CLR Low CLR High

Protein (%) W8 2.11 0.6444 32 1.83 2.58

Protein (%) Control 2.13 . 32 1.82 2.50 1.53 2.79 0.700 4.60

Fat (%) W8 0.162 0.8616 32 0.100 0.250

Fat (%) Control 0.166 . 32 0.100 0.460 0 0.450 0.0200 0.200

Ash (%) W8 0.958 0.8664 32 0.643 1.26

Ash (%) Control 0.951 . 32 0.717 1.49 0.461 1.41 0.440 1.90

Crude Fiber (%) W8 0.469 0.2647 32 0.305 0.670

Crude Fiber (%) Control 0.438 . 32 0.305 0.660 0.190 0.740 0.170 3.50

Carbohydrates (%) W8 16.5 0.0577 32 13.3 20.0

Carbohydrates (%) Control 17.2 . 32 14.5 19.4 13.2 22.1 13.3 30.5

Calories 

(kcal/100g) W8 75.7 0.0599 32 63.7 89.4

Calories 

(kcal/100g) Control 78.8 . 32 68.8 87.7 63.5 97.9 80.0 110

Moisture (%) W8 80.3 0.0652 41 76.9 83.3

Moisture (%) Control 79.7 . 41 77.6 82.0 75.0 83.4 63.2 86.9

Vitamin B3 

(Niacin) (mg/100g) W8 1.86 0.8651 32 1.41 2.30

Vitamin B3 

(Niacin) (mg/100g) Control 1.84 . 32 1.43 2.48 0.768 2.86 0.0900 3.10

Vitamin B6 

(mg/100g) W8 0.120 0.0019 32 0.0960 0.150

Vitamin B6 

(mg/100g) Control 0.132 . 32 0.111 0.150 0.0740 0.150 0.110 0.340

Vitamin C 

(mg/100g) W8 26.7 0.0040 32 18.7 32.2

Vitamin C 

(mg/100g) Control 23.5 . 32 16.9 28.8 11.9 44.5 1 54.0

Copper (%) W8 0.0695 0.4460 32 0.0500 0.120

Copper (%) Control 0.0724 . 32 0.0500 0.110 0.0210 0.130 0.0200 0.700

Magnesium (%) W8 20.9 0.1472 32 17.3 29.1

Magnesium (%) Control 20.1 . 32 16.4 23.5 13.3 29.0 11.3 55.0

Potassium (%) W8 427 0.9262 32 367 481

Potassium (%) Control 428 . 32 367 484 281 578 350 625

1

TI = Tolerance Interval

2

CLR = Combined Literature Range. Literature ranges are from, Lisinska and Leszczynski (1989); Rogan et al. (2000); Horton and 

Anderson (1992); Talburt and Smith (1987).

P-values indicating significant differences with control are bold and underlined. 

Many values fell below detection limits and were adjusted to the limit for statistical analysis.
J.R. Simplot Company JRS06 Page 111 of 196

Table 8-2: Total Amino Acids in W8 and Control

Compound Variety Mean P-value N

Range 

Min

Range 

Max TI Low TI High CLR Low CLR High

Alanine 

(mg/100g) W8 70.4 0.0004 32 58.0 87.7

Alanine 

(mg/100g) Control 63.8 . 32 41.3 75.2 38.9 95.1 39.2 95.2

Arginine 

(mg/100g) W8 104 0.1463 32 83.7 157

Arginine 

(mg/100g) Control 98.4 . 32 62.5 119 52.9 142 70.0 138

Aspartic Acid 

(mg/100g) W8 255 <.0001 32 189 331

Aspartic Acid 

(mg/100g) Control 454 . 32 278 636 203 686 339 738

Glutamic Acid 

(mg/100g) W8 478 <.0001 32 389 583

Glutamic Acid 

(mg/100g) Control 310 . 32 232 373 185 482 292 604

Glycine 

(mg/100g) W8 58.1 0.0079 32 45.7 70.6

Glycine 

(mg/100g) Control 52.8 . 32 40.8 62.4 38.5 76.3

Histidine 

(mg/100g) W8 33.1 0.7008 32 26.2 41.6

Histidine 

(mg/100g) Control 32.7 . 32 24.2 42.7 18.4 46.8 13.3 46.9

Isoleucine 

(mg/100g) W8 72.5 0.5519 32 60.0 95.3

Isoleucine 

(mg/100g) Control 71.2 . 32 47.9 86.9 46.9 98.4 52.5 95.3

Leucine 

(mg/100g) W8 110 0.0162 32 81.4 137

Leucine 

(mg/100g) Control 102 . 32 72.1 123 67.2 149 68.5 138

Lysine 

(mg/100g) W8 100.0 0.1590 32 83.6 118

Lysine 

(mg/100g) Control 95.7 . 32 73.2 115 63.7 136 68.7 137

Methionine 

(mg/100g) W8 41.4 0.2150 32 34.2 51.6

Methionine 

(mg/100g) Control 39.9 . 32 29.8 49.4 24.7 52.0

Phenylalanine 

(mg/100g) W8 81.9 0.7842 32 68.4 100

Phenylalanine 

(mg/100g) Control 81.2 . 32 54.5 94.8 55.5 115 55.2 109

Proline 

(mg/100g) W8 63.1 0.0399 32 23.2 104

Proline 

(mg/100g) Control 52.9 . 32 11.8 72.9 11.0 127 35.5 146
J.R. Simplot Company JRS06 Page 112 of 196

Table 8-2 (continued): Total Amino Acids in W8 and Control

Compound Variety Mean P-value N

Range 

Min

Range 

Max TI Low TI High CLR Low CLR High

Serine 

(mg/100g) W8 74.0 0.0175 32 56.8 91.5

Serine 

(mg/100g) Control 68.7 . 32 49.1 80.1 48.5 94.0 50.0 102

Threonine 

(mg/100g) W8 72.9 0.0020 32 56.0 91.5

Threonine 

(mg/100g) Control 65.3 . 32 46.7 79.2 41.8 95.2 43.6 85.5

Tryptophan 

(mg/100g) W8 21.5 0.7800 32 17.4 26.8

Tryptophan 

(mg/100g) Control 21.9 . 32 16.1 26.8 13.5 29.1 11.4 28.2

Tyrosine 

(mg/100g) W8 80.3 <.0001 32 68.8 95.1

Tyrosine 

(mg/100g) Control 65.2 . 32 47.3 77.7 39.3 96.5 45.7 94.2

Valine 

(mg/100g) W8 112 0.2126 32 93.0 141

Valine 

(mg/100g) Control 108 . 32 73.6 127 67.7 143 75.2 145

1

TI = Tolerance Interval

2

CLR = Combined Literature Range. Literature ranges are from, Talley et al. (1984); Rogan et al. (2000).

P-values indicating significant differences with control are bold and underlined. 

Many values fell below detection limits and were adjusted to the limit for statistical analysis.
J.R. Simplot Company JRS06 Page 113 of 196

Compositional Analysis of Russet Burbank Event W8 at Harvest

Tubers of Event W8 were equivalent to those of the control except for the following observed changes:

 Event W8 tubers contained, on average, 0.12 mg/100g vitamin B6 as compared to the control, 

which contained 0.13 mg/100g (p = 0.0019). The mean for Event W8 was within the tolerance 

interval and combined literature range.

 Event W8 tubers contained, on average, 26.7 mg/100g vitamin C as compared to the control, 

which contained 23.4 mg/100g (p = 0.004). The mean for Event W8 was within the tolerance 

interval and combined literature range.

 Event W8 tubers contained, on average, 70.4 mg/100g total alanine as compared to the control, 

which contained 63.8 mg/100g (p = 0.0004). The mean for Event W8 was within the tolerance 

interval and combined literature range.

 Event W8 tubers contained, on average, 255.1 mg/100g total aspartic acid as compared to the 

control, which contained 477.8 mg/100g (p < 0.0001). The mean for Event W8 was within the 

tolerance interval and combined literature range. This was expected based on the intended 

reduction in asparagine.

 Event W8 tubers contained, on average, 477.8 mg/100g total glutamic acid as compared to the 

control, which contained 310.3 mg/100g (p < 0.0001). The mean for Event W8 was within the 

tolerance interval and combined literature range. This was expected based on the intended 

reduction in asparagine, which results in an increase in glutamic acid.

 Event W8 tubers contained, on average, 58.1 mg/100g total glycine as compared to the control, 

which contained 52.8 mg/100g (p = 0.0079). The mean for Event W8 was within the tolerance 

interval.

 Event W8 tubers contained, on average, 109.8 mg/100g total leucine as compared to the 

control, which contained 101.7 mg/100g (p = 0.0162). The mean for Event W8 was within the 

tolerance interval and combined literature range.

 Event W8 tubers contained, on average, 63.1 mg/100g total proline as compared to the control, 

which contained 52.9 mg/100g (p = 0.0399). The mean for Event W8 was within the tolerance 

interval and combined literature range.

 Event W8 tubers contained, on average, 74.0 mg/100g total serine as compared to the control, 

which contained 68.7 mg/100g (p = 0.0175). The mean for Event W8 was within the tolerance 

interval and combined literature range.

 Event W8 tubers contained, on average, 72.9 mg/100g total threonine as compared to the 

control, which contained 65.3 mg/100g (p = 0.0020). The mean for Event W8 was within the 

tolerance interval and combined literature range.
J.R. Simplot Company JRS06 Page 114 of 196

 Event W8 tubers contained, on average, 80.3 mg/100 g total tyrosine as compared to the 

control, which contained 65.2 mg/100g (p < 0.0001). The mean for Event W8 was within the 

tolerance interval and combined literature range.

The changes in vitamin B6, vitamin C, and total amino acids: alanine, aspartic acid, glutamic acid, glycine, 

leucine, proline, serine, threonine, and tyrosine, result in a very small modification in composition that is 

still standard for potato and will not significantly alter potential dietary intake levels. All values for Event 

W8 were within the tolerance interval or combined literature range and are thus still at normal levels for 

potatoes. It can be concluded that tubers of Event W8 are substantially equivalent to the control 

regarding their nutritional value for food and feed.

8.2 Glycoalkaloids

Results of Glycoalkaloid testing in Event W8

W8 and the control had mean glycoalkaloid ranges from 6.4 to 7.2 mg/100g, respectively (Table 8-3); all 

below the 20mg/100g safety limit described by Sinden (1987). Since the mean value for Event W8 was 

not significantly different from the control and was within the tolerance interval, it was concluded that 

glycoalkaloid levels in event W8 are no different from the Russet Burbank control.

This research confirmed that event W8 is as safe as controls and did not contain increased levels of 

natural glycoalkaloids, which are toxins commonly found in Solanaceous crops, including potato. The 

principal glycoalkaloids in potatoes are -solanine and -chaconine. For food safety purposes an upper 

limit for glycoalkaloid content of 20 mg per 100g of potato is generally accepted (Sinden 1987). 

Table 8-3: Event W8 and Control Glycoalkaloids

Compound Variety Mean P-value N

Range 

Min

Range 

Max

TI1

Low TI1

High

Glycoalkaloids 

(mg/100g) W8 7.2 0.1615 32 5.0 19.0 . .

Glycoalkaloids 

(mg/100g) Control 6.4 . 32 5.0 13.2 5.0 14.4

1

TI = Tolerance Interval 

P-values indicating significant differences with control are bold and underlined.

8.3 Methods for Compositional Assessment

Field Trial Locations. Tubers for the compositional assessment were collected from Simplot Plant 

Sciences studies SPS-PAT-12-02 in 2012 and 13-02-SPS-ENV in 2013. The locations are summarized in 

Table 11-2 from Chapter 11: Agronomic Performance. Each combination of year, site, material, and 

replicate would represent one sample of six tubers in the compositional assessment.

Potato varieties and test events grown in field trials are summarized in Table 8-4. In 2012, only test and 

control varieties were grown at all sites. In 2013, test, control and reference varieties were grown at all 

sites. Reference varieties are commercially-available varieties that provide a range of values common to 

conventional potatoes.
J.R. Simplot Company JRS06 Page 115 of 196

Table 8-4. Varieties in 2012 and 2013 Field Trials

Variety or Test Type Trait/Genotype Seed Type1Seed Source

Events in 2012 Field Trials

Russet 

Burbank Control N/A Greenhouse-grown Mini-Tubers Simplot Plant Sciences - Boise, ID

W8 Test pSIM1278+pSIM1678

Events in 2013 Field Trials

Russet 

Burbank Control N/A

NFT Mini-tubers CSS Farms - Colorado City, CO

W8 Test pSIM1278+pSIM1678

Golden 

Sunburst Reference N/A

Bintje Reference N/A

Nicolet Reference N/A

TX2782Reference N/A

Red Thumb2Reference N/A

1

Greenhouse-grown mini-tubers were grown from tissue culture plantlets in the Simplot Plant Sciences greenhouse to produce nuclear seed. 

NFT mini-tubers were produced at CSS Farms in Colorado City, CO, using nutrient film technique to hydroponically produce a large number of 

seed.

2

At WI-GRA, Red Thumb was used in place of TX278 due to a seed shortage.

Testing Laboratories. Analytical testing was conducted by Covance Laboratories, Inc. in Madison, WI or 

Greenfield, IN. 

Sample preparation. Samples were obtained by randomly selecting 6 mid-sized tubers (at harvest) from 

each site and replicate (rep). Samples were powdered in an industrial blender with liquid nitrogen and 

stored at -70°C until analysis.

Protein. Protein was determined using the Kjeldahl method, approximating protein by multiplying 

Nitrogen by 6.25, as per Covance protocol PGEN_S:4 (AOAC 2005m; AOAC 2005n).

Fat. Fat was determined by Acid Hydrolysis using Covance protocol FAAH_S:7(AOAC 2005i; AOAC 2005j).

Ash. Ash levels were determined by Covance Laboratories using Covance protocol ASHM_S:5 (AOAC 

2005e).

Crude Fiber. Crude fiber was determined by Covance Laboratories using Covance protocol CFIB_S:2

(AOAC 2005f).

Carbohydrates. Carbohydrate levels were determined by Covance Laboratories using Covance protocol 

CHO:6 (USDA 1973). 

Calories. Total calories were determined by Covance Laboratories using Covance protocol CALC:4 (USDA 

1975).

Moisture. Moisture levels were determined by Covance Laboratories using Covance protocol 

M100T100_S:4 (AOAC 2005c; AOAC 2005d).

Vitamin B3 (Niacin). Niacin was determined by Covance Laboratories using Covance protocol NIAP_S:11

(AOAC 2005k; AOAC 2005l).
J.R. Simplot Company JRS06 Page 116 of 196

Vitamin B6 (Pyridoxine). Pyridoxine was determined by Covance Laboratories using Covance protocol

B6A_S:11 (AOAC 2005o; Atkin 1943).

Vitamin C. Vitamin C levels were determined by Covance Laboratories using protocol VCF_S:5 (AOAC 

2005b).

Elements by ICP Emission Spectrometry. The minerals Copper (Cu), Magnesium (Mg),and Potassium (K) 

were determined by Covance Laboratories using Covance protocol (ICP_S:13) (AOAC 2005g; AOAC 

2005h).

Tryptophan. Tryptophan levels were determined by Covance Laboratories using the Covance protocol 

TRPLC_S:3 (AOAC 2005a).

Total amino acids. Total amino acid levels were determined by Covance Laboratories using the Covance 

protocol TAALC_S:6 (Schuster 1988; Henderson 2000; and Barkholt and Jensen 1989).

Glycoalkaloids. Glycoalkaloid levels were determined by Covance Laboratories using protocol COID_S:2 

(AOAC 2006).

8.4 Statistical Analysis for Compositional Assessment

Statistical Methods. The statistical analysis was performed by Simplot using SAS 9.3. All data were 

analyzed using the following linear mixed model:

Yijkl = i + j + k(j) + ( )l + ijkl

•  = mean of treatment (fixed)

•  = effect of site (random)

•  = rep[site] (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the effect of the jth site 

(random effect), k(j) denotes the random rep effect (within site), ( )ik denotes the interaction between 

the i

th treatment and random kth site effect, and ijkl denotes the residual random error.

A significant difference was established with a p-value < 0.05. Every effort was made to generate pvalues to aid in the interpretation of the data. Some departures from the assumptions of normality and 

equal variances were allowed since the results were always interpreted in the context of variation 

observed in the conventional varieties.

The following varieties, all grown in the various field trial sites, were used to calculate tolerance 

intervals: Bintje, Golden Sunburst, Nicolet, Ranger Russet, Red Thumb, Russet Burbank, and TX278 

(Table 8-5). The tolerance intervals were calculated to contain, with 95% confidence, 99% of the values 

in the population. This statistical tolerance interval and the combined range of values for each analyte 
J.R. Simplot Company JRS06 Page 117 of 196

available from the published literature (Tables 8-6 and 8-7) were used to interpret the composition 

results. In interpreting the data, emphasis was placed on the analyte means; means that fell within the 

tolerance interval and/or the combined literature range were considered to be within the normal 

variability of commercial potato varieties.

Table 8-5. Number of data points for each test, control and reference line

Line N Per Attribute Used in Tolerance Interval?

W8 32 No

Russet Burbank Control 32 Yes

Ranger Russet Control 12 Yes

Bintje 32 Yes

Golden Sunburst 30 Yes

Nicolet 32 Yes

Red Thumb 4 Yes

TX278 28 Yes

Total N 202 170 in Tolerance Interval
J.R. Simplot Company JRS06 Page 118 of 196

Table 8-6. Combined Literature Ranges (CLR) for Proximates, Vitamins, Minerals

 

Compound Value Units Reference Conversion Formula Used1CLR End Units

Protein low 0.70 % Lisinska and Leszczynski 1989 NA 0.70 %

Protein high 4.60 % Lisinska and Leszczynski 1989 NA 4.60 %

Fat low 0.02 % Lisinska and Leszczynski 1989 NA 0.02 %

Fat high 0.20 % Lisinska and Leszczynski 1989 NA 0.20 %

Ash low 0.44 % Lisinska and Leszczynski 1989 NA 0.44 %

Ash high 1.90 % Lisinska and Leszczynski 1989 NA 1.90 %

Crude fiber low 0.17 % Lisinska and Leszczynski 1989 NA 0.17 %

Crude fiber high 3.50 % Lisinska and Leszczynski 1989 NA 3.50 %

Carbohydrates low 13.30 % Talburt and Smith 1987 NA 13.30 %

Carbohydrates 

high 30.53 % Talburt and Smith 1987 NA 30.53 %

Calories low 70.00 kcal/100g Horton and Anderson 1992 NA 80.00 kcal/100g

Calories high 551.00 kcal/100g dry Horton and Anderson 1992 kcal/100g x 0.2 110.20 kcal/100g

Moisture low 63.20 % Talburt and Smith 1987 NA 63.20 %

Moisture high 86.90 % Talburt and Smith 1987 NA 86.90 %

Niacin (B3) low 0.18 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 0.09 mg/100g

Niacin (B3) high 6.20 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 3.10 mg/100g

Vitamin B6 

(Pyridoxine) low 0.26 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g x 0.823 0.11 mg/100g

Vitamin B6 

(Pyridoxine) high 0.82 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g x 0.823 0.34 mg/100g

Vitamin C low 1.00 mg/100g Lisinska and Leszczynski 1989 NA 1.00 mg/100g

Vitamin C high 54.00 mg/100g Lisinska and Leszczynski 1989 NA 54.00 mg/100g

Copper low 0.03 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 0.02 mg/100g

Copper high 1.40 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 0.70 mg/100g

Magnesium low 22.50 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 11.25 mg/100g

Magnesium high 110.00 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 55.00 mg/100g

Potassium low 700.00 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 350.00 mg/100g

Potassium high 1250.00 mg/200g Rogan et al. 2000 mg/200g x 0.5mg/100g 625.00 mg/100g

1

0.2 = approximate conversion from dry weight to fresh weight (internal communication), 0.823 is the conversion

 factor from Pyridoxine free base to Pyridoxine HCl (internal communication)

NA = no conversion necessary
J.R. Simplot Company JRS06 Page 119 of 196

Table 8-7. Combined Literature Ranges (CLR) for Total Amino Acids

Compound Value Units Reference

Formula 

Wt (FW) Conversion Formula Used1

CLR

(mg/ 

100g)

Alanine high 53.4 μmol/g Talley et al. 1984 89.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 95.2

Alanine low 22.0 μmol/g Talley et al. 1984 89.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 39.2

Arginine high 39.7 μmol/g Talley et al. 1984 174.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 138.3

Arginine low 20.1 μmol/g Talley et al. 1984 174.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 70.0

Aspartic acid high 1476 mg/200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 738.0

Aspartic acid low 677 mg/200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 338.5

Glutamic acid high 1207 mg/200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 603.5

Glutamic acid low 583 mg/200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 291.5

Histidine high 15.1 μmol/g Talley et al. 1984 155.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 46.9

Histidine low 4.3 μmol/g Talley et al. 1984 155.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 13.3

Isoleucine high 36.3 μmol/g Talley et al. 1984 131.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 95.3

Isoleucine low 20.0 μmol/g Talley et al. 1984 131.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 52.5

Leucine high 52.7 μmol/g Talley et al. 1984 131.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 138.3

Leucine low 26.1 μmol/g Talley et al. 1984 131.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 68.5

Lysine high 46.8 μmol/g Talley et al. 1984 146.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 136.8

Lysine low 23.5 μmol/g Talley et al. 1984 146.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 68.7

Phenylalanine high 32.9 μmol/g Talley et al. 1984 165.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 108.7

Phenylalanine low 16.7 μmol/g Talley et al. 1984 165.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 55.2

Proline high 63.6 μmol/g Talley et al. 1984 115.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 146.4

Proline low 15.4 μmol/g Talley et al. 1984 115.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 35.5

Serine high 48.6 μmol/g Talley et al. 1984 105.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 102.2

Serine low 23.8 μmol/g Talley et al. 1984 105.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 50.0

Threonine high 35.9 μmol/g Talley et al. 1984 119.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 85.5

Threonine low 18.3 μmol/g Talley et al. 1984 119.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 43.6

Tryptophan high 6.9 μmol/g Talley et al. 1984 204.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 28.2

Tryptophan low 2.8 μmol/g Talley et al. 1984 204.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 11.4

Tyrosine high 26.0 μmol/g Talley et al. 1984 181.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 94.2

Tyrosine low 12.6 μmol/g Talley et al. 1984 181.2 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 45.7

Valine high 61.9 μmol/g Talley et al. 1984 117.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 145.0

Valine low 32.1 μmol/g Talley et al. 1984 117.1 μmol/g x mol/106μmol x FW x 1000mg/g x 100g x 0.2 75.2

1

0.2 is the approximate conversion from dry weight to fresh weight (internal communication)
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8.5 Compositional Assessment Conclusions

This compositional assessment study, evaluating proximates, vitamins, minerals, amino acids, and 

glycoalkaloids was conducted on Russet Burbank Event W8 to 1) show equivalence to the 

untransformed control, and 2) compare W8 to conventional variety ranges and literature ranges, where 

applicable. Ultimately, results of this composition study showed food safety equivalence between the 

event and the untransformed Russet Burbank control. Because tubers of Event W8 are substantially 

equivalent to tubers from the control, the potatoes will be as safe as the Russet Burbank control for use 

as food and feed. 
J.R. Simplot Company JRS06 Page 121 of 196

8.6 References

AOAC. (2005a). Official Methods of Analysis of AOAC International, 18th Ed., Method 988.15. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC. (2005b). Official Methods of Analysis of AOAC International, 18th Ed., Method 967.22. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005c). Official Methods of Analysis of AOAC International, 18th Ed., Method 925.09. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005d). Official Methods of Analysis of AOAC International, 18th Ed., Method 926.08. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC. (2005e). Official Methods of Analysis of AOAC International, 18th Ed., Method 923.03. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005f). Official Methods of Analysis of AOAC International, 18th Ed., Method 962.09. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005g). Official Methods of Analysis of AOAC International, 18th Ed., Method 984.27. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005h). Official Methods of Analysis of AOAC International, 18th Ed., Method 985.01. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005i). Official Methods of Analysis of AOAC International, 18th Ed., Method 922.06. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC. (2005j). Official Methods of Analysis of AOAC International, 18th Ed., Method 954.02. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC. (2005k). Official Methods of Analysis of AOAC International, 18th Ed., Method 944.13. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC. (2005l). Official Methods of Analysis of AOAC International, 18th Ed., Method 960.46. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005m). Official Methods of Analysis of AOAC International, 18th Ed., Method 955.04. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005n). Official Methods of Analysis of AOAC International, 18th Ed., Method 979.09. Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 

AOAC. (2005o). Official Methods of Analysis of AOAC International, 18th Ed., Method 961.15.Association 

of Official Agricultural Chemists International, Gaithersburg, MD, USA. 
J.R. Simplot Company JRS06 Page 122 of 196

AOAC. (2006). Official Methods of Analysis of the AOAC International, 19th Edition, Method 997.13. 

Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

Atkin, L., Schultz, A.S., Williams, W.L., and Frey. C.N. (1943). Yeast micro- biological methods for 

determination of vitamins. Industrial and Eng Chem, Analytical Ed. 15: 141-144.

Barkholt, V., and Jensen, A.L. (1989). Amino acid analysis: determination of cystine plus half-cystine in 

proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Analytical Biochem.

177: 318-322.

Henderson, J.W., Ricker, R.D., Bidlingmeyer, B.A., and Woodward, C. (2000) Rapid, accurate, sensitive, 

and reproducible HPLC analysis of amino acids, amino acid analysis using Zorbax Eclipse-AAA columns 

and the Agilent 1100 HPLC. Agilent Publication.

Horton, D.E., Anderson, J.L. (1992). Potato production in the context of the world and farm economy. In: 

The potato crop: the scientific basis for improvement (Ed. Harris PM) Chapman and Hall, London, pp. 

803.

Lisinska, G., Leszczynski, W. (1989). Potato science and technology. Chapter 2: Potato tubers as a raw 

material for processing and nutrition. Elsevier Applied Science, London, pp 11-128.

Rogan, G.J., Bookout, J.T., Duncan, D.R., Fuchs, R.L., Lavrik, P.B., Love, S.L., Mueth, M., Olson, T., Owens,

E.D., Raymond, P.J., and Zalewski, J. (2000). Compositional analysis of tubers from insect and virus 

resistant potato plants. J Agric Food Chem: 48: 5936-5945.

Schuster, R. (1988). Determination of amino acids in biological, pharmaceutical, plant and food samples 

by automated precolumn derivitization and HPLC. J Chromatography. 431: 271-284. 

Sinden, S.L. (1987). Potato glycoalkaloids. Acta Hort. 207: 41-48.

Talburt, W.F., and Smith, O. (1987). Potato processing. Van Nostrand Reinhold, New York.

Talley, E.A., Toma, R.B., Orr, P.H., (1984). Amino acid composition of freshly harvested and stored 

potatoes. Am Pot Journal. 61: 267-279.

USDA. (1973). United States Department of Agriculture, Energy Value of Foods. Agricultural Handbook, 

No. 74, pp. 2-11.

USDA. (1975). United States Department of Agriculture, Composition of Foods Agricultural Handbook, 

No. 8, pp. 159-160.
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9.0 Safety of Russet Burbank W8 Potato

The rationale for safety of Russet Burbank event W8 is based on the following evidence: 1) phenotype 

through agronomic studies, 2) compositional equivalence, 3) bioinformatics assessment for toxins and 

allergens, 4) glycoalkaloid testing, 5) dietary risk assessment of VNT1 protein, 6) low levels of VNT1 

protein, 7) safety of nucleic acids, 8) literature review on gene silencing through RNAi, and 9) previous 

regulatory actions for plant incorporated protectants similar to the late blight resistance gene. 

9.1 Phenotype through Agronomic Studies

Agronomic studies for W8 included complete assessments of phenotype at a total of 11 field trials 

during 2012 and 2013. Although some significant agronomic differences were observed between events 

and controls, most fell within the conventional variety range. For example, some differences in yield and 

tuber size were observed in W8, and these attributes will be investigated further in commercial trials. In 

addition, with the exception of the expected late blight resistance, W8 was not significantly different 

from the control when considering disease susceptibility. Thus, we concluded that there are no major 

differences in agronomic characteristics, susceptibility to diseases, yield and grading, and ecological 

interactions between the untransformed Russet Burbank variety and W8 that would contribute to 

increased weediness or plant pest potential. Results of these studies contribute to the evidence that W8 

has similar agronomic and phenotypic traits to the Russet Burbank control and therefore W8 would be 

as safe as the controls. 

9.2 Compositional Equivalence

The compositional assessment study, evaluating proximates, vitamins, minerals, amino acids, and 

glycoalkaloids was conducted on Russet Burbank W8 to 1) show equivalence to the untransformed 

control, and 2) compare W8 to conventional variety ranges and literature ranges, where applicable. 

Ultimately, results of this composition study showed food safety equivalence between W8 and the 

untransformed control. Because tubers of W8 are either statistically equivalent to tubers from the 

control or within the tolerance interval or combined literature range, the W8 potatoes will be as safe as 

their untransformed control for use as food and feed. 

9.3 A Bioinformatics Approach to Allergen and Toxin Assessment

The bioinformatics approach to assessing toxins and allergens in W8 specifically investigates possible 

changes related to the DNA inserts and flanking regions. Other evidence that W8 would not contain 

new allergens or toxins comes from the compositional assessment as mentioned above in section 9.2: 

Compositional Equivalence. Based on the equivalent composition of W8 and the Russet Burbank control,

additional allergies would not be expected. Some allergies have been detected in children as a result of 

patatin contained in potatoes. Patatin is a storage glycoprotein that displays lipase activity and makes 

up about 40% of the soluble protein in tubers (Mignery et al. 1988). Allergic reactions to processed 

potatoes are considered to be very uncommon and have been reported for children only (De Swert et al.

2002; De Swert et al. 2007), with patatin (Sol t 1) identified as the major allergen involved in this 

reaction (Astwood et al. 2000). Because potato protein naturally contains a relatively large proportion of 

patatin, those who are allergic to potatoes would avoid them and changes to patatin levels would not be 

expected to alter that behavior. 
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The scope of this study included a search of the DNA insert sequence and flanking regions for possible 

open reading frames (ORFs) followed by an assessment of potential allergens or toxins. A general 

consideration is that introduction of DNA into the plant’s genome could result in disruption of a native 

gene or introduce sequence capable of expressing unexpected proteins that may act as allergens or 

toxins. A comprehensive bioinformatics analysis of each insertion site was performed to investigate this 

possibility (Appendix C: Allergen and Toxin Assessment).

BLAST searches indicated that there were no potato transcripts associated with the flanking regions of 

the inserts, and the available genome annotation lacked evidence of a known potato transcript 

associated with the insertion loci. A list of all ORFs present within the sequences of the inserts and their 

flanking regions were identified using the ORF Finder web application (Stothard 2000) available through 

the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/orf_find.html). The search 

parameters were defined to identify all ORFs with at least 19 amino acids located between any two 

contiguous stop codons regardless of whether a canonical start codon was present. This method 

identified a multitude of ORFs including 1) small ORFs naturally present in any DNA sequence, 2) those 

ORFs that specifically cover the junctions between native plant DNA and the inserted DNA (i.e. potential 

fusion proteins), and 3) the Rpi-vnt1 gene (VNT1 protein) present in the pSIM1678 cassette.

To assess the potential for any of these ORFs to act as allergens, they were compared with known 

allergens from the repository available through the Allergy Resource Research Program (FARRP) via the 

University of Nebraska (available at http://www.allergenonline.org/databasefasta.shtml). The ORFs were 

analyzed to identify any 80 amino acid sequence within an ORF that possessed greater than 35% 

homology to a documented allergen or shared sequence identity over any 8 amino acid stretch. Only two 

allergen matches were identified following analysis of 240 ORFs.

One match was based upon an open reading frame containing the 8 amino acid sequence (LPLLLLLL), 

which is also present in an endochitinase protein (GI:3201547) from Persea americana (avocado). This 

polypeptide is just one of 319 distinct 8-mers present in the full-length endochitinase protein, and there 

is no evidence it contributes to the allergenicity as the LPLLLLLL peptide was not found in a database of 

epitopes (http://www.iedb.org/). This 8-mer is present in numerous proteins found in potatoes and 

humans. Thus, even if it was expressed in Russet Burbank W potatoes, it is highly unlikely to pose an 

allergenic risk. Collectively, these data suggest the peptide identified is a false positive and would not be 

a potential allergen, consistent with concerns previously expressed over the high false-positive rates 

associated with identifying 8-mer matches (Goodman 2008).

The second match (8-mer and 80-mer searches) corresponded to a vacuolar acid invertase protein

(GI:18542113) present in tomatoes, which is also found in other Solanum species, such as potatoes. The 

presence of this partial ORF was by design in W8 potatoes as the vacuolar invertase (VInv) gene was the 

target of one of the introduced silencing cassettes. This silencing cassette will prevent expression of 

both the native INV protein and the ORF present in the pSIM1678 insert. Thus, silencing of the native 

vacuolar invertase gene may actually decrease the allergenic potential of Russet Burbank W8 potatoes 

relative to the control.

To assess the potential for these same ORFs to act as toxins, we performed BLAST searches against 

custom databases containing annotated protein toxins found in nature. We did not identify any 

significant matches (E-value < 1) for any of the ORFs. 
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In summary, using a number of well-established bioinformatics tools, we performed a comprehensive 

analysis of potential toxins and allergens associated with our DNA inserts and flanking regions, including 

the Late Blight Resistance Protein, VNT1. We did not identify any significant homology or identity 

between VNT1 and known allergens or toxins. Of the two matches identified from ORFs present in our 

DNA insert and flanking regions, neither appears to be significant, although one was associated with the 

VInv gene silencing cassette designed to limit expression of this gene and shown to be functional in 

tubers (see Chapter 7: Characterization of Gene Silencing and Target Gene Expression). If this sequence 

functioned as an allergen, the protein level would be reduced by the silencing of invertase resulting in 

even lower levels of a possible allergen. Overall, no allergen-related safety concerns were identified for 

the Russet Burbank W8 potato, and it is expected to be at least as safe as the Russet Burbank control.

9.4 Glycoalkaloid Testing

Potato plants naturally produce certain glycoalkaloids (solanine and chaconine) to defend against 

insects, disease, and predators which find those compounds toxic (Friedman 2006), and in response to 

environmental stress, such as physical damage and mishandling. Potato leaves, stems and shoots 

contain naturally higher levels of glycoalkaloids than potato tubers(Friedman and Dao 1990), but the 

relative tissue-specific levels vary depending on potato variety (Friedman et al. 1997). Most 

glycoalkaloids are present in the skin, or immediately below it, with gradually lower and lower levels of 

glycoalkaloids toward the center of the potato (Friedman and Dao 1990; Kozukue et al. 1987). Localized 

production of glycoalkaloids can also be found around eyes or sprouts(Friedman et al. 1997).

Sometimes, glycoalkaloid production is associated with a green color on the potato, typically when the 

tuber is exposed to light. This green color is because of the harmless light-induced production of 

chlorophyll. Depending on the variety, this green color may coincidentally correspond with increased 

glycoalkaloid production, but such color changes have not been found to reliably correlate with a higher 

level of glycoalkaloid content(Friedman 2006; Petersson et al. 2013; Friedman et al. 2003; Patil et al. 

1971; and Edwards et al. 1998). Peeling potatoes and removing any green tissue or sprouts can reduce 

glycoalkaloid levels by 30-95% compared to the levels found in potatoes that remain unpeeled

(Friedman and Dao 1990; Friedman et al. 2003; and Elzbieta 2012).

Glycoalkaloid Content in Commercial Varieties. The total glycoalkaloid content (TGA) of potato tubers 

varies widely. Values between 2 and 410 mg/kg FW were found (Lisinska and Leszczynski 1989), but in 

most cases the TGA concentration in whole tubers is between 10 and 150 mg/kg FW (van Gelder 1990). 

95% of the total glycoalkaloids in potato tubers consists of a-chaconine (solanidine-glucose-rhamnoserhamnose) and a-solanine (solanidine-galactose-glucose-rhamnose).

Most U.S. commercial potato varieties contain less than 120 parts-per-million (ppm) glycoalkaloids with 

ranges typically between 20-130 ppm on an unpeeled fresh weight basis (Friedman and Dao 1990;

Friedman et al. 2003; and Sinden and Webb 1972). Whole russet potato varieties typically contain less 

than 60 ppm (Friedman and Dao 1990) which is far less than the accepted upper limit of 200 ppm 

(Friedman 2006; JECFA 1992; and JECFA 1993). Indeed, a joint World Health Organization and Food and 

Agriculture Committee on glycoalkaloid safety concluded that “the large body of experience with the 

consumption of potatoes, frequently on a daily basis, indicated that normal glycoalkaloid levels (20-100 

mg/kg [=20-100 ppm]) found in properly grown and handled tubers were not of concern (JECFA 1993).

Bitterness and Glycoalkaloids. One sensory characteristic that correlates with glycoalkaloid levels is 

bitterness: potato varieties that are naturally bitter typically contain higher levels of glycoalkaloid than 

non-bitter varieties. Glycoalkaloid levels greater than 140 ppm are reported to taste distinctly bitter and 

levels greater than 200 ppm cause a burning sensation in the throat and mouth (Jansky 2010; Ross et al. 
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1978; Sinden et al. 1976; and Zitnak and Filadelfi 1985). The bitter taste and appearance of such 

potatoes makes it simple to exclude or set apart these types of potatoes from consumption and is one 

reason why the incidence of glycoalkaloid-related poisoning reports is low.

The main symptoms of glycoalkaloid illness are vomiting and diarrhea, but high acute consumption is 

necessary to produce such symptoms, e.g., 1.25mg/kg body weight (Hellenas et al. 1992; Mensinga et 

al. 2005; and Ruprich et al. 2009). For example, a 150 lb. (68 kg) person would need to eat about 1 lb. 

(425g) of peel-on potatoes having the maximum accepted levels of glycoalkaloids to show signs of 

illness. By contrast, he or she would have to consume as much as nine times that, i.e., 9 lbs. (3.8 kg), of

peeled potatoes before such symptoms may arise.

Current Industry Practices Mitigate Risk. Notwithstanding the low levels of glycoalkaloids found in 

commercial varieties, appropriate pre- and post-harvest strategies can help further mitigate any 

potential health issues. Growers and processors follow storage and handling procedures to minimize 

glycoalkaloid production, such as: keeping tubers covered with soil while growing; harvesting mature 

tubers; discarding damaged, sunburned, or defective potatoes; peeling; minimizing mechanical injury; 

suppressing sprouting; and storing harvested tubers in the dark under appropriate conditions of 

temperature and humidity.

Glycoalkaloid testing of W8. Event W8 and the control had mean glycoalkaloid levels of 7.2 and 6.4 

mg/100g (72 and 64 ppm), respectively; all well below the accepted safety limit of 20mg/100g (200 

ppm) (Sinden 1987; Friedman 2006; JECFA 1992; and JECFA 1993). Since the mean value for Event W8 

was not significantly different from the control and was within the tolerance interval, it was concluded 

that glycoalkaloid levels in event W8 are no different from the Russet Burbank control.

9.5 Dietary Risk Assessment of VNT1 protein

The similarity of the Rpi-vnt1 gene sequence to disease-resistance (R) genes found in edible plant 

species was examined (Appendix D: Dietary Exposure to Rpi-vnt1.1). A Basic Local Alignment Search 

Tool (BLAST) homology search was conducted to identify edible plant species containing DNA sequences 

or amino acid sequences with similarity to the Potato Late Blight Resistance gene (Rpi-vnt1) sequence. 

Homology of Rpi-vnt1 with tomato genes identified in the search was >90% with very low E-values, 

indicating significant homology with Rpi-vnt1.

The overall results of the nucleotide BLAST searches show that the Rpi-vnt1 (FJ423044.1) gene shares 

greater than 90% sequence identity with tomato mosaic virus (ToMV) resistance genes. In addition, the 

VNT1 protein (ACJ66594.1) is greater than 75% homologous to ToMV Tm-2 proteins from S. 

lycopersicum (tomato) and S. tuberosum (potato) at the amino acid level. Taken together, results from 

the bioinformatics analyses suggest that genes highly similar to Rpi-vnt1 at the nucleic acid and protein 

levels are present in the human diet as constituents of S. tuberosum L. (potato) and S. lycopersicum

(tomato) / Lycopersicon esculentum (tomato, older nomenclature). Similar R-genes have been used in 

classical breeding for late blight resistance for over 50 years (Malcolmson and Black 1966).

Given the high degree of homology between Rpi-vnt1 and R-proteins (e.g. ToMV Tm-2) present in 

tomato, existing dietary exposure to tomato varieties with the ToMV Tm-2 resistance gene could be 

relied upon to establish the history of dietary exposure to the protein product of the Rpi-vnt1 gene. 

Although a literature search did not yield information about the levels of ToMV resistance proteins in 

tomatoes or in diets, ToMV resistant tomato varieties and rootstocks are readily available and grown in 

the US for fresh market tomato production. Given the large per capita consumption of fresh market 
J.R. Simplot Company JRS06 Page 127 of 196

tomatoes in the United States, consumers have likely been exposed to tomatoes containing homologous 

ToMV resistant genes and associated proteins in their diets.

Widely consumed plant species that are reported to contain disease-resistance (R) genes include the 

Solanaceae family, Zea mays (maize), Glycine max (soybeans), Oryza sativa L. (rice), and Triticum 

aestivum L. (common wheat) (Bakker et al. 2011). Potato belongs to the Solanaceae family, which 

includes eggplant (S. melongena L.), pepper (Capsicum annum L.), and tomato (S. lycopersicum L.). In 

addition to these common cultivated crops, the Rpi-vnt1 gene is identical to the Rpi-phu1 gene 

(Rodewald and Trognitz 2013) found in some S. phureja species, a potato species consisting of many 

varieties which are currently consumed by indigenous people in South America.

In summary, the presence of similar or identical genes in frequently consumed tomatoes and potatoes 

that have not had negative impacts on human health establish a history of safe use. 

9.6 Low levels of VNT1 protein

A sensitive method was developed for detection of the S. venturii derived R-protein, Rpi-vnt1, using 

polyclonal antibodies raised against VNT1 peptides and the purified VNT1-LRR domain. VNT1 confers 

strong resistance against P. infestans, the causal agent of potato late blight. The method is able to 

detect low picogram levels of VNT1 protein. Using immunoblot and quantitative western blot analyses,

the VNT1 protein is below 30 ppb in potatoes transformed with the Rpi-vnt1 gene.

A recent review discusses a need to consider alternative methods of safety assessment for intractable 

proteins, including R-proteins (Bushey et al. 2014). Intractable proteins are defined as those with 

properties that make it extremely difficult or impossible with current methods to express in 

heterologous systems, isolate, purify, or quantitate due to low levels. A specific example is given for the

protein product of the Rpi-blb1 gene, which also confers resistance to late blight. The limit of detection 

for the BLB1 protein was found to be 100 ppb, and the protein was not detectable using an ELISA 

method. The same authors recommend a weight of evidence approach, like we have presented here, to 

establish safety of this type of protein.

9.7 Safety of Nucleic Acids

In 2001, EPA established an exemption from the requirement for a tolerance for residues of nucleic acids 

that are part of a PIP (40 C.F.R. 174.507) under the Federal Food, Drug, and Cosmetic Act (FFDCA), noting 

that “[n]ucleic acids are ubiquitous in all forms of life, have always been present in human and domestic 

animal food and are not known to cause any adverse health effects when consumed as part of food” (66 

Fed. Reg. 37817, July 19, 2001). FDA reached a similar conclusion, stating that nucleic acids were “generally 

recognized as safe” for purposes of FFDCA (57 Fed Reg. 22984, 22990, May 29, 1992).

9.8 Safety of Gene Silencing Methods

Many crops, including tomato, squash, soybean, papaya, potato, and plum, with traits that resulted 

from RNAi, have been deregulated by APHIS and evaluated for food safety by the FDA. In many of these 

products, a small piece of RNA interferes with production of an enzyme, and thus influences a quality or 

nutritional trait. The Russet Burbank event W8 contains gene silencing cassettes for Asn1, Ppo5, R1, 

PhL, and VInv, all of which result in small RNAs that regulate gene expression. Such small RNA (sRNA), 

including siRNA, miRNA, and piRNA, in plants and animals are generally involved in regulating 
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endogenous gene expression, repressing transposons, or targeting invading pathogens for destruction. 

The sRNA are ubiquitous in nature, including prokaryotes where sRNA have also been associated with 

the antiviral CRISPR pathway (Karginov and Hannon 2010). All of these pathways rely upon an RNase III 

endonuclease to process a double-stranded RNA (dsRNA) precursor into small effector RNAs that can be 

used to target RNA or DNA for modification or destruction. Due to bacterial colonization of our 

intestines and our daily diets of plants, animals, and fungi expressing their own spectra of sRNA, we are 

constantly exposed to a multitude of sRNA.

A publication by Chen-Yu Zhang’s team claimed that a plant-derived miRNA had the potential to survive 

substantial obstacles to elicit a biological activity in the liver of humans and mice (L. Zhang et al. 2012). 

The implications of these findings led to a number of studies aimed at reproducing the author’s study. 

However, these claims have not been substantiated (Dickinson et al. 2013; Snow et al. 2013; Witwer et 

al. 2013; Y. Zhang et al. 2012), and have been challenged by many experts in the field leading to selfcorrection of the scientific literature through publication of these numerous failed replication studies

(Editorial 2013). 

The results of the Zhang manuscript were central to the argument put forth by Jack Heinemann and 

colleagues in a communication calling for more rigorous safety testing of RNAi-based biotech products 

due to potential off-target effects of sRNA (Heinemann et al. 2013). The concerns of Heinemann and 

colleagues were thoughtfully considered by fellow scientists associated with the bi-national 

governmental regulatory agency, Food Standards Australia New Zealand (FSANZ), which evaluates food 

safety requirements for biotech foods. In their formal response, FSANZ concluded, “The weight of 

scientific evidence published to date does not support the view that small dsRNA in foods are likely to 

have adverse consequences for humans” (FSANZ 2013). 

The history of safe use, complexity of the human GI tract, irreproducibility of the cited controversial 

manuscript (L. Zhang et al. 2012), our compositional and nutritional data, and the unique characteristics 

of InnateTM potatoes collectively establish these potatoes as safe for human consumption. In fact, there 

is no scientific rationale to suggest that sRNA present in GM-foods are any less safe than those naturally 

abundant and safely consumed in our current diets. 

Stability through Microvesicles or Protein Complexes. Another possible mechanism to increase 

stability of siRNA would be for the plant to package them into microvesicles or apoptotic bodies or bind 

them to large protein or lipid-protein complexes. Plants are not thought to package cellular material 

within apoptotic bodies or microvesicles for destruction by other cells, as is done in animals. Instead, 

during programmed cell death, they concomitantly shrink their protoplasm while destroying cellular 

contents in an effort to contain a pathogen within the original cell structure to maintain structural 

integrity (van Doorn et al. 2011). A large amount of programmed cell death would thus be associated 

with sick plants that are not included in the food production process.

However, the biological activity of sRNA is linked to association with RNA induced silencing (RISC)

complexes. A number of distinct cellular pathways exist in plants and animals for processing sRNA and 

executing their biological activities, where each pathway includes protein complexes that bind to longer 

dsRNA, siRNA duplexes, or the sRNA species (Pumplin and Voinnet 2013). These protein complexes are 

considered critical for stabilizing sRNA as unincorporated sRNA (i.e. passenger strand) are more rapidly 

turned over. Turchinovich and colleagues found that the vast majority (>97%) of miRNAs identified in 

their culture media and plasma samples were not contained within vesicles, but were instead protected 

from degradation by a protein involved in the RISC complex (Turchinovich et al. 2011). 
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In summary, there is evidence that siRNA may be protected by association with RISC complexes within 

and outside of cells. However, since protein transport across cell membranes is highly regulated, these 

complexes may protect sRNA from degradation and prevent their indiscriminate uptake by human cells. 

The challenges of packaging sRNA have been realized by the pharmaceutical industry who have spent 

considerable amounts of time and effort attempting to develop techniques aimed at optimizing the 

stability, delivery, distribution, and pharmokinetics of sRNA for use as orally delivered therapeutics with

limited success (Castanotto and Rossi 2009; Scaggiante et al. 2011). In fact, one of the groups that 

rebutted the work by the Zhang group, miRagen Therapeutics, could have benefitted from confirmation 

of those studies.

Uptake of sRNA in Animals. While genes in some simple organisms can be targeted through feeding 

upon organisms expressing double-stranded RNA (dsRNA), this is highly unlikely in higher organisms, 

such as humans. Humans have complex GI tracts that present numerous obstacles to the uptake of 

dietary RNA, have many more cells to prevent non-specific accumulation, and lack components of the 

RNAi pathway (e.g. RNA-dependent RNA polymerases) that could amplify and sustain a non-specific 

response (Petrick et al. 2013). In addition to the plethora of sRNA consumed through a normal diet, 

humans possess trillions of microbes within their digestive tracts that can both absorb and secrete their 

own sRNA, which have also been detected in human plasma samples (Wang et al. 2012). 

Bioactivity of plant-derived sRNA in mammalian cells. The Lam lab investigated plants as a delivery 

system for siRNAs that could target viruses in consumers (Zhou et al. 2004), whereas the Lee lab

explored the potential of using plants as an economical factory for production of siRNA (Chau and Lee 

2007). These conflicting datasets are the only mammalian studies we are aware of that address 

bioactivity of plant sRNA in mammalian cells, but a study was performed in the model organism, 

Caenorhabditis elegans, which is a highly-sensitive system for inducing and detecting RNA interference 

activity. Consistent with the results of Chau et al., they did not find biological activity of plant-derived 

siRNA (Boutla et al. 2002; Chau and Lee 2007). Interestingly, they were able to induce an RNAi-mediated 

phenotype when injecting longer dsRNA derived from plants. These results may suggest the structure of 

plant siRNA are inconsistent with animal systems or that exogenous siRNA are much less efficient at 

inducing a biological phenotype than dsRNA being processed by the cell’s own machinery. It remains 

unclear whether the very modest phenotype reported by the Lam group is dependent upon RNAi as 

they were treating cells with impure samples, including longer dsRNA that may have activated a cellular 

immune response.

Processing of dsRNA from inverted repeats in plants can produce multiple classes of sRNA, including 21-

22 nt and 24 nt species. The 24 nucleotide population is especially unlikely to have RNAi activity in 

animals as they are not involved in degradation of target transcripts even in plants (Fusaro et al. 2006). 

Summary of RNAi safety. In summary, humans consist of cells, tissues, and organs that remain 

homeostatic in the presence of varying diets consisting of abundant sRNA. It is highly unlikely that a 

sufficient quantity of these sRNA would survive the GI tract and accumulate in a given human cell 

resulting in a short-term, let alone a long-term, biological effect. In addition, the human body possesses 

a number of immune regulatory pathways dedicated to specifically detecting and destroying exogenous 

dsRNA as a means of protecting against foreign invaders.

Scientific rationale of the safety of orally ingested siRNA(s) derived from InnateTM potatoes. As 

described previously, the scientific literature does not support a model whereby sRNA present in 
J.R. Simplot Company JRS06 Page 130 of 196

consumed food pose a safety risk to humans following consumption (Petrick et al. 2013). In contrast 

there is a long record of safe consumption of sRNA within our natural diet. There are a number of 

important characteristics of our InnateTM potatoes and their use of RNAi:

 The InnateTM potatoes rely upon potato genomic DNA to initiate gene silencing using the plant’s 

endogenous pathway. The inverted repeat sequence is derived from the sequence of the genes 

that are already being expressed in the potato.

 Many common potato preparation or cooking practices involve heating at high temperatures, 

which result in the conversion of asparagine with sugar into acrylamide, which has been 

associated with health concerns (Health Canada 2013). InnateTM potatoes use RNAi to reduce 

the accumulation of the precursor asparagine to limit acrylamide potential. Thus, InnateTM

potatoes provide a consumer product with potentially enhanced safety.

 Processing of potatoes by consumers or the food industry involves treatments that are likely to 

limit the amount of sRNA in the final product. In addition to high temperature heating, 

treatments such as blanching, frying, dehydration, and freezing are commonly used, which lead 

to degradation and fragmentation of double-stranded genomic DNA. A similar fate is expected 

for sRNA as was shown in processed milk (Chen et al. 2010).

 The InnateTM potatoes under consideration do not target an evolutionarily conserved exogenous 

animal gene as might be the case when RNAi is used as a plant incorporated protectant. Since 

RNAi in InnateTM potatoes exclusively target plant genes, they are less likely to have adverse offtarget effects in animals. 

 We have performed rigorous compositional, nutritional, and agronomic analyses and have not 

observed any evidence of off-target effects in the plant where expression of sRNA was the 

highest and the potential for off-target effects greatest.

Numerous physiological barriers have impeded introduction of nucleic acid through oral uptake (O’Neill 

et al. 2011), and as noted previously, there is a long history of safe use associated with eating foods 

containing sRNA due to its ubiquitous presence in nature (Ivashuta et al. 2009; Jensen et al. 2013; 

Petrick et al. 2013). Mechanistic studies of a number of cultivars have shown plants selected for 

agronomic traits using conventional techniques are using RNA interference to silence their own genes 

through expression of inverted repeats (Della Vedova et al. 2005; Kusaba et al. 2003; Tuteja et al. 2004).

Comments presented to the EPA’s Scientific Advisory Panel Public Meeting on RNAi technology as a 

pesticide, held January 28, 2014, included support for the safety of dsRNA by experts in the field (Mello 

2014). Dr. Mello reported that oral uptake of dsRNA has proven unfeasible as a drug delivery route, 

thus unlikely to cause off-target effects when used for gene silencing in plants. He also reported that 

ingested RNA is rapidly metabolized in the gut where it is converted to nutrients, thus proposing that 

bioinformatics testing for similar sequences in humans would be unnecessary. Also, RNA is digested 

rapidly, suggesting that digestibility assays would be unnecessary for dsRNA.

In summary, we believe the history of safe use, the irreproducibility of the cited controversial 

manuscript (L. Zhang et al.2012), the submitted compositional and nutritional data, and the unique 

characteristics of InnateTM potatoes collectively establish these potatoes as safe for human 

consumption.
J.R. Simplot Company JRS06 Page 131 of 196

9.9 Previous regulatory actions for plant incorporated protectants

The safety assessment of the Potato Late Blight Resistance Gene (also known as Rpi-vnt1) is based partly 

on previous regulatory actions for similar resistance genes that are considered plant incorporated 

protectants by EPA. For example, a rationale similar to that used for Coat Protein Gene of Plum Pox 

Virus (US EPA 2010) and Potato Leaf Roll Virus (PLRV) Resistance Gene (US EPA 2000) could be 

considered for safety of late blight resistance gene. Like both Coat Protein Gene of Plum Pox Virus and 

PLRV Resistance Gene, late blight resistance has been part of the food supply for many years without 

adverse effects. There is a long history of safe consumption of similar resistance genes throughout the 

food supply. Also, like the Coat Protein Gene of Plum Pox Virus and PLRV resistance, safety is based in 

part on a history of safe consumption of nucleic acids, and dietary prevalence of similar genes. The 

mechanism of action for both Plum Pox Virus and PLRV resistance resulted from gene silencing. In 

contrast, late blight resistance from Rpi-vnt1 relies on synthesis of a protein, however, the VNT1 protein 

levels are too low to detect. 

The EPA’s Biopesticides Registration Action Document (BRAD) for the Coat Protein Gene of Plum Pox 

Virus (US EPA 2010) includes a summary of Data Waivers for Toxicology and the Environmental 

Assessment. Toxicology waivers were granted based on 1) “There is a long history of consumption of 

plant virus particles in food without any known toxicity or other deleterious health effects, and 2) Nonoccupational exposure is minimal to non-existent since the gene is only expressed within plant tissue.” 

The waivers for Non-target Organisms included a rationale that Prunus species are difficult to breed with 

domestic or wild relatives and result in few hybrids with low vigor and fertility. Also, there is a long 

history of consumption of plant viruses in foods consumed by animals without deleterious effects or 

evidence of toxicity. Exposure to nontarget aquatic species or terrestrial insects, including honey bees, 

is not expected since the gene is only expressed within the plant genome. Even if the plum pox virus 

coat protein was produced in plum, the coat protein does not match known toxins or allergens.

EPA’s approval of the Potato Leaf Roll Virus Resistance Gene also included data waivers for toxicology 

and the environmental assessment. The justification for waivers for toxicology was “The long history of 

consumption of virus-infected plants without any reports in the scientific literature of toxicity or other 

harm caused to the general population are sufficient to support the registration of the active ingredient 

potato leaf roll virus resistance gene (also known as orf1/orf2 gene).” Waivers of the data requirements 

for the environmental assessment were based on “the long history of virus-infected food plants as part 

of the domestic animal food supply without any reports of adverse effects.” Also, the environmental 

fate studies were waived because of the lack of toxicity/pathogenicity associated with the active 

ingredient and lack of significant levels of expression. Requirements for nontarget insects, fish, and 

other wildlife were waived because “the lack of toxicity/pathogenicity and mitigating label language for 

aquatic exposure result in minimal to non-existent risk to wildlife.” 

Like the plum species, Prunus domestica, potatoes are difficult to breed because they are tetraploid, 

highly heterozygous and subject to inbreeding depression. The most popular potato variety in the US, 

Russet Burbank, is sterile. Potatoes will not readily breed with other varieties or wild relatives, and in 

the unlikely case where that could occur, true seeds are unlikely to survive, since only the potato tubers 

are typically kept for “seed”.
J.R. Simplot Company JRS06 Page 132 of 196

9.10 Conclusions: Safety of Russet Burbank W8 Potato

In summary, we present a rationale for the safe use of W8 potatoes containing the Potato Late Blight 

Resistance Gene (also known as Rpi-vnt1). Among the supporting evidence are the phenotypic data 

collected in a robust field trial program at 11 sites over two crop years. From those studies, a 

compositional assessment shows the W8 potatoes substantially equivalent to Russet Burbank controls. 

Open reading frames associated with the DNA inserts, including the late blight resistance gene itself,

were screened for allergen and toxin potential, but no safety concerns were identified. The W8 potatoes 

were tested for glycoalkaloids and found no different from the Russet Burbank controls and well below 

the accepted safety limit. Similarity of the Rpi-vnt1 to other gene sequences in related species was 

assessed and greater than 90% homology discovered between this late blight resistance gene and the 

Tomato Mosaic Virus resistance gene (ToMV Tm-2) identified in tomatoes, which has been widely bred 

into tomato varieties in the fresh market and is consumed by humans. 

Among the risk considerations is the presence of the VNT1 protein, and although effective at controlling 

late blight, levels in potato tubers are below our detection limit of 30 ppb. As noted in Section 9.7, 

nucleic acids have long been considered safe under the FFDCA by EPA and FDA. An in depth review is 

included on the topic of the safety of using RNA interference for gene silencing. Regulatory actions 

supporting safe use of the Coat Protein Gene of Plum Pox Virus and Potato Leaf Roll Virus Resistance

Gene provide additional evidence of the safety of W8. 

Accordingly, based on the results of the studies presented here, there is no reason to believe that the 

VNT1 protein will have any impact to human health through allergenicity or toxicity, or any 

environmental impacts to non-target mammals, birds, fish, or insects due to the low expression and lack 

of toxic effects of the protein. There is negligible risk of environmental contamination and no 

persistence in the environment because of the low expression of the VNT1 protein in potato tubers. The 

prevalence of similar resistance genes throughout edible crops suggests that extremely low levels of 

similar proteins are widespread in nature and unlikely to pose risk to human health, non-targets or the 

environment. 

Based on the foregoing rationale for safety, the J.R. Simplot Company respectfully requests that it 

should be granted deregulated status for Russet Burbank event W8 containing the Potato Late Blight 

Resistance Gene (also known as Rpi-vnt1).
J.R. Simplot Company JRS06 Page 133 of 196

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10.0 Trait Efficacy

Introduction

Russet Burbank Event W8 was created using InnateTM technologies in order to address the need of the 

potato industry to improve quality by increasing resistance to late blight, reducing expression of the 

enzyme responsible for black spot, reducing acrylamide and lowering levels of reducing sugars. In the 

following discussion, we provide evidence for the efficacy of these traits in Russet Burbank Event W8. 

Results of the trait efficacy studies are presented first, with methods grouped together following the 

results.

10.1 Composition and Field Results

10.1.1 Potato Free Amino Acids Results

W8 tubers contained less free asparagine, but more aspartic acid, glutamine, and glutamic acid than the 

control (Table 10-1). This change was expected because of silencing Asn1 and is linked to a reduced 

acrylamide-forming potential. The mean values of asparagine, aspartic acid, glutamine, and glutamic 

acid for W8 were all within the tolerance intervals and combined literature ranges, and therefore 

considered within the normal range for potatoes. A review of the biosynthetic pathway for glutamine 

and asparagine in Figure 10-1 illustrates how a reduction in asparagine could lead to increases in these 

other amino acids. 

Table 10-1. Potato Free Amino Acids at Harvest

Compound Variety LSMean P-value

3

N

Range TI1CLR2

Min Max Min Max Min Max

Asparagine 

(mg/100g) W8 87.8 <.0001 41 62.1 140 . . . .

Asparagine 

(mg/100g) Burbank 300 . 41 198 469 60.0 482 31.2 698

Aspartic Acid 

(mg/100g) W8 45.2 0.0032 41 32.8 63.9 . . . .

Aspartic Acid 

(mg/100g) Burbank 40.4 . 41 27.9 52.7 12.6 72.8 6.4 75.2

Glutamine 

(mg/100g) W8 252 <.0001 41 203 335 . . . .

Glutamine 

(mg/100g) Burbank 139 . 41 95.7 198 23.3 260 44.0 540

Glutamic Acid 

(mg/100g) W8 51.3 <.0001 41 27.9 75.5 . . . .

Glutamic Acid 

(mg/100g) Burbank 40.8 . 41 26.0 72.0 10.2 80.3 45.0 74.2

1

TI = Tolerance Interval

2

CLR = Combined Literature Range. Literature ranges are from, Davies (1977); Shepherd et al. (2010); Lisinska and Leszczynski (1989)

3

P-values indicating significant differences between W8 and control are in bold and underlined. 
J.R. Simplot Company JRS06 Page 139 of 196

Figure 10-1. Biosynthesis of GLN and ASN in plants

Figure 10-1 is from New World Encyclopedia, http://www.newworldencyclopedia.org/entry/Asparagine.

10.1.2 Reducing Sugars and Invertase Silencing

The W8 event contains expression cassettes that could lower levels of reducing sugars in tubers by 

multiple mechanisms. Through the transformation with pSIM1278, we introduced a silencing cassette 

for the promoters of the starch associated gene (R1) and the phosphorylase-L gene (PhL) whereas 

transformation with pSIM1678 introduced a silencing cassette for the invertase gene (Ye et al. 2010). 

Together, these traits function by slowing the conversion of starch and sucrose to reducing sugars 

(glucose and fructose). Although silencing of R1 and PhL resulted in lowered levels of reducing sugar

when analyzed at one month after harvest (Collinge and Clark 2013), the major reduction in reducing 

sugar appears to be related to invertase silencing. Overall benefits of silencing R1, PhL, and VInv include 

improved quality, especially relating to color control, and thus contributing to the desired golden brown 

colors required by most french fry or chip customers. Also, the reducing sugars react with amino acids, 

such as asparagine, to produce Maillard products including acrylamide.

Invertase Activity

The VInv gene silencing cassette in pSIM1678 results in decreased levels of vacuolar invertase, an 

enzyme which converts sucrose into glucose and fructose. When levels of invertase are decreased in 

potatoes, reducing sugars glucose and fructose remain at low levels during storage while sucrose 

increases, especially when held below typical storage temperatures of 46 - 48°F for french fry potatoes. 

Before testing for invertase activity, tubers were stored at 39°F for one month. Three replicates for each 

of W8 and Russet Burbank control were used for the assay which was measured by the accumulation of 

glucose. W8 showed an 85% reduction in vacuolar invertase activity in cold-stored tubers compared to 

the control (Figure 10-2). The reduced vacuolar invertase activity in W8 tubers is associated with

reduced RNA accumulation from the VInv gene (Chapter 7: Characterization of Gene Silencing and 

Target Gene Expression, Figure 7-2) and lower levels of reducing sugars glucose and fructose (Tables 10-

2, 10-3).
J.R. Simplot Company JRS06 Page 140 of 196

Figure 10-2. Enzymatic Activity of Invertase in W8 and Russet Burbank Tubers

Potato tubers were stored at 4°C for 30 days. Three independent tubers of each variety were used for the assay. The activity 

was measured by the accumulation of glucose in units of nmol·min-1·mg tuber-1.

Changes in Reducing Sugars and Sucrose

Long-term cold storage is necessary to maintain an adequate supply of high quality potatoes for yearround processing into french fries and potato chips, but also leads to cold-induced sweetening (CIS). CIS 

causes unwanted side effects in potato products processed at high temperatures including flavor 

changes, unwanted dark colors and elevated amounts of acrylamide. Vacuolar acid invertase (VInv) is an 

enzyme that is critically important in the CIS process in increasing the amount of glucose and fructose in 

tubers stored at very low temperatures (Zrenner et al. 1996). W8 has suppressed expression of the VInv

gene and therefore has reduced glucose and fructose levels in cold storage and less CIS compared to the 

Russet Burbank control. In order to demonstrate efficacy of the traits leading to lowered reducing sugar, 

field-grown tubers of W8 and the untransformed control were analyzed at harvest and at normal (46°F) 

and cold (38°F) storage temperatures.

Reducing sugars, glucose plus fructose, and the non-reducing sugar, sucrose, were tested in W8 at the 

time of harvest and then after 3, 6, and 9 months of storage. Two different storage temperatures were 

used, 46 ̊F, which is typically used for Russet Burbank potatoes destined for frozen french fries, and 

38 ̊F, a lower temperature enabled by silencing VInv, possibly allowing for better quality without high 

levels of reducing sugars. At harvest and all storage time points and temperatures, W8 tubers contained

lower levels of reducing sugars compared with the control (Tables 10-2 and 10-3). The decrease in 

reducing sugars was expected from silencing the VInv gene. All sugar values for W8 at the time of 

harvest were within the tolerance interval, indicating compositional equivalence to the controls.

8.86

1.37

0

1

2

3

4

5

6

7

8

9

10

Russet Burbank W8

nmole glu/min/mg tuber
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Table 10-2. Potato Sugar Levels at Harvest and After Storage at 46°F

Timing Variety LS Mean P-value1N

Range Tolerance Interval

Min Max Min Max

Fructose + Glucose (mg/100g)

Fresh W8 38.4 0.0002 41 9.68 106 1.00 424 Control 146 . 41 14.0 406

Month 3 W8 122 0.0056 9 54.1 210 1.00 996 Control 483 . 9 298 598

Month 6 W8 116 <.0001 9 20.8 310 1.00 640 Control 261 . 9 153 459

Month 9 W8 106 0.032 9 79.7 160 1.00 648 Control 224 . 9 105 372

Sucrose (mg/100g)

Fresh W8 395 <.0001 41 161 775 1.00 512 Control 241 . 41 113 558

Month 3 W8 651 <.0001 9 520 738

1.00 1125 Control 148 . 9 56.2 228

Month 6 W8 202 0.0021 9 177 229 1.00 345 Control 97.6 . 9 80.1 144

Month 9 W8 146 <.0001 9 105 201 10.4 103 Control 56.9 . 9 44.8 77.3

1

P-values indicating significant differences between W8 and control are in bold and underlined.

Table 10-3. Potato Sugar Levels When Stored at 38°F

Timing1Variety LSMean P-value3N

Range TI2

Min Max Min Max

Fructose + Glucose (mg/100g)

Month 6

W8 91.7 0.0002 3 83.7 97.4

1.00 1586

Control 640 . 3 590 726

Month 9

W8 151 <.0001 3 102 188

183 1586

Control 754 . 3 703 788

Sucrose (mg/100g)

Month 6

W8 963 <.0001 3 945 986

1.00 661

Burbank 182 . 3 138 206

Month 9

W8 645 <.0001 3 598 714

1.00 325

Burbank 152 . 3 137 163

1

Testing occurred at 6 and 9 months only because the purpose of the study was to evaluate long term storage at 38°F.

2

TI = Tolerance Interval

3

P-values indicating significant differences between W8 and control are in bold and underlined. 

All W8 tubers contained more sucrose (Table 10-2 and 10-3) than control samples at harvest and after 

multiple storage time points at both 38 ̊F and 46 ̊F. The increase in sucrose was expected from silencing 

the VInv gene, thus inhibiting the conversion of sucrose into the reducing sugars glucose and fructose. 

The mean levels of sucrose for W8 were within the tolerance interval and combined literature ranges, 

and therefore would be considered substantially equivalent to the controls.

The net result of silencing the VInv gene in W8 is lower levels of reducing sugars and higher levels of 

sucrose. These changes are observed at the time of harvest and throughout the storage period of up to 
J.R. Simplot Company JRS06 Page 142 of 196

9 months. Reducing sugars in W8 increase with storage time, but remain consistently lower than the 

Russet Burbank control. Much lower levels of reducing sugars were observed in W8 compared with 

controls when stored at 38 ̊F, suggesting that lower temperature storage could be feasible for W8. In all 

cases significant decreases in reducing sugars are coupled with higher levels of sucrose. It would be 

expected that lower temperature storage results in less shrink from respiration, but also would reduce 

losses from disease.

10.1.3 Acrylamide

Reduced asparagine levels in W8 (Table 10-1) result from the intended silencing of the asparagine 

synthase-1 gene (Asn1). Lowered asparagine, fructose, and glucose levels lead to an overall reduction of 

acrylamide in processed potato products because they are reactants in the formation of acrylamide. In 

order to demonstrate the efficacy of reducing acrylamide, field-grown tubers of W8 and the control 

were analyzed at harvest and at normal (46°F) and cold (38°F) storage temperatures.

At the time of harvest, french fries made with W8 tubers contained 85% less acrylamide than the control

(Table 10-4). When potatoes were stored throughout nine months at 46 ̊F, acrylamide levels in W8 

were 78 to 83.7% lower than control Russet Burbank (Table 10-4). Acrylamide levels in W8 potatoes 

were somewhat higher after storage at 38 ̊F for 6 to 9 months (Table 10-5), but consistently much lower 

than controls. These significantly lower acrylamide levels in W8 were expected from silencing the Asn1

and VInv genes, thus reducing the reactants free asparagine and reducing sugars.

Table 10-4. French Fry Acrylamide Levels (ppb) at Harvest and After Storage at 46°F

Timing Compound Variety LSMean

(ppb) P-value3

Percent 

Reduction2

N

Range (ppb) TI1(ppb)

Min Max Min Max

Fresh Acrylamide W8 75.3 <.0001 85.0 41 32.7 185 10.0 1035 Burbank 503 . . 41 229 971

Month 3 Acrylamide W8 86.1 <.0001 80.9 9 74.5 94.3 10.0 599

Burbank 450 . . 9 393 514

Month 6 Acrylamide W8 68.3 0.0011 83.7 9 50.4 96.2 10.0 688 Burbank 420 . . 9 330 528

Month 9 Acrylamide W8 115 0.0013 78.2 9 90.7 156 10.0 1047

Burbank 528 . . 9 429 740

1

TI = Tolerance Interval

2

Percent Reduction in Acrylamide is relative to control Russet Burbank at same storage time.

3

P-values indicating significant differences between W8 and control are in bold and underlined. 
J.R. Simplot Company JRS06 Page 143 of 196

Table 10-5. French Fry Acrylamide Levels at Harvest and After Storage at 38°F

Timing1Compound Variety LSMean

(ppb) P-value4

Percent 

Reduction3

N

Range (ppb) TI2(ppb)

Min Max Min Max

Month 6 Acrylamide 

W8 203 <.0001 86.2 3 199 207

1155 1792

Burbank 1473 .

. 3 1450 1500

Month 9 Acrylamide

W8 212 <.0001 90.8 3 201 234

761 3839

Burbank 2300 .

. 3 2160 2380

1

Testing occurred at 6 and 9 months only because the purpose of the study was to evaluate long term storage at 38°F.

2

TI = Tolerance Interval

3

Percent Reduction in Acrylamide is relative to control Russet Burbank at the same storage time.

4

P-values indicating significant differences between W8 and control are in bold and underlined. 

10.1.4 Black Spot

Black spot is a discoloration affecting potato tubers and is a result of leakage of polyphenol oxidase 

(PPO) from damaged plastids into the cytoplasm. In the cytoplasm, the enzyme oxidizes polyphenol, 

which then form dark precipitants. In order to reduce black spot in W8, the potato Ppo5 gene, was 

silenced. To demonstrate efficacy of this trait, an indirect method was used to test for black spot 

tolerance based on applying catechol to the cut surfaces of tubers.

Black Spot Analysis Results

Results of the catechol assay to determine PPO activity are presented in Figure 10-3. All W8 tubers 

remained unchanged after addition of catechol. All Russet Burbank control tubers turned darker in color 

after addition of catechol. These results indicate that W8 tubers are more resistant to black spot than 

tubers of the control, and support the efficacy of the reduced black spot trait.
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Figure 10-3. Catechol Assay for Polyphenol Oxidase Activity

Enzymatic Assay of PPO Activity in W8

Additional tests were conducted to show that reductions in Ppo5 could be measured by enzymatic 

assay. An enzymatic assay shows that W8 has a 90% decrease in PPO activity (0.025 μmol·min-1·mg dw 

tuber-1), compared to the Russet Burbank control (Figure 10-4). 

The reduced activity in W8 tubers was intentional and associated with reduced black spot through 

silencing of the Ppo5 gene (Chapter 7: Characterization of Gene Silencing and Target Gene Expression, 

Figure 7-2).
J.R. Simplot Company JRS06 Page 145 of 196

Figure 10-4. PPO Activity in Russet Burbank and W8 Tubers

PPO Activity in Russet Burbank and W8 Tubers. Conversion of L-DOPA to dopachrome was monitored over time by 

measuring A474nm. ΔA474nm·min-1, which was converted to units of μmol·min-1·mg dw tuber-1.

10.1.5 Late Blight Resistance

The Potato Late Blight Resistance Gene (also known as Rpi-vnt1) has been added to W8, successfully 

conferring late blight resistance. To demonstrate the efficacy of late blight resistance, studies were 

conducted by inoculating both foliage and tubers.

Foliar Late Blight Test Results

At the end of the trial period, the last rating from each site is summarized in Table 10-6 to show the

effect of the potato late blight resistance gene on foliar resistance in W8. Each trial site had different 

strain inoculum that included US8, US22, or US23, depending on the strains that were found in that 

area.

Table 10-6. Percent Foliar Late Blight Infection at Last Rating

Variety Mean Percent Foliar Late Blight 

Infection P-value2

CVR1

Control 58.3 <.0001 18.8 - 100

W8 0.50 . .

1

CVR = Conventional variety range. The range of mean values of conventional varieties.

2

P-values indicating significant differences between W8 and control are in bold and underlined. 

. = Not applicable

A significant reduction in percent foliar late blight infection was detected for W8 compared to the 

Russet Burbank control (Table 10-6). This supports the conclusion that the potato late blight resistance 

gene confers resistance in W8 and is efficacious in the foliage.

Tuber Late Blight Results

Tuber infection rate determined by percent infection is summarized in Table 10-7.

0.0258

0.0018

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Russet Burbank W8

μmol/min/mg dw tuber
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Table 10-7. Percent Tuber Late Blight Infection

Isolate Variety Mean Percent Late Blight Tuber Infection P-value1

US-22 Control 100.0 .

US-22 W8 51.0 <0.0001

US-8 Control 67.0 .

US-8 W8 21.1 <0.0001

1

P-values indicating significant differences between W8 and control are in bold and underlined. 

. = Not applicable

A significant reduction in percent late blight infection in tubers was detected for W8 compared to the 

control (Table 10-7) for both US-22 and US-8 isolates. 

10.2 Conclusions on Trait Efficacy

Significant decreases in free asparagine and reducing sugars in W8 contribute to substantial reductions 

in acrylamide in french fries compared with the control. In addition, silencing of invertase significantly 

lowers reducing sugars, potentially allowing for long term storage of potatoes at colder temperatures 

such as 38 ̊F. An enzymatic assay for vacuolar invertase activity confirmed a reduction of 85% in tubers 

of W8 compared to the control. Ultimately, results of this study demonstrate the efficacy of the 

constructs to lower reducing sugars, free asparagine, and acrylamide at harvest and after storage at 

normal and cold temperatures for at least 9 months. 

Samples of W8 tubers from multiple field trial sites all showed strong resistance to black spot as 

demonstrated through the catechol assay. In addition, the enzymatic assay showed a 90% reduction in 

PPO activity in tubers from W8 compared to the control.

Tests with late blight inoculum on tubers and foliage support the conclusion that the Potato Late Blight 

Resistance Gene confers strong resistance in both foliage and tubers of W8. The results of the studies 

with Russet Burbank Event W8 show the expected late blight resistance, along with reductions in black 

spot, reducing sugars, and acrylamide. Significant reductions in reducing sugars and acrylamide were 

observed at harvest and sustained throughout long term storage at both 38 ̊F and 46 ̊F. Lastly, activity 

for enzymes associated with the VInv and Ppo5 genes are reduced as expected from gene silencing.

10.3 Analytical Methods for Trait Efficacy and Composition

10.3.1 Analytical Methods for Free Amino Acids, Sugars, Acrylamide

Sample Source, Preparation, and Analysis

Tubers were analyzed at harvest and after 3, 6, and 9 months of storage to determine the efficacy of the 

traits leading to decreased reducing sugars and acrylamide at harvest and throughout storage at normal 

and cold temperatures. Tubers for compositional analyses were collected from all sites in 2012 and 2013 

at harvest. Tubers for compositional analyses at 3, 6, and 9 months at normal storage temperature of

46 ̊F (normal) were collected from all 2012 sites and tubers at the cold temperature storage 

temperature of 38°F (cold) were collected from Grant County, Washington. These locations have been 

summarized in Table 8-1 of the Compositional Analyses Chapter. Samples were obtained by randomly 

selecting 6 mid-sized tubers at harvest and 3 mid-sized tubers at 3, 6, or 9 months of storage from each 

replicate of W8 and control. Samples were powdered in an industrial blender with liquid nitrogen and 
J.R. Simplot Company JRS06 Page 147 of 196

stored at -70°C until analysis. For acrylamide testing, twenty pound samples (combined across 

replicates) of the potatoes were processed into french fries prior to analysis, using standard practices. 

Analytical testing was conducted by Covance Laboratories, Inc. in Madison, WI or Greenfield, IN. Free 

amino acid levels were determined by Covance Laboratories using the Covance protocol FAALC_S:6 

(Schuster 1988; Henderson 2000; and Barkholt and Jensen 1989). Sugar levels were determined by 

Covance Laboratories using High Performance Anion Exchange Chromatograph (HPAEC) equipped with a 

Pulsed Amperometric Detector (PAD) and following Covance protocol SWET_S:9 (Lilla et al. 2005; Dionex 

Technical Note 20). Acrylamide levels were determined by Covance Laboratories in Greenfield, IN using 

the Covance protocol ACMS_GRN_S:4 (FDA 2003; Musser 2003; FDA 2003). 

Statistical Methods Free Amino Acids, Sugars, Acrylamide

The statistical analysis was performed by using SAS 9.3. 

Data were analyzed using the following linear mixed model:

Yij = i + j + ij

•  = mean of treatment (fixed)

•  = rep (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the random rep effect, and ij

denotes the residual random error.

Interpretation of Free Amino Acids, Sugars, Acrylamide Results

Although trait efficacy is the focus of the present discussion, free amino acids and sugars were 

compared with the normal levels in potatoes as additional evidence to establish substantial equivalence 

between W8 and controls. The majority of the evidence for substantial equivalence is found in Chapter 

8: Compositional Assessment. A significant difference was established with a p-value < 0.05. Significant 

differences in the amount of a compound between W8 and the control were considered important for 

trait efficacy. For substantial equivalence, mean values were first compared with tolerance intervals

calculated from commercial controls and reference varieties (Table 8-5), followed by comparison with 

combined literature range values found in the scientific literature (Table 10-8). If the mean values were 

within either the tolerance interval or the combined literature range, they were considered within the 

normal range for potato.
J.R. Simplot Company JRS06 Page 148 of 196

Table 10-8. Combined Literature Ranges (CLR) for Free Amino Acids

1

0.2 is the approximate conversion from dry weight to fresh weight (internal communication)

2

Free glutamine from Davies 1977 was amended to reduce the high level from 1824.4 to 539.6 due to a perceived outlier.

10.3.2 Black Spot Analysis by Catechol Assay

An indirect method to test for black spot tolerance, the polyphenol oxidase activity assay, was used to 

demonstrate trait efficacy. In this assay, two slices each from 10 tubers of W8 and the control were 

tested (Table 10-9). To conduct this polyphenol oxidase assay, 1-ml catechol (25 mM in 50 mM MOPS, 

pH6.5) was pipetted onto the cut surfaces of randomly chosen tubers for W8 and the control. The PPOdependent development of a dark brown precipitate was assessed after 20 minutes. 

Table 10-9. Origin of Tubers from 2013 Field Trials for PPO Assay

Site Code Material Tested Trial Design1

Minidoka County, ID W8, Control RCB, 4 reps

Grant County, WA W8, Control RCB, 4 reps

Grand Forks County,ND W8, Control RCB, 4 reps

1

RCB=Randomized Complete Block design. Number of blocks was equal to the number of reps.

10.3.3 Foliar Late Blight Resistance Test Methods

Late Blight Field Trial Locations

During 2013, plants of W8 and the control were grown at several locations detailed in Table 10-10. With 

the exception of fungicide applications, normal pest control and maintenance practices consistent with 

potato production for the area were used to produce the crop. The entire trial was treated with the 

same agronomic inputs and pesticide and fertilizer applications to ensure uniformity from pre-season 

through harvest. Any fungicide treatment did not affect P. infestans infection and was discussed with 

and approved by study coordinator before application. 

Compound Value Units Reference

Formula Wt 

(FW) Conversion Formula Used1

CLR

(mg/100g)

Asparagine low 11.8 mmol/kg

Shepherd et al. 

2010 132.1

mmol/kg x mol/103mmol x FW x 

1000mg/g x 0.1kg/100g x 0.2 31.2

Asparagine high 3490.0 mg/100g Davies 1977 132.1 mg/100g x 0.2 698.0

Aspartic acid low 32.0 mg/100g Davies 1977 133.1 mg/100g x 0.2 6.4

Aspartic acid high 376.0 mg/100g Davies 1977 133.1 mg/100g x 0.2 75.2

Glutamine low 220.0 mg/100g Davies 1977 146.1 mg/100g x 0.2 44.0

Glutamine high22968.0 mg/100g Davies 1977 146.1 mg/100g x 0.2 539.6

Glutamic acid low 225.0 mg/100g Davies 1977 147.1 mg/100g x 0.2 45.0

Glutamic acid high 371.0 mg/100g Davies 1977 147.1 mg/100g x 0.2 74.2
J.R. Simplot Company JRS06 Page 149 of 196

Table 10-10. Late Blight Study Information

Year Study Site 

State

Site 

County

Material Tested Trial Design1

2012 Late Blight-Tubers WA Grant W8, Control RCB, 3 Reps

2013 Late Blight-Foliar MI Ionia W8, Control, TX2782

, Bintje2, 

Nicolet2, Golden Sunburst2 RCB, 4 Reps

2013 Late Blight-Foliar PA Centre W8, Control, TX2782

, Bintje2, 

Nicolet2, Golden Sunburst2 RCB, 4 Reps

2013 Late Blight-Foliar ID Boundary W8, Control, TX2782

, Bintje2, 

Nicolet2, Golden Sunburst2 RCB, 4 Reps

1

RCB=Randomized Complete Block design. Number of blocks was equal to the number of reps.

2

Reference varieties used as a part of the conventional variety range calculation.

Plot Inoculation and Foliar Infection Rating

Susceptible spreader rows were inoculated with P. infestans after row closure. Plots were inoculated 

with zoospores at a concentration of 8-12x104 sporangia/mL. Details on inoculation at each site are 

listed in Table 10-11. After inoculation, plots were irrigated regularly to induce a humid environment 

and facilitate spread of disease (Personal Communication with Joe Coombs, Michigan State University). 

Table 10-11. Plot Inoculation Information for Late Blight

Site Code Inoculation Date Genotype of Late Blight Inoculum

Iona County, MI 7/26/2013 US-22 and US-23

Centre County, PA 8/8/2013 US-23

Boundary County, ID 8/1/2013 US-8

Foliar evaluations were carried out by agronomists and scientists with experience in evaluation of late 

blight infection in the field. Foliar infection of plots was analyzed approximately every week after the 

first symptoms of infection by estimating the percentage of foliar area affected. Observations were 

made until plant senescence conditions progressed enough to possibly interfere with disease ratings. 

The last foliar rating at each site is analyzed in this report to determine the effect of the potato late 

blight resistance gene on foliar late blight resistance in W8 (Personal Communication with Joe Coombs, 

Michigan State University). 
J.R. Simplot Company JRS06 Page 150 of 196

Statistical Methods for Foliar Late Blight Test

The statistical analysis was performed by using SAS 9.3. 

Data were analyzed using the following linear mixed model:

Yijkl = i + j + k(j) + ( )l + ijkl

•  = mean of treatment (fixed)

•  = effect of site (random)

•  = rep[site] (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the effect of the jth site 

(random effect), k(j) denotes the random rep effect (within site), ( )ik denotes the interaction between 

the i

th treatment and random kth site effect, and ijkl denotes the residual random error.

A significant difference was established with a p-value < 0.05. Some departures from the assumptions of 

normality and equal variances were allowed since the results were always interpreted in the context of 

variation observed in the conventional varieties.

10.3.4 Tuber Late Blight Resistance Test Methods

Tuber Late Blight Inoculation Methods

In order to analyze tuber late blight resistance, samples of the event and control were obtained by 

randomly selecting 15 mid-sized tubers from each field replicate from the 2012 field site in Grant 

County, WA (described in Table 11-2). Four replicates of five tubers for each event and control were 

tested with P. Infestans genotypes US-22, US-8, and a negative control (water) for a total of 60 tubers of 

each event and control being tested for disease resistance. Tuber late blight development was evaluated 

using whole tuber sub–peridermal inoculation. All tubers were washed in distilled water to remove soil. 

The tubers were then surface-sterilized by soaking in 2% sodium hypochlorite solution for 4 hours. 

Tubers were dried in a controlled environment with forced air ventilation at 5950 l min1 at 15oC in dry air 

(30% relative humidity) for four hours prior to inoculation. To inoculate the tubers, a single plug (4 mm 

diameter) was removed from the surface of the test tuber at the apical end about 1 cm from the 

dominant sprout to a maximum depth of 1 cm and a plug of mycelium plus agar about 5 x 4 mm (length 

x diameter) was placed into the cavity and the tuber tissue plug replaced. The wound was sealed with 

petroleum jelly. Each sub-peridermal inoculation contained about 2 x 10-5sporangia per plug. Negative 

control tubers were inoculated with cold (4oC) sterile distilled H2O. The non-inoculated control tubers 

were inoculated with an agar plug. After inoculation, tubers were placed in the dark in sterilized covered 

plastic crates and returned to controlled environment chambers [Percival Incubator (Model I-36LLVL, 

Geneva Scientific, LLC, PO Box 408, Fontana, WI)]. The chambers were set at 10oC and 95% humidity and 

the sample tubers were incubated for 40 days until evaluation (Personal Communication with Dr. Willie 

Kirk, Michigan State University).

Tuber Late Blight Infection Rating 

Tubers were evaluated visually for percent late blight disease infection. Twenty tubers were cut 

longitudinally from apical to basal ends and the amount of internal and external late blight disease was 
J.R. Simplot Company JRS06 Page 151 of 196

assessed on a 0-100% percent scale for each tuber (Personal Communication with Dr. Willie Kirk, 

Michigan State University). 

Statistical Methods for Tuber Late Blight Test

Data were analyzed using the following linear mixed model:

Yij = i + j + ij

•  = mean of treatment (fixed)

•  = rep (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the random rep effect, and ij

denotes the residual random error. A significant difference was established with a p-value < 0.05. 

10.3.5 Enzymatic Assays for PPO and Invertase

PPO Assay

Tubers were ground under liquid N2 to a fine power and stored at -80 °C. Protein was extracted by 

vortexing ~100 mg of powdered material in Tricine buffer (pH 7.5) followed by centrifugation @ 21,100 x 

g. The protein extracts from Burbank control lines were diluted 1:10 to maintain activity within a linear 

range. L-DOPA conversion to dopachrome was monitored spectrophotometrically by measuring A474 nm

at 8 sec intervals over a 10 min time course. The ΔA474nm·min-1 was converted to moles·min-1using ε = 

3700 M-1·cm-1for L-DOPA (Behbahani et al. 1993). Rates were determined by non-linear regression 

fitting to a single exponential using Sigma Plot 8.02 (SPSS Science, Inc.). All experiments were done with 

three biological replicates. L-DOPA conversion to dopachrome was monitored over time by measuring 

A474nm. ΔA474nm·min-1then converted to units of μmol·min-1·mg dw tuber-1. 

Invertase Activity Assay

One gram of tuber samples was ground in liquid nitrogen to a fine powder. Approximately 500 mg of 

sample was transferred to a tube containing 1 ml of extraction buffer (50 mM HEPES-KOH, pH 7.5, 5 mM 

MgCl2·6H2O, 1 mM EDTA, 1 mM EGTA, 0.1% (w/v) Triton X-100, 10% (w/v) glycerol, 5 mM dithiothreitol 

, and 1 mM phenylmethlsulfonyl fluoride). Crude extracts were centrifuged at 15,000 rpm for five 

minutes and 1 ml of supernatant was desalted using PD MidiTrap G-25 columns (GE Healthcare) and 

eluted in 1.5 mls of extraction buffer. The extracts were shaken for 90 min rapidly enough to generate 

foaming to minimize the activity of invertase inhibitors (Brummell et al. 2011). 20 l of protein extract 

was added to a tube containing 60 ul of reaction buffer (133 mM Suc and 26.7 mM Na-acetate, pH 4.7) 

and incubated at 300C for 60 minutes (Bhaskar et al. 2010). Reactions were stopped by the addition of 8 

l of 1M Na-phosphate, pH 7.4 and heating samples at 970C for 3 minutes. Controls were first 

inactivated by heating samples at 970C for 3 minutes followed by the addition of 8 l of 1M Naphosphate, pH 7.4 and 60 l of reaction buffer. Glucose formed was measured using the MyQubit 

Amplex Red Glucose Assay (Life Technologies) following the manufactures instructions. Activity was 

expressed as nmol glu min-1 mg-1protein.
J.R. Simplot Company JRS06 Page 152 of 196

10.4 References

Barkholt, V., and Jensen, A.L. (1989). Amino acid analysis: determination of cystine plus half-cystine in 

proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Analytical Biochem.

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Dopaquinone and Dopachrome Assays Using Diode-Array Spectrophotometry: Dopachrome Formation 

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Jiang, J. (2010). Suppression of the Vacular Invertase Gene Prevents Cold-Induced Sweeting in Potato. 

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(2011). Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced 

sweeting resistance and includes spliced hybrid mRNA variants. Journal of Experimental Botany. 62: 

3519-3534.

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Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).

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carbohydrates” using high-performance anion exchange chromatography. J of AOAC International. 88: 

714-719.

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raw material for processing and nutrition. Elsevier Applied Science, London, pp 11-128.

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J.R. Simplot Company JRS06 Page 154 of 196

11.0 Agronomic Evaluation

11.1 Agronomic Performance

The purpose of the agronomic trials was to confirm that Russet Burbank Event W8 has an equivalent 

phenotype compared to the control Russet Burbank, when grown at multiple locations representing the 

major areas for potato production in the US, including Russet Burbank. Observations throughout the 

growing season allowed for a thorough assessment of (1) growth, (2) disease and pest susceptibility, and 

(3) tuber yield and quality. The field assessments confirmed that W8 has the intentionally incorporated 

new traits and maintains all the benefits of the conventional Russet Burbank parent variety. These 

assessments also made it possible to ensure that addition of the DNA insert did not result in unintended 

effects associated with weediness or pest-like characteristics. Results of the studies are presented first 

followed by detailed descriptions of the test methods.

11.1.1 Typical Agronomic Practices

The agronomic practices and pest control measures used were location-specific and were typical for 

potato cultivation. They were recommended by both regional potato extension specialists and 

agronomists and they related to all aspects of soil preparation, fertilizer application, irrigation, and 

pesticide-based control methods. An example of typical inputs for Russet Burbank potato production is 

given in Table 11-1. W8 and untransformed control received identical inputs and treatments within each 

site. The trial sites selected for the agronomic evaluations were different agricultural zones and 

represented the main production areas for potatoes in the US, including Russet Burbank.

Table 11-1. Example of Agronomic Inputs for Russet Burbank Potatoes

Planting Date April 1 to May 10

Planting Rate 15,000 - 18,000 seed pc or 17 – 23 cwt/A

Row Spacing 34-36” between rows 

Seed Spacing 10-12” within row

Fertilizer For 600 cwt/A yields and optimum soil test levels: 250 lb N-100 lb P2O5- 330 lb K2O/ 

 acre

Yield/Plant 2-4 lb

Yield/Acre 400-700 cwt/A

Harvest Date September 1 to October 15

11.1.2 Field Trial Locations

During 2012 and 2013, plants of W8 and the Russet Burbank control were grown at several locations 

representing the major production areas for potatoes in the US including Russet Burbank. Specific 

details regarding the field trial sites are summarized in Table 11-2. All field trials were conducted in 

accordance with applicable USDA APHIS notification requirements at 7 CFR 340.3.
J.R. Simplot Company JRS06 Page 155 of 196

Table 11-2. Field Trial Locations

Year

USDA 

Notification 

#

Site 

State

Site 

County

Material 

Tested

Trial 

Design1

Rows x

Planted 

Tubers/ 

Rep

Seed 

Type

Sticky 

Traps 

Deployed

Regional Specifics

2012 12-066-

102n ID Canyon W8, Control RCB,3 reps 4x20 Minitubers Y

Typical for Southwest Idaho, an important 

potato-growing region in the Northwest that 

produces about 120 million cwt/year, mainly for 

the french fry industry. Careful management is 

needed to limit or prevent heat-associated 

agronomic issues.

2012 12-121-

101n ID Minidoka W8, Control RCB,3 reps 4x20 Minitubers N

2013 13-079-

106n ID Canyon

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 2x20

NFT 

MinitubersY

2013 13-072-

112n ID Minidoka

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 6x20

NFT 

Minitubers

Y

Typical for Southern Idaho, an important potatogrowing region that produces about 120 million 

cwt/year, mainly for the french fry industry, with 

harvests in Fall. The growing season is relatively 

short because of cooler temperatures.

2012 12-066-

102n WA Grant W8, Control RCB,3 reps 4x20 Minitubers N Typical for Washington, a state in the Northwest 

that produces about 85 million cwt/year, mainly 

for the french fry industry, with harvests in Fall. 

Ideal growing conditions give rise to very high 

2013 yields per acre.

13-072-

112n WA Grant

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 6x20

NFT 

MinitubersY

2013 13-072-

112n ID Jerome

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 2x20

NFT 

MinitubersY

Typical for Southern Idaho, an important potatogrowing region that produces about 120 million 

cwt/year, mainly for the french fry industry, with 

harvests in Fall. The growing season is relatively 

short because of cooler temperatures.

2013 13-072-

112n WA Adams

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 2x20

NFT 

MinitubersN

Typical for Washington, a state in the Northwest 

that produces about 85 million cwt/year, mainly 

for the french fry industry, with harvests in Fall. 

Ideal growing conditions give rise to very high 

yields per acre.

1

RCB=Randomized Complete Block design
J.R. Simplot Company JRS06 Page 156 of 196

Table 11-2 continued. Field Trial Locations

Year

USDA 

Notification 

#

Site 

State

Site 

County

Material 

Tested1

Trial 

Design1

Rows x 

Planted 

Tubers/ 

Rep

Seed 

Type

Sticky 

Traps 

Deployed

Regional Specifics

2013 13-079-

107n ND Grand Forks

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 6x20

NFT 

MinitubersY

Typical for North Dakota, a Midwest state that 

produces about 20 million cwt/year, mainly for 

the french fry industry, with harvests in Fall. The 

Red River Valley is the 3rd largest potato growing 

region in the US.

2013 13-072-

112n MN Sherburne

W8, Control, 

Nicolet, Bintje, 

TX278, Golden 

Sunburst

RCB, 4 reps 2x20

NFT 

MinitubersN

Typical for Minnesota, a Midwest state that 

produces about 20 million cwt/year for the 

french fry and chip industry, with harvests in the 

Fall. Sherburne county produces about 35-40% of 

the potatoes grown in Minnesota.

2013 13-079-

102n WI Adams

W8, Control, 

Nicolet, Bintje, 

Red Thumb, 

Golden 

Sunburst

RCB, 4 reps 2x20

NFT 

MinitubersN

Typical for Wisconsin, a Midwest state that 

produces about 20 million cwt/year, for both the 

chip and fresh potato industry, with harvests in 

Fall. Large areas are dominated by muck soils.

1

RCB=Randomized Complete Block design

11.1.3 Field Performance and Tuber Assessment Results

Summaries of evaluations of agronomic characteristics, yield and grading characteristics, and ecological 

interactions of W8 and controls grown in 2012 and 2013 are shown in Tables 11-3, 11-4, and 11-5.

Overall, the results confirm that there are no major differences between W8 and the control with 

respect to these characteristics.

Agronomic Characteristics

The agronomic characteristics for W8 and the control are shown in Table 11-3. No statistically significant 

differences were detected between W8 and the control for final emergence, stems per plant, or vine 

desiccation. For early emergence, W8 was lower than the control (39.5 vs 61.5 %), for plant vigor, W8

was less vigorous than the control (3.0 vs 3.7), and for plant height, W8 was shorter than the control 

(40.4 vs 45.1 cm). All of the values for W8 for which differences were detected were within the CVR.

Although W8 showed delayed emergence, lower vigor, and was shorter than the control, these 

characteristics do not indicate increased plant pest potential in W8 compared to the control.
J.R. Simplot Company JRS06 Page 157 of 196

Table 11-3. Agronomic Characteristics

1

P-values in bold and underlined indicate statistically significant differences.

2

SD = standard deviation.

3

90% CI = 90% confidence interval.

4

CVR = Conventional variety range. The range of mean values of conventional varieties.

Yield and Grading Results

The yield and grading characteristics of W8 and the control are shown in Table 11-4. There were no 

statistically significant differences detected for total yield, US#2 yield, tubers per plant, tubers <4 oz, 

tubers 4-6 oz, tubers 6-10 oz, tubers >14 oz, specific gravity, fry 1, fry 3, fry 4, and total internal defects. 

Compared to the control, W8 had fewer tubers in the 10-14 oz group (14.0 vs. 19.0 %), fewer fry strips 

with high sugar (1.4 vs. 11.0 %), fewer strips with sugar ends (3.3 vs. 19.7 %), and more strips with light 

color defined as fry 0 (94.9 vs. 76.5%), and fewer scored as fry 2 (1.7 vs. 9.9%). All of the values for W8

for which differences were detected were within the CVR.

W8 had fewer large tubers than the control, which could be associated with lower yield, however yield 

was not significantly different. Other characteristics, such as fewer sugar ends and high sugar fries and 

more fries with lighter fry color are positive indicators of potato and fry quality and are an expected 

result of invertase silencing. None of these differences in size or quality would indicate increased plant 

pest potential.

Characteristic Variety N Mean P-Value1SD290% CI3CVR4

Early Emergence 

(%)

Control 41 61.5 . 14.8 57.1 64.9

0.0 93.1

W8 40 39.5 0.0001 23.3 31.8 44.3

Final Emergence 

(%)

Control 41 87.1 . 10.7 84.4 90.0

10.6 98.1

W8 40 80.3 0.2060 20.2 74.4 85.1

Stems Per Plant (#)

Control 39 1.7 . 0.7 1.4 1.8

1.0 3.1

W8 36 1.7 0.8868 0.7 1.4 1.8

Plant Vigor (1-5 

Scale)

Control 37 3.7 . 0.9 3.4 3.9

2.0 5.0

W8 35 3.0 0.0065 0.8 2.8 3.2

Plant Height (cm) Control 41 45.1 . 14.0 42.7 50.0

31.8 71.6

W8 38 40.4 0.0098 12.5 38.0 44.9

Vine Desiccation 

(%)

Control 37 44.0 . 29.3 37.0 53.3

3.8 100.0

W8 35 35.5 0.1471 30.7 29.4 46.9
J.R. Simplot Company JRS06 Page 158 of 196

Table 11-4. Yield and Grading Characteristics

Characteristic Variety N Mean P-Value1SD290% CI3CVR4

Total Yield 

(cwt/a)

Control 41 445.7 . 149.7 397.1 475.9

135.6 733.2

W8 38 417.9 0.4080 165.4 360.8 451.3

US#2 Yield 

(cwt/a)

Control 41 375.7 . 147.5 328.8 406.3

118.9 653.7

W8 38 312.2 0.0506 144.1 264.2 343.1

Tubers Per 

Plant (#)

Control 41 7.8 . 2.5 7.0 8.3

2.5 19.5

W8 38 8.4 0.4657 3.2 7.2 8.9

Tubers <4 oz 

(%)

Control 9 8.2 . 5.3 4.9 11.5

. .

W8 9 15.9 0.2434 6.9 11.7 20.2

Tubers 4-6 

oz (%)

Control 41 18.5 . 8.6 16.5 21.0

4.6 41.1

W8 41 20.5 0.3148 7.1 18.8 22.6

Tubers 6-10 

oz (%)

Control 41 31.1 . 8.7 28.9 33.5

15.3 41.2

W8 41 28.6 0.3435 8.2 26.1 30.5

Tubers 10-14 

oz (%)

Control 41 19.0 . 8.4 16.7 21.1

1.0 26.8

W8 41 14.0 0.0071 8.1 11.8 16.0

Tubers >14 

oz (%)

Control 41 14.3 . 16.9 9.3 18.2

0.0 45.5

W8 41 8.6 0.0554 11.0 5.8 11.5

Specific 

Gravity

Control 41 1.077 . 0.0 1.1 1.1

0.7 1.2

W8 41 1.073 0.8058 0.0 1.1 1.1

High Sugar

(%)

Control 41 11.0 . 16.9 6.0 14.9

0.0 84.8

W8 41 1.4 0.0337 7.3 0 3.4

Sugar Ends

(%)

Control 41 19.7 . 18.5 14.7 24.4

0.0 52.6

W8 41 3.3 <.0001 6.5 1.2 4.6

Fry 0

Control 32 76.5 . 30.9 67.2 85.7

0.0 100.0

W8 32 94.9 0.0272 19.4 89.1 100.0

Fry 1

Control 32 2.9 . 10.5 0.0 6.0

0.0 29.7

W8 32 1.5 0.6142 6.2 0.0 3.4

Fry 2 Control 32 9.9 . 18.9 4.3 15.6

0.0 28.4

W8 32 1.7 0.0173 7.3 0.0 3.9

Fry 3

Control 32 1.9 . 3.7 0.8 3.0

0.0 23.1

W8 32 0.8 0.4899 3.4 0.0 1.8

Fry 4

Control 32 6.3 . 14.8 1.8 10.7

0.0 79.7

W8 32 1.0 0.2316 5.9 0.0 2.8

Total 

Internal 

Defects (%)

Control 41 1.6 . 2.5 0.8 2.1

0.0 15.5

W8 41 1.2 0.7205 2.2 0.6 1.8

1

P-values in bold and underlined indicate statistically significant differences.

2

SD = standard deviation.

3

90% CI = 90% confidence interval.

4

CVR = Conventional variety range. The range of mean values of conventional varieties.
J.R. Simplot Company JRS06 Page 159 of 196

Insect, Disease, and Abiotic Stressor Assessments

The insect, disease, and abiotic stressor evaluations for W8 and the control are shown in Table 11-5.

Insect, disease, and abiotic stressors were chosen by the principal investigator at each site and varied 

from site to site based on which stressors were present or expected to be present. Stressors were rated 

at early season, midseason, and late season on a 0 to 3 scale, where 0 = stressor was not present, 1 =

symptoms were observed, 2 = moderate symptoms were present, and, 3 = severe symptoms were 

observed. Stressor evaluations were intended to be categorical and were not statistically analyzed. The 

range of ratings for W8 and the control were compared for each observation and an observed difference 

occurred when the range of W8 did not overlap with the range of the control. Three differences were 

noted between W8 and the control during 92 individual observations of seven insect stressors. Eight 

differences were noted between W8 and the control during 95 individual observations of disease 

stressors. Three differences between W8 and the control were observed during 69 observations of the 

eight abiotic stressors. Observed differences in stressor evaluations between W8 and the control are 

detailed below in Table 11-5. In two disease stressor observations out of twenty one, W8 had more late 

blight symptoms than controls. However, multiple replicated field trials with inoculated strains of late 

blight confirmed significant late blight resistance in W8 compared with the Russet Burbank control as 

tested by expert plant pathologists at Michigan State University (Chapter 10: Trait Efficacy). All 

differences in stressor evaluations between W8 and the control were within the CVR. Evaluation of 

insect, disease, and abiotic stressors in W8 compared to the control support a conclusion of no altered 

environmental interactions.

Table 11-5. Stressor Observations in W8 and the Control

Stressor

Total # 

Observations

# Observed

Differences1

Discussion

Insect

Aphid 30 0 -

Colorado Potato 

Beetle (CPB) 32 2

W8 was higher for 2 observations - 1 at 

Minidoka County, ID and 1 at Adams County, 

WI. CPB values in W8 were within the CVR.

Grasshopper 2 0 -

Leaf Hopper 8 0 -

Loopers (LP) 7 1

W8 was higher for 1 observation - Minidoka 

County, ID. LP values in W8 were within the 

CVR.

Psyllids 11 0 -

Stink Bugs 2 0 -

Insect Totals 92 3 -

Disease

Black Dot 2 0 -

Black Leg 5 0 -

Early Blight (EB) 29 4

W8 was lower in 2 observations, both in Grant 

County, WA. W8 was higher in 2 observations -1 

at Jerome County, ID and 1 at Adams County, 

WA. EB values in W8 were within the CVR.

Fusarium 1 0 -

Grey Mold 2 0 -
J.R. Simplot Company JRS06 Page 160 of 196

Table 11-5. continued. Stressor Observations in W8 and the Control

Stressor

Total # 

Observations

(Obs)

# Observed

Differences1

Discussion

Disease

Late Blight (LB) 21 2

W8 was higher in 2 observations – 1 at Jerome 

County, ID and 1 at Adams County, WI. LB 

values in W8 were within the CVR.

Leaf Roll Virus 3 0 -

Psyllids 2 0 -

Rhizoctonia 3 0 -

Sclerotinia 3 0 -

Stem Rot 3 0 -

Verticillium (VC) 8 2

VC in W8 was higher in 2 observations, 1 at 

Canyon County, ID and 1 at Grant County, WA. 

VC values in W8 were within the CVR.

White Mold 13 0 -

Disease Totals 95 8

Abiotic

Cold Stress 7 0 -

Drought 13 0 -

Excessive Moisture 5 0 -

Hail 2 0 -

Heat Stress (HS) 25 2

HS in W8 was higher in 2 observations, 1 at 

Canyon County, ID and 1 at Minidoka County, 

ID. HS values in W8 were within the CVR.

Nutrient Imbalance

(NI) 6 1

NI in W8 was higher during 1 observation at 

Adams County, WI. NI values in W8 were within 

the CVR.

Sun Scald 3 0 -

Wind Damage 8 0 -

Abiotic Stress Totals 69 3

1

Stressor evaluations were intended to be categorical and were not statistically analyzed. The range of ratings for each variety and its control 

were compared for each observation and an observed difference occurred when the range of the variety was outside the range of the control. 

-

= no differences observed.

Arthropod Abundance 

The results of the arthropod abundance monitoring are presented in Tables 11-6 and 11-7. No 

significant differences were observed between W8 and the control for any of the arthropods collected. 

Because there were no damsel bugs found in control samples, there is no p-value available for a 

comparison between damsel bug abundance in W8 and the control. The presence of 0.3 damsel bugs, 

on average in W8 compared to an average of 0.0 damsel bugs present in the control could be seen as a 

significant difference even though this comparison lacks a p-value. In both W8 and control, the presence 

of damsel bugs, one of the beneficial insects, was very low. The overall lack of differences in 15 of 16 

insect types observed supports a conclusion that the environmental interactions of Russet Burbank 

Event W8 potatoes were not altered as a result of the introduction of the biotechnology-derived traits 

compared to conventional potatoes. While these data do not show differences in overall arthropod 
J.R. Simplot Company JRS06 Page 161 of 196

fitness, the fact that there are few differences between Russet Burbank Event W8 and the control show 

that the insertions of the traits do not have an impact on abundance within the potato agro-ecosystem.

Table 11-6. Beneficial Arthropod Abundance in Field Trials 

Arthropod Variety N

Average

Abundance P-value1

SD290% Confidence Interval

Spiders Control 112 2.9 . 3.9 2.4 3.6

W8 112 3.3 0.4045 4.4 2.7 4.1

Big-eyed Bugs Control 15 0.6 . 1.0 0.2 1.1

W8 15 0.7 0.9142 1.4 0.1 1.4

Lacewings Control 85 2.4 . 3.3 2.0 3.2

W8 85 2.3 0.8100 3.8 1.8 3.2

Ladybird Beetles W8 132 10.0 . 16.9 9.3 14.2

Control 132 9.5 0.7447 17.5 8.7 13.7

Minute Pirate Bugs Control 95 4.1 . 10.2 3.1 6.5

W8 95 4.0 0.9116 8.1 3.3 6.1

Damsel Bug Control 4 . . 0.0 0.0 0.0

W8 4 0.3 NA 0.5 0.0 0.8

Syrphid Flies Control 70 1.6 . 3.8 1.2 2.7

W8 70 1.5 0.8152 2.7 1.3 2.4

Tachinid Flies Control 28 0.6 . 1.1 0.3 1.0

W8 28 0.9 0.4334 1.7 0.4 1.5

Trichogramma 

Wasps

Control 98 12.3 . 35.3 7.5 19.3

W8 98 11.0 0.7096 29.3 7.1 17.0

1

P-values in bold and underlined indicate statistically significant differences.

2

SD = standard deviation.

Table 11-7. Pest Arthropod Abundance in Field Trials

Arthropod Variety N

Average

Abundance P-value1

SD290% Confidence Interval

Aphids Control 132 54.6 . 68.5 38.5 58.2

W8 132 68.0 0.3213 96.2 47.3 75.0

Flea Beetles Control 57 3.1 . 10.4 2.0 6.6

W8 57 2.8 0.8565 9.8 1.9 6.3

Potato Leafhoppers Control 82 47.5 . 247.8 15.7 106.7

W8 82 52.6 0.8781 257.8 19.0 113.7

Psyllids Control 129 5.4 . 7.0 3.9 5.9

W8 129 6.6 0.3157 9.5 4.7 7.5

Potato Tuberworm Control 12 0.8 . 1.2 0.2 1.5

W8 12 0.5 0.6561 0.8 0.2 1.0

Click Beetle Control 4 0.0 . 0.0 0.0 0.0

W8 4 0.0 1.0000 0.0 0.0 0.0

Tarnished Plant 

Bugs

Control 94 2.6 . 4.6 2.5 4.1

W8 94 2.9 0.5133 4.3 2.9 4.4

1

P-values in bold and underlined indicate statistically significant differences.

2

SD = standard deviation.
J.R. Simplot Company JRS06 Page 162 of 196

11.2 Conclusions: Agronomic Characteristics

Results of these agronomic trials confirmed that W8 had an equivalent phenotype compared to the 

control when grown at multiple locations representing the major areas for potato production in the US. 

Observations throughout the growing season demonstrated equivalence of growth, disease and pest 

susceptibility, and tuber yield and quality. The field assessments confirmed that W8 has the intentionally 

incorporated new traits and maintains all the benefits of the conventional Russet Burbank parent 

variety. 

Phenotypes that could indicate enhanced weediness or plant pest potential are any characteristic where 

the test products were more vigorous than controls. Although there were significant agronomic 

differences, none of them would signal enhanced survival compared with control and, all fell within the 

conventional variety range. For example, at the time plants were observed for early emergence, fewer

W8 plants had emerged than the control. However, when final emergence data were collected, W8 was 

no different from the control. 

W8 had fewer large tubers than the control, which could be associated with lower yield, however yield 

was not significantly different. Other characteristics, such as fewer sugar ends and high sugar fries and 

more fries with lighter fry color are positive indicators of potato and fry quality and are an expected 

result of invertase silencing. None of these differences in size or quality would indicate increased plant 

pest potential.

Thus, we concluded that no biologically meaningful differences that would contribute to increased 

weediness or plant pest potential were observed in W8 for any of the agronomic characteristics, yield 

and grading characteristics, or ecological interactions such as plant-insect interactions, plant-disease 

interactions, and plant interactions with abiotic stressors. It can be concluded Russet Burbank Event W8 

is not different from its untransformed control in terms of agronomy, tuber yield and grade, and 

susceptibility to insects or diseases other than late blight. Therefore, W8 would not have enhanced 

weediness or plant pest potential.

11.3 Agronomic and Phenotypic Methods

This summary includes agronomic evaluations of W8 and the Russet Burbank control at geographically 

distinct sites that represent most of the main production areas for potatoes destined for french fry 

production in the US, including Russet Burbank. All agronomic trials were conducted under 

Biotechnology Quality Management System (BQMS) standards and USDA-APHIS compliance. The 

agronomic evaluations relate to both field observations and tuber assessments, both at harvest and 

during storage.

Varieties grown in field trials are summarized in Table 11-8. In 2012, test and control varieties were 

grown at all sites. In 2013, test, control, and reference varieties were grown at all sites. Reference 

varieties are commercially-available varieties that provide a range of values common to conventional 

potatoes.
J.R. Simplot Company JRS06 Page 163 of 196

Table 11-8. Varieties in Field Trials

Variety Type Trait/Genotype Seed Type1Seed Source

Varieties in 2012 Field Trials

Russet Burbank Control N/A

Greenhouse-grown Mini-Tubers Simplot Plant Sciences - Boise, ID

W8 Test pSIM1278+pSIM1678

Varieties in 2013 Field Trials

Russet Burbank Control N/A

NFT Mini-tubers CSS Farms - Colorado City, CO

W8 Test pSIM1278+pSIM1678

Golden Sunburst Reference N/A

Bintje Reference N/A

Nicolet Reference N/A

TX2782Reference N/A

Red Thumb2Reference N/A

1

Greenhouse-grown mini-tubers were grown from tissue culture plantlets in the Simplot Plant Sciences greenhouse. NFT mini-tubers were 

produced at CSS Farms in Colorado City, CO, using nutrient film technique.

2

At Adams County, WI, Red Thumb was used in place of TX278.

11.3.1 Agronomic Trial Experimental Design 

The experiments were established in a randomized complete block design (RCBD). The RCBD is typical 

for the evaluation of new potato varieties and events. In 2012, each plot consisted of four rows 

approximately 20 feet long, each planted with 20 mini-tubers, 12” apart. There were three replicates at 

each site. The mini-tubers were either mechanically or hand planted to a depth of 2-3 inches. In 2013, 

three sites had plots that consisted of six rows and five sites had plots that consisted of two rows. Each 

row was approximately 20 feet long, planted either mechanically or by hand with 20 mini-tubers, 12” 

apart and to a depth of 2-3 inches, as in 2012. There were four replicates at each site in 2013.

Agronomic Trial Seed

For the 2012 field trials, greenhouse grown mini-tubers for W8 and the control were planted. Seed for 

the field trials was grown in Simplot Plant Sciences greenhouse. Seed piece size varied and to minimize 

within site variability, the largest seed pieces were planted at Canyon County, ID, medium sized pieces 

were planted at Minidoka County, ID, and the smallest pieces were planted in Grant County, WA. 

For the 2013 field trial evaluations, minitubers were produced using nutrient film technology (NFT) minitubers for W8 and the control. This seed was grown at CSS Farms in Colorado City, Colorado. Plants 

from tissue culture were used to grow mini-tubers using NFT, which refers to seed that is hydroponically 

propagated using water enriched with dissolved nutrients. In this system, a large number of small seed 

can be produced because multiple seed harvests from each plant are possible.

11.3.2 Phenotypic Assessments 

The phenotypic characteristics evaluated are listed in Table 11-9.
J.R. Simplot Company JRS06 Page 164 of 196

Table 11-9. Characteristics Evaluated

Characteristic Evaluation 

Timing1 Description Units/Scale

Early Emergence Early season Count of emerged plants Percent based on number of seed pieces planted

Final Emergence Early season Count of emerged plants Percent based on number of seed pieces planted

Insect Stressors Early, mid, & 

late season

Visual estimate of damage 0 to 3 scale: 0=not present, 1=slight, 2=moderate, 

3=severe

Disease 

Stressors

Early, mid, & 

late season

Visual estimate of damage 0 to 3 scale: 0=not present, 1=slight, 2=moderate, 

3=severe

Abiotic Stressors Early, mid, & 

late season

Visual estimate of damage 0 to 3 scale: 0=not present, 1=slight, 2=moderate, 

3=severe

Stems Per Plant Early season Count of stems per plant Count

Plant Vigor Mid season Visual estimate of vigor 1 to 5 scale: 1=poor vigor, 3=average vigor, 

5=excellent vigor

Plant Height Mid season

Plant height of 10 non-systematically selected plants 

per plot measured from the soil surface to the top of 

the uppermost leaf

Average height (cm)

Vine Desiccation Late season Visual estimate of percent natural vine desiccation 

(prior to chemical or mechanical vine desiccation) Percent

Total Yield Yield Total yield including all tubers Hundredweight per acre (cwt/a)

US#2 Yield Yield

Yield of tubers meeting or exceeding the US#2 

standard (consists of potatoes or usable pieces which 

meet the following requirements: similar varietal 

characteristics, moderately firm, not seriously 

misshapen, free from freezing, disease, or insect 

damage, not less than 1 1/2 inches in diameter or 

usable pieces not less than 4 ounces in weight.)

Hundredweight per acre

(cwt/a)

Tubers Per Plant Yield Average count of tubers per plant from one entire row

per plot Count

Tubers <4 oz.2Grading Tubers weighing <4 oz. Percent by weight

Tubers 4-6 oz. Grading Tubers weighing 4-6 oz. Percent by weight

Tubers 6-10 oz. Grading Tubers weighing 6-10 oz. Percent by weight

Tubers 10-14 oz. Grading Tubers weighing 10-14 oz. Percent by weight

Tubers >14 oz. Grading Tubers weighing >14 oz. Percent by weight

Specific Gravity Grading Tuber sample weight in air/(weight in air - weight in 

water) Specific gravity ratio

High Sugar Grading Color rating of fried strips based on Munsell Color 

Chart for french fried potatoes

Percent of tubers with fry strips which, when 

compared with the Munsell Color Chart for french 

fried potatoes, has on the darkest side a 

predominate color of a number 3 or darker.

Sugar Ends Grading Color rating of the end of fried strips based on Munsell 

Color Chart for french fried potatoes

Percent of tubers with fry strips which has an end 

1⁄4 inch long or longer on the darkest side of the 

strip, for the full width of the strip, testing 

number 3 color or darker when compared with 

the Munsell Color Chart for french fried potatoes.

Fry 0, Fry 1, Fry 

2, Fry 3, Fry 4 Grading Color rating of fried strips based on Munsell Color Chart for french fried potatoes

Percent of tubers with fry strips which, when the 

predominate color of the darkest side is 

compared with the Munsell Color Chart for french 

fried potatoes, is determined to be a color 

reading of 0, 1, 2, 3, or 4.

Total Internal

Defects Grading

Tubers showing evidence of defects including hollow 

heart, vascular necrosis, brown center, internal 

discoloration, insect, internal brown spot, nematode, 

and other internal defects

Percent

1

Early season observations were made when emergence was complete (about 30-80 days after planting). Midseason observations were made 

during the early bloom stage (about 60-100 days after planting). Late season observations were made during the crop senescence stage (about 

100-140 days after planting).

2

Data on tubers <4oz. were obtained in 2012 only. In 2013, tubers <4 oz. were grouped with all unusable tubers because tubers <4 oz. are not 

taken into account when calculating US#2 yield.
J.R. Simplot Company JRS06 Page 165 of 196

Early emergence (at approximately 50% emergence) and final emergence (at complete emergence) 

were evaluated by determining the number of plants that emerged out of 20 mini-tubers planted in the 

middle two rows of each plot (in the case of two-row plots, “middle rows” refer to both rows). 

Emergence rates vary among conventional varieties and are dependent on both the physiological age of 

the “seed” and environmental factors such as temperature and moisture levels.

Stems per plant were evaluated by counting the number of stems of 10 plants in the middle rows of 

each plot.

At approximately mid-season, plant vigor was visually assessed in the middle rows of each plot using the 

following 1 to 5 comparative scale based on the principal investigators’ experience of the potato 

varieties being grown:

1 = less vigor – plants are less vigorous than the varietal average

2 = intermediate to 1 and 3

3 = normal vigor - plants are similar in vigor to the varietal average

4 = intermediate to 3 and 5

5 = more vigor - plants are more vigorous than the varietal average

At approximately mid-season, plant height was measured in centimeters from the soil surface at the top 

of the hill, to the top of the uppermost leaf of 10 non-systematically selected plants per plot from the 

middle rows in each plot.

At approximately late season, vine desiccation was assessed by visually estimating the percent of vines 

desiccated in the middle rows of each plot prior to chemical or mechanical vine desiccation.

11.3.3 Insect, Disease, and Abiotic Stressors 

Each plot was evaluated at approximately early season, mid-season, and late season for the presence of 

insects, diseases, and abiotic stressors using a 0 to 3 rating scale described in Table 11-9. Examples of 

common potato disease and insect symptoms can be found in Table 11-10. An observation is defined as 

looking for a specific insect, disease, or abiotic stressor during a point in time. Even if no stressors were 

present, zeroes were recorded because the stressors were looked for and comparisons can be made 

between the event and the conventional control.
J.R. Simplot Company JRS06 Page 166 of 196

Table 11-10. Common Potato Disease and Insect Symptoms1

Insect or Disease Agent Symptom

Emposasca fabae (Potato Leafhopper) Leaf feeding damage

Epitrix species (Flea Beetle) Shot-holes in leaves

Leptinotarsa decemlineata (Colorado Potato Beetle) Defoliation

Limonius californicus (Wireworm) Bored holes in tubers and shoots

Ostrinia nubilalis (European Corn Borer) Severe vine wilting above point of injury

Bactericera (Paratrioza) cockerelli (Potato Psyllid) Yellows

Phthorimaea operculella (Tuberworm) Foliar and tuber damage

Various aphid spp. Leaf suckling damage

Aster Yellows MLO Purple top disease

Potato Leafroll Virus Rolling of leaves and net necrosis

Potato Spindle Tuber Viroid Potato spindle tuber disease

Potato Virus A,M, X, Y Mosaic symptoms

Tobacco Rattle Virus Stem mottling

Erwinia carotovora Blackleg, aerial stem rot and tuber soft rot

Corynebactium sepedonicum Bacterial ring rot

Ralstonia solanacearum Brown rot

Phytophthora infestans Late blight

Phytophthora erythroseptica Pink rot

Verticillium spp. Early dying

Sclerotinia sclerotiorum Sclerotinia stalk rot

Rhizoctonia solani Causes cankers

Streptomyces scabies Scab

Fusarium spp. Dry rot

Pythium ultimum Water rot, shell rot, Pythium leak

Alternaria solani Early blight

Botrytis cinerea Gray mold

1

All stressors shown here were not necessarily observed at all sites or observation timings. This table is meant to give the reader an accurate list 

of insects and diseases that may impact potatoes.

Arthropod Abundance

At sites with arthropod collection, Pheromone-free sticky traps were deployed in every plot during the 

2012 and 2013 field seasons in order to monitor arthropod abundance. Sticky traps were deployed at 

canopy height, collected approximately every two weeks (every week in 2012) and shipped to the 

University of Arkansas for identification and enumeration. Arthropods were broken into 2 groups: 

beneficial which is defined as an arthropod that has characteristics that are beneficial to potatoes, such 

as those that consume pests; and pest which is defined as an arthropod that has characteristics that may 

be damaging to potatoes, such as vectoring diseases.

11.3.4 Tuber Grading and Yield

Tuber Grading

Tubers were harvested during early fall. At harvest, all tubers from one row of each plot were 

transported to the State of Idaho Department of Agriculture Grading Facility in Caldwell, ID. The grading 

methods employed were identical to those used to grade commercial potatoes intended for the 

production of fries.

Size profiles were determined by weighing tubers from the sample sorted by size. The size groups 

included tubers <4 oz (in 2012 only), tubers 4-6 oz, tubers 6-10 oz, tubers 10-14 oz, and tubers >14 oz. 

Potatoes greater than six-ounces produce optimal french fries and allow the french fry processor to 

meet most customer specifications at the highest potential recovery. 
J.R. Simplot Company JRS06 Page 167 of 196

The specific gravity was determined by using a weight in air/weight in water measurement. Sub-samples 

of tubers were first weighed in air and then weighed submerged under water at room temperature. 

From the two measurements, specific gravity was calculated using the following formula: specific 

gravity = weight in air/ (weight in air - weight in water). Specific gravity is the industry standard for 

measuring solids and is thus an important characteristic to compare the event to the conventional 

control.

High Sugar and Sugar Ends

Fried potato strips were prepared by cutting several (10-20) tubers into approximately 3/8 inch strips. 

The center strip was selected from each tuber and fried in cooking oil for three minutes at 375 F. The 

color of the fried strips was compared to a USDA Munsell color chart. High sugar is the percentage of 

tubers with fry strips which, when compared with the Munsell Color Chart for french fried potatoes, has 

on the darkest side a predominate color of a number 3 or darker. Sugar ends is the percentage of tubers 

with fry strips which has an end 1⁄4 inch long or longer on the darkest side of the strip for the full width of 

the strip, testing number 3 or darker color (USDA AMS 1969). Fry 0 – Fry 4 is the percent of tubers with 

fry strips which, when the predominate color of the darkest side is compared with the Munsell Color 

Chart for french fried potatoes is determined to be a color reading of 0-4, respectively. Therefore, the 

reported numbers are the percentage of Fries that score 0, 1, 2, 3, or 4 on the Munsell chart.

Yield and Defects

Total yield was determined by weighing each single row sample and US#2 yield was determined by 

subtracting the weight of undersize and unusable tubers. Tubers per plant was determined by counting 

the total number of tubers in each single-row sample and dividing by the total number of plants in the 

sample row. 

Tubers were checked for internal defects which included hollow heart, vascular necrosis, brown center, 

internal discoloration, insect, internal brown spot, nematode, and other internal defects. Total internal

defects represents the percentage of tubers affected by any internal defect.

11.3.5 Statistical Methods for Agronomic Studies

The statistical analysis for agronomic, grading, stressor, and arthropod abundance data was performed 

by Simplot using SAS 9.3. All data were analyzed using the following linear mixed model:

Yijkl = i + j + k(j) + ( )l + ijkl

•  = mean of treatment (fixed)

•  = effect of site (random)

•  = rep[site] (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the effect of the jth site 

(random effect), k(j) denotes the random rep effect (within site), ( )ik denotes the interaction between 

the i

th treatment and random kth site effect, and ijkl denotes the residual random error.
J.R. Simplot Company JRS06 Page 168 of 196

A significant difference was established with a p-value < 0.05. Every effort was made to generate pvalues to aid in the interpretation of the data. Some departures from the assumptions of normality and 

equal variances were allowed since the results were always interpreted in the context of variation 

observed in the conventional varieties.

Interpretation of the Results

For each comparison between W8 and the control, the following logic was employed to assess the 

results. First, when p-values were available, statistical significance was determined for each comparison. 

If the p-value indicated no statistical significance, it was unlikely that there was a difference that would 

be biologically meaningful or indicative of increased plant pest potential. Next, if the p-value indicated 

statistical significance or if a p-value was not present, the mean value of W8 was compared to the 

conventional variety range (CVR), which represents the minimum and maximum mean values of all 

conventional varieties included in the experiments. If the value for W8 was within the CVR, it was 

concluded that W8 exhibited characteristics within the natural variation of potatoes and that the 

difference was unlikely to be biologically meaningful or indicative of increased plant pest potential. If the 

value of W8 was outside the CVR, further consideration was given to the difference in the context of 

agronomic impact and increased plant pest potential. 

The CVR for the agronomic, yield, and grading characteristics used the range of mean values from one 

conventional control at three sites during 2012, four conventional reference varieties at eight sites 

during 2013, one conventional control at eight sites during 2013, and a second conventional control at 

five sites during 2013.

11.3.6 BQMS Quality Management Systems

All agronomic trials were conducted according to USDA Biotechnology Quality Management System 

(BQMS) standards and were USDA-APHIS compliant. Compliance with the applicable USDA requirements 

described in 7 CFR part 340 included: 

(1) Providing details on the introduced DNA in the submitted Design Protocol.

(2) Confirming that plants and tubers are free of Agrobacterium.

(3) Monitoring eight critical control points (CCPs) in the process of transporting, evaluating, 

harvesting, storing, and eliminating potatoes that are no longer needed. The associated 

documents were developed in collaboration with APHIS/BQMS and include: (1) Record of 

Transport indicating the USDA movement permit number, (2) Record of Storage and Inventory 

Change, (3) Record of Planting, (4) Record of Spatial Isolation, (5) Record of Harvest, (6) Record 

of Processing, (7) Record of Post-Harvest Monitoring, which commits to monitoring trials for the 

establishment of volunteers during the two years following an experiment, and (8) Record of 

Corrective Action. 

Both internal and external audits were conducted to ensure adherence to the guidelines of BQMS. The 

BQMS guidelines were followed to ensure that regulated plants or tubers were not lost or mixed with 

other plants or tubers during the conduct of the study. After the studies, all unused regulated material 

was devitilzed and discarded as prescribed in the J.R. Simplot Company Design Protocol which was 

approved by USDA APHIS. Internal audits were conducted by third party auditors that are approved by 

APHIS for the BQMS program. The external audits were conducted by USDA auditors. In addition, many 
J.R. Simplot Company JRS06 Page 169 of 196

of the regulated field trials referenced in this chapter were inspected by USDA inspectors and no 

compliance infractions were received.

11.3.7 Survival in Fields

Any potatoes that produce tubers, including all varieties such as Russet Burbank can persist in fields for 

several years after planting, including W8 and the Russet Burbank control. In most Northern areas, 

potatoes are rotated with other crops which minimizes the survival in subsequent years and the chance 

of accidental mixing in subsequent potato plantings. 

After conducting field trials, we routinely look for potatoes in the field the following year. We have not 

found any potatoes to persist in test plots containing W8 from 2012 trials after monitoring and 

destroying volunteers. Fields will be checked in 2014 for 2012 and 2013 field trials and in 2015 for 2013 

field trials, as potatoes have a 2-year volunteer monitoring cycle. These field trials consisted of small 

plots that are carefully monitored by expert researchers and represent a well-controlled environment. 

11.4 Disease Susceptibility Assessment

As part of the agronomic assessment, W8 was tested for evidence of trait efficacy and specificity. 

Resistance to late blight in W8 is conferred by the Potato Late Blight Resistance Gene (Rpi-vnt1) which 

expresses the VNT1 protein. This protein, with 891 amino acids, induces resistance to late blight with a 

broad spectrum against P. infestans isolates (Foster et al. 2009). Also, researchers have investigated the 

relationship between polyphenoloxidase (PPO) and disease resistance (Valentines et al. 2005; Li and 

Steffens 2002; Hakimi et al. 2006); with some proposing that enhanced PPO may increase resistance to 

disease, while others claim that reduced PPO could increase resistance. Considering that some evidence 

exists for a relationship between PPO and diseases and that the VNT1 protein is directly related to 

disease resistance, we chose to test W8 and the Russet Burbank control for trait specificity by testing for 

resistance to 3 common potato foliar diseases, one common potato stem and stolon disease, and 6 

common potato tuber diseases.

The purpose of these studies was to evaluate the susceptibility of W8 to common foliar, stem, stolon,

and tuber diseases in order to determine if W8 has altered disease susceptibility other than the 

intended increased resistance to late blight. This study addresses the environmental safety issues 

associated with the spread of plant diseases. All disease trials were conducted according to USDA 

Biotechnology Quality Management System (BQMS) standards and were USDA-APHIS compliant.

11.4.1 Disease Study Locations

Diseases that were evaluated affect a variety of tissues and studies were designed using the most 

relevant susceptible parts of the plant. Tests on foliage were conducted for Atlernaria alternata (early 

blight), Alternaria solani (brown leaf spot), and Botrytis cinerea (botrytis leaf spot). Stolons and stems, 

from field grown potato plants were tested for resistance to Rhizoctonia solani. Additionally, field grown 

tubers were tested for resistance to R solani (black scurf), Fusarium sambucinum (dry rot), Phytophthora

erythroseptica (pink rot), Pythium ultimum (pythium leak), Pectobacterium carotovora (soft rot), and

Streptomyces scabies (common scab). Details of each disease specificity trial are listed in Table 11-11.
J.R. Simplot Company JRS06 Page 170 of 196

Table 11-11. 2012 and 2013 Disease Study Details

1

RCB=Randomized Complete Block design

2

Reference varieties used as a part of the conventional variety range calculation.

Year Study USDA 

Notification #

Site 

State

Site 

County Material Tested Trial Design1

Rows x

Planted 

Tubers/ 

Rep

Seed 

Type Regional Specifics

2013

Early Blight, 

Brown Leaf 

Spot, 

Botrytis Leaf 

Spot

13-079-108n MI Ionia

W8, Burbank 

Control, TX2782, 

Bintje2, Nicolet2, 

Golden Sunburst2

RCB,

4 reps 2X5

NFT 

Minitubers

Typical for Michigan, a Midwest state 

that produces about 15 million cwt/year, 

for both the chip and fresh potato 

industry, with harvests in Fall. The 

climate is characterized by mild 

temperatures and ample rain.

2013 Rhizoctonia

solani 13-079-108n MI Ionia

W8, Burbank 

Control, TX2782, 

Bintje2, Nicolet2, 

Golden Sunburst2

RCB,

4 reps 1X10

NFT 

Minitubers

Typical for Michigan, a Midwest state 

that produces about 15 million cwt/year, 

for both the chip and fresh potato 

industry, with harvests in Fall. The 

climate is characterized by mild 

temperatures and ample rain.

2012

Dry Rot, Pink 

Rot, Pythium 

Leak, Soft 

Rot- Tubers

12-066-102n WA Grant W8, Burbank 

Control

RCB,

3 reps 4x20

Greenho

usegrown 

Minitubers

Typical for Washington, a state in the 

Northwest that produces about 85 

million cwt/year, mainly for the french fry 

industry, with harvests in Fall. Ideal 

growing conditions give rise to very high 

yields per acre.

2013 Common 

Scab 13-079-108n MI Montcalm

W8, Burbank 

Control, TX2782, 

Bintje2, Nicolet2, 

Golden Sunburst2

RCB,

4 reps 1X5

NFT 

Minitubers

Typical for Michigan, a Midwest state 

that produces about 15 million cwt/year, 

for both the chip and fresh potato 

industry, with harvests in Fall. The 

climate is characterized by mild 

temperatures and ample rain.
J.R. Simplot Company JRS06 Page 171 of 196

11.4.2 Disease Susceptibility Assessment Results

Symptoms of diseases caused by Atlernaria alternata, Alternaria solani, Botrytis cinerea, Rhizoctonia

solani, Fusarium sambucinum, Phytophthora erythroseptica, Pythium ultimum, and Pectobacterium 

carotovora were not significantly different between W8 and the control (Table 11-12). For all these 

diseases, we would expect a similar response in W8 as in the Russet Burbank controls.

Common scab caused by Streptomyces scabies was significantly different between W8 and the control 

for mean percent coverage of tubers and mean severity of lesion type. W8 had less scab than the 

control as measured by mean percent coverage and lesions were less severe than the control (Table 11-

12). Altered response to S. scabies was not expected in W8, but would be considered a positive finding 

if future experience confirms such resistance. Also, this unexpended result would not enhance the plant 

pest potential of W8.

Overall, the results of the disease studies show that W8 has similar response to potato diseases as 

Russet Burbank control, with the exception of increased resistance to P. infestans as shown in Chapter 

10:Trait Efficacy

11.5 Conclusions: Disease Susceptibility

Out of 3 foliar diseases, 1 stem and stolon disease, and 6 tuber diseases analyzed, W8 showed no 

difference in susceptibility compared to the control with the exception of a reduction in tuber coverage 

and severity of common scab caused by S. scabies. This unexpected difference in S. scabies

susceptibility will be monitored in future field and storage studies to determine whether this pattern 

persists in commercial production. Overall, these data support the claim that the Ppo5 silencing and the 

Rpi-vnt1 genes are not linked to altered disease susceptibility in foliage, stolons, stems, and tubers. The 

results provide evidence of no increased plant pest potential in W8 and also show specificity to the 

Potato Late Blight Resistance Gene, Rpi-vnt1. Thus, we concluded that no biologically meaningful 

differences that would contribute to increased weediness or plant pest potential were observed in W8 

for altered susceptibility or resistance to diseases other than late blight when compared to the Russet 

Burbank control. Therefore, W8 does not have enhanced weediness or plant pest potential.
J.R. Simplot Company JRS06 Page 172 of 196

Table 11-12. Disease Susceptibility Assessment Results

Pathogen Test and Data Variety Mean 

Value P-Value CVR1

Foliar Assessment

Early Blight and Brown Leaf Spot (Mean Percent 

Infection)

Control 7.5 .

0.5 17.5

W8 0.0 0.1316

Botrytis Leaf Spot (Mean Percent Infection) Control 5.00 .

3.75 5.00

W8 5.00 1

Stem Infection Assessment

Rhizoctonia solani (Mean Percent Infection) Control 27.5 .

0.0 80.0

W8 45.0 0.4114

Stolon Infection Assessment

Rhizoctonia solani (Mean Percent Infection) Control 37.5 .

20.7 57.5

W8 54.0 0.1499

Tuber Assessment

Rhizoctonia solani (Mean Severity of Infection) Control 0.20 .

0.2 0.9

W8 0.50 0.5254

Rhizoctonia solani (Mean Percent Incidence of

Infection)

Control 2.5 .

2.5 8.75

W8 5.0 0.5776

Tuber Dry Rot (Mean Percent Infection by Area) Control 15.0 .

. .

W8 18.6 0.3404

Pink Rot (Mean Percent Infection) Control 35.0 .

. .

W8 45.0 0.1639

Pythium Leak (Mean Percent Infection) Control 75.0 .

. .

W8 75.0 .

Soft Rot Infection (Mean Percent Weight) Control 57.4 .

. .

W8 47.9 0.1715

Common Scab (Mean Percent Coverage of 

Tuber)

Control 2.65 .

2.48 4.58

W8 1.90 0.034

Common Scab (Lesion Type, Table 11-13) Control 3.18 .

3.18 5.95

W8 1.35 <.0001

1

CVR = Conventional variety range. The range of mean values of conventional varieties.

Data in this table are from 2013 field trials in Montcalm County, MI.

P-values in bold and underlined indicate statistically significant differences.

. = Not applicable
J.R. Simplot Company JRS06 Page 173 of 196

11.6 Disease Susceptibility Methods

The following assessments were conducted to determine if the insertion of the late blight resistance 

trait altered disease susceptibility of other common potato diseases:

Foliar Alternaria alternata, Alternaria solani, and Botrytis cinerea

The trial was planted at the Michigan State University Horticultural Experimental Station in Clarksville, 

MI, as a RCBD consisting of four replicates. Each plot was five feet in length and consisted of two rows of 

five plants. Plots were irrigated as needed with sprinklers and were hilled immediately before sprays 

began. Potato late blight was prevented from movement into the plots from adjacent plots inoculated 

with P. infestans with weekly fungicide applications that prevent late blight infection but do not affect A. 

alternaria, A. solani, or B. cinerea. Plots were rated visually for percentage foliar area affected by A. 

alternaria, A. solani, and B. cinerea on August 30, September 5, and September 10, 2013. A. alternaria

and A. solani developed slowly during August and untreated controls reached about 15% foliar infection 

by September 10. B. cinerea developed slowly during August and untreated controls reached about 5% 

foliar infection by September 10 (Personal Communication with Dr. Willie Kirk, Michigan State 

University).

Rhizoctonia solani in Stems, Stolons, and Tubers

The trial was planted at the Michigan State University Horticultural Experimental Station in Clarksville, 

MI as a RCBD consisting of four replicates of ten plants per plot. Each plot was one row and ten feet in 

length. A five foot unplanted alley separated single-row beds form other trials. Four plants per plot were 

harvested 90 days after planting and the percentage of stems and stolons with greater than 5% of the 

total surface area affected by R. solani infection were counted. The rest of the plots were harvested on 

October 7, 2013, and potatoes were stored for 35 days in the dark at 50°F. After 35 days of storage, 20 

tubers from each plot were assessed for R. solani incidence (%) and severity. Severity of R. solani was 

measured as an index calculated by counting the number of tubers (n = 20) falling into each class 0 = 0; 1 

= 1 - 5; 2 = 6 - 10; 3 = 11 - 15; 4 >15% surface area of tuber covered with sclerotia. The number in each 

class was multiplied by the class number and summed. The sum was multiplied by a constant to express 

as a percentage. Indices of 0 - 25 represent 0 - 5%; 26 - 50 represent 6 - 10%; 51 - 75 represent 11 - 15% 

and 75 - 100 >15% surface area covered with sclerotia. R. solani symptoms were mild in this trial 

because this was the first year that potatoes had been planted at that site and the seed tubers were 

clear of black sclerotia at planting. Despite this, some symptoms developed on stems, stolons and tubers 

harvested and stored (Personal Communication with Dr. Willie Kirk, Michigan State University).

Fusarium sambucinum in Tubers

Samples of W8 and the control were obtained by randomly selecting mid-sized tubers across field 

replicates from the 2012 field site in Grant County, WA. Four replicates of five tubers each for W8 and 

the control were tested with F. sambucinum inoculum and a negative control (water). All tubers were 

washed in distilled water to remove soil. The tubers were surface-disinfected for 10 min in 0.5% sodium 

hypochlorite and rinsed twice in sterile water. The tubers were then surface-sterilized by soaking in 2% 

sodium hypochlorite solution for four hours. Tubers were dried in a controlled environment with forced 

air ventilation at 5950 L min-1 at 15oC in dry air (30% relative humidity) for four hours prior to 

inoculation. Tubers were injected with 20 μl of a conidial suspension (approximately 106conidia/ml as 

determined by a hemocytometer) of the Fusarium isolate grown on PDA for 7 days. Negative control 

tubers were each injected with 20 μl sterile distilled water. After inoculation, tubers were placed in the 

dark in sterilized covered plastic crates and returned to controlled environment chambers [Percival 

Incubator (Model I-36LLVL, Geneva Scientific, LLC, PO Box 408, Fontana, WI)]. The chambers were set at 
J.R. Simplot Company JRS06 Page 174 of 196

10oC and 95% humidity and the sample tubers were incubated for 30 days until evaluation (Personal 

Communication with Dr. Willie Kirk, Michigan State University). 

After 30 days of incubation, tubers were cut in half through the point of inoculation and evaluated for 

the development of symptoms typical of potato dry rot: dry, necrotic areas discolored light to dark 

chocolate brown on the tuber surface. Tubers were subdivided into 4 replications of 5 tubers and placed 

in a boxes arranged in a randomized complete block design. To assess the virulence level of the 

Fusarium isolates, images of the symptomatic areas on the inoculated tuber surfaces were generated 

from a method adapted from Niemira et al. described below. The freshly-cut tuber sections were placed 

on a piece of glass (30 cm X 40 cm X 2 mm) with the cut tuber surface facing down. A ruler was placed 

underneath the glass, and used for measurements during image analysis. The glass and ruler were 

transferred to a flatbed scanner (HP Scan-Jet 4c; Hewlett Packard Co., Houston, TX) for image 

processing. Scanner control software (DeskScan II Version 2.4; Hewlett Packard Co.) generated an image 

of the cut tuber surfaces against a black background. The image files created were first loaded into 

Adobe Photoshop CS3 (Version 10.01, 2007, Adobe Systems Incorporated, Pittsburg, PA) from which the 

lesions were selected on each image and “painted” white using the “fill” tool. The images were then 

loaded into SigmaScan Pro 5 Version 1987-1999, (SPSS Inc., Chicago, IL) to determine the area of the 

lesion. The lesion length and width (mm) were measured to calculate the dimension and area (mm2) of 

the lesions, and calibrated to convert image pixels to a unit of measurement using the ruler within the 

image. The measurement “fill’ was then adjusted to a threshold so that the lesion was a lighter color 

than the rest of the tuber surface. The area of the lesion was then measured according to the SigmaScan 

manufacturer’s protocol (Personal Communication with Dr. Willie Kirk, Michigan State University). 

Pythium erythroseptica and Pythium ultimum in Tubers

Samples of W8 and the control were obtained by randomly selecting 80 mid-sized tubers across field 

replicates from the 2012 field site in Grant County, WA. Four replicates of five tubers for W8 and the 

control were tested with P. erythroseptica, P. ultimum, and a water negative control. All tubers were 

washed in distilled H2O to remove soil. The tubers were each surface-disinfested for 10 min in 0.5% 

sodium hypochlorite and rinsed twice in sterile water. The tubers were then surface-sterilized by 

soaking in 2% sodium hypochlorite solution for 4 hours. Tubers were dried in a controlled environment 

with forced air ventilation at 5950 l min-1 at 15oC in dry air (30% relative humidity) for four hours prior to 

inoculation. Tubers were immersed in a water/oospore/sporangial suspension (approximately 104

propagules/ml) for 24 hours; control tubers were immersed in distilled water for a corresponding 

period. After inoculation, tubers were placed in the dark in sterilized covered plastic crates and returned 

to controlled environment chambers [Percival Incubator (Model I-36LLVL, Geneva Scientific, LLC, PO Box 

408, Fontana, WI)]. The chambers were set at 10oC and 95% humidity and the sample tubers were 

incubated for 30 days until evaluation (Personal Communication with Dr. Willie Kirk, Michigan State 

University). 

Tubers were cut longitudinally into four slices and evaluated for presence of symptoms and/or signs of 

the target pathogens. Tubers with symptoms or signs of the individual disease were counted and disease 

incidence determined as percentage of symptomatic tubers relative to the total number of tubers in 

each replicate (Personal Communication with Dr. Willie Kirk, Michigan State University). 

Pectobacterium carotovora in Tubers

Samples of W8 and the control were obtained by randomly selecting 40 mid-sized tubers across field 

replicates from the 2012 field site in Grant County, WA. Four replicates of five tubers for W8 and the 

control were tested with P. carotovora and a negative control (water). All tubers were washed in 
J.R. Simplot Company JRS06 Page 175 of 196

distilled H2O to remove soil. The tubers were then surface-sterilized by soaking in 2% sodium 

hypochlorite solution for 4 hours. Tubers were dried in a controlled environment with forced air 

ventilation at 5950 l min-1 at 15oC in dry air (30% relative humidity) for four hours prior to inoculation. 

The washed, surface-sterilized tubers were inoculated by a sub-peridermal injection of a bacterial 

suspension of 1x1010 ml (delivering about 1000 bacterial cells per inoculation) with a hypodermic syringe 

and needle at the apical end of the tuber about 1 cm from the dominant sprout to a maximum depth of 

1 cm. The control tubers were inoculated with cold sterile distilled water. The wound was sealed with 

paraffin wax. After inoculation, tubers were placed in the dark in sterilized covered plastic crates and 

returned to controlled environment chambers [Percival Incubator (Model I-36LLVL, Geneva Scientific, 

LLC, PO Box 408, Fontana, WI)]. The chambers were set at 10oC and 95% humidity and the sample tubers 

were incubated for 40 days until evaluation (Personal Communication with Dr. Willie Kirk, Michigan 

State University). 

Tubers were weighed prior to inoculation and the weight recorded on a spreadsheet and written on the 

tuber surface. The tubers were subdivided into four replications of five tubers and placed in a boxes 

arranged in a randomized complete block design. After incubation, the site of the inoculation was cut 

longitudinally and the rotted area removed with a stream of water. The remaining tubers tissue was 

weighed and the percentage tissue lost was calculated (Personal Communication with Dr. Willie Kirk, 

Michigan State University).

Streptomyces scabies in Tubers

A trial was planted in the Potato Common Scab Disease Nursery at Michigan State University. The field 

was inoculated with S. scabies from aggressive Michigan isolates and was cultivated for high disease 

pressure for six years. Potatoes are grown every year in this field and organic matter is added to 

promote disease development. The trial was planted as a randomized complete block design consisting 

of four replications of five plants per plot. S. scabies-susceptible potato varieties were used as markers 

between plots. Standard cultivation practices were used for field preparation, planting, and field 

maintenance under irrigation conditions. The plots were harvested with a one-row digger and laid on 

top of the soil for evaluation of disease severity. A pathology rating scale was used to evaluate S. 

scabies. Each tuber was rated separately for lesion type and severity of coverage. From each plot, 10-20 

tubers were scored based on the rating scale described in Table 11-13 (Personal Communication with 

Joe Coombs, Michigan State University).

Table 11-13. Scab Severity Rating Scale

Rating Lesion Type Percent Coverage (%)

0 No scab lesions 0

0.5 Brown-like lesions (small, star-shaped) 1

1 Superficial lesions, discrete. 1-2

2 Superficial lesions, coalescing 2.1-5

3 Raised lesions, discrete 5.1-10

4 Raised lesions, coalescing 10.1-25

5 Pitted lesions, discrete 25.1-50

6 Pitted lesions, coalescing >50
J.R. Simplot Company JRS06 Page 176 of 196

11.6.1 Statistical Methods for Disease Studies

The statistical analysis for disease studies was performed by Simplot using SAS 9.3. All data were 

analyzed using the following linear mixed model:

Yij = i + j + ij

•  = mean of treatment (fixed)

•  = rep (random)

•  = residual random error

Where i denotes the mean of the i

th treatment (fixed effect), j denotes the random rep effect, and ij

denotes the residual random error.

A significant difference was established with a p-value < 0.05. Some departures from the assumptions of 

normality and equal variances were allowed since the results were always interpreted in the context of 

variation observed in the conventional varieties.

Interpretation of the Results

For each comparison between W8 and the control, the following logic was employed to assess the 

results. First, when p-values were available, statistical significance was determined for each comparison. 

If the p-value indicated no statistical significance, it was unlikely that there was a difference that would 

be biologically meaningful or indicative of increased plant pest potential. Next, if the p-value indicated 

statistical significance or if a p-value was not present, the mean value of W8 was compared to the 

conventional variety range (CVR), which represents the minimum and maximum mean values of all 

conventional varieties included in the experiments. If the value for W8 was within the CVR, it was 

concluded that W8 exhibited characteristics within the natural variation of potatoes and that the 

difference was unlikely to be biologically meaningful or indicative of increased plant pest potential. If the 

value of W8 was outside the CVR, further consideration was given to the difference in the context of 

agronomic impact and increased plant pest potential. 

The CVR for the field disease specificity studies used the range of mean values from three conventional 

controls and four conventional reference varieties at one site per study during 2013.

11.7 Field Test Reports

The J.R. Simplot Company has adhered to USDA compliance regulations by submitting the field test 

reports for each notification (Table 11-14). 
J.R. Simplot Company JRS06 Page 177 of 196

Table 11-14. Field release notifications for agronomic and disease susceptibility studies

USDA 

Reference 

Number

Field Trial 

Year

Effective Start and Expiration 

Date

Release Sites (by 

State) Covered by 

Notification

Field Test Report 

Submitted 

(Yes or No)

12-066-102n 2012 3/28/2012 - 4/2/2013 ID, WA Yes

12-121-101n 2012 4/30/2012 – 4/30/2013 ID Yes

13-079-106n 2013 3/29/2013 – 4/8/2014 ID No

13-072-112n 2013 4/2/2013 – 4/2/2014 ID, WA, MN, PA No

13-079-107n 2013 4/10/2013 – 4/10/2014 ND, ID No

13-079-102n 2013 4/10/2013 -4/10/2014 WI No

11.8 Conclusion on Agronomic Performance, Yield and Grading, and Disease Susceptibility

The purpose of the agronomic trials was to confirm that Russet Burbank Event W8 has an equivalent 

phenotype compared to the Russet Burbank control, when grown at multiple locations representing the 

major areas for potato production in the US including Russet Burbank. 

Results of the trials confirmed that W8 had an equivalent phenotype compared to the control. 

Phenotypes that could indicate enhanced weediness or plant pest potential are any characteristic where 

the test products were more vigorous than controls. Although there were significant agronomic 

differences, none would signal enhanced survival compared with control. For example, at the time 

plants were observed for early emergence, the percent of W8 plants emerged was less than the control. 

However, when final emergence data were collected, W8 was no different from the control. 

W8 had fewer large tubers than the control, which could be associated with lower yield, however yield 

was not significantly different. Other characteristics, such as fewer sugar ends and high sugar fries and 

more fries with lighter fry color are positive indicators of potato and fry quality and are an expected 

result of invertase silencing. None of these differences in size or quality would indicate increased plant 

pest potential.

Out of 3 foliar diseases, 1 stem and stolon disease, and 6 tuber diseases analyzed, W8 showed no 

difference in susceptibility compared to the control with the exception of a reduction in tuber coverage 

and severity of common scab caused by S. scabies. If W8 has lower susceptibility to common scab, it 

would not likely be associated with increased plant pest potential. Overall, these data support the claim 

that the Ppo5 silencing and the Rpi-vnt1 genes are not linked to altered disease susceptibility in foliage, 

stolons, stems, and tubers. The results provide evidence of no increased plant pest potential in W8 and 

also show specificity to the Potato Late Blight Resistance Gene, Rpi-vnt1. 

Thus, no differences were observed that would contribute to increased weediness or plant pest 

potential in W8 for any of the agronomic characteristics, yield and grading characteristics, or ecological 

interactions such as plant-insect interactions, plant-disease interactions, and plant interactions with 

abiotic stressors. It can be concluded Russet Burbank Event W8 is no different from the control in terms 

of agronomy, tuber yield and grade, and susceptibility to insects or diseases other than late blight.
J.R. Simplot Company JRS06 Page 178 of 196

11.9 References

Foster, S.J., Park, T.H., Pel, M., Brigneti, G., Sliwka, J., Jagger, L., van der Vossen, E., and Jones, J.D.G. 

(2009). Rpi-vnt1. 1, a Tm-22 Homolog from Solanum Venturii, Confers Resistance to Potato Late Blight. 

Molecular Plant-Microbe Interactions 22. 589–600.

Hakimi, S.M., Krohn, B.M., and Stark, D.M.: Monsanto Technology LLC (2006) United States Patent: 

Method of imparting disease resistance to plants by reducing polyphenol oxidase activities. Patent No.: 

US 7,122,719 B2.

Li, L., and Steffens, J.C. (2002). Overexpression of polyphenol oxidase in transgenic tomato plants results 

in enhanced bacterial disease resistance. Planta. 215: 239-247.

Niemira, B.A., Kirk, W.W., and Stein, J.M. (1999). Screening for late blight susceptibility in potato tubers 

by digital analysis of cut tuber surfaces. Plant Dis. 83: 469-473.

USDA AMS. (1969). United States Department of Agriculture Agricultural Marketing Service. Grading 

Manual for Frozen French Fried Potatoes. 

http://www.ams.usda.gov/AMSv1.0/getfile?dDocName=STELPRD3106128 (Accessed 26 April 2013).

Valentines, M.C., Vilaplana, R., Torres, R., Usall, J., and Larrigaudiere, C. (2005). Specific roles of 

enzymatic browning and lignifications in apple disease resistance. Postharvest Biotech and Tech. 36: 

227-234.
J.R. Simplot Company JRS06 Page 179 of 196

12.0 Environmental Safety Assessment

The environmental safety of Russet Burbank W8 is supported by extensive testing including agronomic 

performance, trait efficacy and specificity, genetic characterization, compositional assessment, VNT1 

protein evaluation, and bioinformatics screening for toxins and allergens. Throughout these studies, the 

concept of familiarity of potato was considered. Familiarity has been widely used to describe the 

interactions that a plant may have in nature by considering the biology of the crop, the introduced trait, 

the receiving environment, and the interactions with all of these factors (Hokanson et al. 1999). This 

concept allows regulatory decision-makers to draw upon past experience with introduction of plants 

into the environment and compare genetically modified plants to their non-modified counterparts.

Information on W8 has been reviewed to determine the potential risk to the environment using the 

following five criteria: (1) potential to become a weed of agriculture or to be invasive of natural habitats, 

(2) potential for gene flow to sexually compatible plants, (3) potential to become a plant pest, (4) 

potential impact on non-target species including humans, and (5) potential impact on biodiversity.

12.1 Potential to become a weed of agriculture or to be invasive of natural habitats

Weediness is a term used to describe the ability of a plant to become a weed (survive and thrive) 

outside of cultivation. Multiple field trials with W8 did not provide any evidence for altered growth 

characteristics such as accelerated tuber sprouting, increased plant vigor, increased tuber set, delayed 

senescence, or other key agronomic characteristics associated with weediness or survival outside of 

cultivation. Additionally, all field sites were monitored for 2 years after harvest for volunteer activity as 

required by USDA-BRS compliance. Through two years of field studies and up-to-date volunteer 

monitoring of every site, no differences have been observed that would lead us to believe that W8 has 

properties that would increase the survivability compared to conventional potato.

Potato is a poor competitor and does not thrive in a non-cultivated environment (Love 1994). Due to 

modern agricultural practices, it is highly unlikely that potatoes would persist in a field from one crop 

cycle to the next, particularly since potatoes are typically grown as a rotational crop. Also, in the 

northern U.S. and Canada, most production areas experience deep frost penetration in the soil, 

minimizing the likelihood of over-winter survival (CFIA 1996). Results of the agronomic assessment

show no differences between W8 and its conventional control that would alter survivability, and thus it

is unlikely that W8 possesses increased potential to become a weed of agriculture or to be invasive of 

natural habitats.

12.2 Potential for gene flow to sexually compatible plants

Gene flow from Russet Burbank Event W8 is nonexistent because the variety is sterile. Generally, the 

potential for gene transfer in any potatoes through outcrossing within the species is minimal for several 

reasons: 1) a high percentage of fertile potatoes are self-pollinated and are not frequented by 

honeybees due to a lack of nectar; 2) pollen transfer between plants tends to be limited to about 20 

meters making transfer between commercial-scale fields unlikely; 3) it is unlikely that true potato seeds 

produced through outcrossing would grow into mature potatoes since potato seeds are not saved and 

propagated in a typical farming operation; and 4) potatoes are almost always clonally propagated using 

“seed potatoes”, thus removing the potential for further propagation of seed produced through 

outcrossing.
J.R. Simplot Company JRS06 Page 180 of 196

In the unlikely event that outcrossing was to occur between W8 and untransformed plants in the field, 

the impact would be negligible to future plantings since tubers rather than seed are removed from the 

field. If seedlings did arise from an outcrossing event, establishment would be nearly impossible since 

potatoes are grown in rotation and are poor competitors compared to other crop and weed species 

(Love 1994; CFIA 1996). If seedlings were to establish after harvest, they would be easily identified and 

eliminated as part of the standard agricultural practices in potato production. In either case, seedlings 

could be identified and eliminated to prevent them from entering the commercial stream.

The tetraploid species S. fendleri and diploid S. jamesii are the only wild species that grow within the 

borders of the United States (Bamberg et al. 2003; Spooner et al. 2004). These species mostly occur 

outside of the major potato growing regions in Texas, New Mexico, Utah, Colorado, and Arizona. These 

species reside in dry forests, scrub desert, and sandy areas at altitudes of 5,000 to 10,000 feet, well 

isolated from most commercial production areas (Bamberg and del Rio 2011). Therefore, pollen flow 

from commercial events to wild species is extremely unlikely simply because of the geographic location. 

In addition, the Atlas of Wild Potatoes, (Hijmans et al. 2012) makes the statement that no wild potato 

species occur in Canada.

Based on conversations with Bamberg, Love reported that no one has ever reported finding hybrids 

between native and cultivated potatoes in the U.S., although gene transfer has been accomplished using 

special laboratory techniques (Love 1994). Love concluded that, based on the barriers that exist, 

including geographic isolation as described above, endosperm imbalances, and multiple ploidy levels, 

natural hybridization is highly unlikely, and introgression by cross hybridization over multiple 

generations, is impossible or at least highly improbable (Love 1994). 

The US EPA has concluded that, based on its review of the scientific literature, successful gene 

introgression between native and cultivated potatoes in the U.S. is virtually excluded due to constraints 

of geographical isolation and other barriers to natural hybridization. These barriers include 

incompatible, meaning unequal, endosperm balance numbers that lead to endosperm failure and 

embryo abortion, multiple ploidy levels and incompatible mechanisms that do not express reciprocal 

genes to allow fertilization to proceed. No natural hybrids have been observed between these species 

and cultivated potatoes in the U.S. (US EPA 2011).

In conclusion, the Russet Burbank potato variety and W8 produce few flowers and are male sterile. 

Other factors limiting outcrossing include the tendency for most fertile varieties to be self-pollinated, an 

inability to attract honey bees because they lack nectar, a limited pollen transfer range of about 20 

meters, and the fact that true seeds would be unlikely to grow into mature potatoes since potato seeds 

are not saved and propagated in a typical farming operation. If potatoes were grown from true potato 

seed, the offspring would be so diverse that they would not be useful as commercial potatoes. In 

addition, potatoes are not known to escape from commercial fields or show weediness potential. Wild 

potato varieties are rare in North America and for the most part geographically isolated from 

commercial production areas, further reducing concerns about cross-pollination with wild species (CFIA 

1996). No biologically relevant differences were identified in W8 which could contribute to increased 

potential for gene flow.
J.R. Simplot Company JRS06 Page 181 of 196

12.3 Potential to become a plant pest

Data collected and reported in the agronomic performance section show that in multi-year and multisite field trials, no specific differences leading to increased weed or plant pest potential were present. 

Weediness and invasiveness are already considered above and the data on abiotic and biotic stressors 

included in the agronomic performance section indicate that there are no meaningful differences in 

susceptibility to pests and diseases common to potatoes except for the intended resistance to late 

blight. The agronomic data supplied in this submission show no evidence of an altered response to a 

wide range of pests and diseases. Thus, no increased potential to become a plant pest would likely result 

from cultivation of W8.

12.4 Potential impact on non-target species including humans

The modifications to Russet Burbank that resulted in W8 were intended to enhance both the late blight 

resistance and the quality of potatoes. In the agronomic performance section, the data show that W8 is 

effective against late blight but responds like Russet Burbank control to a panel of other diseases. Also, 

no differences were observed for insects or other arthropods interacting within the potato ecosystem 

indicating a lack of altered impact on non-target organisms during cultivation. The methodology used to 

develop W8 does not result in the expression of new polypeptides in the plants other than the VNT1 

protein responsible for late blight resistance. A bioinformatics assessment showed no new toxins or 

allergens that could affect humans or other non-target species. The compositional analysis did not 

identify any altered nutritional components that would be biologically significant and there were no 

changes in glycoalkaloids. Thus there is no indication that the impact on humans or other non-target 

organisms would be altered by cultivation of W8. 

In conclusion, no altered potential for a negative impact on non-target organisms, including humans, 

from the cultivation of Russet Burbank Event W8 is expected. In fact, the reduction of acrylamide levels 

in food products made with these potatoes could be beneficial to humans considering the potential 

negative health effects associated with ingestion of acrylamide (NTP 2012).

12.5 Potential impact on biodiversity

The modifications to Russet Burbank resulting in W8 were intended to enhance late blight resistance 

and quality, not agronomic characteristics, of potatoes. Planting, cultivation, management and 

harvesting techniques were not affected by the incorporated traits, with the exception that late blight 

resistant potatoes should require less fungicide use. No other natural defense mechanisms have been 

activated; therefore, the modified potatoes have no new properties to enhance their weediness, 

invasiveness, or pest resistance and therefore display no traits which would have a direct impact on 

endangered species or biodiversity. The changes in potato quality intentionally introduced into these 

events have no more impact on biodiversity than any other commercial potato variety as shown by our 

multi-site and multi-year field trials. Furthermore, the W8 event is likely to be planted in areas that are 

already growing potatoes and would not result in a significant expansion of planted acres. For these 

reasons, it is unlikely that the cultivation of W8 would have any greater impact on biodiversity than 

conventional potatoes.
J.R. Simplot Company JRS06 Page 182 of 196

12.6 Summary of the Environmental Safety Assessment

The environmental safety of W8, for which detailed information is included in this submission, was 

assessed by comparing W8 with the Russet Burbank control variety. Familiarity of potato was 

considered during evaluation of the data, including agronomic performance, trait efficacy, specificity, 

compositional assessment, toxicology, and allergenicity.

Information on W8 has been reviewed to determine the potential risk to the environment using the 

following five criteria: (1) potential to become a weed of agriculture or to be invasive of natural habitats, 

(2) potential for gene flow to sexually compatible plants, (3) potential to become a plant pest, (4) 

potential impact on non-target species including humans, and (5) potential impact on biodiversity.

Regulated field trials over multiple years with W8 did not provide any evidence for increased plant pest 

characteristics such as accelerated tuber sprouting, increased plant vigor, increased tuber set, or 

delayed senescence. Due to modern agricultural practices it is highly unlikely that potatoes would 

persist in a field from one crop cycle to the next, particularly since potatoes are typically grown as a 

rotational crop. Wild potato varieties are rare in North America and for the most part geographically 

isolated from commercial production areas, reducing concerns about cross-pollination with wild species. 

Field studies confirmed there were no differences in ecological interactions between W8 and the Russet 

Burbank control. Planting, cultivation, management and harvesting techniques were not affected by the 

incorporated traits, with the exception that late blight resistant potatoes should require reduced 

fungicide. The introduction of W8 would not result in a significant expansion of planted acres or a 

change in the areas where potatoes would normally be grown. Therefore, the potential to impact 

insects and other nontarget organisms, weed or disease susceptibility, endangered species, or 

biodiversity is negligible from the cultivation of W8.

The data presented here demonstrate that introduction of Russet Burbank Event W8 will have a similar 

environmental impact when compared to untransformed potatoes and poses no increased risk to the 

environment. The unconfined introduction and cultivation of these potatoes is not expected to cause 

any adverse environmental or biological impacts.
J.R. Simplot Company JRS06 Page 183 of 196

12.7 References

Bamberg, J.B., del Rio, A., Huaman, Z., Vega, S., Martin, M., Salas, A., Pavek, J., Kiru, S., Fernandez, C., 

and Spooner, D. (2003). A decade of collecting and research on wild potatoes of the southwest. Am J of 

Potato Res. 80: 159-172.

Bamberg, J.B., and del Rio, A. (2011) Use of native potatoes for research and breeding. HortScience. 46: 

1444-1445.

CFIA. (1996). Biology Document BIO1996-09: The Biology of Solanum tuberosum (L.) (Potatoes). 

Canadian Food Inspection Agency. http://www.inspection.gc.ca/plants/plants-with-noveltraits/applicants/directive-94-08/biology-documents/solanum-tuberosum-l-

/eng/1330982063974/1330982145930 (Accessed 23 August 2012).

Hijmans RJ, Spooner DM, Salas AR, Guarina L, and de la Cruz J (2012) Atlas of Wild Potatoes 

http://www.vcru.wisc.edu/spoonerlab/pdf/Potato%20Atlas%20Final.pdf (Accessed 30 August 2012).

Hokanson K, Heron D, Gupta S, Koehler S, Roseland C, Shantharam S, Turner J, White J, Schechtman M, 

McCammon S, Bech R (1999) The concept of familiarity and pest resistant plants. Proceedings of a 

workshop on: Ecological Effects of pest resistance genes in managed ecosystems. January 31- February 

3, 1999, Bethesda, MD. USDA-APHIS Plant Protection and Quarantine, Scientific Services.

Love, S. (1994). Ecological risk of growing transgenic potatoes in the United States and Canada. Am 

Potato J, 71: 647-658.

NTP. (2012) Technical Report on the Toxicology and Carcinogenesis Studies of Acrylamide in F344/N Rats 

and B6C3F1 Mice. National Toxicology Program. National Institutes of Health. Public Health Service. 

U.S. Dept. of Health and Human Services.

Spooner, D.M., van den Berg, R.G., Rodriguez, A., Bamberg, J.B., Hijmans, R.J., and Cabrera, S.I.L. (2004).

Wild potatoes (Solanum section Petota: Solanaceae) of North and Central America. Syst Bot Monogr. 68: 

1-209.

US EPA. (2011). United States Environmental Protection Agency. Introduction to Biotechnology 

Regulation for Pesticides. http://epa.gov/oppbppd1/biopesticides/regtools/biotech-reg-prod.htm 

(Accessed 27 January 2012).
J.R. Simplot Company JRS06 Page 184 of 196

13.0 Crop Production and the Potato Industry

Russet Burbank Event W8 has multiple traits that provide benefits to potato processors, growers and 

consumers including late blight resistance, low acrylamide potential, reduced black spot, and lowered 

reducing sugars. These benefits were added to Russet Burbank potatoes through tools of modern 

biotechnology such as gene silencing using potato genomic DNA and in the case of late blight resistance,

genes were obtained from a wild potato species. Integrating these traits simultaneously through 

conventional breeding would not be possible because potatoes are tetraploid, highly heterozygous, and 

are sensitive to inbreeding depression. Additionally, in the case of Russet Burbank, the variety is sterile. 

Commercially grown potato varieties such as the Russet Burbank are vegetatively propagated to 

maintain desirable traits developed over many years. 

13.1 Benefits of Potatoes with Late Blight Resistance

Historically, late blight has long been a problem in potatoes, and was the cause of the Irish potato 

famine in the 1840s. It remains one of the most devastating diseases in modern potato production. The 

potato late blight oomycete overwinters in tubers infected from previous seasons which can give rise to 

infected potato seedlings. In infected plants, 1 potato leaf can hold 10,000 late blight spores, which can 

be spread through wind or animal movement. Late blight is prevalent in many areas of the United States 

and is now found in nearly every leading potato state (Figure 13-1).

Figure 13-1. Areas of late blight infection in 2012 are shaded.

The late blight pathogen spreads quickly through infected fields, causing widespread foliar necrosis 

resulting in plant death and significant yield reduction. Infected tubers can cause rot that destroys entire 

lots in storage. If late blight is not detected early or before pressure is high, susceptible varieties may 

require fungicide treatment every 3 to 5 days (for a total of 5 to 15 total applications before harvest) 
J.R. Simplot Company JRS06 Page 185 of 196

and still incur yield losses of 20% to 70%. In terms of pesticide use, fall harvested potatoes require 23% 

of the total pesticides used for major crops including corn, soybean, cotton and wheat, which is second 

only to corn at 40% of the pesticide use (O’Donahue et al. 2011). Considering that potatoes are planted

on a little over 1 million acres and corn is planted on about 90 million acres in the US, pesticide use per 

acre is much higher for potatoes than for corn. The average cost to control late blight in 2000 was 

estimated to be $205/ acre for a total cost of $77.1 million in the US. In addition, lost revenues related 

to poor yield from potato plants decimated by late blight resulted in an additional $210.7 million in 2000 

(Guenthner et al. 2001). 

A more recent study (Context 2014) suggests that annual fungicide use on potatoes costs about $90 

million in the US, with about $60 million specifically for late blight management. This same study 

recommends that late blight resistant potatoes like W8 may require some fungicide applications, but 

could result in savings of $29 million per year with reductions of 290 million lbs of fungicide active 

ingredient. Other savings would be realized from labor, water, fuel, equipment maintenance, and the 

benefit of continual plant manufactured resistance that would not be subject to loss of effectiveness 

from rain or other environmental factors.

As one of the most important potato pathogens, resistance to late blight could have significant benefits 

with respect to reduced use of pesticides, but also could effectively improve yield compared with 

diseased potatoes. One result could be less chemical residue in food, land, and waste water. Even with 

pesticide application, some disease tends to persist in potato fields so effective resistance could result in 

healthier plants and better yield. Late blight affects both foliage and tubers. Evidence suggests that 

tuber damage is far more likely to occur if the plant foliage is infected than by picking up the disease 

agent only through soil contact. Tuber infection with late blight results in lesions, rot, and ultimately 

economic loss in storage. The loss of entire crops due to late blight is not uncommon in fields and 

storages in developed countries as well as in developing countries.

13.2 Benefits of Potatoes with Reduced Acrylamide Potential

Based on a Federal Register notice published in 2013, FDA has proposed guidance for industry on the 

reduction of acrylamide levels in food products (FDA 2013). In FDA’s Draft Guidance for Industry on 

Acrylamide in Foods the FDA states that “Reducing acrylamide in foods may mitigate potential human 

health risks from exposure to acrylamide.” An extensive list of potential mitigation techniques were 

summarized in the guidance document including the reduction of sugar levels in potatoes (FDA 2013). 

The list also includes variety development and selection, focusing on varieties that are more resistant to 

cold-induced sweetening, and lower levels of reducing sugars or asparagine. Many of the methods in 

FDA’s Guidance document are consistent with those reported in the Acrylamide Toolbox published by 

Food Drink Europe (2011). 

The introduction of InnateTM potatoes with low acrylamide potential would provide potatoes that are 

largely indistinguishable from existing varieties. The reduction in asparagine and sugars using InnateTM

technologies and the resulting reduction in acrylamide upon heating will address food industry needs 

with respect to the FDA’s Draft Guidance (2013). As such, this biotechnology approach to lowering 

acrylamide was mentioned in the guidance document as a promising method to develop potato varieties 

with reduced acrylamide potential. 

At the time of harvest, french fries made with W8 tubers contained 85% less acrylamide than the 

control. When potatoes were stored throughout nine months at 46 ̊F, acrylamide levels in W8 were 78 
J.R. Simplot Company JRS06 Page 186 of 196

to 84% lower than control Russet Burbank (Tables 10-4 and 10-5 in Chapter 10: Trait Efficacy). The low 

acrylamide potatoes will provide an option that addresses this potential health issue for all sectors of 

the potato processing market and thus should increase the demand for these InnateTM W8 potatoes. 

13.3 Benefits of Potatoes with Reduced Black Spot

The blackening that occurs after potatoes are bruised affects quality and recovery in processing french 

fries and chips. Potatoes that have been damaged and show black spot must be trimmed or could be 

rejected before processing, resulting in quality challenges or economic loss or both. In many instances 

potato growers have contracts that provide incentives for delivering “bruise free” potatoes. A 

significant reduction in black spot could result in higher profit for the farmer as a result of decreased 

visible damage. Also, these black spots are considered defects in potato chip and french fry processing, 

causing economic loss from trimming or culling potatoes with black spot. Potatoes may develop black 

spots from pressure bruising which results from the weight of potatoes in deep piles during storage. 

The weight of the potatoes causes damage to the potato tissue resulting in dark colors that must be 

removed through trimming. 

Bruising and discoloration are also problems in the market for fresh whole potatoes. Potato lots that 

don’t meet USDA bruise tolerance standards are diverted to other uses at lower prices. Another 

possible advantage of silencing black spot could be the enabling of new markets for “freshly cut” 

potatoes, without pre-cooking or using sulfites or other preservatives to maintain color and flavor. The 

convenience of fresh-cut potatoes could boost demand for InnateTM W8 potatoes. Focus group market 

research indicates that consumers will readily accept packaged fresh-cut potatoes in the refrigerated 

produce section of supermarkets.

13.4 Benefits of Potatoes with Lowered Reducing Sugars

In breeding programs for processing potatoes, one of the most desired characteristics is low levels of the 

reducing sugars glucose and fructose. High levels of reducing sugars lead to undesirable dark colors and 

bitter flavors in fries and chips. High sugar levels can result from any stress to the plants during growing 

or harvesting such as drought or heat stress (Bethke et al 2009), but develop rapidly with low 

temperature storage (Driskill et al. 2007). Typical storage temperatures for potatoes for the frozen fry 

market are 46 - 48 ̊F (Driskill et al. 2007) to maintain sugar levels while decreasing potato storage 

disease potential. If potatoes could be stored at a lower temperature without adversely affecting sugar 

level, the result would be less disease damage and less weight-loss from shrinkage due to lower 

respiration rates. 

With introduction of pSIM1278 into Russet Burbank potatoes (Collinge and Clark 2013), we found 

silencing of the promoters for R1 and PhL to result in slight reductions in reducing sugars at the time of 

harvest or after one month of storage. The silencing cassette contained in pSIM1678 results in silencing 

of VInv in W8, which results in lower levels of reducing sugars throughout the storage period. VInv

silencing may also allow for lower temperature storage which will reduce yield loss from respiration and 

disease. Many potatoes must be stored for as long as 3 to 12 months before processing, and the ideal 

storage potato maintains a low level of reducing sugar throughout storage. A consistent focus of 

breeders for processing potatoes would be to have low reducing sugars at the time of harvest that 

remain low throughout their storage life (Driskill et al. 2007). There is also evidence that VInv silencing 

reduces the incidence of high sugar potatoes and sugar ends (see Chapter 11: Agronomic Performance,

Table 11-4), both quality attributes that result in economic loss by causing potatoes to be rejected by 
J.R. Simplot Company JRS06 Page 187 of 196

french fry and chip processors, resulting in lower prices as they are only suitable for making dehydrated 

potatoes or cattle feed. Additionally, significantly lower levels of acrylamide were found after frying (see 

Chapter 10: Trait Efficacy, Table 10-4). 

13.5 Potato uses and exports

Potatoes are grown commercially in 36 of the 50 states in the US. Total US production in 2012 was 467 

million hundred weight (cwt), with total value of $3.91 billion, planted on 1.15 million acres (USDA ERS 

2013). The commercial uses include 36% as frozen, 26% sold fresh, 15% chips and shoestrings (julienne 

cut crispy snacks), 11% dehydrated, 6% seed potatoes, 4% other frozen products, and 1% canned (NPC 

2013). Annual per capita potato use in the US in 2012 was 117 lbs per person (USDA ERS 2013). 

The states with the largest volume and sales value in 2012 were Idaho, Washington, Wisconsin, North 

Dakota, Colorado, Oregon, Minnesota, Michigan, California, and Maine (NPC 2013). These data 

represent the combined markets including all processing and fresh varieties.

US exports of all potatoes and potato products for the 2013 fiscal year (July 2012-June 2013) were 

1,561,896 metric tons (MT) valued at $1.64 billion, which are both 4 % increases over fiscal year 2012

(USPB 2013). US exports of frozen potato products for fiscal year 2013 increased by 2 % for volume and 

6 % by value over fiscal year 2012, with 926,553 MT and a value of just over $1 billion, respectively. US 

exports of fresh potatoes increased by 13 % in fiscal year 2013 to 456,366 MT, but value was reduced by 

4 % at $198 million compared to fiscal year 2012. The decline of value was mainly due to a reduction in 

value of exports to Canada. Finally, US exports of dehydrated potatoes decreased by 3 percent in fiscal 

year 2013 to 114,408 MT, but the value stayed about the same at $192 million compared to fiscal year 

2012 (USPB 2013). Overall, 2013 was the most valuable year to date with over $1 billion in value for the 

first time.

A majority of the potato exports were frozen fries at 62% of the export market, fresh potatoes made up 

13% of the market, chips and dehydrated potatoes each had 12% of the export market, and seed made 

up 1% of the export market (NPC 2013). Finally, the top three export markets for US potatoes in 2012 

were Japan, Canada, and Mexico with values of $404 million, $342 million, and $185 million, 

respectively.

13.6 Submissions to Other Regulatory Agencies

An Experimental Use Permit (EUP) application was submitted to EPA on December 16, 2013, for field 

testing of InnateTM late blight resistant potatoes on more than 10 acres across the United States. An 

EUP, also for late blight resistant potatoes, with a Petition for Temporary Tolerance Exemption was 

submitted February 20, 2014. A Section 3 Registration will be filed after experiments are completed 

under the EUPs. Simplot has initiated and will complete a consultation process for food safety and 

nutrition with the Food and Drug Administration (FDA) prior to commercial distribution of potatoes from 

Russet Burbank W8.

An assessment of the safety of W8 will also be submitted to Health Canada and the Canadian Food

Inspection Agency (CFIA). Furthermore, submissions in support of approval to import products from the

events will be made to the Japan Ministry of Health, Labor and Welfare (MHLW) and the Ministry of 

Agriculture, Forestry, and Fisheries (MAFF). Submissions are anticipated for other key international 

markets including Mexico.
J.R. Simplot Company JRS06 Page 188 of 196

13.7 Impact on the Organic Market

Organic potatoes in 2011 represented less than 1.0% (0.7%) of total potato plantings with 8,273 acres of 

certified organic potatoes (USDA ERS 2014). Of the 13,258 organic acres, California, Colorado, and 

Oregon were the top three organic potato producers with 6,520 acres, 2,138 acres, and 1,654 acres, 

respectively. Potato varietal selection and breeding are similar between organic and conventional 

potato production as commercial potatoes are predominantly planted from tuber seed. Risks to organic 

growers would be most likely to occur with accidental mixing of planting material or of potatoes in 

farming, transportation, or processing channels. These risks are the same as those that organic growers 

already experience when keeping their organically grown potatoes separate from conventionally grown 

potatoes. Because potatoes are clonally propagated and the Russet Burbank variety is sterile, there is no 

risk of contaminating seed supplies through cross-pollination. Organic farmers routinely plant organic 

tuber seed material and any incidence of cross-pollination in production fields will not affect the 

harvested potatoes. Producers of organic true potato seed (TPS) will be able to protect their seed from 

InnateTM material by ensuring that the seed production fields are sufficiently isolated from InnateTM

potato fields. Potential outcrossing can be prevented by separating fields planted for organic TPS 

production by at least 20 meters from fields planted with InnateTM potatoes (Conner and Dale 1996). 

However, outcrossing would not be relevant for Russet Burbank W8 since the variety is sterile.

 

13.8 History of Biotech Potatoes

A review of the history of the introduction of Monsanto’s genetically modified potatoes in 1995 in the 

U.S. (Thornton 2008) indicates the importance of a careful assessment of the performance of W8, 

synchronizing approvals in North American and trading countries, and weathering public acceptance 

issues that may result from activism against food industry members that adopt the new technology. 

This history points to the importance of consumer acceptance when introducing any InnateTM crops and 

products to the market. Since the time when biotech potatoes were on the market briefly, the adoption 

of biotechnology in the food supply has increased consistently. Herbicide tolerant (HT) soybeans went 

from 17 percent of U.S. soybean acreage in 1997 to 68 percent in 2001 and 93 percent in 2013. The 

adoption of HT corn went from 4.3 percent in 1997 to 8 percent in 2001 and then accelerated to 85 

percent of U.S. corn acreage in 2013. Plantings of insect resistant Bt corn grew from about 8 percent of 

U.S. corn acreage in 1997 to 76 percent in 2013 (USDA ERS http://www.ers.usda.gov/dataproducts/adoption-of-genetically-engineered-crops-in-the-us/recent-trends-in-geadoption.aspx#.UnGtr_ns_cA). These two crops provide significant quantities of biotechnology-derived 

ingredients in widely marketed processed food and feed products, such as protein, oils, starches, and 

sweeteners. Many of the conventional processed potato products already contain one or more of these 

biotech ingredients. With widespread adoption of food ingredients from biotech crops, today’s food 

industry should be far more likely to embrace biotech potatoes such as Russet Burbank W8, particularly 

in light of the positive consumer attributes of the W8 potatoes.

13.9 Stewardship of InnateTM W8 Potatoes

Stewardship of Potatoes

Throughout development of the InnateTM W8 potatoes, the Biotechnology Quality Management System 

(USDA-BRS Compliance assistance program) has served as a stewardship program to conduct field trials, 

monitor movement, and complete testing while regulated. As a part of BQMS, qualified and experienced 

potato growers were identified, and contractual agreements were established to ensure compliance and 

conformance with requirements for growing InnateTM potatoes. Processes were developed that 
J.R. Simplot Company JRS06 Page 189 of 196

included Standard Operating Procedures (SOPs) and forms for all critical control points to guide internal 

and external collaborators to conduct activities using InnateTM potatoes. 

The following management procedures were included for effective implementation of BQMS:

1. Document and record control

2. Resource management

3. Training external and internal associates

4. Planning and process realization

5. Conducting internal audits

6. Implement corrective / preventive actions

7. Continuous improvement of the system

8. Resolution of compliance issues

Targeted training programs were used to educate and bring awareness on handling InnateTM W8 

potatoes in storage, transport, planting, harvest, post-harvest, processing, and final disposition to 

ensure that no InnateTM potato material entered food or feed channels prior to de-regulation. Both 

internal and external audits were used to verify that planned activities occurred and personnel were 

adhering to the protocols recommended by BQMS. This stewardship program includes corrective and 

preventive actions to avoid compliance and conformance incidents. While BQMS served as a quality 

management system during product development, with commercial introduction we anticipate a full 

extension of such methods as outlined in the ‘Excellence Through Stewardship®’ (BIO 2007) program.

Identity Preservation 

To help prevent trade disruptions, international approvals will be sought from key trading partner 

countries before the InnateTM W8 potatoes are launched commercially. The initial introduction will build 

up slowly as seed becomes available and will be controlled within existing processing channels to ensure 

that W8 potatoes enter only the intended markets. This will provide an extended period of time to 

assess consumer acceptance and to address grower and industry awareness. A limited introduction in a 

vertically integrated supply chain will be well controlled by grower and processor agreements. In this 

situation, conventional products will be considered “identity preserved” with respect to the wellcontrolled stewardship of the InnateTM crop and its products. As InnateTM potato adoption increases, 

programs for identity preservation will be implemented as needed. It’s expected that development and 

implementation of identity preservation systems will add some cost to the supply chain. The total costs 

will depend upon the type and extent of market penetration. 

Synchronized Regulatory Approvals

We intend to follow the recommended stewardship policy statement released by the Biotechnology 

Industry Organization in May 2007. 

“To help ensure the continued adoption of agricultural biotechnology globally and to continue to have 

products of agricultural biotechnology bring value to the marketplace, BIO’s Food and Agriculture 

Section supports actions that facilitate the flow of goods in commerce and minimize trade disruptions. 

BIO’s Food and Agriculture Section believes that henceforth individual member companies should, prior 

to commercialization meet applicable regulatory requirements in key countries identified in a market 

and trade assessment that have functioning regulatory systems and are likely to import the new 

biotechnology-derived plant products.”
J.R. Simplot Company JRS06 Page 190 of 196

This voluntary guideline was adopted because, according to BIO: “asynchronous authorizations 

combined with importing countries maintaining ‘zero tolerance’ for recombinant-DNA products not yet 

authorized results in the potential for major trade disruptions. The potential occurrences of trade 

disruptions will only increase given the substantial amount of research that will bring many new 

products and combinations of products to market.”

13.10 Conclusions: Crop Introduction and the Potato Industry

Russet Burbank W8 potatoes provide the potato industry the opportunity to keep the highly desired 

characteristics of the leading french fry and fresh potato variety with multiple enhancements that could 

not be added through traditional breeding. Overall improvement in yield and lowered fungicide use 

with late blight resistance should benefit all members of the food value chain by reducing environmental 

impacts associated with fungicide use, and potentially reducing acreage devoted to potato production. 

The combination of low asparagine and reducing sugars results in greater than 70% reductions in 

acrylamide even after extended cold storage, addressing the potential health risk for consumers and the 

food industry. Quality improvements related to lower levels of reducing sugars and black spot provide 

benefits to processors and consumers, and invertase silencing could positively impact quality and yield 

from potato storage. Finally, reduced invertase could enable storage of processing potatoes at 

significantly lower temperatures, decreasing loss from disease and yield losses from higher respiration 

rates related to typical storage at 46 – 48  ̊F. Lastly, the combination of low asparagine and reducing 

sugars results in greater than 70% reductions in acrylamide even after extended cold storage, addressing 

this potential health risk.

Knowledge about potato biology, especially the fact that Russet Burbank is a sterile variety, should 

alleviate potential questions regarding outcrossing to other varieties or organic potatoes. It is 

anticipated that the combination of the planned stewardship program along with approval in key 

international markets will prevent trade disruptions through careful management of the supply chain.
J.R. Simplot Company JRS06 Page 191 of 196

13.11 References

Bethke, P.C., Sabba, R., Bussan, A.J. (2009). Tuber water and pressure potentials decrease and sucrose

contents increase in response to moderate drought and heat stress. Am J Pot Res. 86: 519-532.

Bhaskar, P.B., Wu, L., Busse, J.S., Whitty, B.R., Hamernik, A.J., Jansky, S.H., Buell, R.B., Bethke, P.C., and 

Jiang, J. (2010). Suppression of the Vacular Invertase Gene Prevents Cold-Induced Sweeting in Potato. 

Plant Physiology. 154: 939-948.

BIO. (2007). US Biotechnology Industry Organization Product Launch Stewardship Policy. 

http://www.bio.org/foodag/stewardship/20070521.asp (Accessed 22 May 2011).

Collinge, S. and Clark, P. (2013). Petition for Determination of Nonregulated Status for InnateTM Potatoes 

with Low Acrylamide Potential and Reduced Black Spot Bruise: Events E12 and E24 (Russet Burbank); 

F10 and F37 (Ranger Russet); J3, J55, and J78 (Atlantic); G11 (G); H37and H50 (H). J.R. Simplot Company 

Petition JRS01 (USDA Petition 13-022-01p).

Conner, A.J., and Dale, P.J. (1996), Reconsideration of pollen dispersal data from field trials of transgenic 

potatoes. Theor Appl Genet. 92: 505-508.

Context (2014). Potato Blight Prevention Fungicide Overview. The Context Network. March 3, 2014.

Driskill, E.P., Knowles, L.O., and Knowles, R.N. (2007). Temperature-induced changes in potato 

processing quality during storage are modulated by tuber maturity. Amer J of Potato Res. 84: 367-383.

FDA. (2013). Food and Drug Administration Guidance for Industry, Acrylamide in Foods. Available from: 

http://www.fda.gov/downloads/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/

ChemicalContaminantsMetalsNaturalToxinsPesticides/UCM374534.pdf [Accessed 20 November 2013).

Food Drink Europe. (2011). Food Drink Europe Acrylamide Toolbox 2011. Accessed online at 

http://www.fooddrinkeurope.eu/uploads/publications_documents/Toolboxfinal260911.pdf.

Guenthner, J.F., Michael, K.C., Nolte, P. (2001). The Economic Impact of Potato Late Blight on US 

Growers. Potato Research. 44: 121-125.

NPC. (2013). National Potato Council, 2013 Potato Statistical Yearbook.

NTP. (2012). Technical Report on the Toxicology and Carcinogenesis Studies of Acrylamide in F344/N 

Rats and B6C3F1 Mice. National Toxicology Program. National Institutes of Health. Public Health 

Service. U.S. Dept. of Health and Human Services.

O’Donahue, E.J., Hoppe, R.A., Banker, R.E., Fuglie, K., Korb, P., Livingston, M., Nickerson, C., Sandretto, C.

(2011). The Changing Organization of U.S. Farming. EIB-88. U.S. Department of Agriculture, Econ. Res. 

Serv. December 2011.
J.R. Simplot Company JRS06 Page 192 of 196

Thornton, M. (2008). The rise and fall of NewLeafTM potatoes. 

http://nabc.cals.cornell.edu/pubs/nabc_15/chapters/Thornton.pdf (Accessed 31 May 2011).

USDA ERS. (2014). Organic Production. United States Department of Agriculture Economic Research 

Service. http://search.ers.usda.gov/search?affiliate=ers&query=certified%20organic%20acreage 

(Accessed March 20, 2014).

USDA ERS. (2013). Vegetables and Pulses Outlook. United States Department of Agriculture Economic 

Research Service. March 29, 2013. http://www.ers.usda.gov/publications/vgs-vegetables-and-pulsesoutlook/vgs353.aspx

USPB. (2013). United States Potato Board, US potato exports set another record. 

http://www.uspotatoes.com/pressRoom/pr.php?id=270 (accessed March 25, 2014).

USPB. (2014). United States Potato Board, Export market. 

http://www.uspotatoes.com/downloads/Frozen%20Potato%20Products-Export_NEW.pdf (accessed 

March 25, 2014).

Ye, J., Shakya, R., Shreshtha, P., and Rommens, C.M. (2010). Tuber Specific Silencing of the Acid 

Invertase Gene Substantially Lowers the Acrylamide Forming Potential of Potato. J Agric Food Chem. 58: 

12162-12167.
J.R. Simplot Company JRS06 Page 193 of 196

14.0 Conclusions: Determination of Nonregulated Status for Russet Burbank W8 Potatoes

The potato industry and customers will benefit from introduction of late blight resistant Russet Burbank 

potatoes, with lower reducing sugars, black spot, and acrylamide potential. Considering that the Russet 

Burbank variety is sterile, tools of modern biotechnology such as InnateTM technologies, are ideally 

suited for simultaneously incorporating multiple traits. The propagation of commercial potatoes 

through cloning mitigates concerns about increased plant pest potential such as seed dispersal, survival 

outside of cultivation, or outcrossing. Based on multi-year field trials evaluating composition, agronomic 

performance, and disease stressors, as well as the basic understanding and familiarity of potato biology, 

W8 poses no significant risk of persistence in the environment or altered environmental interactions as 

a result of weediness or increased plant pest potential.

Results of efficacy studies with Russet Burbank Event W8 confirm the expected late blight resistance, 

along with reductions in black spot, reducing sugars, and acrylamide. Significant reductions in reducing 

sugars and acrylamide were observed throughout long term storage. 

Collectively, the ubiquitous nature of resistance genes like Rpi-vnt1 and their encoded resistance 

proteins such as VNT1 in Solanum species, including potatoes, lack of toxins or allergens, mode of action 

not based upon inducing pest toxicity, and extremely low expression levels provides confidence in the 

safe use of potatoes created to express VNT1. 

The data presented here demonstrate that introduction of W8 will have a similar environmental impact 

as control Russet Burbank potatoes and poses no increased risk to the environment. The unconfined 

introduction and cultivation of these potatoes is not expected to cause any adverse environmental or 

biological impacts. 

We now seek nonregulated status for Russet Burbank event W8 based on the weight of evidence 

demonstrating that the W8 potatoes are unlikely to pose a plant pest risk and respectfully submit that

these plants should not be classified as “regulated articles” as defined under 7 CFR Part 340. 
J.R. Simplot Company JRS06 Page 194 of 196

15.0 Acknowledgements

The J.R. Simplot Company would like to acknowledge the efforts of the following Regulatory Scientists 

for their contributions in this submission: Matt Pence, Ph.D., Sathya Adimulam, Stephanie McInerney, 

Jeff Hein, Janet Layne, Erika Roach, Juan Pablo Burzaco, and Eric Rosenbaum. Additionally, we would like 

to acknowledge the R&D and Commercial Teams led by Craig Richael, Ph.D. and Kerwin Bradley, 

respectively.
J.R. Simplot Company JRS06 Page 195 of 196

16.0 Statement of Grounds Unfavorable

JR Simplot is not aware of any information indicating that InnateTM potatoes may pose a greater plant 

pest risk than conventional potatoes. There are no adverse environmental consequences anticipated 

with its introduction based on the available data and information. The benefits of introduction of 

commercial events are provided in this submitted petition.

1 of 22

TITLE

Appendix A: Genetic, Molecular, and Biochemical Methods

AUTHORS

Jingsong Ye, PhD and Jeff Habig, PhD

DATE

March 28, 2014

SUBMITTED BY

J.R. Simplot Co

Plant Sciences

5369 W. Irving St., Boise, ID 83706

PAGE COUNT

1 of 22
2 of 22

Contents

1.0 Genetic Characterization ..................................................................................................................3

1.1 Characterization of Insert Structures............................................................................................3

Confirmation of Internal Band Size and Identity ..................................................................................5

1.2 Characterization of Vector Backbone Sequences.........................................................................8

1.3 Analysis of Genetic Stability........................................................................................................12

2.0 Characterizing Rpi-vnt1 and VNT1 ..................................................................................................13

3.0 Gene Expression Studies.................................................................................................................16

Northern Blot Hybridization....................................................................................................................16

Reverse Transcriptase quantitative PCR (RT-qPCR)................................................................................18

4.0 Sequence Analysis...........................................................................................................................19

5.0 References ......................................................................................................................................22
3 of 22

1.0 Genetic Characterization

The following methods were used to generate the data presented in Chapter 5: Genetic 

Characterization of Russet Burbank Event W8. Methods are included for characterization of the 

pSIM1268 and pSIM1278 insertions, including insert structures, absence of vector backbone, 

identification of flanking regions, and genetic stability.

1.1 Characterization of Insert Structures

Plant Material. Russet Burbank W8 Plants used for characterization of insert DNA were grown in 

Sunshine mix-1 (www.sungro.com) in two-gallon pots in a greenhouse that was controlled for 

temperature (18°C minimum/27°C maximum) and light (16-h photoperiod with an intensity of ~1500 

μmol/m2/s). Russet Burbank control plants were also grown in the greenhouse under the same 

conditions and used as a background control. After one to two months of growth, leaf materials were 

taken from W8 and control plants for genomic DNA isolation.

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a 

mortar using liquid nitrogen. The ground tissue was transferred to a pre-cooled 15 ml conical tube with 

a pre-cooled spatula and stored at -80oC until ready to process. 10 ml extraction buffer (0.35 M 

Sorbitol, 0.1 M Tris-HCl, pH8.0, 0.05M EDTA) was mixed with the powder, and the suspension was then

centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was thoroughly resuspended in 2 

ml extraction buffer containing 200 μg RNase A. After incubating the resuspended DNA at 650C for 20 

min with 2 ml of nuclear lysis buffer (0.2 M Tris-HCl pH7.5, 0.005 M EDTA pH 8.0 and 20 mg/ml CTAB 

Hexadecyl Trimetyl Ammonium Bromide) and 800 μl of 5% Sarcosyl, it was mixed with an equal volume

of chloroform : isoamyl alcohol (24:1), vortexed for about 1 min, and centrifuged at 3000 rpm for 5 min 

at room temperature. The DNA was precipitated with an equal volume of isopropyl alcohol, washed 

with 70% ethanol, air dried, and dissolved in 400-700 μl 1X Tris/EDTA buffer (TE). DNA concentration 

was measured using a spectrophotometer at an OD of 260 nm, whereas quality was confirmed by 

running the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE) for 30-40 min at 80 volts. In some 

cases, an alternative method was used to isolate DNA. The ground tissue from 0.7 g young leaves was 

mixed with 7 ml CTAB buffer (2% CTAB, 1.4 M NaCl, 0.1 M pH 8.0 Tris-HCI, 20 mM pH8.0 EDTA, 100 

μg/ml RNase) and incubated at 55o-65oC for 30 min followed by centrifugation at 3,000 rpm for 15 min. 

DNA was extracted twice with equal volumes of chloroform: isoamyl alcohol (24:1) by shaking for 10 min 

and centrifuged at 3000 rpm for 5 min at room temperature. The DNA was precipitated with equal 

volumes of ethanol and rinsed with 70% ethanol. The resulting pellet was air dried and resuspended in 

TE buffer. The quality and concentration of DNA was determined spectrophotometrically and by running 

a sample on a 0.8% agarose gel. Control and event DNA were always extracted with the same method 

for any given comparison.

DNA Gel (Southern) Blot Analysis. 3 μg of plant DNA was digested overnight in 400 μl final volume with 

at least 5 μl (10 units/μl) restriction enzyme (Invitrogen) at 37oC. Digested DNA was concentrated by 

ethanol precipitation (40 μl of 3M NaOAc, pH5.3 and 1 ml ethanol) at -80oC for 10 min followed by a 

wash with 70% ethanol. The DNA pellet was dissolved in 20 μl 1X TE followed by addition of 2 μl DNA 

gel loading buffer, which consists of 40% sucrose and 0.35% Orange G (Sigma) in water.

Membrane Preparation. Digested plant DNA was loaded on a large 0.7% agarose gel (170 ml) containing 

0.5X tris-borate-EDTA (TBE) buffer and 3-5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 

hrs. The gel was photographed using a gel documentation system from Alpha Innotech (Santa Clara, 
4 of 22

California), and then depurinated by submerging it into 0.25 N HCl for 20 min. After subsequent 

denaturation in 0.5 M NaOH /1.5 M NaCl for 2X15 min and neutralization in 1.5 M NaCl and 0.5 M TrisHCl, pH7.5, for 2 X 15 min on a shaker at room temperature and equilibrated with 10X SSC for 10 min. 

The transfer of DNA to the nylon membrane was carried out using 10X SSC according to a standard 

capillary transfer method. 

Probe Preparation. The labeling of the PCR-derived probe was achieved using Hotmaster Taq enzyme 

and buffer (Fisher BioReagents) according to Roche’s DIG labeling instructions. A standard 50 μl 

reaction consisted of 5 μl of 10x Hotmaster Taq Buffer, 2-5 μl of 10 uM forward primer, 2-5 μl of 10 μM 

reverse primer, 5 μl DIG-labeled dNTP (Roche), 10 ng plasmid template, 0.75 μl Hotmaster Taq

polymerase, and water. The PCR amplification conditions were dependent on each DIG-labeled probe. 

PCR with regular dNTP instead of DIG labeled dNTP was used as control. Quality of the DIG-labeled 

probe was assessed by running a small amount of the probe on 1% agarose DNA gel (it always ran 

slower than control PCR product). The probe was denatured before use by incubating the probe at 

100oC for 5 min, placing on ice for 2 min. For the exact position of probes in the DNA insert, see Figure 

A-1.

Figure A-1. DNA Insert Probes

RV=EcoRV, R1=EcoRI, Sc=ScaI, Hd=HindIII, Xb=XbaI

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.

Hybridization. The nylon membrane carrying transferred DNA was prehybridized in 40 ml pre-warmed 

DIG Easy Hybridization solution (Roche) at 42oC for 1-4 hrs in a bottle in a standard hybridization oven 

(Amerex Instruments Inc.) at 20-25 rpm. Hybridization was carried out by replacing the prehybridization 

buffer with a fresh amount of the same preheated solution, now containing 25-50 μl denatured DIG 

labeled probe, and continuing the incubation at 42oC, 20-25 rpm for about 16 hrs. The hybridization 

solution could be store at -20oC and reused up to 3 times. The reused hybridization solution was heated 

at 68oC for 10 minutes before use.

RB

RI (3718) Sc(8247) Hd (2444) Hd (6674) Hd (7987)

RV (1128)

RV (2353)

RV (3065)RV (5358)RV (6583)

pGbss pGbss Sc (7469)

LB pAgp pAgp (1) (194)

(10148)

tPpo5

pR1

tPpo5 fAsn1 Spacer

fAsn1

pR1

pPhLpPhL

AGP AGP 

pSIM1278

Probe ASN ASN GBS1 R1 R1 GBS1 GBS2 

AGP

LB pVnt1 Vnt1 tVnt1 pAgp Inv pGbss RB

RRI (7457) V (5444) ScRV (6669) (834) Sc (2132) Xb (2832) Xb (7383) Hd (1947) Hd (2608) Hd (3265) Hd (4106) Hd (6760)

Inv

VNT1 INV INV 

pSIM1678

Probe GBS1 GBS2 (1) (9090)
5 of 22

Detection. The hybridization solution was removed and replaced by 100 ml washing solution I (2X 

SSC/0.1% SDS). The membrane was washed twice in washing solution I for 10 min at room temperature. 

This low stringency buffer was poured off and preheated high stringency washing solution II (0.5X 

SSC/0.1% SDS, 600C) was added immediately. The membrane was washed twice in washing solution II at 

60-63oC for 20 min each at 25-30 rpm. This was followed by a brief rinse with 2X SSC to remove SDS. 

The membrane was rinsed with 150 ml of 1X DIG Washing Solution (Roche) in a tray for 2 min and 

incubated in 1X Blocking solution (Roche) for 0.5-3 hrs on a low-speed shaker. The blocked membrane 

was incubated with DIG antibody solution (1:10,000 dilution of Anti-DIG-alkaline phosphate conjugate 

with 1X Blocking solution) for 30 min on a shaker. The membrane was washed twice (15 min each) with 

1X DIG Washing Solution (Roche) and equilibrated with 1X detection buffer. The detection reaction was 

carried out with 2 ml CDP-Star solution (1:100 diluted stock of CDP-Star with 1X detection buffer) for 5 

min. The membrane was wrapped in a plastic film and exposed to the Z-ray film in the dark. Depending 

on the experiment, multiple exposures were taken from 30 sec to 30 min. The films were developed 

with Konica SRX-101A Z-ray film developer. The developed films were scanned to obtain the final 

images. 

Confirmation of Internal Band Size and Identity

The migration rate of many high molecular weight bands (>3kb) isolated from potato plants have a 

tendency to run faster than the molecular weight markers. The discrepancy between band migration 

and markers can make it challenging to accurately determine the sizes of unknown bands, but also raises 

questions about the sizes of expected bands. A number of examples of this issue are presented in Figure 

A-2. In each of these gels, the molecular weight marker (DIGVII) consists of a ladder of lambda DNA 

labeled with DIG. The samples that follow all consist of plant genomic material (Russet Burbank or W8) 

where some samples have been spiked with pSIM1268 plasmid that has been digested with the 

indicated restriction enzymes. As shown in panel a, an EcoRV digest of a both plasmid DNA and W8 

genomic DNA produce a 2.3 kb band, which migrates as expected according the DIGVII markers. 

However, a HindIII digest results in a larger 4.2 kb band in both plasmid and W8. When compared to the 

MW ladder, it appears that the 4.2 kb band migrates faster than expected as it is close to the 3.6 kb 

marker band. The pSIM1278 plasmid has been sequenced to ensure the digested band is actually 4.2 kb 

and it appears to migrate appropriately when it is not spiked into plant DNA. Similarly, an EcoRI/ScaI 

digest generates 3.75 and 2.08 kb bands detected by GBS1 (panel c). The 2.08 kb band is specific to the 

plasmid and does not exist in the W8 structure; however, the 3.75 kb band is found in both where it also 

migrates faster than expected – faster than 3.6 kb. Again, spiked plasmid DNA co-migrates with the W8-

specific band when it is spiked into a sample containing potato DNA. It appears the discrepancy between 

migration of bands in potato samples and the ladders increases as a function of molecular weight and is 

more prevalent when electrophoresis is performed in TBE than in TAE based buffers.
6 of 22

Figure A-2. Southern blots showing anomalous migration of bands

Genomic DNA of Russet Burbank control (WT) and event W8 were digested with EcoRV, HindIII, and EcoR1/Scal and some 

samples were spiked with similarly digested pSIM1278 plasmid DNA. Samples were electrophoresed on 0.7% agarose (TAE) gels 

and hybridized with the GBS1 probe. 

PCR Confirmation of EcoRI/Scal Fragments in W8 

As further evidence that the faster migrating bands are correctly identified, we have PCR amplified and 

sequenced the 3.75 kb band shown in Figure A-2c. As shown in the Figure A-3, four primers were 

designed to amplify two fragments: (A) a 1653-bp fragment including EcoRI site (3717) and EcoRV site 

(5356); (B) a 2125-bp fragment including EcoRV sites (5356, 6581) and Scal site (7467). Since the PCR 

product is unique to the pSIM1278 construct, no amplification product was expected or obtained for 

Russet Burbank samples. As shown in the gel associated with Figure A-3, the expected bands were 

amplified in the W8 and plasmid control samples. Sequence analysis confirmed the identity and size of 

these bands. 

Collectively, these data show that samples isolated from potato plants have a tendency to alter the 

migration of higher molecular weight bands.

GBS1 (EcoRV)

WT WT WT W8

plasmid - + + -

GBS1 (HindIII)

WT WT WT W8

plasmid - + + -

GBS1 (EcoRI/ScaI)

WT WT WT W8

plasmid - + + -

2.3 kb

4.2 kb

10.6 kb

3.75 kb

2.08 kb

kb

2.8

1.52

1.48

8.6 

7.4

6.1

4.9

3.6

1.95

1.88

1.2

1.0

0.7

kb

2.8

1.52

1.48

8.6 

7.4

6.1

4.9

3.6

1.95

1.88

1.2

1.0

0.7

kb

2.8

1.52

1.48

8.6 

7.4

6.1

4.9

3.6

1.95

1.88

1.2

1.0

0.7

a b c
7 of 22

Figure A-3. The primers and PCR fragments to confirm 3753-bp EcoRI/Scal fragment of P-DNA.

Confirmation of a 3753-bp internal fragment of P-DNA in W8

Bands on the ethidium bromide-stained gels are indicative for the presence of fragment A and B. M = 1 kb DNA marker lane 

(Invitrogen), PC = positive control, WT = untransformed recipient variety (“wild type”), NC = negative control. Event W3 not 

included in this petition.

A

B

Fragment Forward primer Reverse primer Length

A CGAATTCGTGATGTGTGGTC GGATGTATGATATCACATGTGTTTG 1653 bp

B CAAACACATGTGATATCATACATCC ACTATTGCAAGAAAATATCAAAGGC 2125 bp

3753 bp

RB

RI (3717)

Sc(8247)

Hd (2444)Hd (6674)Hd (7987)

RV (1128)

RV (2353)

RV (3065)RV (5356)RV (6581)

pGbss pGbss Sc (7467)

LB pAgp pAgp (1) (194)

(10148)

tPpo5

pR1

tPpo5fAsn1

Spacer

fAsn1

pR1

pPhLpPhL

Spacer

3715-5367

5343-7467

bp

3,000

2,000

1,650

1,000

PC M W3 W8 WT NC

A B A B A B A B A B
8 of 22

1.2 Characterization of Vector Backbone Sequences

Plant material. Plants used for DNA gel blot analysis were grown for two months in Sunshine mix-1 

(www.sungro.com) in two-gallon pots in a greenhouse controlled for temperature (18°C minimum/27°C 

maximum) and light (16-h photoperiod with an intensity of ~1500 μmol/m2/s).

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a 

mortar using liquid nitrogen. The ground tissue was transferred to a pre-cooled 15 ml conical tube with 

a pre-cooled spatula and stored at -80oC until ready to process. 10 ml extraction buffer (0.35 M Sorbitol, 

0.1M Tris-HCl, pH 8.0, 0.05M EDTA) was mixed with the powder and the resulting suspension was 

centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was thoroughly re-suspended in 2 

ml extraction buffer containing 200 μg RNase A. After incubating the resuspended DNA at 65oC for 20 

min with 2 ml of nuclear lysis buffer (0.2 M Tris-HCl pH7.5, 0.005 M EDTA pH 8.0 and 20 mg/ml CTAB 

Hexadecyl Trimetyl Ammonium Bromide) and 800 μl of 5% Sarcosyl, it was mixed with an equal volume

of chloroform : isoamyl alcohol (24:1), vortexed for about 1 min, and centrifuged at 3000 rpm for 5 min 

at room temperature. The DNA was precipitated with an equal volume of isopropyl alcohol, washed 

with 70% ethanol, air dried, and dissolved in 400-700 μl 1X Tris/EDTA buffer (TE). DNA concentration 

was measured using a spectrophotometer at an OD of 260 nm, whereas quality was confirmed by 

examining the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE) for 30-40 min at 80 volts. In 

some cases, an alternative method was used to isolate DNA. The ground tissue from 0.7 g young leaves 

was mixed with 7 ml CTAB buffer (2% CTAB, 1.4 M NaCl, 0.1 M pH 8.0 Tris-HCI, 20 mM pH 8.0 EDTA, 100 

μg/ml RNase A) and incubated at 55-65oC for 30 min followed by centrifugation at 3,000 rpm for 15 min. 

DNA was extracted twice with equal volumes of chloroform: isoamyl alcohol (24:1) by shaking for 10 min 

and centrifuged at 3000 rpm for 5 min at room temperature. The DNA was precipitated with equal 

volumes of ethanol and rinsed with 70% ethanol. The resulting pellet was air dried and re-suspended in 

TE buffer. The quality and concentration of DNA was determined spectrophotometrically and by 

examining a sample on a 0.8% agarose gel. Control and event DNA samples were always extracted using 

the same methods.

Southern Blot - Gel Preparation. Digested DNA was electrophoresed on a large 0.7% agarose gel (170 

ml) containing 0.5X Tris-borate-EDTA (TBE) buffer and 3-5 μl ethidium bromide (10 mg/ml) for 18 hrs 

using 25 volts. The gel was photographed using a gel documentation system from Alpha Innotech (Santa 

Clara, California), and then depurinated by submersion in 0.25N HCl for 20 min. After subsequent 

denaturation in 0.5 M NaOH / 1.5 M NaCl for 2 x 15 min and neutralization in 1.5 M NaCl / 0.5 M Tris-HCl

(pH 7.5), for 2 x 15 min on a shaker at room temperature, the gel was equilibrated with 10X SSC for 10 

min. The transfer of DNA to the nylon membrane was carried out using 10X SSC using capillary transfer.

Southern Blot - Probe Preparation. Nylon filters cross-linked with DNA digested with EcoRI were 

hybridized independently with six different probes, each of which covered approximately 1-kb of the 

vector backbone (see Table A-1 for primer sequences and Figure A-4 for the linear arrangements of the 

probes described in Table A-1). Untransformed potato varieties were used as negative controls, and the 

T130 event carrying the entire backbone of pSIM1278 and pSIM1678 vectors provided positive controls. 
9 of 22

Table A-1. Backbone Probes for pSIM1278 and pSIM16781

Backbone

Probe

Size (bp) pSIM1278

Coordinates

Forward Primer (5 ́ to 3 ́) Reverse Primer (5 ́ to 3 ́)

1 1,474 10,149-11,622 ACTAGTTGTGAATAAGTCGCTGTG ATCGGAATCGACTAACAGAACAT

2 1,515 11,591-13,105 CCGGGGCCGATGTTCTGTTAG GCTCGCCGGCAGAACTTGAG

3 1,588 13,054-14,641 GCCGCGTGTTCCGTCCACAC CCTGTCGGGTTTCGCCACCT

4 1,660 14,614-16,273 CAAGTCAGAGGTGGCGAAAC CTTTATGCTCATTGGGTTGAGTA

5 1,074 16,590-17,663 AGTCCACCCGAAATATAAACAAC GGTATGGACCTGCATCTAATTTTC

6 832 18,827-19,658 GCTCTAATATAGCGCATTTCAAG GCTTCCAGCCAGCCAACAGCTC

1Numbering system based on pSIM1278, but backbone sequences identical for pSIM1678. 

Figure A-4. Probes for backbone testing and PCR primers for GapA and GapB amplification

1

Numbering system based on pSIM1278 (Chapter 4: Donor Genes and Regulatory Sequences, Table 4-1 and 

Figure 4-1), but backbone sequences are identical to pSIM1678. The backbone DNA is spanned by the probes 

shown as blue rectangles with two small gaps labeled as Gap A and Gap B detected by PCR using the indicated 

primer sets. The gapped regions exist in the potato genome and can be amplified using the internal primer sets, 

while the flanking set of primers is unique to the backbone DNA.

The labeling of PCR-derived probes were achieved using Hotmaster Taq enzyme and buffer (Fisher 

BioReagents) according to Roche’s DIG labeling protocol. A standard 50 μl reaction consisted of 5 μl of 

10X Hotmaster Taq Buffer, 2-5 μl of 10 μM forward primer, 2-5 μl of 10 μM reverse primer, 5 μl of DIG 

labeled dNTPs (Roche), 10 ng plasmid template, 0.75 μl Hotmaster Taq polymerase, and water. The PCR 

amplification conditions were optimized for each DIG-labeled probe. PCR with regular dNTPs instead of 

DIG labeled dNTPs was used as positive control. Quality of the DIG labeled probe was assessed by 

analyzing a fraction of the product on a 1% agarose gel alongside control (unlabeled) PCR product. The 

probe was denatured before use by incubating the probe at 100oC for 5 min, and then quenched on ice 

for 2 min.

Southern Blot – Hybridization and Exposure. The nylon membrane carrying transferred DNA was 

prehybridized in 40 ml pre-warmed DIG Easy Hybridization solution (Roche) at 42oC for 1-4 hrs in a 

hybridization oven (Amerex Instruments Inc.) rotating at 20-25 rpm. Hybridization was carried out by 

replacing the prehybridization buffer with a fresh amount of the same preheated solution containing 25-

50 μl denatured DIG labeled probe, and continuing the incubation with rotation (20-25 rpm) at 42oC for 

about 16 hrs. The probe-containing hybridization solution was stored (-20oC) and reused up to 3 times. 

1474 bp

Probe 1

Probe 4 Probe 5 Probe 6 Probe 2

1515 bp Probe 3

1588 bp

1660 bp 1074 bp 834 bp

Pat P pBR322 bom

PVS1 Sta

nptIII

pVS1Rep

pBR322 ori

Ubi3T RB LB

UBQmon

ipt

EcoRI (16227)

Ubi7

EcoRI (17793)

Y277 - Y278

Y279 - Y280

Y281 - Y282

Y283 - Y284

• Amplification from Potato

• No Amplification from Potato

Gap A Gap B
10 of 22 Backbone LB DNA insert RB Backbone

377 bp JY725 JY726

215 bp JY915 JY749

587 bp JY719 JY718

503 bp JY1029 JY1030

(Size is not at scale)

pSIM1278

Backbone LB DNA insert RB Backbone

377 bp

JY725 JY726

215 bp

JY915 JY749

405 bp Y179 Y180

pSIM1678

229 bp

Y181 Y182

The reused hybridization solution was heated at 68oC for 10 minutes before use. The hybridization 

solution was removed and replaced by 100 ml washing solution I (2X SSC/0.1% SDS). The membrane was 

washed twice in washing solution I for 10 min at room temperature. This low stringency buffer was 

poured off and preheated high stringency washing solution II (0.5X SSC/0.1% SDS, 600C) was added 

immediately. The membrane was washed twice in washing solution II at 60-63oC for 20 min each at 25-

30 rpm. This was followed by a rinse with 2X SSC to remove SDS. The membrane was rinsed with 150 

ml of 1X DIG Washing Solution (Roche) in a tray for 2 min and incubated in 1X Blocking solution (Roche) 

for 0.5-3 hours on a low-speed shaker. The blocked membrane was incubated with DIG antibody 

solution (1:10,000 dilution of Anti-DIG-alkaline phosphate conjugate with 1X Blocking solution) for 30 

min on a shaker. The membrane was washed twice (15 min each) with 1X DIG Washing Solution (Roche) 

and equilibrated with 1X detection buffer. The detection reaction was carried out with 2 ml CDP-Star 

solution (1:100 diluted stock of CDP-Star with 1X detection buffer) for 5 min. The membrane was 

wrapped in plastic film and exposed to the Z-ray film in the dark. Depending on the experiment multiple 

exposures were taken from 30 sec to 30 min. Films were developed with a Konica SRX-101A Z-ray film 

developer. The developed films were scanned to obtain the final images. 

PCR-Based Identification of Vector Backbone sequences. The standard 30 μl PCR reaction mixture 

consisted of 3 μl of 10X PCR buffer, 0.6 μl of 10 mM dNTPs, 0.6 μl of 10 μM forward primer, 0.6 μl of 10 

μM reverse primer, 100 ng of genomic DNA template, with 0.3 μl of HotMaster Taq polymerase (Fisher 

BioReagents). The PCR was carried out under the following amplification conditions: 1 cycle of 3 min at 

95°C followed by 40 cycles of 30 sec at 94°C, 30 sec at 55°C, 30 sec at 68°C, and finishing with 10 min at 

68°C. Primers were designed to amplify fragments indicative of (1) junctions between DNA insert border 

regions and flanking backbone DNA or (2) regions entirely within the backbone DNA that flank the DNA

insert. The primer pair JY725-JY726 amplified a 377-bp fragment comprising the junction at the Right 

Border region and flanking backbone, and primers JY915-JY749 were used to amplify a 215-bp backbone 

fragment flanking the Right Border of both pSIM1278 and pSIM1678 (see Figure A-5 and Table A-2). The 

primer pair JY718-JY719 amplified a 587-bp fragment comprising the junction at the Left Border region 

and flanking backbone of pSIM1278, and primers JY1029-JY1030 were used to amplify a 503-bp 

backbone fragment flanking the Left Border of pSIM1278 (see Figure A-5 and Table A-2). The primer pair 

Y179-Y180 amplified a 405-bp fragment comprising the junction at the Left Border region and flanking 

backbone of pSIM1678, and primers JY719-Y180 were used to amplify a 307-bp fragment also spanning

the Left Border of pSIM1678 and its flanking backbone sequence (see Figure A-5 and Table A-2).

Figure A-5. PCR primers for detecting backbone adjacent to left and right borders of pSIM1278 and 

pSIM1678
11 of 22

Table A-2. Primer sequences and PCR products for detection of backbone adjacent to left and 

right borders of pSIM1278 and pSIM1678

PCR Primers for Detecting Backbone Adjacent to Left Border of pSIM1278

Name Sequence (5 ́ to 3 ́) Location in pSIM1278

Product

length (bp) Backbone (bp)

JY719 GAGCTGTTGGCTGGCTGGAAG 19,637-1,9657 (backbone)

JY718 GTTGGAAATCAATTATCACTGAG 541-563 (AGP promoter) 587 24

JY1029 CCGTTCTTCCGAATAGCATC 19,507-19,526 (backbone)

503 154

JY1030 CGGGTTATCGGTTCTTAACG 330-349 (AGP promoter)

JY749 GAAGCCGACTGCACTATAGCAG 10,342-10,363 (backbone)

PCR Primers for Detecting Backbone Adjacent to Right Border of pSIM1278 and pSIM1678

Name Sequence (5 ́ to 3 ́) Location in pSIM1278

Product

length (bp) Backbone (bp)

JY725 GCTTCCCGTATACAACATAACATG 9,813-9,836 (GBSS promoter)

JY726 GATCTCAAACAAACATACACAGCG 10,166-10,189 (backbone) 377 41

JY915 ACTAGTTGTGAATAAGTCGCTGTG 10,149-10,172 (backbone)

JY749 GAAGCCGACTGCACTATAGCAG 10,342-10,363 (backbone) 215 215

PCR Primers for Detecting Backbone Adjacent to Left Border of pSIM1678

Name Sequence (5 ́ to 3 ́) Location in pSIM1678

Product

length (bp) Backbone (bp)

Y179 AAAGTCTGCCGCCTTACAAC 18,481-18,500 (backbone)

Y180 GGCGTTCGGTTCGATATTTA 264-283 (vnt1 promoter) 405 bp 122

JY719 GAGCTGTTGGCTGGCTGGAAG 18,579-18,599 (backbone)

Y180 GGCGTTCGGTTCGATATTTA 264-283 (vnt1 promoter) 307 bp 24

Six probes mentioned above were used to detect the backbone of plasmid pSIM1278 and pSIM1678. 

However, these probes do not cover entire backbone and two gaps are shown in the Figure A-4: Gap A 

between probe 4 and probe 5 and Gap B between probe 5 and probe 6. The blast reach revealed that 

the Gap A is potato ubi3 terminator (Garbarino and Belknap, 1995) and the Gap B is part of potato ubi7 

promoter (Garbarino et al. 1994). To confirm that the sequences of the gapped regions are originally 

from potato PCR assays are performed using primers annealing gapped regions and primers annealing 

the backbone sequences outside the gaps (Table A-3). As shown in the Figure 5-25 the entire Gap A and 

Gap B can be amplified by PCR from W8 and its untransformed control Russet Burbank using primers 

Y277/Y278 and Y281/Y282, respectively. But nothing was amplified from all samples except plasmid 

controls when primers (Y279/Y280 for gap A, Y283/Y284 for gap B) just outside the gaps were used. This 

result indicates that the sequences of Gap A and Gap B in W8 are from potato genome rather than from 

backbone of transformation vector pSIM1278. The PCR-amplified fragments were further cloned and 

sequenced. The results of sequence alignment also demonstrated that the sequences of Gap A and Gap 

B are from potato genome.
12 of 22

Table A-3. Primer sequences and PCR products for detection of Ubi3 terminator (GAP A) and partial 

Ubi7 promoter (GAP-B) in the backbone region of pSIM1278 and pSIM1678

Name Sequence (5 ́ to 3 ́) Location in pSIM1278

Product

length (bp)

Y277 CTATTTTTTTACTATATTATACTCAAC 16,232-16,257 (UGP3 terminator in backbone)

355

Y278 TTTTAATGTTTAGCAAATGTCTTATC 16,561-16586 (UGP3 terminator in backbone)

Y279 TTCAATTCCGGTGATATTCTCA 16,005-16,026 (backbone)

Y280 AGTTGTTTATATTTCGGGTGGA 16,592-16,613 (backbone) 609

Y281 GATCCACCTCCACGTAGACGGAG 17,668-17,690 (UGP7 promoter)

Y282 GAAATGCGCTATATTAGAGCATA 18,824-18,846 (UGP7 promoter) 1,179

Y283 TATCGCGGTCGTCGTCTTTC 17,604-17,623 (ipt gene in backbone)

Y284 GGTCATGTTCATGATAAACTTG 18,846-18,867 (UGP7 promoter) 1,264

1.3 Analysis of Genetic Stability

Materials. DNA insert stability was demonstrated in the originally-transformed material (G0) by 

extracting and evaluating DNA from leaves of plants that had been propagated in vitro and never 

planted in soil. For generation 1 (G1) and generation-2 (G2) analyses, leaves of two plants from each 

event and one plant from each control were collected from an Ephrata, WA field trial. For the 

generation 3 (G3) analysis, leaves of two plants from each event and one plant from each control were 

collected from plants grown in a green house. The G2 tubers of unmodified Russet Burbank control and 

W8 were harvested from Rupert ID, Northwood ND, and Ephrata WA field trial sites and were used for 

the catechol assay.

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a 

mortar using liquid nitrogen. The ground tissue was transferred to a pre-cooled 15 ml conical tube with 

a pre-cooled spatula and stored at -80°C until ready to process. 10 ml extraction buffer (0.35 M Sorbitol, 

0.1 M Tris-HCl, pH8.0, 0.05M EDTA) was added to, and mixed with, the powder, and the resulting 

suspension was centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was thoroughly 

re-suspended in 2 ml extraction buffer containing 200 μg RNase A, mixed with 2 ml of nuclear lysis 

buffer (0.2 M Tris-HCl pH7.5, 0.005 M EDTA pH 8.0 and 20 mg/ml CTAB Hexadecyl Trimetyl Ammonium 

Bromide) and 800 μl of 5% Sarcosyl. The resuspended DNA was incubated at 65oC for 20 min, mixed with 

an equal volume of chloroform: isoamyl alcohol (24:1) by vortexing for 1 min, and centrifuged at 3,000 

rpm for 5 min at room temperature. The DNA in the upper layer was precipitated with an equal volume 

of isopropyl alcohol, washed with 70% ethanol, air dried, and dissolved in 400-700 μl 1X Tris/EDTA 

buffer (TE). DNA concentration was measured using Qubit Fluorimetic Quantitation (Life Technologies), 

whereas quality was confirmed by running the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE) 

for 30-40 min at 80 volts. 

Southern Blot Analysis. Extracted genomic DNA from leaves was either digested with EcoRV and 

hybridized with two probes (GBS and AGP) or digested with XbaI and hybridized with two probes (VNT1 

and INV). The probes used in the Southern blot analysis are depicted in Figure A-6. 3 μg of DNA was 

digested overnight in 400 μl final volume with at least 5 μl (10 units/μl) restriction enzyme (Invitrogen) 

at 37°C. Digested DNA was concentrated by ethanol precipitation (40 μl of 3M NaOAc, pH5.3 and 1 ml 

ethanol) at -80oC for 10 min followed by a wash with 70% ethanol. The DNA pellet was dissolved in 20 μl 
13 of 22

1X TE followed by addition of 2 μl DNA gel loading buffer, which consists of 40% sucrose and 0.35% 

Orange G (Sigma) in water.

Figure A-6. Probes Used for the Southern Blot Stability Analysis

Membrane Preparation. Digested DNA was loaded on a large 0.7% agarose gel (170 ml) containing 0.5X 

TBE buffer and 3-5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 hrs. The gel was 

photographed using a gel documentation system from Alpha Innotech (Santa Clara, California), and then 

depurinated by submerging it into 0.25 N HCl for 20 min. After subsequent denaturation in 0.5 M NaOH 

/1.5 M NaCl for 2 X 15 min and neutralization in 1.5 M NaCl and 0.5 M Tris-HCl, pH7.5, for 2X15 min on a 

shaker at room temperature and equilibrated with 10X SSC for 10 min. The transfer of DNA to the nylon 

membrane was carried out using 10X SSC according to a standard capillary transfer method (Sambrook 

and Russell 2001). 

2.0 Characterizing Rpi-vnt1 and VNT1

The following methods were used to generate the data presented in Chapter 6: Characterization and 

Safety of the VNT1 Protein and Appendix B: Characterization of VNT1, as part of the biochemical and 

molecular characterization of the Rpi-vnt1 gene and associated VNT1 protein.

Russet Burbank, W8, and S. venturii Tissue Sources. Greenhouse grown plants were used as source 

materials for all analyzed tissues. S. tuberosum v. Russet Burbank was used as a negative control for 

VNT1 expression and is susceptible to late blight. S. venturii was used as a positive control for VNT1 

expression and is resistant to late blight. W8 expresses the Rpi-vnt1 gene encoding VNT1 and is resistant 

to late blight. Leaf, tuber (or stolon), stem, root, and flower tissues were harvested, ground under liquid 

N2, and stored at -80°C.

Expression Vectors, Primers, Chemicals, and other Reagents. pMAL expression vectors and restriction 

enzymes were purchased from New England BioLabs (NEB) (Ipswich, MA); pCR-Blunt-TOPO TA cloning 

kit and PCR primers from Life Technologies (Grand Island, NY); Mini-prep kits from Qiagen 

(Germantown, MD); DNA sequencing from Sequetech (Mountain View, CA); Complete Protease Inhibitor 

Cocktail Tablets from Roche (Indianapolis, IN); Chromatography columns and systems from GE 

Healthcare Life Sciences (Piscataway, NJ); and Amicon Centrifugal Filter Units from Millipore (Billerica, 

MA). All gel and blotting apparatus were purchased from Bio-Rad (Hercules, CA). All other chemicals, 

L

B

R

B pVnt1 vnt1 tVnt1 pAgp inv inv pGbs

Probes VNT1 AGP INV INV GBS

A) pSIM1278 Insert

B) pSIM1678 Insert

> > <
14 of 22

reagents, and laboratory supplies were purchased from Sigma-Aldrich (St. Louis, MI), Fisher Scientific 

(Pittsburg, PA), or Research Products International (Mt. Prospect, IL).

Construction of E. coli Expression Vectors for Full-length Rpi-vnt1 and Rpi-vnt1 Fragments. The pMALc5x and pMAL-p5x vectors (NEB) were modified to introduce a BsaI restriction site into the multiple 

cloning site. A BsaI-containing PCR fragment with 5 ́-BglII and 3 ́-EcoRI restriction sites was amplified 

from the pMAL vector template using forward primer: 5 ́-GCTGATTTATAACAAAGATCTGCTG-3 ́ (BglII 

restriction site underlined) and reverse primer: 5 ́-

AGAATTCCGATCGAGACCCCGAGGTTGTTGTTATTGTTAT-3 ́ (EcoRI and BsaI restriction sites underlined) 

and ligated into the pCR-Blunt-TOPO vector using a TA cloning kit (Life Technologies). The introduced 

BsaI restriction site and BglII and EcoRI restriction sites were confirmed by DNA sequencing (Sequetech). 

Clones containing the BsaI restriction site were generated by digestion of the pCR-Blunt-TOPO vectors 

and ligation into the pMAL-c5x and pMAL-p5x plasmids using BglII and EcoRI restriction enzymes. This 

resulted in the modified vectors pMAL-c5x-BsaI and pMAL-p5x-BsaI. The Rpi-vnt1 gene and gene 

fragments were obtained by PCR amplification of the Simplot vector pSIM1678, which contains the Rpivnt1 (FJ423044.1) gene, using appropriate primer pairs (Table A-4). The amplified Rpi-vnt1 fragments 

were ligated into the pCR-Blunt-TOPO vector using a TA cloning kit (Life Technologies). Each Rpi-vnt1

fragment was verified by DNA sequencing (Sequetech). Clones containing the Rpi-vnt1 fragments were 

generated by digestion of the pCR-Blunt-TOPO vectors and ligation into the pMAL-BsaI vectors using 

BsaI and EcoRI restriction enzymes. Full-length Rpi-vnt1 contains an internal BsaI cut site and was cloned 

using a 3-piece ligation strategy with fragments containing 5 ́-BsaI/3 ́-PstI and 5 ́-PstI/3 ́-EcoRI 

restriction sites.

Table A-4. PCR Primers for Cloning Rpi-vnt

Rpi-vnt1 Fragment Primer Sequence

Rpi-vnt1 (1-891)-5’ For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-CTTAACATGCCTGCAGTCACCACAAT-3 ́

Rpi-vnt1 (1-891)-3’ For 5 ́-ATTGTGGTGACTGCAGGCATGTTAAG-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Rpi-vnt1 (1-125) For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-TGAATTCTTAATTGGATTGTTGAATTTTTGG-3 ́

Rpi-vnt1 (126-544) For 5 ́-AGGTCTCCATCGAGGGAAGGAAGTTCATTTGTTGCCTTAAGAC-3 ́

Rev 5 ́-TGAATTCTTACCTAGCAACATTGCTAGG-3 ́

Rpi-vnt1 (545-891) For 5 ́-AGGTCTCCATCGAGGGAAGGGTGCGAAGGATTACATTCTAC-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Rpi-vnt1 (1-544) For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-TGAATTCTTACCTAGCAACATTGCTAGG-3 ́

Rpi-vnt1 (126-891) For 5 ́-AGGTCTCCATCGAGGGAAGGAAGTTCATTTGTTGCCTTAAGAC-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Primers for PCR Amplification of Full-Length Rpi-vnt1 (1-891) and Rpi-vnt1 Fragments from pSIM1678. 

BsaI recognition sequence highlighted in gray; EcoRI in blue; PstI in yellow; stop and start codons for 

each Rpi-vnt1 gene fragment shown in red.

Expression and Purification of VNT1. Recombinant VNT1 was expressed and purified as a fusion with 

the Maltose Binding Protein (MBP)-VNT1 for the full-length VNT1 (1-891) and the LRR domain, VNT1-LRR 

(545-891). The pMAL-BsaI vectors containing the Rpi-vnt1 coding sequences were transformed into BL21 
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E. coli cells (NEB). Cells were grown in Luria-Bertani broth supplemented with ampicillin (100 μg/ml) to 

an O.D.600 = 0.5. VNT1 expression was induced by adding 1 mM Isopropyl-β-D-galactopyranoside. 

Expression levels of VNT1 were determined using multiple induction temperatures (17°C and 37°C) and 

times (2-8 hrs. and overnight). Full-length VNT1 (1-891) was maximally expressed by overnight 

incubation at 37°C. VNT1-LRR (545-891) was maximally expressed from 2 hr. incubations at 37°C. Cell 

cultures were spun at 4000 x g at 4°C for 30 min. The pelleted cells were resuspended in 50 mM Tris-HCl 

(pH 7.5) buffer containing 1 mM EDTA, 500 mM NaCl, 10% glycerol and Complete Protease Inhibitor 

Cocktail Tablets (Roche) and lysozyme (2 mg/ml). The cells were sonicated for 2 min. using a Branson 

Probe Sonifier (Branson; Danbury, CT). Extracts were spun at 24,000 x g at 4°C for 30 min. and the 

supernatant loaded onto a 3-ml amylose column. The purified MBP-VNT1 fusion protein was eluted with 

10 mM maltose and concentrated using an Amicon Centrifugal Filter Unit (Millipore) to a volume of ~1 

ml. A final purification step was performed using a HiPrep 16/60 Sephacryl S-200 HR column (GE 

Healthcare).

Transient Expression of VNT1 in N. benthamiana by Agroinfiltration. Infiltration was performed as 

described previously (22). Briefly, N. benthamiana seeds were germinated (1-2 weeks) in a petri dish 

with Whatman filter paper (Sigma-Aldrich, St.Louis, MO). Seedlings were transferred to 4-inch pots with 

Sunshine Professional Growing Mix LC1 (Sun Gro Horticulture; Vancouver, Canada). The plants were 

grown 4-6 weeks at 22°C in 50% humidity with 16- and 8-h light-dark cycling and regular watering. 

Agrobacterium tumefaciens strain AGL1 harboring a VNT1 expression vector was used for infiltration. A. 

tumefaciens cultures were prepared by overnight incubation at 28°C with kanamycin (50 μg/mL) and 

carbenicillin (100 μg/mL). Growth was spectrophotometrically monitored to O.D.600 = 0.1-0.2 (1:10 

dilution). Cells were pelleted by centrifugation and resuspended in 10 mM Tris-HCl (pH 7.5) buffer 

containing 10 mM MgCl2 and washed. Cultures were diluted to O.D.600 = 0.25 in the same buffer and 

injected at 2-6 sites on the underside of N. benthamiana leaves using a syringe. Leaves were harvested 

2-3 days post-infiltration, ground in liquid nitrogen using a mortar and pestle, and stored at -80oC.

Protein Extraction from Potato Tissues. A combination of trichloroacetic acid (TCA)/acetone washes 

together with phenol extraction and ammonium acetate/methanol precipitation was used to extract 

proteins from collected tissue materials. 0.3-0.5 g powered tissue material was washed with acetone, 

dried, and ground with mortar and pestle. The fine powder was washed with 20% TCA/acetone followed 

by 20% TCA/H2O. The pellet was dissolved at a 1:1 ratio in 100 mM Tris-HCl (pH 7.5) buffer containing 

2% SDS, 5% β-mercaptoethanol, and 30% sucrose:phenol. Following centrifugation, the phenol phase 

was removed, mixed with ammonium acetate/methanol, and incubated at -20°C overnight. The 

precipitated proteins were pelleted by centrifugation and washed in ammonium acetate/methanol and 

acetone. After drying the proteins were suspended in Tris-HCl (pH 7.5) buffer containing 1 mM EDTA and

10% glycerol. Protein was quantified using the BCA assay (Thermo Scientific).

Preparation and Characterization of Antibodies. Polyclonal antibodies were generated using New 

Zealand White Rabbits by Anaspec (Fremont, CA) with two animals per antigen. Several peptides of 

VNT1 (MSQKGYQHVTFPKKIG (antibodies 5361, 5362), FHSSSKLPFGVWESKIL (5363, 5364), 

KLKKLFLVQGPNISPISLR (5365, 5366), CRTTYSITDTSNNNDD (5467, 5368), VNTGNGREAESLADDC (5469, 

5370), and AKRTYDGRISSC (5471, 5472) were selected as antigens based on uniqueness to VNT1 

compared to homologous proteins and antigenic properties. Peptides were conjugated to keyhole 

limpet hemocyanin:bovine serum albumin (7.0:1.5 (w/w)) before injection. A standard 70-day protocol 

was extended and final bleeds were collected 100 days post-immunization (initial injection + 6 boosts; 

antibodies 5361-5366). A 28-day “speedy” protocol was followed for antibodies 5467-5472. Final bleeds 

were collected 28 days post-immunization (initial injection + 4 boosts). The final bleeds were affinity 
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purified by peptide-coupled thiopropyl-sepharose 6B chromatography for all antibodies except 5362, 

5364 and 5366. A second strategy for raising a polyclonal antibody against VNT1 used the purified VNT1-

LRR (545-891) as antigen. Antibodies were raised in New Zealand White rabbits (specific-pathogen free) 

by Pierce Biotechnology (Rockland, IL) using the company’s standard 90-day protocol, with two animals 

per antigen. Purified VNT1-LRR (545-891) was run on an SDS-polyacrylamide gel electrophoresis (PAGE) 

gel and a gel slice containing the ~80 kDa VNT1-LRR (545-891) band was cut out and sent for use as 

VNT1 antigen. The first injection used 0.5 mg of antigen. After 14, 42 and 56 days each rabbit was 

boosted with another 0.5 mg of the same adjuvant. Fourteen days following the boost, serum was 

collected and processed from each rabbit. Characterization of all antisera was done by SDSPAGE/immunoelectrophoretic blotting. 

VNT1 Western Blot Detection. Proteins were separated by SDS-PAGE and transferred to PVDF 

membranes using a wet tank transfer system in 25 mM Tris-HCl (pH 8.3) buffer, 192 mM Glycine, and 

20% methanol (transfer buffer). The SDS-polyacrylamide gel was soaked in transfer buffer for 15 min. 

PVDF membrane was soaked in methanol for 5 min. and then in transfer buffer for 10 min. The transfer 

apparatus was assembled according to manufacturer’s instructions and run at 100V for 1.5 hr. or for 50V 

at 3 hr. The membrane was placed in transfer buffer with 0.1% Tween-20 and 5% non-fat milk added 

(blocking buffer) for 1 hr. at room temperature. Three 5 min. washes in transfer buffer + 0.1% Tween-20 

(wash buffer) were performed before addition of primary antibody. For anti-MBP a 1:10,000 (v/v), and 

for anti-VNT1 a 1:1,000 (v/v) dilution was used. The membrane was incubated with primary antibody for 

30 min. and washed three times for 5 min. each in wash buffer. The secondary antibody (anti-rabbit-IgGperoxidase; Sigma-Aldrich) was added at a dilution of 1:20,000 (v/v). The membrane was incubated for 

30 min. and again washed three times for 5 min. each. SuperSignal® West Femto Maximum Substrate 

(Thermo Scientific) was added and after 5 min. blots were imaged with a Bio-Rad Chemi-Doc XRS+ 

system (Bio-Rad).

Identification of MBP-VNT1 (LRR) and GroL from E. coli. An Agilent 1200 HPLC coupled to an Agilent 

6320 Ion-Trap Mass Spectrometer with an ESI ionization source was used for peptide separation and 

analysis. Digested samples of purified VNT1-LRR were separated by reversed-phase chromatography 

using an Aeris Peptide 3.6 μm-C18-250 mm column (Phenomenex). Peptides were eluted at a flow rate 

of 0.3 mL/min. with a 15 min. gradient from 5% to 40% Buffer B (Buffer A = 0.1% formic acid with 

0.001% TFA, Buffer B = 0.1% formic acid in acetonitrile). The mass spectrometer was operated in 

positive ion mode with dry gas at 350C, N2 flow at 10 mL/min., and 30 psi. The data were analyzed 

using the MS/MS Mascot database (Matrix Science, Boston, MA).

3.0 Gene Expression Studies

The following methods were used to generate data presented in Chapter 6: Characterization and Safety 

of the VNT1 Protein and Chapter 7: Characterization of Gene Silencing and Target Gene Expression, to 

characterize expression of the Rpi-vnt1 gene and gene silencing targets.

Northern Blot Hybridization

Plant material. Gene expression levels were determined by carrying out northern blot analyses on 

tubers, roots, stems, leaves, and flowers of field-grown plants of W3, W8 and Russet Burbank controls. 

RNA Isolation. RNA was extracted from 1 g of tuber, and root tissue using Plant RNA reagent 

(Invitrogen, Carlsbad, California), whereas Trizol reagent (Invitrogen) was used to extract RNA from 
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leaves, flowers and stems. The concentration of isolated RNA was measured spectrophotometrically at 

260 nm, and RNA quality was confirmed by running a sample on a 1% agarose gel with 1X MOPS buffer 

(200 mM MOPS, 50 mM NaOAc , 20 mM EDTA , pH 7.0) for 30-60 min at 90 volts. 

RNA transfer. 20-25 g of total RNA was denatured in RNA loading dye for 10 min at 650C followed by 

incubation on ice for 5 min. Denatured RNA was loaded onto a 1% agarose gel (1X MOPS, 0.1-0.25 g/ml 

ethidium bromide and 2% formaldehyde). The gel was run at 80-85 volts for 2-3 hrs and then 

photographed using the gel documentation system from Alpha Innotech (Santa Clara, California). The 

gel was soaked twice in 10X SSC for 15 min to remove the formaldehyde. RNA was transferred from to a 

positively charged nylon membrane (Roche, Indianapolis) by capillary blotting with 10X SSC for 16-18 hrs

and stabilized by UV cross-linking (UVP, Upland, California). The RNA-containing membrane was stored 

at 40C until further use.

Preparation of DIG labeled Probe. A PCR-based method was used to prepare DIG-labeled probes for 

four target transcripts and an internal control of 18S rRNA. The primers used for PCR amplification and 

the lengths of probes are listed in Table A-5. A typical 50 μl labeling reaction consisted of 5 μl HotMaster 

Taq Buffer (Fisher BioReagents), 2-5 μl of 10M forward primer, 2-5l of 10 μM reverse primer, 5μl 

DIG-labeled dNTP (Roche, Indianapolis), 5-30 ng plasmid template, 0.50-0.75 μl HotMaster Taq 

polymerase, and dH2O for a total volume of 50 μl. PCR conditions were specific for each DIG-labeled 

probe. The DIG-labeled probe was checked for size and purity on a 1% agarose gel where it runs slower 

than the control PCR product due to the presence of DIG. The probe was heat denatured before use (5 

min at 1000C) and quenched on ice. 

Hybridization. Nylon membranes containing transferred RNA were pre-hybridized in 40 ml pre-warmed 

DIG Easy Hybridization solution (Roche, Indianapolis) for 1-4 hrs at 20-25 rpm in a hybridization oven set 

at 42OC (Amerex Instruments). The hybridization solution was replaced by a mix of 40 ml fresh prewarmed hybridization solution and 25-50μl of denatured DIG-labeled probe, and the membrane was 

incubated in this mix for 3-16 hrs at 42OC. The hybridization solution was stored at -200C and reused. 

The reused hybridization solution was heated at 680C for 10 minutes before use.
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Table A-5. Primers and Probes Used for Northern Analysis of Potato Genes

Probe 

name

Hybridization 

target

Primer sequences Probe size 

(bp)

ASN1 Asn1 transcript 5 ́-GGTTGATGACTGATGTCCCCTTTG-3 ́

5 ́-TAGTTAGCTCCTTATTGTGAGCTC-3 ́

1115

PPO1Ppo5 transcript 5 ́-ATCTTCCACTCCTAAGCCCTCTCAAC-3 ́

5 ́-CCGCCAAAGAACATCCGAGG-3 ́

864

PHL PhL transcript 5 ́-CAATTCCAGATTCATCCATTTCAC-3 ́

5 ́-CATCCCCAGGGTAGAGTATGTAAC-3 ́

968

R1

VInv

R1 transcript

VInv transcript

5 ́-ACCAGGGATTCCTAACCTCAAC-3 ́

5 ́-CAGGTATATCACTCTTTGTTAC-3 ́

5 ́-ACCCAGTCCAGACACCGTTA-3 ́

5 ́-TACACATTCCTCCCGGATCA-3 ́

852

496

18s 18s rRNA 5 ́-GCATTTGCCAAGGATGTTTT-3 ́

5 ́-GTACAAAGGGCAGGGACGTA-3 ́

694

1

The PPO probe was derived from a cDNA of the Ppo5 gene of S. tuberosum, which is highly expressed in tubers. 

The probe also hybridized with Ppo genes expressed in other tissues. The sequence in pSIM1278 used to silence 

the Ppo5 gene consists of the untranslated trailer of a Ppo5 gene from S. verrucosum.

Detection. The hybridization solution was removed and replaced by 100 ml washing solution I (2X 

SSC/0.1% SDS). Nylon membranes were washed twice in washing solution I for 10 min at RT in the 

hybridization oven at 25-30 rpm. This low stringency buffer was poured off and preheated high 

stringency washing solution II (0.5X SSC/ 0.1% SDS) was immediately added. The membrane was washed 

twice in washing solution II at 600C for 20 min each at 25-30 rpm. This was followed by a wash for 20 

min with washing solution III (0.1X SSC/ 0.1% SDS) at 650C and a brief rinse with 2X SSC to remove SDS. 

The membrane was rinsed with 100 ml 1X DIG Washing Solution (Roche, Indianapolis) in a tray at room 

temperature for 2 min. and incubated in 1X Blocking solution (Roche, Indianapolis) for 30 min-3 hrs on a 

shaker. The blocked membrane was incubated with DIG antibody solution (1:10,000 dilution of Anti-DIGalkaline phosphate conjugate with 1X Blocking solution, Roche, Indianapolis) for 30 min on a shaker. The 

membrane was washed twice (15 min each) with 1X DIG Washing Solution and equilibrated with 1X 

detection buffer. The detection reaction was carried out with 2 ml CDP-Star solution (1:100 diluted stock 

of CDP-Star with 1X detection buffer) for 5 min. The membrane was wrapped in a plastic film and 

exposed to Z-ray film in the dark. Depending on the experiment multiple exposures were taken from 30 

sec to 10 min. The films were developed with Konica SRX-101A X-ray film developer. The x-ray films 

were scanned to obtain electronic images. 

Reverse Transcriptase quantitative PCR (RT-qPCR) 

Plant Materials. Greenhouse grown materials (stem, root, tuber, leaf and flower) were collected from 

W8 and untransformed Russet Burbank plants and used for RNA isolation. 

RNA Isolation. RNA was isolated as above and then treated with DNase I (Qiagen). Approximately 20 μg 

RNA was added to 10 μl RDD Buffer (Qiagen), 4.0 μl DNase I (Qiagen) and DEPC water up to 100 μl and 

allowed to sit at room temperature for 10 minutes. RNA was cleaned using the RNeasy Kit (Qiagen) 

according to the manufacturer’s instructions. 

cDNA Synthesis. cDNA and negative controls (No-RT) were made by combining 16 μl 5X iScript reverse 

transcription reaction mix or No-RT Control Mix (Bio Rad), 3 μg RNA and DEPC water to 80 μl on ice. 

Reactions were then incubated at 25oC for 5 minutes, 42oC for 30 minutes, and 85oC for 5 minutes and 

cDNA concentration was measured with the Qubit ssDNA Assay Kit (Invitrogen). 
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RT-qPCR. To establish the baseline expression of Rpi-vnt1 in these materials, RT-qPCR was performed 

using primers specific to endogenous reference genes, Elongation Factor 1α and α-Tubulin, in addition 

to primers for Rpi-vnt1 (Table A-6). RT-qPCR reactions included 10 ng of cDNA, 10 μl of 2X SSO 

Advanced SYBR Green Supermix (Bio Rad), 0.4 μM final concentration of each primer and HPLC-grade

water up to 20 μl. RT-qPCR cycling parameters were 98.0°C for 2 minutes followed by 35 cycles of 

98.0°C for 5 seconds, 50.0°C for 15 seconds and 56.0°C for 60 seconds. Reactions were then heated to 

95.0°C for 10 seconds and a melting curve analysis was performed from 65.0°C to 95.0°C, increasing in 

increments of 0.5°C every 5 seconds. No-RT controls and no template controls were included to ensure 

that there was no contaminating DNA in the reactions. All samples included three technical replicates 

along with a standard curve to ensure qPCR parameters were appropriate. Expression was normalized 

to the endogenous reference genes. 

Table A-6. Primers Used for qPCR

Probe name Primer sequences

Elongation Factor 1α 5 ́- GTTGGTGTTATCAAGAGT -3 ́

5 ́- CATAGCAAGTTCTCGTAA -3 ́

α-Tubulin 5 ́- AATTTGTCGACTGGTGTCCT -3 ́

5 ́- GTCAATGCGAGAGAAGACCT -3 ́

Rpi-vnt1 5 ́- AGAGACCCTGGATATATTTCATAGCTCT -3 ́

5 ́- ATCAGCATTGAGCCCTGTGCCTAGAGCG -3 ́

4.0 Sequence Analysis

Potato material. Leaves from greenhouse-grown event W8 and untransformed Russet Burbank plants 

were used to extract DNA and characterize the DNA insert junctions.

DNA Extraction Method. DNA was isolated from leaf material using a modified CTAB protocol. Briefly, 

1g leaf tissue was ground under liquid nitrogen then suspended in extraction buffer containing 50mM 

EDTA, 0.1M Tris-HCl, pH 8.0, and 0.35M Sorbitol. Tissue was pelleted by centrifugation, rinsed with 

extraction buffer, and resuspended in 2mL extraction buffer with 100 μg/mL RNase A. An equal volume 

of lysis buffer containing 0.2M Tris-HCl, pH 8.0, 50mM EDTA, 2M NaCl, 2% CTAB, and 2% Sarcosyl was 

added and incubated 20 min. at 65 °C. DNA was extracted with two rounds of 24:1 chloroform: isoamyl 

alcohol followed by precipitation in Isopropanol, centrifugation, and a single wash with 70% Ethanol. 

Purified genomic DNA was resuspended in TE, pH 8.0 and quantified using a Qubit 2.0 Fluorometer 

(Molecular Probes) with the dsDNA Broad Range Assay kit (Molecular Probes).

Adapter ligation-mediated PCR Junction fragments were amplified by PCR using digested DNA ligated 

with adapter primers AP1 and AP2 as described by O’Malley et al. (2007) and detailed below. The 

amplified product containing a DNA insert primer binding site was selected by a secondary PCR reaction. 

The sequences of DNA insert primers used to analyze RB and LB junctions are shown in Tables A-1 and 

A-2, respectively. Plant DNA was isolated using either the CTAB method or the Qiagen Plant DNAeasy kit: 

200-500 ng DNA was digested for 3-5 hours with a restriction enzyme for which an adapter had been 

designed (EcoRI, HindIII, BamHI, AseI/NdeI, etc.) in a total volume of 20 μl. The digested DNA (3 μl) was 

ligated with its respective adapter (1 μl) in a mixture containing 0.5 μl T4 DNA ligase, 0.3 μl ATP, 1.0 μl 

10X ligation buffer, and 4.2 μl water. The primary PCR was carried out using the first DNA insert specific 
20 of 22

primer and AP1 with Hot Master Taq polymerase (Fisher BioReagents) under the following amplification 

conditions: 1 cycle of 3 min at 95°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 60°C, 2.5 min at 

68°C, and finishing with 10 min at 68°C. A 1 μl aliquot of the primary PCR product was used for 

secondary reactions ( 1 cycle of 3 min at 95°C; 35 cycles of 30 sec at 94°C, 30 sec at 62°C, 2.5 min at 

68°C; 1 cycle of 10 min at 68°C) with the second DNA insert specific primer and AP2. This protocol was 

performed at the same time for non-transformed control plants and the events. Products of the 

secondary PCR were run on 1% agarose in TAE buffer. Bands unique for the events were gel-extracted 

using a Qiagen QIAquick Gel Extraction kit, cloned into pGEM-T Easy vector (Promega, Madison WI), and 

sequenced. Primers specific for DNA insert-flanking DNA were used, together with a DNA insert primer, 

to amplify junction fragments.

Thermal asymmetric interlaced (TAIL) PCR. This method was used as an alternative to adapter ligationmediated PCR. Three series of reactions using AD (Arbitrary Degenerate) and SP (nested sequencespecific) primers were carried out to obtain DNA insert junction fragments. The standard 20 μl reaction 

mixture consisted of 2 μl 10X PCR buffer, 0.4 μl 10 mM dNTP, 0.4 μl 10 μM SP1, 5 μl 8-16 μM AD, 1 μl 

template, with either 0.1 μl HotMaster Taq polymerase (extension temperature 68 °C) or TaKaRa Ex 

TaqTM (TAKARA BIO Inc). A 1 μl aliquot of 50ng/μl leaf DNA was used as template for the first PCR; 1 μl of 

a 1:50 dilution of the first reaction was used as template for the second PCR; and 1 μl of 1:50 dilution of 

the second reaction was used for the third PCR. The second and third reactions were run on a gel, and 

bands with sizes that changed between the second and third reactions were cloned into pGEM-T Easy 

vector (Promega, Madison WI) and sequenced. Primers specific for DNA insert-flanking DNA were used, 

together with DNA insert-specific primers to clone junction fragments that were sequenced to confirm 

their identity.

Agilent SureSelect Probe design. A concatenated fasta sequence file was created from pSIM1278 and 

pSIM1678 for probe design. Virtual, potential probes were assessed for possible repetitive hybridization 

using lastz alignment tool, within the Galaxy package, performed on the Amazon Cloud. Since the 

regions that are highly repetitive are short (<400bp) they can be excluded from the probe design 

without negatively affecting resequencing coverage. One exception is the LB-AGP junction, which is 

~600bp long and therefore this exclusion will allow only a small chance of assembling >100bp of 

sequence data upstream of the LB insertion point. After repetitive sequences were “masked”, or 

removed from the probe design, 53 probes with >70% GC were “boosted”, or replicated to counteract 

low sequence coverage generally seen with high GC content regions. The order was placed with Agilent 

and received as synthesized, biotinylated RNA probes 6 weeks later.

Illumina library preparation and SureSelect capture HMW gDNA was sheared by Bioruptor (Diagenode) 

to ~650bp and size-selected by agarose gel electrophoresis and QiaexII (Qiagen) purification. Libraries 

were end-repaired and A-tailed (New England Biolabs NEBNext reagents and protocol), before ligation 

to custom barcoded Illumina-compatible adapters. Barcoded identification prior to PCR amplification is 

important to minimize cross-contamination. Ligation products were size-selected by agarose gel 

electrophoresis and PCR amplified. The library was prepared and captured (Agilent SureSelect reagents 

and protocol), using homemade potato Cot1 blocking DNA. Illumina-compatible libraries were 

quantified and pooled to mix barcodes, and submitted for sequencing externally. Paired-end 150bp and 

paired-end 100bp sequencing was conducted by HiSeq (Illumina) at two academic core sequencing 

facilities.

Bioinformatics analysis. Illumina data was processed by splitting samples by barcode and trimming for 

quality. The reads were aligned with CLC Genomics Workbench to Sanger sequenced PCR products to 
21 of 22

confirm support for novel junctions in the candidate transgenic line, compared with other transgenic 

lines prepared at the same time. Independent PCR products are distinguished from clonal PCR amplicons 

due to the random shearing prior to library preparation.
22 of 22

5.0 References

Garbarino JE and Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato 

and expression of its promoter in transgenic plants. Plant Molecular Biology 24:119-127.

Garbarino JE, Oosumi T, and Belknap WR (1995) lsolation of a Polyubiquitin Promoter and Its Expression 

in Transgenic Potato Plants. Plant Physiol 109: 1371-1378.

O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated PCR method for 

high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nature Protocols, 2:2910-2917.

Sambrook J, Russell DW (2001) Southern Hybridization. In: Molecular Cloning: A Laboratory Manual. (Ed. 

Argentine J) Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY (pp. 6.33-6.38). 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 1

J.R. SIMPLOT COMPANY

Appendix B: Characterization of VNT1

A Detection Method for the 

Solanum venturii R-Protein 

VNT1

Report No. 13-37-SPS-MOL

Matthew Pence, Nikolaos Georgelis, Roshani Shakya, Kelsey Wood, Alexi Balmuth

and Jingsong Ye

3/28/2014
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 2

ABBREVIATIONS

ATP Adenosine Triphosphate

CC Coiled-Coil

EDTA Ethylenediaminetetraacetic Acid

ETI Effector Triggered Immunity

LRR Leucine Rich Repeat

MAPK Mitogen Activated Protein Kinase

MBP Maltose Binding Protein

MCS Multiple Cloning Site

NB-ARC Nucleotide Binding Domain shared by APAF-1, R-proteins, and CED-4

PAGE Polyacrylamide Gel Electrophoresis

PAMP Pathogen Associated Molecular Pattern

PRR Pattern Recognition Receptor

PTI PAMP Triggered Immunity

PVDF Polyvinylidene Flouride

STAND Signal Transduction ATPases with Numerous Domains

TFA Trifluoroacetic Acid

TIR Toll-like/Interleukin-1 Receptor
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 3

INTRODUCTION

Plants utilize a two-part innate immune response to detect and combat invading microbes such as 

bacteria, fungi, and oomycetes, as well as viruses and insects (Jones and Dangl, 2006). Plants detect 

microbes and potential pathogens by recognizing pathogen-associated molecular patterns (PAMPs) that 

bind trans-membrane pattern recognition receptors (PRRs) at the cell surface (Jones and Dangl, 2006; 

Monaghan and Zipfel, 2012). Pathogen recognition initiates mitogen-activated protein kinase (MAPK) 

signaling cascades that mediate physiological changes such as calcium influx, oxidative burst, increased

phytoalexin production, and other immune responses (GroBkinsky et al., 2012; Herman and Williams, 

2012; Monaghan and Zipfel, 2012; Ranf et al., 2011; Spoel and Dong, 2012). This initial immune 

response, known as PAMP-triggered immunity (PTI), protects plants from most microbial diseases. 

However, successful pathogens overcome PTI by secreting effector proteins that suppress the PAMPtriggered immune response (Jones and Dangl, 2006). A secondary layer of defense relies upon recognition 

of pathogen effector proteins either directly or indirectly by resistance protein receptors (R-proteins). Rprotein mediated immunity, known as Effector Triggered Immunity (ETI), results in a form of 

programmed cell death called a hypersensitive response (Morel and Dangl, 1997).

Late blight is a devastating disease among cultivated Solanaceae species, including tomato and 

potato. In potato, late blight affects foliage and tubers causing rapid necrosis and crop loss if left untreated 

(Haverkort et al., 2008). Expensive and environmentally harsh fungicide treatments are used to control 

the spread of the disease. The causal agent of late blight is the oomycete Phytophthora infestans. P. 

infestans spreads rapidly by clonal propagation of zoospore filled sporangia which are aerially dispersed 

and infect susceptible plant tissue (Fry, 2008). The genome sequence of P. infestans revealed at least 560 

effector proteins potentially encoded and secreted by the pathogen. The majority of these genes exist in 

sparse gene regions of the genome underscoring a high variability and rapid evolution (Haas et al., 2009). 

The effectors secreted by the pathogen are recognized by potato R-proteins in resistant plants. The ability 

of P. infestans to evolve new effectors unrecognized by R-proteins allows the pathogen to overcome 

resistant Solanum species and has caused several epidemics worldwide (Rodewald and Trognitz, 2013).

Plant R-proteins are members of the STAND ATPase protein family with homologs found in all 

domains of life (Leipe et al., 2004). They function as signaling activators to mediate a hypersensitive 

response, a critical role in plant immunity. To avoid spontaneous apoptosis, R-protein activity is tightly 

regulated (Spoel and Dong, 2012). R-protein expression levels are extremely low and an auto-inhibitory 

mechanism blocks signaling except in the presence of pathogen effectors (Ayliffe et al., 1999; Tan et al., 

2007). The potato genome encodes for at least 430 R genes, many of these share > 80% sequence identity

(Jupe et al., 2012). R genes are mainly found in gene clusters on specific potato chromosomes where 

higher rates of recombination contribute to maintaining an evolutionary arms-race against evolving 

pathogens (Spoel and Dong, 2012; Xu et al., 2011). R-proteins have a conserved domain structure 

organized with a central nucleotide-binding—APAF-1, R-proteins, and CED-4 (NB-ARC) domain, 

flanked by C-terminal leucine-rich repeats (LRR), and an N-terminal toll-like/interleukin-1 (TIR) or 

coiled-coil (CC) domain (Takken and Goverse, 2012). Of these three domains, the LRR is the least 

conserved and has been hypothesized to function in effector recognition. Studies of R-proteins from 

several species, including Arabidopsis thaliana (RPS5) and potato (Rx), have demonstrated a mechanism 

for regulating R-protein activity (Moffett et al., 2002; Qi et al., 2012; Rairdan et al., 2008). R-proteins are

maintained in a perpetual “off-state” through inhibitory intermolecular interactions. Upon recognition of 

the effector, a conformational change produces an “on-state” and initiation of downstream signaling 

events.

Genetic engineering is a powerful technology with potential to increase the specificity and 

durability of R-protein mediated disease resistance. One challenge in utilizing R-proteins for effective 

disease resistance is the inability to produce, either recombinantly or in planta, large quantities of protein 

for safety testing. R-proteins are naturally low abundance and are estimated to be at concentrations as low 

as 18 ppt with low exposure levels in humans (3.6 ng protein/year) (Bushey, 2014). It is possible that 

difficult to work with proteins may be unstable or rendered inactive during digestion or processing
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 4

making regulatory requirements for safety testing expensive, impractical, or impossible using current 

technologies for overexpression and purification of adequate protein quantities. A potentially beneficial 

approach would be to use a variety of indirect assessment tools that collectively provide evidence for safe

use (Bushey, 2014).

VNT1 is an 891 amino acid R-protein of the CC-NB-LRR class and a homolog of the Tm-2

2

tomato mosaic virus disease resistance protein (Foster et al., 2009). VNT1 confers resistance to late blight 

with a relatively broad resistance spectrum against P. infestans isolates (Foster et al., 2009). Using 

Simplot InnateTM technology, the Rpi-vnt1 (FJ423044.1) gene has been transformed into commercially 

important potato varieties successfully conferring late blight resistance. We describe a method for the 

detection and quantification of VNT1 in InnateTM potato lines and demonstrate that the VNT1 protein is 

expressed at lower than detectable levels.

MATERIALS AND METHODS

Materials—pMAL expression vectors and restriction enzymes were purchased from New England 

BioLabs (NEB) (Ipswich, MA); The pCR-Blunt-TOPO TA cloning kit and PCR primers from Life 

Technologies (Grand Island, NY); Mini-prep kits from Qiagen (Germantown, MD); DNA sequencing 

from Sequetech (Mountain View, CA); Complete Protease Inhibitor Cocktail Tablets from Roche 

(Indianapolis, IN); Chromatography columns and systems from GE Healthcare Life Sciences 

(Piscataway, NJ); and Amicon Centrifugal Filter Units from Millipore (Billerica, MA). All gel and 

blotting apparatus were purchased from Bio-Rad (Hercules, CA). All other chemicals, reagents, and 

laboratory supplies were from Sigma-Aldrich (St. Louis, MI), Fisher Scientific (Pittsburg, PA), or 

Research Products International (Mt. Prospect, IL).

Construction of E. coli Expression Vectors for Full-length Rpi-vnt1 and Rpi-vnt1 Fragments—pMAL-c5x 

and pMAL-p5x vectors (NEB) were modified to introduce a BsaI restriction cut site at the multiple 

cloning site (Fig. 1A). A BsaI-containing PCR fragment with 5 ́-BglII and 3 ́-EcoRI restriction sites was 

amplified from the pMAL vector template using forward primer: 5 ́-

GCTGATTTATAACAAAGATCTGCTG-3 ́ (BglII restriction site underlined) and reverse primer: 5 ́-

AGAATTCCGATCGAGACCCCGAGGTTGTTGTTATTGTTAT-3 ́ (EcoRI and BsaI restriction sites 

underlined) and ligated into the pCR-Blunt-TOPO vector using a TA cloning kit (Life Technologies) (Fig. 

1B, Lane 2). The introduced BsaI restriction site and BglII and EcoRI restriction sites were confirmed by 

DNA sequencing (Sequetech). Clones containing the BsaI restriction site were generated by digestion of 

the pCR-Blunt-TOPO vectors and ligation into the pMAL-c5x and pMAL-p5x plasmids using BglII and 

EcoRI restriction enzymes to generate the modified vectors pMAL-c5x-BsaI and pMAL-p5x-BsaI. The 

Rpi-vnt1 gene and gene fragments were obtained by PCR amplification of the Simplot vector pSIM1678, 

containing the Rpi-vnt1 (FJ423044.1) gene, using appropriate primer pairs (Fig. 1B and 1C; Table 1). The 

amplified Rpi-vnt1 fragments were ligated into the pCR-Blunt-TOPO vector using a TA cloning kit (Life 

Technologies). Each Rpi-vnt1 fragment was verified by DNA sequencing (Sequetech). Clones containing 

the Rpi-vnt1 fragments were generated by digestion of the pCR-Blunt-TOPO vectors and ligation into the 

pMAL-BsaI vectors using BsaI and EcoRI restriction enzymes. Full-length Rpi-vnt1 contains an internal 

BsaI cut site and was cloned using a 3-piece ligation strategy (Fig. 1D and Table 1).

Expression and Purification of VNT1—Recombinant full-length VNT1 (1-891) and the LRR domain, 

VNT1-LRR (545-891), was expressed and purified as Maltose Binding Protein (MBP)-VNT1 fusion 

proteins. The pMAL-BsaI vectors containing the Rpi-vnt1 coding sequences were transformed into BL21 

E. coli cells (NEB). Cells were grown in Luria-Bertani broth supplemented with ampicillin (100 μg/ml) to 

an O.D.600 = 0.5. VNT1 expression was induced by adding 1 mM isopropyl-β-D-galactopyranoside. 

Expression levels of VNT1 were determined at different induction temperatures (17 °C and 37 °C) and 

times (2-8 hr. and overnight). Full-length VNT1 (1-891) was maximally expressed by overnight
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 5

Figure 1. pMAL Vector Modification and Rpi-vnt1 Cloning. A, Scheme showing modification of 

pMAL vectors introducing a BsaI cut site at the 5 ́-end of the MCS. B, PCR amplification of Rpi-vnt1

gene fragments using pSIM1678 template and appropriate primers pairs (Table 1) resulted in bands of 

predicted size. C, Six fragments of Rpi-vnt1 including: full-length Rpi-vnt1 (2676 bp; amino acids (aa) 

1-891), individual CC (378 bp; aa 1-125), NBS (1260 bp; aa 125-544), and LRR (1044 bp; aa 544-

891) domains, and combinations of the CC-NBS (1635 bp; aa 1-544) and NBS-LRR (2301 bp; aa 

125-891) domains were cloned into the pMAL E. coli expression vector. D, Scheme showing the 

ligation strategy for the full-length Rpi-vnt1 (1-891) gene fragment (which contains an internal BsaI 

cut site) using BsaI, PstI, and EcoRI restriction enzymes. 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 6

Rpi-vnt1 Fragment Primer Sequence

Rpi-vnt1 (1-891)-5’ For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-CTTAACATGCCTGCAGTCACCACAAT-3 ́

Rpi-vnt1 (1-891)-3’ For 5 ́-ATTGTGGTGACTGCAGGCATGTTAAG-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Rpi-vnt1 (1-125) For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-TGAATTCTTAATTGGATTGTTGAATTTTTGG-3 ́

Rpi-vnt1 (126-544) For 5 ́-AGGTCTCCATCGAGGGAAGGAAGTTCATTTGTTGCCTTAAGAC-3 ́

Rev 5 ́-TGAATTCTTACCTAGCAACATTGCTAGG-3 ́

Rpi-vnt1 (545-891) For 5 ́-AGGTCTCCATCGAGGGAAGGGTGCGAAGGATTACATTCTAC-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Rpi-vnt1 (1-544) For 5 ́-AGGTCTCCATCGAGGGAAGGATGAATTATTGTGTTTACAAGACT-3 ́

Rev 5 ́-TGAATTCTTACCTAGCAACATTGCTAGG-3 ́

Rpi-vnt1 (126-891) For 5 ́-AGGTCTCCATCGAGGGAAGGAAGTTCATTTGTTGCCTTAAGAC-3 ́

Rev 5 ́-TGAATTCTTATAGTACCTGTGATATTCTCA-3 ́

Table 1. Primers for PCR amplification of Rpi-vnt1 (1-891) and Rpi-vnt1 fragments from pSIM1678. 

BsaI recognition sequence highlighted in gray; EcoRI in blue; PstI in yellow; stop and start codons for 

each Rpi-vnt1 gene fragment shown in red.

incubation at 37 °C. VNT1-LRR was maximally expressed from 2 hr. incubations at 37 °C. Cell cultures 

were spun at 4000 x g at 4 °C for 30 min. The pelleted cells were suspended in 50 mM Tris-HCl (pH 7.5) 

buffer containing 1 mM EDTA, 500 mM NaCl, 10 % glycerol and Complete Protease Inhibitor Cocktail 

Tablets (Roche) and lysozyme (2 mg/ml). The cells were sonicated for 2 min. using a Branson Probe 

Sonifier (Branson; Danbury, CT). Extracts were spun at 24,000 x g at 4 °C for 30 min. and the 

supernatant loaded onto a 3-ml amylose column. The purified MBP-VNT1 fusion protein was eluted with 

10 mM maltose and concentrated using an Amicon Centrifugal Filter Unit (Millipore) to a volume of ~1 

ml. A final purification step was performed using a HiPrep 16/60 Sephacryl S-200 HR column (GE 

Healthcare).

Transient Expression of VNT1 in Nicotiana benthamiana by Agroinfiltration—Infiltration was performed 

as described previously (22). Briefly, N. benthamiana seeds were germinated (1-2 weeks) in a petri dish 

with Whatman filter paper (Sigma-Aldrich, St.Louis, MO). Seedlings were transferred to 4-inch pots with 

Sunshine Professional Growing Mix LC1 (Sun Gro Horticulture; Vancouver, Canada). The plants were 

grown 4-6 weeks at 22 °C in 50% humidity with 16- and 8-h light-dark cycling and regular watering. 

Agrobacterium tumefaciens strain AGL1 harboring a VNT1 expression vector was used for infiltration. A. 

tumefaciens cultures were prepared by overnight incubation at 28 °C with kanamycin (50 μg/mL) and 

carbenicillin (100 μg/mL). Growth was monitored until the O.D.600 = 0.1-0.2 (1:10 dilution). Cells were

pelleted by centrifugation and suspended in 10 mM Tris HCl (pH 7.5) buffer containing 10 mM MgCl2

and washed once. Cultures were diluted to O.D.600 = 0.25 in the same buffer and injected at 2-6 different 

sites on the underside of N. benthamiana leaves using a syringe. Leaves were harvested 2-3 days postinfiltration, ground in liquid nitrogen using a mortar and pestle, and stored at -80 oC.

Protein Identification of MBP-VNT1 (LRR) and GroL from E. coli—An Agilent 1200 HPLC coupled to 

an Agilent 6320 Ion-Trap Mass Spectrometer with an ESI ionization source was used in peptide 

separation and analysis. Digested samples of purified VNT1-LRR were separated by reverse-phase 

chromatography using an Aeris Peptide 3.6 μm-C18-250 mm column (Phenomenex). Peptides were 

eluted at a flow rate of 0.3 mL/min. with a 15 min. gradient from 5% to 40% Buffer B (Buffer A = 0.1% 

formic Acid with 0.001% TFA, Buffer B = 0.1% formic acid in acetonitrile). The mass spectrometer was 

operated in positive ion mode with dry gas at 350 C, N2 flow at 10 mL/min., and 30 psi. The data were 

analyzed using MS/MS Mascot search (Matrix Science, Boston, MA).
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 7

Preparation and Characterization of Antibodies—Polyclonal antibodies were raised in New Zealand 

White rabbits by Anaspec (Fremont, CA) with two animals per antigen. Several peptides of VNT1

(MSQKGYQHVTFPKKIG (antibodies 5361, 5362), FHSSSKLPFGVWESKIL (5363, 5364),

KLKKLFLVQGPNISPISLR (5365, 5366), CRTTYSITDTSNNNDD (5467, 5368), 

VNTGNGREAESLADDC (5469, 5370), and AKRTYDGRISSC (5471, 5472) were selected as antigens 

based on uniqueness to VNT1 compared to homologous proteins and antigenic properties. Peptides were 

conjugated to keyhole limpet hemocyanin:bovine serum albumin (7.0:1.5 (w/w)) before injection. A 

standard 70-day protocol was extended and final bleeds were collected 100 days post-immunization

(initial + 6 boosts; peptide antibodies 5361-5366). A 28-day “speedy” protocol was followed for 

antibodies 5467-5472. Final bleeds were collected 28 post-immunization (initial + 4 boosts). The final 

bleeds were affinity purified by peptide-coupled thiopropyl-sepharose 6B chromatography for all 

antibodies except 5362, 5364 and 5366. A second strategy for raising polyclonal antibodys against VNT1 

used purified VNT1-LRR as antigen. Antibodies were raised in New Zealand White rabbits (specificpathogen free) by Pierce Biotechnology (Rockland, IL) using the company’s standard 90-day protocol, 

with two animals per antigen. VNT1-LRR was run on an SDS-polyacrylamide gel electrophoresis

(PAGE) gel and a gel slice containing the ~80 kDa VNT1-LRR band was cut out and sent for use as 

VNT1 antigen. The first injection was with 0.5 mg of antigen. After 14, 42 and 56 days each rabbit was 

boosted with another 0.5 mg of the same adjuvant. Fourteen days following the boost, serum was 

collected and processed from each rabbit. Characterization of all antisera was done by SDSPAGE/immunoelectrophoretic blotting.

Protein Extraction from N. Benthamiana and Potato Tissues—Proteins from N. benthamiana, Russet 

Burbank (RB), and W8 tissue materials were extracted using different protocols to compare yields. A 

non-denaturing extraction in 50 mM MOPS (pH 7.0) buffer, containing 0.15% NP-40, 1 mM EDTA, 1% 

PVP, 20 mM DTT, and Protease Inhibitor (PI) (Sigma-Aldrich). Samples were incubated on ice and 

centrifuged at 20,000 x g for 5 min. at 4 °C. The supernatant was mixed with Laemmli loading buffer 

(Bio-Rad) containing 100 mM DTT and heated at 80 oC for 5 min. The protein samples were aliquoted 

and stored at -20 oC. Alternatively, proteins were extracted (denatured) in 40 mM Tris-HCl (pH 8.5) 

buffer containing 2% SDS, 60 mM DTT, and 20% glycerol. Samples were incubated at 80 oC for 10 min.

and centrifuged at 20,000 x g for 5 min. at room temperature. The supernatant was mixed with Laemmli 

loading buffer (Bio-Rad) containing 100 mM DTT and heated at 80 oC for 5 min. The protein samples 

were aliquoted and stored at -20 oC. A TCA/acetone and phenol extraction was also performed. Tissue 

materials were washed several times in acetone followed by additional washes in 10% TCA/acetone and 

10% TCA/H2O. After drying, the pellet was suspended in 0.1 M Tris-HCl (pH 7.5) buffer containing 2% 

SDS, 30% sucrose, and 5% β-mercaptoethanol. Proteins were extracted with the addition of an equal 

volume of phenol. After centrifugation, the upper phenol phase was precipitated in 100 mM ammonium 

acetate/methanol at -20 oC overnight. The proteins were pelleted by centrifugation at 20,000 x g for 5 

min., washed in 100 mM ammonium acetate/methanol and acetone, and after drying, resuspended in 0.1 

M Tris-HCl (pH 7.5) buffer containing 0.5 M NaCl, and 0.1 M EDTA. Protein concentration was 

determined by BCA assay before addition of SDS loading buffer.

Detection of VNT1 by Western Blot—Proteins were separated by SDS-PAGE and transferred to PVDF 

membranes using a wet tank transfer system in 25 mM Tris-HCl (pH 8.3) buffer, 192 mM Glycine, and 

20% methanol (transfer buffer). The SDS-polyacrylamide gel was soaked in transfer buffer for 15 min. 

PVDF membrane was wetted in methanol for 5 min. and then in transfer buffer for 10 min. The transfer

apparatus was assembled according to manufacturer’s instructions and run at 100V for 1.5 hr. or for 50V 

at 3 hr. The membrane was placed in transfer buffer with 0.1% Tween-20 and 5% non-fat milk added

(blocking buffer) for 1 hr. at room temperature. Three 5 min. washes in transfer buffer + 0.1% Tween-20 

(wash buffer) were performed before addition of primary antibody. For anti-MBP a 1:10,000 (v/v), and 

for anti-VNT1 a 1:1000 (v/v) dilution was used. The membrane was incubated with primary antibody for

30 min. and washed three times for 5 min. each in wash buffer. The secondary antibody (anti-rabbit-IgG-
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 8

peroxidase; Sigma-Aldrich) was added at a dilution of 1:20,000 (v/v). The membrane was incubated for 

30 min. and again washed three times for 5 min. each. SuperSignal ® West Femto Maximum Substrate 

(Thermo Scientific) was added and after 5 min. blots were imaged with a Bio-Rad Chemi-Doc XRS+

system (Bio-Rad).

RESULTS

Domain Structure of VNT1—Sequence alignments and structural models were used to assign VNT1 

amino acid residues 1-125 as the coiled-coil (CC) domain, residues 126-544 as the nucleotide binding 

(NBS) domain, and residues 545-891 as the leucine-rich repeat (LRR) domain. The domain sequences 

were determined by comparing sequence alignments of known VNT1 homologs and analyzing predicted 

structural models generated using I-TASSER and Phyre2 protein structure prediction servers (Kelley and 

Sternberg, 2009; Zhang, 2008). The VNT1 protein fragments tested for expression in E. coli were fulllength VNT1 (1-891), individual CC (1-125), NBS (126-544), and LRR (545-891) domains, and 

combinations of the CC-NBS (1-544) and NBS-LRR (126-891) domains (Fig. 1). BLASTP searches 

performed against the complete potato genome database (Spud DB Potato Genomics Resource; 

http://potato.plantbiology.msu.edu/integrated_searches.shtml) and the S. tuberosum non-redundant 

protein sequences (nr) database at NCBI, using VNT1 (ACJ66594.1) as a query, revealed that VNT1 has 

72% amino acid identity to Tm-2

2

from S. tuberosum (ABM05492.1) and high similarity to several 

putative genes in the double monoploid S. phureja complete potato genome sequence.

Expression and Purification of Recombinant VNT1—High expression levels of VNT1 (1-891) were 

observed with 6 hr. to overnight inductions at 37 °C. The full-length VNT1 (1-891) fusion protein eluted 

from the amylose resin as a 145 kDa protein observable by Coomassie and silver stained SDS-PAGE. 

However, breakdown products of smaller molecular weights were also observed (Fig. 2). Several of the 

smallest molecular weight bands were successfully removed using gel filtration chromatography. The 

highest level of expression of VNT1-LRR was observed with a 2 hr. induction at 37 °C. The protein 

(predicted to be ~80 kDa) eluted from the amylose resin as two prominent bands of 80 and 60 kDa and 

several smaller molecular weight bands observed on SDS-PAGE (Fig. 3). Using gel filtration 

chromatography the 80 and 60 kDa bands were eluted together at early void volumes indicating that they 

formed a complex in solution while the small molecular weight bands were successfully separated away.

Attempts to separate the 80 and 60 kDa proteins using ion-exchange chromatography, hydrophobic 

interacting chromatography, and hydroxyapatite chromatography were unsuccessful. Mass spectrometric 

analysis confirmed that the 80 kDa band was VNT1-LRR and revealed the 60 kDa band to be E. coli

chaperone protein GroL (WP_001417209). GroL is a homolog of heat shock protein 60 (HSP60) which is 

known to bind the LRR domains of several CC-NB-LRR proteins. Heat shock proteins have been 

implicated to play a role in plant immune responses (Shirasu, 2009). Amylose-purified MBP used as an 

empty vector control was observed, as expected, a 42 kDa band by SDS-PAGE (Fig. 4).

Polyclonal Antibodies against VNT1—Three of the tested peptide antibodies: MSQKGYQHVTFPKKIG, 

FHSSSKLPFGVWESKIL, and KLKKLFLVQGPNISPISLR detect both N. benthamiana expressed 

VNT1 and VNT1-LRR (Fig. 5). Western Blots were used to test the sensitivity and specificity of the

antibody. FHSSKLPFGVWESKIL had the highest specificity of all antibodies tested and was used in 

further analyses for detection of VNT1 in InnateTM lines.

Limit of Detection and Quantitation for VNT1—9 pg was the lowest amount of VNT1-LRR detectable by 

quantitative Western Blot analysis using the FHSSSKLPFGVWESKIL antibody (Fig. 6). The 

concentration of E. coli expressed VNT1-LRR/GroL complex was determined by Bradford Assay and 

VNT1-LRR determined to be 33.3 % of the total protein based on densitometric analysis of the gel bands

(Fig. 6). The result indicates that the complex is in a stoichiometric ratio of two copies GroL chaperone to 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 9

one copy VNT1-LRR. The concentration of VNT1-LRR was used to estimate the concentration of fulllength VNT1 (1-891) expressed in N. benthamiana by comparative immunoblot analysis (data not 

shown). Protein extracts from 0.5 mg (leaf and flower), 1 mg (tuber and root), or 1.5 mg (stem) tissue 

were spiked with known amounts of full-length N. benthamiana expressed VNT1 (Fig. 7). Quantitation

limits for VNT1 were determined to be 60 ppb in leaf, 30 ppb in tuber, 20 ppb in stem, 15 ppb in root, and 

60 ppb in flower. Calibration curves, made from densitometric analysis of the gel bands, demonstrate the 

linear dynamic range of the assay.

VNT1 is Undetectable in InnateTM Lines—VNT1 was not detected in crude protein extracts from InnateTM

W8 tissues using the FHSSSKLPFGVWESKIL antibody (Fig. 8). The W8 line is resistant to late blight in 

leaf inoculation studies (Fig. 8), however, based on the measured LOQ for VNT1 the amount of VNT1 

protein expressed in Innate

TM lines is lower than 60 ppb (60 ng VNT1 per g of tissue) in all tissues tested.

CONCLUSIONS

A method for the detection of the S. venturii derived R-protein, VNT1, was developed using

antibodies raised against VNT1 peptides. VNT1 confers strong resistance against P. infestans, the causal 

agent of potato late blight. The method detects low picogram levels of purified VNT1 protein, but was 

unsuccessful at detecting VNT1 protein expressed in Simplot InnateTM transformed potato line W8. 

Results of quantitative Western Blot analyses show that VNT1 is expressed at levels below the 

quantitation limit of 30 ppb in tubers, and between 15-60 ppb in all other tissue types, and demonstrate 

that the late blight resistance of InnateTM lines is independent of high R-protein expression. InnateTM lines 

appear otherwise phenotypically equivalent to untransformed Russet Burbank.
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 10

Figure 2. Full-length VNT1 (1-891) Purification. A, Chromatograms showing purification of VNT1 

(1-891) with amylose column (top) and gel filtration column (bottom). Collected fractions are shown 

in red bars. B, Coomassie-stained SDS-PAGE gel showing purified VNT1 (1-891) as a band running 

at ~145 kDa. Lane 1: Molecular Weight (MW) marker; Lane 2: MBP expressed and purified from 

empty pMAL vector (see Fig. 4); Lane 3: Fraction collected from amylose column; Lane 4-5: 

Fraction collected from gel filtration column before and after being concentrated (see MATERIALS 

AND METHODS). Several small molecular weight bands (VNT1 breakdown products) were 

successfully removed using gel filtration chromatography. 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 11

Figure 3. VNT1-LRR Purification. A, Chromatograms showing purification of VNT1-LRR with 

amylose (top) and gel filtration columns (bottom). Collected fractions are shown in red bars. B, 

Coomassie-stained SDS-PAGE gel showing the purified VNT1-LRR fusion protein as an ~75 kDa 

band and several VNT1 breakdown products eluted from the amylose column (top). Lane 1: MW 

marker; Lane 2: empty vector; Lane 3: fraction from amylose column. Coomassie-stained SDS-PAGE 

gel showing the purified VNT1-LRR after size exclusion chromatography (bottom). Several small 

molecular weight bands (VNT1 breakdown products) were successfully removed. A 60 kDa protein, 

GroL (E. coli chaperone) co-elutes with the VNT1-LRR domain. Both proteins form a complex in 

solution.
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 12

Figure 4. pMAL Empty Vector Purification. A, Chromatogram showing amylose resin purification of 

MBP. Collected fraction shown in red bars. B, Coomassie-stained SDS-PAGE gel showing the 

purified MBP as a ~42 kDa band.
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 13

Figure 5. Western Blots of Full-Length VNT1 (1-891) and VNT1-LRR (545-891). A, Western Blots 

of VNT1-LRR using anti-MBP and three anti-VNT1 peptide antibodies, MSQKGYQHVTFPKKIG, 

FHSSSKLPFGVWESKIL, and KLKKLFLVQGPNISPISLR. The results demonstrate the high 

sensitivity of the FHSSSKLPFGVWESKIL antibody for VNT1. Lane 1: VNT1-LRR, Lane 2: fulllength VNT1 (1-891), Lane 3: MBP, Lane 4: MW marker. B, Western Blots of full-length VNT1 (1-

891) using anti-MBP and anti-VNT1 (FHSSSKLPFGVWESKIL) antibodies. Lane 1: MBP, Lane 2: 

10 μL full-length VNT1 (1-891), Lane 3: 5 μL full-length VNT1 (1-891). VNT1 appears as a band 

~145 kDa. All other bands are breakdown products of VNT1.
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 14

Figure 6. Limit of Detection for anti-VNT1 antibody FHSSSKLPFGVWESKIL. Densitometric 

analysis of Coomassie-stained SDS-PAGE gel was used to determine the VNT1-LRR concentration as 

33.3% of the total protein. Western Blot analysis was used to determine an LOD of 9 pg for purified 

VNT1-LRR. A calibration curve shows linearity between 9 and 288 pg. 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 15

Figure 7. Limit of Quantitation for VNT1 in Russet Burbank Tissues. Protein extracts from 0.5, 1, 

1.5, 1, and 0.5 mg of leaf, tuber, stem, root, and flower tissue, respectively, were spiked with 0, 7.5, 

15, 30, 50, 100, and 150 pg full-length N. benthamiana expressed VNT1. Amounts of spiked VNT1 

are shown. Calibration curves show the assay is within the linear dynamic range. LOQ values of 60, 

30, 20, 15, and 60 ppb were determined for leaf, tuber, stem, root, and flower tissues, respectively. 
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 16

Figure 8. Detection and Quantitation of VNT1 in InnateTM W8 Tissues. Western Blot analysis using 

antibody FHSSSKLPFGVWESKIL demonstrates that VNT1 is below detectable limits in all W8 

tissues. Protein was extracted from 0.5 mg of leaf, 1 mg of tuber, 1mg of root, 1.5 mg of stem, and 0.5 

mg flower. 30 pg of VNT1 were spiked into leaf, tuber, stem, and flower extracts and 15 pg of VNT1

were spiked into root extracts. LOQ values are shown in ppb. A detached leaf assay demonstrates late 

blight resistance in W8 compared to susceptible Russet Burbank.
A Detection Method for the Solanum venturii R-Protein VNT1, J.R. Simplot Co. Page 17

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J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 1

Appendix C: Allergen and Toxin Assessment for Russet Burbank W8

Bioinformatics Evaluation of the Inserts and Flanking Sequences

AUTHORs

Jeff Habig, PhD and Jingsong Ye, PhD

Date

March 28, 2014

SUBMITTED BY

J.R. Simplot Co. Plant Sciences

5369 W. Irving St., Boise, ID 83706

PAGE COUNT

1 of 14
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 2

Contents

Introduction ..................................................................................................................................................3

Stop-to-stop ORF Identification ....................................................................................................................4

Allergenicity Evaluation ................................................................................................................................5

8-mer identity search................................................................................................................................5

80-mer homology search..........................................................................................................................6

Toxicity evaluation........................................................................................................................................7

pSIM1278 locus.........................................................................................................................................8

pSIM1678 locus.........................................................................................................................................9

Summary .....................................................................................................................................................11

Methods......................................................................................................................................................12

ORF detection .........................................................................................................................................12

Database and software resources ..........................................................................................................12

Allergenicity database searches..............................................................................................................12

Toxin database searches.........................................................................................................................13

Native gene disruption............................................................................................................................13

References ..................................................................................................................................................14

Certification.................................................................................................... Error! Bookmark not defined.
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 3

Introduction

The purpose of this study was to assess the safety of Russet Burbank W8 resulting from inserting DNA 

into the genome of the potato. This evaluation considers the remote potential to disrupt a native gene 

or introduce an unexpected toxin or allergen through expression of a novel open reading frame (ORF). 

This report describes our analysis of possible native gene disruption, toxicity, and allergenicity for the 

Russet Burbank W8 event using the standard bioinformatics techniques summarized in Table D-1.

Disruption of native genes and expression of unexpected toxins or allergens are all highly unlikely 

events, particularly in InnateTM potatoes that rely almost exclusively upon potato genomic DNA. In 

addition to promoter and enhancer elements, expression of unexpected proteins would require 

productive transcription through the insertion region with post-transcriptional processing producing a 

properly structured and polyadenylated messenger RNA transcript, followed by translation into a stable 

protein. The genetic inserts added to Russet Burbank to create the W8 potato consist of multiple 

cassettes including inverted repeats driven by opposing promoters, which limit transcriptional read 

through from either direction. The end result, by design, is small complementary RNAs or doublestranded hairpins that are processed by the RNA interference pathway into small interfering RNAs, 

preventing them from being translated. The pSIM1678 plasmid also contains the Rpi-vnt1 gene, which 

drives expression of the R-protein, VNT1. This analysis evaluates all potential open reading frames 

(ORFs) consisting of at least 19 amino acids for both insertion events. We show the absence of

allergenicity and toxicity potential of our events using a number of well-established bioinformatics 

techniques (Ladics, 2007; Goodman, 2008; Terrat, 2013).

Table D-1. Overview of bioinformatics analyses

Analysis Purpose Approach

Stop-to-stop ORF Analysis Identify all potential proteins that 

may be expressed by DNA insert 

and surrounding sequence. 

ORF Finder: systematically identify 

all ORFs (≥19 amino acids) located 

between contiguous stop codons 

from all frames. Used for 

subsequent toxin and allergen 

analyses.

Allergenicity Analysis Ensure that sequences capable of 

expressing known allergens have 

not been introduced by 

transformation.

AllergenOnline: find small regions 

of identity (8 amino acids) or 

larger regions of similarity (80 

amino acid, ≥35% homology) 

compared with known allergens.

Toxicity Analysis Ensure sequences capable of 

producing proteins similar to 

known toxins have not been 

introduced by transformation.

NCBI protein BLAST (blastp): 

identify proteins homologous to 

known toxins contained in target 

databases (E-value ≤ 1).

Native gene disruption Verify that a novel protein has not 

been generated by integration 

within a native gene.

NCBI nucleotide BLAST (blastn): 

identify any native genes 

containing the flanking sequence 

as an indication of gene 

disruption.
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 4

Stop-to-stop ORF Identification

Open reading frames (ORFs) can be identified as the contiguous sequence located between a canonical 

start codon and a downstream stop codon or, more generally, all sequence located between two stop 

codons in the same reading frame. As the latter definition will identify more potential ORFs, we used it 

as the basis for our analysis. The ORF Finder algorithm associated with the Sequence Manipulation Suite 

(Stothard, 2000) was used to identify all ORFs. As shown in Figure D-1, the analysis for each event was 

performed using the insert with the known flanking sequences. 

Figure D-1. ORF Analysis Scheme

As the W8 event was developed through transformation with two plasmids, both inserts were 

collectively analyzed. The ORFs identified included any and all ORFs that extend across the junctions 

between flanking and insert DNA. The ORFs overlapping the known junction sites were included within 

each analysis. 

As summarized in Table D-2, 132 potential ORFs were identified with the pSIM1278 insertion locus and 

108 with the pSIM1678 insertion locus when a start codon was not required. These ORFs were 

converted into a FASTA-formatted file and used for the subsequent toxicity and allergenicity analyses.

Table D-2. Stop to Stop ORF Analysis Summary for W8

Construct Insertion Number of ORFs Mean ORF size* Largest ORF size*

pSIM1278 132 35 141

pSIM1678 108 46 893

 *sizes refer to number of amino acids within each ORF
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 5

Allergenicity Evaluation

Allergenicity potential of these ORFs was analyzed using a web-based tool 

(http://www.allergenonline.org/databasefasta.shtml) provided by the Food Allergy Research and 

Resource Program (FARRP). This tool allowed us to identify small regions (8-mers) of sequence identity

between any ORFs found in each of the events and those of known allergens as these eight-amino acid 

matches could theoretically represent a B-cell or T-cell epitope, which is the part of the protein that is 

specifically recognized by the immune system (Metcalfe, 1996). The allergen database contains the 

sequence of known allergens, but the specific sequence responsible for allergenicity is not necessarily 

known, nor is it known whether an 8-mer is capable of inducing an allergic response (Goodman, 2008). 

8-mer identity search

As shown in Table D-3, a FASTA search led to identification of a single polypeptide (LPLLLLLL) that was 

common to both constructs and a known allergen, endochitinase (GI:3201547) from Persea Americana

(avocado). There were two independent ORFs containing this sequence within our pSIM1278 insert and 

one ORF in our pSIM1678 insert because the sequence is derived from an open reading frame contained 

within the GBSS promoter, which is native to the potato and coopted to drive expression of the silencing 

constructs contained in the insert. It is highly unlikely that this polypeptide is responsible for the 

allergenicity of the endochitinase protein given that it represents only one of 319 distinct 8-mers 

present in the full-length, allergenic endochitinase protein. As further confirmation, we verified that the 

putative epitope, LPLLLLLL, was not found in a separate database of epitopes (http://www.iedb.org/). 

The presence of an ORF does not predict that it will be expressed, but even if it were the LPLLLLLL 

sequence is present in both potato and numerous human proteins. Collectively, these data suggest the 

peptide identified is a false positive and would not be a potential allergen, consistent with concerns over 

the high false-positive rate when looking for 8-mer matches (Goodman, 2008).

An extended ORF was identified in the pSIM1678 plasmid that had high sequence identity with a 

documented minor allergen found in tomatoes (Table D-3). This allergen is the vacuolar invertase gene, 

which is not only found in tomatoes, but is also found in other Solanum species, such as potatoes. The 

presence of this partial ORF was by design as the potato vacuolar invertase (VInv) gene was the target of 

one of our silencing cassettes. Thus, the VInv silencing cassette will prevent expression of a protein from 

the native VInv gene as well as from the pSIM1678 insert. Overall, no allergen-related safety concerns 

were identified for the Russet Burbank W8 potato event. In fact, silencing of the native vacuolar 

invertase gene may decrease the allergenicity potential of the potatoes.
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 6

Table D-3. Summary of Allergenicity Findings for W8

Construct 8-mer 

hits

8-mer sequence 8-mer allergen target

pSIM1278 1 LPLLLLLL Endochitinase

GI:3201547

pSIM1678 175 Not shown Minor allergen beta-fructofuranosidase 

precursor - vacuolar invertase(Solanum 

lycopersicum)

GI:18542113

GI:18542115

pSIM1678 1 LPLLLLLL Endochitinase

GI:3201547

80-mer homology search

A second analysis performed using the FARRP web-tool identifies larger regions of similarity between 

the ORFs and known allergens. This analysis compares all 80 amino acid sequences within an ORF and 

identifies any matches with greater than 35% homology to known allergens. The algorithm only 

identified one potential allergen corresponding to an ORF in our pSIM1678 insert (Table D-4). Similar to 

the 8-mer match, the target identified was the vacuolar invertase gene. As discussed earlier, there is no 

allergen-relatated safety concern associated with this ORF or the inserts, in general.

Table D-4. 80-mer Sliding Window Search Results for Query A

Query* Match

Best

%ID

# Hits

> 35%

Full Alignment Links

E-val %ID length NCBI

Minor allergen betafructofuranosidase precursor -

vacuolar invertase(Solanum 

lycopersicum)

100.00% 206 of 226 1.1e-098 96.50% 226

gi|18542113

gi|18542115

*>226 AA Putative ORF

IPKIPSITRRVYQSYLWSSRKFRQNQVYKVTLDISIIKLKISAVIQVQSHSTTHSLVERS

VDNKLVNGSTFLPDQPDSGHRKSLKIISGIFLSSFLLLSVAFFPILNNQSPDLQSNSRSP

APPSRGVSQGVSDKTFRDVVNASHVSYAWSNAMLSWQRTAYHFQPQKNWMNDPNGPLYHK

GWYHLFYQYNPDSAIWGNITWGHAVSKDLIHWLYLPFAMVPDQWYDINGVWTGSATILPD

GQIMMLYTGDTDDYVQVQNLAYPTNLSDPLLLDWVKYKGNPVLVPPPGIGVKDFRGIQRT

QSRHR
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 7

Toxicity evaluation

Unlike with allergenicity studies, we are not aware of a formal method for establishing toxin-related 

safety in genetically-modified organisms, so we modeled our analysis after the rigorous approach used 

for the allergenicity studies. We consulted with experts at NCBI and Uniprot to develop methods to 

generate and search toxin-specific databases for homology between our ORFs and known protein toxins. 

We took advantage of two repositories of annotated toxins for this analysis. The first database (mvirDB) 

consists of known bacterial toxins, virulence factors, and antibiotic resistance genes, which is a publiclyavailable repository maintained as a combination of at least eight independent database sources (Zhou, 

2006). A second BLAST-compatible database was created to supplement the bacterial toxin database by 

extracting all eukaryotic proteins annotated as toxins within the Uniprot repository. These smaller and 

more specific databases make toxicity assessment more straightforward than non-specific searches 

against NCBI databases.

Table D-5. Summary of Toxicity Database Queries for W8

Construct mvirDB hits uniprotToxin hits NCBI nr hits

pSIM1278 0 0 513

pSIM1678 0 0 1380

Using the blastp algorithm within the BLAST suite of applications available through NCBI, we initially 

blasted all of our ORFs against the NCBI non-redundant protein database. Since the vast majority of the 

ORFs were small (35 amino acid average), many non-specific hits are generated making it a challenge to 

objectively assess toxicity although, as expected, most matches (E-value ≤ 1.0) were of potato origin. 

However, the blastp algorithm did not identify any proteins with significant homology when the toxin 

databases were queried with the ORF sequences (Table D-5). Thus, no toxin-related safety concerns 

were identified for any of the InnateTM potato events.

Native gene disruption

The potato (Solanum tuberosum) genome presents some unique challenges for analysis of native gene 

disruption. Most potato cultivars, including the Russet Burbank parent of W8, are tetraploid, highly 

heterozygous, and include a significant amount of redundant and /homologous DNA. These challenges 

have prevented the research community from sequencing the genome of these cultivars directly. 

Instead, they sequenced a unique homozygous form of the potato, doubled monoploid, derived using 

tissue culture techniques (The Potato Genome Sequencing Consortium). This sequence was later used to 

integrate sequence from a heterozygous diploid line. Together, these efforts have provided a genome 

sequence that is valuable for investigating the evolution and genome organization of potatoes, but is 

still lacking for detailed analysis of individual loci in commercial cultivars, which are mostly tetraploid. It 

is not uncommon to BLAST known sequences against the current potato genome only to identify 

numerous hits scattered across chromosomes, or more perplexing, to retrieve no hits at all.
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 8

It is highly unlikely that a gene disruption would have a functional impact on the plant or tuber due to its 

tetraploid nature, which means that a single gene disruption would leave the plant with up to three 

functional copies of the native gene. These traits highlight the power of using biotechnology in potatoes 

as native gene disruptions are both uncommon and unlikely to result in a measurable or observable 

changes in phenotype. In the event that a native gene is disrupted by an InnateTM construct, it is 

generally going to produce a truncated version of the native protein as opposed to a novel fusion 

protein since the ORFs contained in the construct are extremely short on average and would halt 

translation. Also, native gene disruption that significantly altered plant health would be identified in 

early stages of line selection, where only those plants matching the parent phenotype are advanced to 

the final stages of product development.

In an effort to identify the insertion site of the construct, we performed a BLAST (blastn) search against 

the NCBI and MSU potato genome databases to identify the likely insertion location and any annotated 

genes that may have been disrupted. When possible, the locus was further evaluated using the graphical 

viewers made available by NCBI and MSU to visually inspect the locus for disrupted genes. We also 

performed BLAST (blastx) searches, which translates the input nucleic acid sequences in all six reading 

frames and searches for protein matches. The results for each event are summarized below.

pSIM1278 locus

There is strong homology between the right and left flanking sequences in a locus on chromosome 2 

(left flank, 29305660:29305587, right flank) according to the Michigan State University (MSU) genome 

database

(http://potato.plantbiology.msu.edu/integrated_searches.shtml).(http://potato.plantbiology.msu.edu/i

ntegrated_searches.shtml).

Neither the blastn nor blastx searches against the NCBI database identified annotated genes disrupted 

by the flanking sequences. A BLAST search against the “PGSC S. tubersum group Phureja DM1-3 

Transcripts (v3.4)” dataset did not reveal an expressed transcript disrupted by the pSIM1278 integration 

event. Similarly, visual inspection of the tracks covering the locus on chromosome 2 using the MSU 

graphical genome browser did not identify any candidates for native gene disruption. Collectively, we 

found no evidence of native gene disruptions associated with the insertion of pSIM1278 in W8 for 

further consideration.

Figure D-2. W8 pSIM1278 insertion on chromosome 2

pSIM1278 Insert Left flank 

(Chr2:29305660)

Right flank 

(chr2:29305587) 
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 9

pSIM1678 locus

The insertion site for pSIM1678 is more difficult to conclusively map than the integration site for 

pSIM1278. The flanking regions have weak homology within the reference genome, but a locus on 

chromosome 10 (left flank, 48535311:48535313, right flank) had homology with both flanking regions, 

independently and is considered the best candidate for integration of pSIM1678. 

Neither the blastn nor blastx searches against the NCBI database identified annotated genes disrupted 

by the flanking sequences. A BLAST search against the “PGSC S. tubersum group Phureja DM1-3 

Transcripts (v3.4)” dataset did not reveal an expressed transcript disrupted by the pSIM1278 integration 

event. Similarly, visual inspection of the tracks covering the locus on chromosome 10 using the MSU 

graphical genome browser did not identify any candidates for native gene disruption. Collectively, we 

found no evidence of native gene disruptions associated with the insertion of pSIM1678 in W8 for 

further consideration.

Figure D-3. W8 pSIM1678 insertion on chromosome 10

The sequences for the left (L1-L6) and right junctions (R1-R6) were analyzed as part of the analysis 

described above and are summarized in more detail in Tables D-5 and D-6. As shown, no potential toxins 

or allergens were identified associated with the ORFs specifically covering the junction regions.

pSIM1678 Insert Left flank 

(Chr10:48535311) 

Right flank

(chr10:48535313)
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 10

Table D-5. Summary of toxicity and allergenicity of pSIM1278 junction bands 

Frame ORF sequences Toxins Allergens

L1 ANFLVVSSPTKGPNSKSKTLYTKWYKYNYLIYYRVID* None None

L2 ENSVRPIFWLYPALQKAQTLSQRLYIQNGINIII* None None

L3 NIKYRKNKKTRLGQFFGCIQPYKRPKL* None None

L4 LRVWAFCRAGYNQKIGLTEFSYFFGILYFKSSNN* None None

L5 SLGLL* None None

L6 LYLYHFVYKVFDLEFGPFVGLDTTKKLA* None None

R1 LGVGFIPSWTTVSHFQVLELRKLYSVI* None None

R2 NYENYIR* None None

R3 IIISGAHRYAINFIYLVGSRLYTELDYGQSLSSPRTTKIIFGNIIQEFFLLF* None None

R4 FS* None None

R5 IILPNIIFVVLGLESD* None None

R6 KKFLNYITEYNFRSSRT* None None

Table D-6. Summary of toxicity and allergenicity of pSIM1678 junction bands 

Frame ORF sequences Toxins Allergens

L1 ILENQIVLLQQQLKKA* None None

L2 KKPNFHGTYKLSLIIAQTHSHEPKLGKVLQDVHQTVRKHKMALDI* None None

L3 IVKHMVMFNINTRKSNSTTTTTIKKSLIFMEHTN* None None

L4 KLGFF* None None

L5 RHFMFPNCLMNVLQNFTQFGFMAVSLGYYETQFVCSMKIRLFLIVVVV

VLFDFRVFMLNITMCLTIYCIS*

None None

L6 AFFNCCCSSTI* None None

R1 *EPISNRDSRFVSYSIKLIFGQFISVNLIFDWELREVIWSHVVPDFSS* None None

R2 IIISGAHRYAINFIYLVGSRLYTELDYGQSLSSPRSQFQIAIPASFLIRLS* None None

R3 LGVGFIPSWTTVSHFQALGANFKSRFPLRFLFD* None None

R4 NWLLGLESD* None None

R5 SNKKRSGNRDLKLAPRA* None None

R6 ETKRESRFEIGS* None None

- R1 ORF does not cross the junction as the flanking sequence contains a stop codon at the 

junction. Sequence shown is entirely within pSIM1678 insert.
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 11

Summary

Using a number of well-established bioinformatics tools, we have performed a comprehensive analysis 

on the loci including each of the inserts in Russet Burbank W8. We did not find evidence of a native gene 

disruption from either insert in the plant genome. Being that commercial potatoes are tetraploid, a 

native gene disruption is unlikely to have an effect on the physiology of the potato and would only be a 

concern if a novel allergen or toxin was produced as a result of the insertion. However, our 

bioinformatics analysis did not find any evidence of allergens or toxins associated with inserts that pose 

a safety concern. If anything, our Russet Burbank W8 event is predicted to have reduced allergen 

potential due to silencing of the vacuolar invertase protein, which is listed as a mild allergen in tomato. 

The comprehensive bioinformatics analysis covered: (1) the VNT1 protein being expressed by the 

cassette containing the Rpi-vnt1 gene, (2) all ORFs spanning junctions between the flanking regions and 

insert sequence for pSIM1278 and pSIM1678, and (3) any ORF consisting of at least 19 amino acids, 

whether or not it contained a start codon.

Collectively, our bioinformatics studies did not identify any safety concerns associated with Russet 

Burbank W8 as a result of the transformation. 
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 12

Methods

ORF detection

All potential open reading frames (ORFs) created as a result the event-specific, integration event were 

identified using the ORF Finder web application (Stothard, 2000) available through the Sequence 

Manipulation Suite (http://www.bioinformatics.org/sms2/orf_find.html). The search parameters were 

defined to identify all ORFs with at least 20 amino acids located between two contiguous stop codons. A 

nucleotide sequence has the potential to be translated in up to three reading frames consisting of 

contiguous codon triplets from RNA transcribed from either direction on the chromosome. Thus, ORFs 

were identified from all six reading frames for input consisting of the insert sequence flanked by 

sufficient plant genomic sequence on each side where available. The 500 nucleotides of flanking 

sequence is more than enough to account for any potential ORFs encompassing the junction sequences.

The results were converted into FASTA-formatted files for further analysis.

Database and software resources

The most recent Blast software suite (v2.2.28; Altschul, 1997) was downloaded from NCBI 

(ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) and installed locally. A number of BLASTcompatible databases were generated and/or downloaded to support the toxin analysis described 

herein. The most recent NCBI non-redundant (nr) protein database (August 26, 2013 post date) was 

downloaded using the update_blastdb.pl script provided with the BLAST application suite. Searches 

against this database are comprehensive, but non-specific and produce data that can be challenging to 

associate with putative toxins. In order to perform more directed searches, we took advantage of two 

toxin-specific resources. The first is a microbial database of toxins, virulence factors, and antibiotic 

resistance genes (Zhou et al., 2006) that was downloaded from the Lawrence Livermore National 

Laboratory Virulence Database Home Page (http://mvirdb.llnl.gov/) and installed locally (mvirdb). This 

database comprises known microbial protein toxins from numerous publically available sources, 

including Tox-Prot, SCORPION, PRINTS, VFDB, TVFac, ARGO, and VIDA. A second eukaryote-specific 

(taxid: 2759) toxin database was generated by downloading all FASTA-formatted sequences annotated 

with the keyword=toxin from the uniprot database using a browser script provided by Uniprot technical 

support at the SIB Swiss Institute of Bioinformatics: 

http://www.uniprot.org/uniprot/?query=taxonomy%3a2759+keyword%3atoxin&force=yes&format=fas

ta&include=yes

This toxin dataset was converted into a BLAST-compatible database (uniprot_toxin) using the 

makeblastdb.exe utility provided in the BLAST software suite.

Allergenicity database searches

Allergenicity potential was evaluated using the public, allergen-specific search engine 

(http://www.allergenonline.org/databasefasta.shtml) available through the Food Allergy Research and 

Resource Program at the University of Nebraska. The ORFs associated with the insert and flanking 

regions were analyzed using two approaches: (1) 80-mer sliding region homology search and (2) 8-mer 

identity search. Only protein sequences consisting of 28 or more amino acids were analyzed using the 
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 13

80-mer sliding window as this is the minimum sequence size capable of reaching the lower threshold 

target of 35% homology. However, all ORFs were analyzed using the 8-mer match. All searches were 

performed using database version 13, dated February 12, 2013. 

Toxin database searches

The comprehensive set of ORFs predicted by the ORF Finder application was used as input for BLAST

(blastp) searches against the databases described above; nr, mvirdb, or uniprot_toxin. When performing 

blastp searches it is common to consider the Expect value (E-value) as a measure of statistical 

significance where values less than 0.1 or 0.05 are generally considered biologically significant (BLAST 

help). That is, the E-value describes the number of hits that one might “expect” to see by chance when 

searching a database of a particular size. In order to consider any potential homology between the ORFs 

and the sequences in the toxin-specific databases, we analyzed our BLAST searches using a higher Evalue threshold of 1.0. However, since the E-value calculation is dependent upon the size of the search 

space, we consulted with a BLAST application expert at NCBI to determine the appropriate search space 

(–searchsp) parameter for our toxin database searches. BLAST reported an effective search space of

roughly 1 x 1011 when the ORFs were blasted (blastp-short) against the NCBI nr database. Our searches 

used a search space that was an order of magnitude smaller. Thus, our database searches used the 

following command line input:

>blastp –task blastp-short –query ORFs.fa –db database –out db_output.txt –searchsp 10000000000 –

outfmt 6 –evalue 1.01

The database string was replaced by nr, mvirdb, or uniprot_toxin depending on the specific search. The 

nr searches served as a control to ensure our searches were performing as expected. The algorithm 

applies the PAM30 scoring matrix with gap penalties of 9 for existence and 1 for extension as a default 

associated with the blastp-short search parameter.

Native gene disruption

Where available, the sequences identified as the left and right flanking regions were used as input for a 

BLAST (blastn) search against the NCBI potato genome to identify the likely integration site and any 

genes associated with the flanking regions (E-value cutoff of 1 x 10-6). The same sequences were used to 

search the more frequently updated potato genome housed at Michigan State University (MSU)

(http://potato.plantbiology.msu.edu/integrated_searches.shtml). All searches were performed against 

the most recent version of the database (v 4.03). The sequences were also provided as input for BLAST 

(blastx) searches to identify any homologous proteins to the nucleotide coding sequence. This algorithm 

compares the translational products of the nucleotide sequence from all six reading frames to proteins 

in the database. These searches were also performed against the NCBI potato database (E-value cutoff 

of 0.01). 
J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 14

References

Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., and D.J. Lipman (1997), "Gapped 

BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 

25:3389-3402.

Goodman R.E., Vieths S., Sampson H.A., Hill D., Ebisawa M., Taylor S.L., and R. van Ree, “Allergenicity 

assessment of genetically modified crop-what makes sense?”, Nature Biotechnology, 2008, 26(1):73-81.

Ladics G.S., Bannon G.A., Silvanovich A., and R.F. Cressman, “Comparison of conventional FASTA identity 

searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities 

to known allergens”, Mol. Nutr. Food Res., 2007, 51:985-998.

Metcalfe D.D., Astwood J.D., Townsend R., Sampson H.A., Taylor S.L., and RL Fuchs, “Assessment of the 

allergenic potential of foods derived from genetically modified crop plants.”, Crit. Rev. Food Sci. Nutr., 

1996, 36 Suppl:S165-186.

The Potato Genome Sequencing Consortium, “Genome sequence and analysis of the tuber crop potato,” 

Nature, 2011, 475:189-197.

Stothard P., “The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting 

protein and DNA sequences”, Biotechniques, 2000, 28:1102-1104.

Terrat Y. and F. Ducancel, “Are there unequivocal criteria to label a given protein as a toxin? Permissive 

versus conservative annotation processes”, Genome Biology, 2013, 14:406-412.

Zhou C.E., Smith J., Lam M., Zemla A., Dyer M.D., and T. Slezak, “MvirDB-a microbial database of protein 

toxins, virulence factors and antibiotic resistance genes for bio-defence applications”, Nucleic Acid 

Research, 2007, 35:D391-D394.

BLAST help: 

http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=ProgSelectionGu

ide
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 1

TITLE

Dietary Exposure to Rpi-vnt1.1 –

A Bioinformatics Review of Genes Homologous to Rpi-vnt1.1 in Edible Plants

AUTHORS

Susan Collinge, PhD and Pete Clark, PhD

Report No. 13-38-SPS-MOL

SUBMITTED BY

J.R. Simplot Co. Plant Sciences

5369 W. Irving St., Boise, ID 83706

PREPARED WITH:

Exponent

1150 Connecticut Ave., NW

Suite 1100

Washington, DC 20036

Date

March 28, 2014

PAGE COUNT

1 of 29
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 2

Table of Contents

List of Tables .................................................................................................................................................3

List of Figures................................................................................................................................................3

Acronyms and Abbreviations........................................................................................................................4

Introduction ..................................................................................................................................................5

Background ...................................................................................................................................................5

Late Blight Potato Breeding History..........................................................................................................5

Rpi-Vnt1 History/Origin ............................................................................................................................5

Mode of action..........................................................................................................................................6

VNT1 Similarities to other Dietary Proteins – Blast Homology Search.........................................................6

General BLAST Search.................................................................................................................................11

Methods..................................................................................................................................................11

Results.....................................................................................................................................................11

DNA Homology....................................................................................................................................11

Protein Homology ...............................................................................................................................17

Specific BLAST Search and Results..............................................................................................................18

ToMV RESISTANCE PROTEINS LEVEL EXPRESSION......................................................................................19

Literature Search.....................................................................................................................................19

Results.....................................................................................................................................................19

Database Search .....................................................................................................................................20

Results.....................................................................................................................................................20

Consumption of Tomatoes with ToMV Resistance Gene .......................................................................21

Conclusions.................................................................................................................................................21

Literature Cited...........................................................................................................................................22
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 3

List of Tables

Table 1. DNA Sequences from Edible Plants Homologous to Rpi-vnt1.1 (FJ423044.1). 12

Table 2. Protein Sequences with Significant Homology to Rpi-vnt1.1 (ACJ66594.1) Protein 

Sequence 17

Table 3. Fresh Tomato Varieties with ToMV Resistance Gene 26

List of Figures

Figure 1. Rpi-vnt1.1 Gene Sequence (FJ4230441.1) 7
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 4

Acronyms and Abbreviations

BLAST Basic Local Alignment Search Tool

BLASTn Standard Nucleotide BLAST

BLASTp Protein- Protein BLAST

BLASTx Translated BLAST

CC Coiled-Coil

DDBJ DNA Databank of Japan

EMBL The European Molecular Biology Laboratory

EST Expressed Sequence Tags

HTGS High-Throughput Genome Sequence

GSS Genome Survey Sequences

NB-LRR Nucleotide-Binding Site and Leucine- Rich Repeat

NCBI National Center for Biotechnology Information

PDB Protein Data Bank

Rpi-vnt1 R-gene that is resistant to Phytophtora infestans (Rpi) from 

Solanum venturii

STS Sequence-Tagged Sites

TIR Toll / Interleukin 1 Receptor

Tomato Currently known as Solanum lycopersicum, previously known as 

Lycopersicon esculentum. May be referred to as Lycopseriscon 

esculentum in older papers and database searches such as BLAST.

VNT1 Protein expressed from Rpi-vnt1 gene
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 5

Introduction

A resistance gene to the most important potato pathogen, Phytopthora infestans, referred to as Rpivnt1, was introduced into commercial potato varieties using Agrobacterium-mediated transformation. 

The Rpi-vnt1 gene confers resistance to late blight, a devastating disease to potatoes and tomatoes. A

bioinformatics review of genes that are homologous to the gene Rpi-vnt1.1 (FJ423044.1) in edible plants 

was conducted to establish existing dietary exposure to this gene. This report summarizes the 

bioinformatics review, literature search, and results. In addition, the report provides evidence to 

support the safe introduction of this gene into the diet.

Background

Late Blight Potato Breeding History 

Resistance to late blight (Phytophthora infestans) occurs in many tuber-bearing wild Solanum species 

that belong to the highly diverse section Petota Dumort. Early potato breeding programs from the midtwentieth century used Solanum demissum, a common species in central Mexico and a great source of 

resistance against late blight. In total, eleven S. demissum resistance (R) genes designated R1-R11 are 

distinguished in a potato differential set made by Black and Mastenbroek (Black et al. 1953; Malcolmson 

and Black, 1966). R1, R3, and R10, and to a lesser extent R2 and R4, have been widely used for 

introgression in European breeding programs (Hoekstra and van Loon 2000)) to help control late blight. 

The introduction of new cultivars containing these R genes was initially successful, but rapidly evolving

populations of P. infestans reduced their efficacy (Fry 2008; McDonald and Linde 2002; Pink and 

Puddephat 1999; Wastie 1991). However, durability in the field of a particular R gene is variable (Leach 

et al. 2001), and additional novel resistance genes against P. infestans (Rpi-genes) are being discovered 

from other wild Solanum species. To date, 68 Rpi-genes, from wild Solanum species, have been 

characterized (Rodewald and Trognitz, 2013) and about 20 of them have been cloned. In addition, 

germplasm from those wild Solanum species has been integrated into numerous cultivars through 

breeding, for example but not limited to: S. bulbocastanum (Park et al. 2005; Ramanna and Hermsen 

1971), S. stoloniferum (Hutten and van Berloo 2001, referred to as sto or CPC 2093), S. microdontum

(Tan et al. 2008) and S. phureja (Sliwka et al. 2010; 2013). 

Rpi-Vnt1 History/Origin

Recently, the first Rpi-gene (Rpi-vnt1) from a South America wild Solanum species, S. venturii, was 

cloned (Pel et al. 2009; Foster et al. 2009). Three functional alleles of the gene were identified, Rpivnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 alleles. The gene was first located on chromosome 9 (same locus as 

Tm-2(2) from S. lycopersicum which confers resistance against Tomato mosaic virus (ToMV)) of the 

potato wild species S. venturii. The gene was identified by a classical genetic and physical mapping 

approach including nucleotide binding site profiling and bulked segregant analysis, as well as a Tm-2(2) 

based allele mining strategy (Pel et al. 2009; Foster et al. 2009). Transgenic potato and tomato plants 

carrying Rpi-vnt1.1 were shown to be resistant to P. infestans. Of 11 P. infestans isolates tested, only 

isolate EC1 from Ecuador was able to overcome Rpi-vnt1.1 and cause disease on the inoculated plants 

(Pel 2010). However, Rpi-vnt1.1 remains of major importance since it brings resistance against the most 

dominant late blight isolate in Europe, Blue 13, by responding to the presence of the cognate effector 

AVR-VNT1 (Cooke et al. 2012). Alleles of Rpi-vnt1 (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) which differed 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 6

by only a few nucleotides were found in other late blight resistant accessions of S. venturii. The late 

blight resistance gene Rpi-phu1 from S. phureja (Sliwka et al. 2006) has been shown to be identical to 

Rpi-vnt1.1 (Pel 2010; Sliwka et al. 2013), suggesting either that this strong resistance gene has been 

maintained since a common ancestor, due to selection pressure for blight resistance, or that genetic 

exchange between S. venturii and S. phureja has occurred at some time. In addition, a collection 

containing 200 wild Solanum species with 5 genotypes each was screened. Rpi-vnt1 alleles were found 

in S. venturii, as expected, S. mochiquense and S. weberbaueri (Pel 2010; Vleeshouwers et al. 2011; 

SOLRgene database). Moreover, Rpi-phu1 (also called Rpi-vnt1) was introgressed into S. tuberosum

(tetraploid potato varieties) from an interspecific cross between Solanum phureja and Solanum 

stenotomum (known as pinta boca in Peru and Bolivia). Both of these species are edible and research 

programs are underway in Europe to introgress Rpi-phu1/Rpi-vnt1 (identical genes) in cultivated 

potatoes (Sliwka et al. 2010; 2013).

Mode of action

Expression of Rpi-vnt1 in wild and cultivated potato confers broad-spectrum resistance to late blight 

strains caused by P. infestans. Resistant potato parts include leaves, stems and tubers. This resistance is 

based on the recognition of a pathogen-specific protein (effector protein) by a host-specific protein 

(resistance or R protein). Upon infection, AVR-VNT1, an effector protein produced by P. infestans, is 

recognized by the R-protein VNT1 expressed in potato (Pel, Dissertation, 2010). Consequently, VNT1

activates a signal transduction pathway that leads to localized plant cell death or hypersensitive 

response. Death is restricted to a few plant cells and limits the growth and spread of P. infestans

throughout the rest of the plant.

VNT1 Similarities to other Dietary Proteins – Blast Homology Search 

The similarity of the Rpi-vnt1.1 gene (GenBank number FJ423044.1, GI: 215401988 from Solanum

venturii) to disease-resistance (R) genes found in edible plant species was examined. Widely consumed 

plant species that are reported to contain disease-resistance (R) genes include the Solanaceae family, 

Zea mays (maize), Glycine max (soybeans), Oryza sativa L. (rice), and Triticum aestivum L. (common 

wheat) (Bakker et al. 2011). Potato belongs to the Solanaceae family, which includes eggplant (Solanum 

melongena L.), pepper (Capsicum annum L.), and tomato (Solanum lycopersicum L.). 

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site 

(NBS or NB) and leucine-rich repeat (LRR) domain containing protein (Lozano et al., 2012). Jupe et al. 

(2012) performed an amino acid motif based search of the annotated potato genome and identified 438 

NB-LRR type genes among approximately 39,000 potato gene models. There are two classes of Rproteins with distinct motifs: N-terminal toll/interleukin 1 receptor (TIR)(TIR-NB-LRR) and N-terminal 

coiled-coil (CC-NB-LRR). Of the predicted genes, 77 contain a TIR-NB-LRR-like domain and 107 of the 

remaining 361 non-TIR genes contain a CC-NB-LRR N-terminal domain. The Solanum venturii late blight 

resistance protein VNT1 has all the sequence features of a CC-NB-LRR protein. The tomato mosaic virus 

resistance gene Tm-2(2) from Lycopersicon esculentum consists of a single gene that encodes an 861 

amino acid polypeptide, which belongs to the CC-NB-LRR class of resistance proteins (Lanfermeijer et al., 

2003). 

A Basic Local Alignment Search Tool (BLAST) homology search was conducted, via the National Center 

for Biotechnology Information (NCBI), to identify edible plant species containing DNA sequences or 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 7

amino acid sequences with similarity to the Rpi-vnt1.1 gene sequence (FJ423044.1, as described above). 

Searches conducted were optimized by selecting the megablast program for highly similar sequences, 

discontiguous megablast for more dissimilar sequences, and blastn for somewhat similar sequences. 

Because Rpi-vnt1 belongs to the super-family that harbors a nucleotide binding and a leucine-richrepeat domain (NB-LRR) (Bakker et al. 2010; Jupe et al. 2012), additional BLAST analyses were 

conducted to identify edible plant species that contain disease resistance NB-LRR genes that may be 

homologous to the Rpi-vnt1.1 gene sequence. The full entry for the Rpi-vnt1.1 (FJ423044.1) gene 

sequence is provided below in Figure 1 as it appears in the NCBI nucleotide database (available at 

http://www.ncbi.nlm.nih.gov/nucleotide/). 

Figure 1. Rpi-vnt1.1 Gene Sequence (FJ4230441.1)

Solanum venturii late blight resistance protein (Rpi-vnt1.1) gene

GenBank: FJ423044.1

LOCUS FJ423044 4310 bp DNA linear PLN 23-AUG-2010

DEFINITION Solanum venturii late blight resistance protein (Rpi-vnt1.1) gene,

 complete cds.

ACCESSION FJ423044

VERSION FJ423044.1 GI:215401988

KEYWORDS .

SOURCE Solanum venturii

 ORGANISM Solanum venturii

 Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;

 Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons;

 asterids; lamiids; Solanales; Solanaceae; Solanoideae; Solaneae;

 Solanum.

REFERENCE 1 (bases 1 to 4310)

 AUTHORS Foster,S.J., Park,T.H., Pel,M., Brigneti,G., Sliwka,J., Jagger,L.,

 van der Vossen,E. and Jones,J.D.

 TITLE Rpi-vnt1.1, a Tm-2(2) homolog from Solanum venturii, confers

 resistance to potato late blight

 JOURNAL Mol. Plant Microbe Interact. 22 (5), 589-600 (2009)

 PUBMED 19348576

REFERENCE 2 (bases 1 to 4310)

 AUTHORS Pel,M.A., Foster,S.J., Park,T.H., Rietman,H., van Arkel,G.,

 Jones,J.D., Van Eck,H.J., Jacobsen,E., Visser,R.G. and Van der

 Vossen,E.A.

 TITLE Mapping and cloning of late blight resistance genes from Solanum

 venturii using an interspecific candidate gene approach

 JOURNAL Mol. Plant Microbe Interact. 22 (5), 601-615 (2009)
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 8

 PUBMED 19348577

REFERENCE 3 (bases 1 to 4310)

 AUTHORS Foster,S.J., Park,T.-H., Pel,M., Brigneti,G., Sliwka,J., Jagger,L.,

 van der Vossen,E. and Jones,J.

 TITLE Direct Submission

 JOURNAL Submitted (27-OCT-2008) John Innes Centre, The Sainsbury

 Laboratory, Colney Lane, Norwich NR4 7UH, United Kingdom

FEATURES Location/Qualifiers

 source 1..4310

 /organism="Solanum venturii"

 /mol_type="genomic DNA"

 /specimen_voucher="CGN18108"

 /db_xref="taxon:610331"

 /chromosome="9"

 /note="originally submitted as Solanum okadae;

 linked to TG35 and TG551"

 gene 710..>3385

 /gene="Rpi-vnt1.1"

 CDS 710..3385

 /gene="Rpi-vnt1.1"

 /codon_start=1

 /product="late blight resistance protein"

 /protein_id="ACJ66594.1"

 /db_xref="GI:215401989"

 /translation="MNYCVYKTWAVDSYFPFLILTFRKKKFNEKLKEMAEILLTAVIN

 KSIEIAGNVLFQEGTRLYWLKEDIDWLQREMRHIRSYVDNAKAKEVGGDSRVKNLLKD

 IQQLAGDVEDLLDEFLPKIQQSNKFICCLKTVSFADEFAMEIEKIKRRVADIDRVRTT

 YSITDTSNNNDDCIPLDRRRLFLHADETEVIGLEDDFNTLQAKLLDHDLPYGVVSIVG

 MPGLGKTTLAKKLYRHVCHQFECSGLVYVSQQPRAGEILHDIAKQVGLTEEERKENLE

 NNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKIGSRIIITSRNSNVGRYIGG

 DFSIHVLQPLDSEKSFELFTKKIFNFVNDNWANASPDLVNIGRCIVERCGGIPLAIVV

 TAGMLRARGRTEHAWNRVLESMAHKIQDGCGKVLALSYNDLPIALRPCFLYFGLYPED

 HEIRAFDLTNMWIAEKLIVVNTGNGREAESLADDVLNDLVSRNLIQVAKRTYDGRISS

 CRIHDLLHSLCVDLAKESNFFHTEHNAFGDPSNVARVRRITFYSDDNAMNEFFHLNPK

 PMKLRSLFCFTKDRCIFSQMAHLNFKLLQVLVVVMSQKGYQHVTFPKKIGNMSCLRYV

 RLEGAIRVKLPNSIVKLKCLETLDIFHSSSKLPFGVWESKILRHLCYTEECYCVSFAS

 PFCRIMPPNNLQTLMWVDDKFCEPRLLHRLINLRTLCIMDVSGSTIKILSALSPVPRA

 LEVLKLRFFKNTSEQINLSSHPNIVELGLVGFSAMLLNIEAFPPNLVKLNLVGLMVDG

 HLLAVLKKLPKLRILILLWCRHDAEKMDLSGDSFPQLEVLYIEDAQGLSEVTCMDDMS

 MPKLKKLFLVQGPNISPISLRVSERLAKLRISQVL"
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 9

ORIGIN 

 1 agttatacac cctacattct actcgagtca ttatgatgat gtctcacgac caaatcaaat

 61 caaagttaaa taaatatcga accgaacgcc cactctgtat gagtatggca aaagattttg

 121 agagaatcaa gttgcataaa agcctaattt tcatggaaca tacaaattga gtctcataat

 181 agcccaaact cacagccatg aacccaaatt gggtaaagtt ttgcaagacg ttcatcaaac

 241 agttaggaaa cataaaatgg cgctagatat ataataaatt tttttaacat atggtgtgat

 301 tgatagttat atactaaaga tgtttgctta gttacgtaat tttttcaaaa aaaaaaggta

 361 cattatcaat catcagtcac aaaatattaa aagttactgt ttgtttttta aattccatgt

 421 cgaatttaat tgaatgacac ttaaattggg acgaacggtg taatttcttt tgactattct

 481 actagtatct atccacagca cgtgttgttc ctttcttctt tcgtttttca tttacttgac

 541 attattagga gacttggccc tgaactccaa ctattctaag ctgacctttc ttttccttta

 601 ccaattatct tcttctttct aatttcgttt tacgcgtagt actgcctgaa ttttctgact

 661 ttcaacgttt gttattcatg cttgaaaacg aaataccagc taacaaaaga tgaattattg

 721 tgtttacaag acttgggccg ttgactctta ctttcccttc ctcatcctca catttagaaa

 781 aaagaaattt aacgaaaaat taaaggagat ggctgaaatt cttctcacag cagtcatcaa

 841 taaatcaata gaaatagctg gaaatgtact ctttcaagaa ggtacgcgtt tatattggtt

 901 gaaagaggac atcgattggc tccagagaga aatgagacac attcgatcat atgtagacaa

 961 tgcaaaggca aaggaagttg gaggcgattc aagggtgaaa aacttattaa aagatattca

 1021 acaactggca ggtgatgtgg aggatctatt agatgagttt cttccaaaaa ttcaacaatc

 1081 caataagttc atttgttgcc ttaagacggt ttcttttgcc gatgagtttg ctatggagat

 1141 tgagaagata aaaagaagag ttgctgatat tgaccgtgta aggacaactt acagcatcac

 1201 agatacaagt aacaataatg atgattgcat tccattggac cggagaagat tgttccttca

 1261 tgctgatgaa acagaggtca tcggtctgga agatgacttc aatacactac aagccaaatt

 1321 acttgatcat gatttgcctt atggagttgt ttcaatagtt ggcatgcccg gtttgggaaa

 1381 aacaactctt gccaagaaac tttataggca tgtctgtcat caatttgagt gttcgggact

 1441 ggtctatgtt tcacaacagc caagggcggg agaaatctta catgacatag ccaaacaagt

 1501 tggactgacg gaagaggaaa ggaaagaaaa cttggagaac aacctacgat cactcttgaa

 1561 aataaaaagg tatgttattc tcttagatga catttgggat gttgaaattt gggatgatct

 1621 aaaacttgtc cttcctgaat gtgattcaaa aattggcagt aggataatta taacctctcg

 1681 aaatagtaat gtaggcagat acataggagg ggatttctca atccacgtgt tgcaacccct

 1741 agattcagag aaaagctttg aactctttac caagaaaatc tttaattttg ttaatgataa

 1801 ttgggccaat gcttcaccag acttggtaaa tattggtaga tgtatagttg agagatgtgg

 1861 aggtataccg ctagcaattg tggtgactgc aggcatgtta agggcaagag gaagaacaga

 1921 acatgcatgg aacagagtac ttgagagtat ggctcataaa attcaagatg gatgtggtaa

 1981 ggtattggct ctgagttaca atgatttgcc cattgcatta aggccatgtt tcttgtactt

 2041 tggtctttac cccgaggacc atgaaattcg tgcttttgat ttgacaaata tgtggattgc

 2101 tgagaagctg atagttgtaa atactggcaa tgggcgagag gctgaaagtt tggcggatga

 2161 tgtcctaaat gatttggttt caagaaactt gattcaagtt gccaaaagga catatgatgg

 2221 aagaatttca agttgtcgca tacatgactt gttacatagt ttgtgtgtgg acttggctaa

 2281 ggaaagtaac ttctttcaca cggagcacaa tgcatttggt gatcctagca atgttgctag
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 10

 2341 ggtgcgaagg attacattct actctgatga taatgccatg aatgagttct tccatttaaa

 2401 tcctaagcct atgaagcttc gttcactttt ctgtttcaca aaagaccgtt gcatattttc

 2461 tcaaatggct catcttaact tcaaattatt gcaagtgttg gttgtagtca tgtctcaaaa

 2521 gggttatcag catgttactt tccccaaaaa aattgggaac atgagttgcc tacgttatgt

 2581 gcgattggag ggggcaatta gagtaaaatt gccaaatagt attgtcaagc tcaaatgtct

 2641 agagaccctg gatatatttc atagctctag taaacttcct tttggtgttt gggagtctaa

 2701 aatattgaga catctttgtt acacagaaga atgttactgt gtctcttttg caagtccatt

 2761 ttgccgaatc atgcctccta ataatctaca aactttgatg tgggtggatg ataaattttg

 2821 tgaaccaaga ttgttgcacc gattgataaa tttaagaaca ttgtgtataa tggatgtatc

 2881 cggttctacc attaagatat tatcagcatt gagccctgtg cctagagcgt tggaggttct

 2941 gaagctcaga tttttcaaga acacgagtga gcaaataaac ttgtcgtccc atccaaatat

 3001 tgtcgagttg ggtttggttg gtttctcagc aatgctcttg aacattgaag cattccctcc

 3061 aaatcttgtc aagcttaatc ttgtcggctt gatggtagac ggtcatctat tggcagtgct

 3121 taagaaattg cccaaattaa ggatacttat attgctttgg tgcagacatg atgcagaaaa

 3181 aatggatctc tctggtgata gctttccgca acttgaagtt ttgtatattg aggatgcaca

 3241 agggttgtct gaagtaacgt gcatggatga tatgagtatg cctaaattga aaaagctatt

 3301 tcttgtacaa ggcccaaaca tttccccaat tagtctcagg gtctcggaac ggcttgcaaa

 3361 gttgagaata tcacaggtac tataaataat tatttacgtt taatatccat gattttttta

 3421 aatttgtatt tagttcatca actaaatatt ccatgtctaa taaattgcag ggatgccttt

 3481 gaaaatgatt ctgtgttgga gagaatcttc tgatgcctgt tggtattata atactaataa

 3541 taagagaaaa agtttgatta ctgtttcaag ttaattgctt gtgatttgta aaaacaaatt

 3601 acttttatat ttctctttgt tttattttat gtttatttat ctttaattaa tggagtaata

 3661 aaataaaaat cttattttca atagaaaaaa gtagacctta tttgtggtgc atgtatggta

 3721 tctttttgaa atttttgata tatttgctct ttgattcgaa tttcttgctt atatgatgat

 3781 ttgcataaat ataaaatatt atacaaatac ctatgggttg gaaaatatag aaatatgcca

 3841 atcaaatgta tacaaaaatc attaatagat agaatcgtaa aagatataca aatgagaaat

 3901 gcttgactaa gaagcttcgt gcaacctctc acactgagca caatgcattt ggtgatctcg

 3961 gcactattgc tgttacttgt aagactacgt tccccaataa gtctttccaa acggcttgca

 4021 aagctgagaa tatgaaaatc tcataggtta gtttgctgcg ttaattattt acatttaata

 4081 tgctcgataa ggtgatttta aaaaaatttg tactagttaa ttcatgaact aaatatttca

 4141 tttaatactc cataattctg aatatggaaa ataaataata tttaataaca agaataaaat

 4201 gataaattat tcattgattt tataaattgg ataaatatta ttaaatattc ttaaataata

 4261 taatgaacaa gtgaagatga acggagggag tatgaagcct cttttcaaag
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 11

General BLAST Search 

Methods 

A basic BLAST search was performed using the DNA sequence for Rpi-vnt1.1 (FJ423044.1) (see Figure 1). 

The basic nucleotide BLAST search interface was utilized (available: 

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=

BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome). The purpose of the basic BLAST search was 

to determine DNA sequences which are homologous to the Rpi-vnt1 sequence and amino acid 

sequences that would result from the Rpi-vnt1.1 gene. 

To determine homology to the DNA sequence, nucleotide BLAST analysis was performed. The Rpi-vnt1.1

DNA sequence (Figure 1) was subjected to three standard nucleotide BLAST analyses. First, the highly 

similar sequences option (megablast) was selected; second, a nucleotide BLAST (blastn) was performed 

to optimize for somewhat similar sequences; and lastly, a discontiguous megablast was performed to 

optimize for more dissimilar sequences. 

To determine proteins in the NCBI databases that have amino acid sequences highly similar to that of Rpivnt1.1 VNT1 protein, a translated BLAST (blastx) and a standard protein BLAST (blastp) search was 

performed. Blastp uses an amino acid sequence to query protein databases. The protein homology 

searches were performed using the VNT1 protein sequence that has 100% homology with Solanum 

venturii late blight resistance protein Rpi-vnt1.1 gene (FJ423044.1). 

For all DNA and protein BLAST searches, the “nr” database option was selected for databases against 

which to search. Due to constraints of computational cost, the “nr” database collection is no longer nonredundant; however, this was not anticipated to have affected the search output. Databases included 

under the broader “nr” database included the following:

 All of GenBank

 EMBL (The European Molecular Biology Laboratory)

 DDBJ (DNA Databank of Japan)

 PDB (RSCB Protein Data Bank, not including EST (expressed sequence tags), STS (sequencetagged sites), GSS (genome survey sequences), or phase 0, 1, or 2 HTGS (high-throughput 

genome) sequences.

Results

DNA Homology

Results from the BLAST analyses show a number of homologous DNA sequences in closely related 

potato species. Sequences and database entries for plants that contain genes that are greater than 70% 

homologous to Rpi-vnt1.1 (FJ423044.1) are found in tomato species with high homology to the Rpi-vnt1 

gene. A summary of these results, indicating DNA sequences in the NCBI GenBank that have greater 

than 70% homology with Rpi-vnt1.1 (FJ423044.1), are summarized in Table 1. 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 12

Table 1. DNA Sequences from Edible Plants Homologous to Rpi-vnt1.1 (FJ423044.1).

Description Organism 

common 

name

Accession 

number

Max.

Identity

Expect 

value

Search Method

Solanum lycopersicum

cDNA, 

clone:LEFL2055C17, 

HTC in fruit

Tomato AK328455.1 96%, 

96%, 

96%

9e-22, 

2e-21, 

2e-21

Megablast, 

Discontiguous*, 

Blastn

Solanum lycopersicum

genomic DNA, 

chromosome 8, clone: 

C08HBa0005L01, 

complete sequence

Tomato AP009261.1 96%, 

96%, 

96%

9e-22, 

2e-21, 

2e-21

Megablast, 

Discontiguous, 

Blastn

Lycopersicon 

esculentum cultivar 

Craigella GCR236 Tm2(Tm-2) gene, partial 

cds

Tomato AY765394.1 92%, 

92% 

92%

2e-52, 

1e-53, 

1e-53

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar VFNT Cherry 

LA1221 disease

resistance protein 

Tm2^2 (Tm-2) gene, 

Tm-2^2 allele, partial

cds

Tomato FJ817597.1 91%, 

91%, 

91%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar LA3433 disease

resistance protein Tm-2 

(Tm-2) gene, partial cds

Tomato FJ817598.1 91%, 

91%, 

91%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar Bush Celebrity 

disease resistance 

protein Tm-2^2 (Tm-2) 

gene, Tm-2^2 allele, 

partial cds

Tomato FJ817592.1 91%, 

91%

3e-18, 

3e-18

Discontiguous, 

Blastn
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 13

Solanum lycopersicum

cultivar LA0656 disease 

susceptible protein tm2 (tm-2) gene, partial 

cds > gb[FJ817599.1] 

Solanum lycopersicum 

cultivar Peto 95-43 

LA3528 disease 

susceptible protein tm2(tm-2) gene, partial 

cdsSolanum venturii

late blight resistance 

(Rpi-vnt1.2) gene, 

complete cds

Tomato FJ817596.1 91%, 

90%, 

90%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar Anahu LA0655 

disease susceptible 

protein tm-2(tm-2) 

gene, partial cds

Tomato FJ817595.1 91%, 

90%, 

90%

0.0, 

0.0,

0.0

Megablast, 

Discontiguous, 

Blastn

Lycopersicon 

esculentum cultivar 

ATV840 tm-2 (tm-2) 

gene, partial cds

Tomato AY765396.1 90%, 

90%

6e-08, 

6e-08

Discontiguous, 

Blastn 

Solanum lycopersicum

cultivar Yukang 2 ToMV 

resistance protein (Tm2nv) gene, 5’ UTR and 

partial cds

Tomato HM027885.1 90%, 

90%

6e-08, 

6e-08

Discontiguous, 

Blastn 

Solanum tuberosum

Tm-2 ToMV resistance 

protein mRNA, 

complete cds

Potato EF137705.1 88%, 

87%, 

87%

0.0, 

0.0, 0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

chromosome 9 clone 

C09HBa0165P17, 

complete sequence

Tomato EF647605.1 87%, 

84%, 

84%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum 

strain Heinz 1706 

chromosome 9 clone 

sle-16j3 map9, 

complete sequence

Tomato AC253987.8 86%, 

85%, 

85%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 14

Lycopersicon 

esculentum ToMVresistance locus, Tm-2 

resistance allele, 

complete cds

Tomato AF536200.1 86%, 

85%,

85%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Lycopersicon 

esculentum ToMVresistance locus, Tm2^2 resistance allele, 

complete cds

Tomato AF536201.1 86%, 

85%,

85%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Lycopersicon 

esculentum ToMVresistance locus, tm-2 

susceptible allele, 

complete cds

Tomato AF536199.1 86%, 

85%, 

85%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

strain Heinz 1706, 

chromosome 9, clone 

slm-15o9, map 9, 

complete

Tomato AC254102.4 86%, 

85%, 

85%

0.0, 

0.0,

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum lycopersicum

strain Heinz 1706 

chromosome 10 clone 

hba-74I20 map 10, 

complete sequence

Tomato AC244714.3 86% 4e-04 Discontiguous

Lycopersicon 

esculentum cultivar 

Yukang 2 Tm-2 ToMV 

resistant protein (Tm2nv) gene, complete cds

Tomato AY742887.1 85%, 

85%, 

85%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Capsicum annuum

clone pSJS1093 Tm-2 

protein-like gene, 

complete sequence

Pepper FJ605517.1 82%, 

81%, 

81%

0.0, 

0.0, 

0.0

Megablast, 

Discontiguous, 

Blastn

Solanum tuberosum

strain P6/210 contig R1, 

complete sequence

Potato EF514212.1 82% 2e-07 Blastn

Solanum lypersicum

strain Heinz 1706 

chromosome 7 clone 

sle-22m17 map 7, 

complete sequence

Tomato AC253643.6 79% 7e-07 Blastn
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 15

Solanum lypersicum

strain Heinz 1706 

chromosome 7 clone 

sle-42o4 map 7, 

complete sequence

Tomato AC253675.4 79% 7e-07 Blastn

Solanum lypersicum

strain Heinz 1706 

chromosome 7 clone 

sle-71m4 map 7, 

complete sequence

Tomato AC253630.3 79% 7e-07 Blastn

Solanum lycopersicum

cDNA, clone: 

LEFL2048L12, HTC in 

fruit

Tomato AK247925.1 79% 3e-17 Blastn

Solanum lypersicum

strain Heinz 

chromosome 10 clone 

hba-31k6 map 10, 

complete sequence

Tomato AC244575.3 79% 3e-17 Blastn

Solanum lypersicum

strain Heinz 

chromosome 10 clone 

hba-62p19 map 10, 

complete sequence

Tomato AC244733.4 79% 3e-17 Blastn

Solanum lypersicum

strain Heinz 1706 

chromosome 7 clone 

sle-18e18 map 7, 

complete sequence

Tomato AC253606.5 79% 7e-07 Blastn

Solanum tuberosum

MADS-box transcription 

factor MADS11 gene, 

complete cds

Potato EU664445.1 78% 5e-09 Blastn

Glycine max cDNA, 

clone: GMFL01-04-K22

Soybean AK244481.1 77% 0.001 Discontiguous

Solanum lycopersicum

cultivar LA0656 disease 

susceptible protein Tm2 (Tm-2) gene, partial 

cds > gb [FJ817607.1] 

Solanum lycopersicum

cultivar Peto 95-43 

LA3528 disease 

susceptible protein Tm2 (Tm-2) gene, partial 

cds

Tomato FJ817601.1 76%, 

76%

6e-141, 

6e-141

Discontiguous, 

Blastn
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 16

Solanum lycopersicum

cultivar Anahu LA0655 

disease susceptible 

protein Tm-2(Tm-2) 

gene, partial cds

Tomato FJ817600.0 76%, 

76%

6e-141, 

6e-141

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar Cherry LA1221 

disease resistance 

protein Tm-2^2 (Tm-2) 

gene, Tm-2^2 allele, 

partial cds

Tomato FJ817602.1 75%, 

75%

5e-129, 

5e-129

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar LA3433 disease 

resistance protein Tm-2 

(Tm-2) gene, partial cds

Tomato J817605.1 75%, 

75%

5e-129, 

5e-129

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar LA3432 disease 

resistance protein Tm-2 

(Tm-2) gene, partial cds

Tomato FJ817604.1 75%, 

75%

5e-129, 

5e-129

Discontiguous, 

Blastn

Solanum lycopersicum

cultivar Royal Red 

Cherry LA2088 disease 

resistance protein Tm2^2 (Tm-2) gene, Tm2^2 allele, partial 

cds>gb[FJ817606.1] 

Solanum lycopersicum

disease resistance 

protein Tm-2^2 (Tm-2) 

gene, Tm-2^2 allele, 

partial cds

Tomato FJ817603.1 75%, 

75%

5e-129, 

5e-129

Discontiguous, 

Blastn

Solanum lycopersicum

chromosome 11 clone 

C11Hba0139J14, 

complete sequence

Tomato AC212436.2 75% 8e-13 Blastn

Capsicum annuum

clone BAC CaCM421A08 

genomic sequence

Pepper GU108230.1 71% 7e-07 Blastn

* “Discontiguous” denotes discontiguous megablast

The DNA homology results are summarized as follows:

 The majority of plants with genes homologous to Rpi-vnt1.1 (FJ423044.1) are wild Solanum and 

are not edible.

 Homologous genes returned in the search that were from tomato were largely divided into two 

categories: one group of 17 genes for resistance to the tomato mosaic virus (called ToMV, Tm-2, 

or Tm-2^2) and the other group of 15 being submissions to NCBI as a result of the International 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 17

Tomato Genome Sequencing Project (also referred to in the NCBI database as entries from the 

Tomato Genome Consortium). 

 There were 3 results captured for potato, one of which is a transcription factor gene 

(EU664445.1) that may not be related to disease resistance. The two remaining potato results 

are for disease resistance genes (EF137705.1, EF514212.1).

 The percent identity measure of homology for tomato genes identified in the search was 

generally high with very low e-values1, indicating significant homology of the returned genes 

with Rpi-vnt1.1 (FJ423044.1) according to the algorithm used by the BLAST program.. 

 Two genes from pepper were returned in the search, one of which is for ToMV resistance, and 

the other is a submission from genome mapping efforts (a BAC clone). 

 In general, for BAC clones and for sequences entered as part of genome sequencing efforts, 

further confirmation will need to be made in order to determine whether the DNA in these 

homologous regions is ultimately translated and expressed as protein.

 The single entry returned for soybean, although greater than 70% in maximum identity value, 

has a high e-value and is a relatively short sequence, indicating poor overall homology despite 

the maximum identity value of 77%. 

Protein Homology

A protein BLAST search was performed using the Blastp program by searching for proteins homologous 

to the Rpi-Vnt1.1 protein sequence (ACJ66594.1). Results indicating proteins with greater than 70% 

amino acid sequence homology with the Rpi-vnt1.1 protein product (ACJ66594.1) are reported in Table 

2. Overall, results indicate significant homology between the protein product of Rpi-vnt1.1 (ACJ66594.1) 

and Tm-2 resistance genes in tomato and potato, as indicated by very low e-values and relatively high 

maximum identity values.

Table 2. Protein Sequences with Significant Homology to Rpi-vnt1.1 (ACJ66594.1) Protein Sequence 

Description Common name Accession 

number

Maximum 

Identity

Expect value

Disease resistance protein 

Tm-2, partial (Solanum 

lycopersicum)

Tomato ACO52371.1 88% 6e-115

Tm-2 ToMV resistance protein 

(Solanum tuberosum)

Potato ABM05492.1 77% 0.0

Tm-2 ToMV Resistance

protein (Solanum 

lycopersicum)

Tomato AAQ10735.1 76% 0.0

 

1

E-value (or Expect value) is a parameter that describes the number of hits one can "expect" to see just 

by chance when searching a database of a particular size. It decreases exponentially with the Score (S) 

that is assigned to a match between two sequences. Essentially, the e-value describes the random 

background noise that exists for matches between sequences.
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 18

Description Common name Accession 

number

Maximum 

Identity

Expect value

Tm-2 ToMV Resistance

protein (Solanum 

lycopersicum)

Tomato AAV87531.1 75% 0.0

Specific BLAST Search and Results

Following the broad BLAST searches described above, several specific BLAST searches were conducted 

which searched the genomes of several select Solanum family members against the Rpi-vnt1.1 gene 

(FJ423044.1). This search was conducted to ensure that Solanum family members containing genes 

homologous to Rpi-vnt1.1 (FJ423044.1) were not over-looked among the other entries in the general 

BLAST output, which contained many hits, most of which were not edible Solanum. While the basic 

concept of this search is the same as that of the general BLAST search, in this search the Rpi-vnt1.1

(FJ423044.1) gene sequence is only compared to genes in the GenBank database for the specific plant 

species selected. 

Plant species that were searched in this specific search included Capsicum annum L. (pepper), and 

Solanum melongena (eggplant, or aubergine) and Solanum aculeatissimum (Dutch eggplant). Megablast, 

discontiguous megablast, and blastn searches were performed for Rpi-vnt1.1 (FJ423044.1) against the 

full collection of genes in GenBank for the three organisms. Unlike the tomato, entire genome 

sequences for pepper and eggplant have not been deposited in GenBank. A search of the database for 

genes from eggplant or aubergine using the three search methods did not reveal any homologous genes 

to Rpi-vnt1.1 (FJ423044.1). The search performed for genes from Dutch eggplant returned a gene for an 

NBS-coding resistance protein-like protein RGA3 () with an e-value of 2e-173 in the megablast search, 

and 0.0 in the discontiguous megablast and blastn searches. Maximum identity values for these genes 

were 88% in all three searches. Finally, the search for homologous genes from pepper returned one 

entry for a homologous Tm-2 resistance gene (FJ605517.1), however, this entry was already captured in 

the main BLAST search described above.

The Rpi-vnt1 gene is a member of a key class of genes that comprises the vast majority of plant 

resistance (R) genes. These genes, found in Solanum family members, contain a nucleotide-binding (NB) 

and leucine-rich repeat (LRR) domain, and are collectively known as NB-LRR genes (Jupe et al., 2012). In 

order to determine if the Rpi-vnt1.1 (FJ423044.1) sequence is similar to some LRR or NB-LRR disease 

resistance genes present in widely consumed edible plant species, additional specific BLAST analyses 

were conducted. The Rpi-vnt1.1 (FJ423044.1) DNA sequence was compared with two sample LRR genes

from edible organisms: Lycopersicon pimpinellifolium (currant tomatoes) cf-9 resistance gene cluster 

(Jones et al., 1994) and Oryza sativa (rice) receptor kinase-like protein Xa21 (Song et al., 1995); and with 

a sample NB-LRR gene, the L6 rust resistance gene from Linum usitatissimum (flax). No significant 

similarities were found when the Rpi-vnt1.1 (FJ423044.1) sequence was compared with the cf-9 

resistance gene in currant tomatoes , XA21 in rice or the L6 rust resistance gene from flax. 

Specific BLAST searches against the genomes of the following plant species were also performed:

including Oryza sativa (rice), Glycine max (soybeans), and Triticum aestivum L. (common wheat). 

Searches were conducted using the megablast, discontiguous megablast, and blastn options. Although 

some homologous sequences were returned in these searches, overall results do not provide evidence 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 19

that Oryza sativa L. , Triticum aestivum L, and Glycine max L. contain genes that are similar to Rpi-vnt1.1

(FJ423044.1). Although maximum identity values are greater than 70% for some of the captured 

sequences, the e-values were generally poor and the lengths of homologous sequence region were very 

short, generally less than 100 base pairs. This indicates that despite relatively favorable maximum 

identity values, the overall homology between Rpi-vnt1.1 (FJ423044.1) and genes within these three 

commonly consumed organisms is quite low. 

ToMV RESISTANCE PROTEINS LEVEL EXPRESSION 

Results of the BLAST searches indicated a high homology between Rpi-vnt1.1 (FJ423044.1) and Tm-2 

resistance genes to ToMV and related proteins. Several searches were conducted to determine if levels 

of ToMV resistance proteins were reported in food and therefore could be used to assess dietary 

exposure to these related proteins.

Literature Search

A literature search via PubMed, Google Scholar, and AGRICOLA was conducted to identify publications 

that provide information on the levels of tomato mosaic virus resistance proteins that are expressed in 

plant’s fruit tissues both at basal levels and in response to the presence of pathogen. The PubMed 

search was performed and subsequently updated on 2/19/2013 with search terms including: “tomato 

mosaic virus” AND “resistance” AND “protein”. The AGRICOLA search was performed using the Keyword 

Search function and updated on 2/14/2013. Search terms included Keyword Search of all words and 

phrases “tomato mosaic virus” AND “resistance” AND “protein”. 

Results

The PubMed search returned 23 citations. Three of these were of potential relevance (Lanfermeijer et 

al. 2003, 2005; and Weber and Pfitzner 1998). However, after screening the three potential 

publications, no relevant information on expression levels of resistance protein were found. The 

Google scholar search used the same terms as the PubMed search and found two additional 

publications (Kang et al. 2005 and Whitham et al. 1996); however, upon further review these 

publications also did not contain useful protein expression level data for the resistance proteins. There 

were 76 citations captured in the AGRICOLA search, and of these citations, those that appeared as 

relevant were already captured in the PubMed or Google Scholar searches previously performed.

In summary, this literature search did not yield any papers providing information about the levels of 

ToMV resistance proteins in tomatoes or in the diets. Some publications do point to the similarity 

between ToMV and Rpi-VNT1, confirming the findings of the BLAST search results presented above.
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 20

Tomato Varieties with ToMV Resistance Gene 

Database Search 

In addition to searching for literature that would provide levels of the ToMV resistance protein, a search 

was conducted of potato and tomato varieties that contain the virus resistance. Databases containing 

tomato or potato varieties and details on pathogen resistance of each variety were located using the 

Google search engine. Both tomato and potato variety databases were identified; however, potato 

varieties did not appear to display the trait of ToMV resistance, so the potato variety search was 

abandoned. Tomato variety databases and listings which contained information on ToMV resistance of 

each particular variety included a publication by North Carolina State University (January 2010 revision, 

http://ipm.ncsu.edu/Production_Guides/Tomatoes/AG-405Web.pdf), a table compiled by the Cornell 

Department of Plant Pathology (dated April 2012, 

http://vegetablemdonline.ppath.cornell.edu/Tables/TomatoTable.html), and a publication generated by 

the University of Florida, Horticultural Sciences Department (Santos et al., 2013). 

To determine the availability of ToMV resistant tomato varieties for planting, marketing and 

consumption, the following retail web sites were also reviewed between February 11-22, 2013:

 Burpee

 Harris Seed

 Harris Morgan

 High Mowing Seeds

 Johnny Seeds

 Jung Seed

 Paramount Seeds

 Reimer Seeds

 Seedway

 Siegers

 Simply Seeds and Plants

 Tradewind Fruits

Results

Seventy eight (78) varieties and 2 rootstocks of ToMV resistant tomato were found in the databases and 

publications that were searched. Of the 78 tomato varieties identified as ToMV resistant tomato 

varieties, all are considered fresh market tomatoes and fifty five of them are available on the various 

retail web sites (see Table 3). Many of the ToMV resistant tomato varieties are recommended by the 

university agriculture extension offices to commercial producers and individual gardeners as being 

suitable varieties to grow in their areas. The Seminis’s Cheong Gang tomato rootstock is ToMV resistant 

and recommended for grafting purposes for fresh market growers. 

The U.S. tomato processing industry, comprised primarily of tomato pastes, sauces, and canned tomato 

products, is distinctly separate from the fresh-market industry. Nearly 100 percent of processing 

tomatoes are produced and sold under contract between growers and processing firms. Approximately 

34 percent of the world’s processed tomatoes are grown using Heinz proprietary tomato seeds. None of 

these processed varieties appear to have been bred for ToMV resistance as the breeding focus has been 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 21

on other traits such as total soluble solids and sugar content. However, there were two hits for a 

chromosome 9 clone for Solanum lycopersicum strain Heinz, when Rpi-vnt1.1 (FJ423044.1) gene was 

subjected to a BLAST search. One of the Campbell Soup Company’s seeds developed for processing 

tomatoes, CXD 253, has been bred for ToMV resistance but has only recently been introduced 

commercially and is not considered part of the 2012 top 50 California processed tomato list. 

Consumption of Tomatoes with ToMV Resistance Gene 

California is the leading producer of all tomatoes in the United States, accounting for 96 percent of U.S. 

processing tomato output and one-third of the fresh crop. Other major fresh-market tomato-producing 

States include Florida, Virginia, Georgia, Ohio, Tennessee, North Carolina, New Jersey and Michigan 

(USDA-ERS, 2012). Greenhouse/hydroponic products for the fresh-tomato retail market have grown 

significantly in the United States. Mexico now accounts for 71 percent of the United States import 

market for greenhouse tomatoes, while Canada has approximately 27 percent of the import greenhouse 

market (Cook and Calvin, 2005).

There are hundreds of fresh market tomato varieties grown in the United States and, in addition to the 

traditional slicer round or globe tomatoes, they include types such as cherry tomatoes, grape tomatoes, 

heirloom, saladette, beef-steak, vine ripe cluster. They are also available in a variety of colors including 

red, yellow, pink, purple, and green. Unlike the processed tomato industry, the fresh market tomato 

growers obtain their seed or plants from a variety of sources and many are available through their web 

sites. Per capita consumption of fresh tomatoes has been increasing in recent years. Average annual 

per capita consumption in 1981 was 12.3 pounds and has steadily increased to 18.5 pounds in 2008 

(USDA-ERS, 2008).

Conclusions

The overall results of the nucleotide BLAST searches demonstrate that the Rpi-vnt1.1 (FJ423044.1) gene 

shares greater than 90% sequence identity with tomato mosaic virus (ToMV) resistance genes. In 

addition, the VNT1 protein (ACJ66594.1) is greater than 75% homologous to Tm-2 ToMV proteins from 

Solanum lycopersicum (tomato) and Solanum tuberosum (potato) at the amino acid level. Taken 

together, results from the bioinformatics analyses suggest that genes highly similar to Rpi-vnt1.1

(FJ423044.1) are present in the human diet as constituents of S. tuberosum L. and S. lycopersicum . 

Given the greater than 90% homology between Rpi-vnt1.1 and ToMV Tm-2 resistance genes and greater 

than 75% homology for the related proteins, existing dietary exposure to tomato and potato varieties 

with the ToMV Tm-2 resistance gene could be relied upon to establish the history of dietary exposure to 

the protein product of the Rpi-vnt1.1 gene. Several searches to determine the levels of ToMV resistance 

proteins in food and existing dietary exposure to tomato varieties with the ToMV resistance gene were 

carried out. A literature search did not yield any papers providing information about the levels of ToMV

resistance proteins in tomatoes or in the diets. However, ToMV resistant tomato varieties and 

rootstocks are readily available and grown in the US for fresh market tomato production. 

Given the large per capita consumption of fresh market tomato consumption in the United States,

consumers are very likely to have been exposed to ToMV resistant tomatoes in their diet. Since the Rpivnt1.1 gene is substantially similar to the ToMV resistance gene (approximately 90% homologous), it is 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 22

reasonable to conclude that US consumers have dietary exposure to proteins that are substantially 

similar to the Rpi-VNT1.1 protein product without negative impacts. 

Widely consumed plant species that are reported to contain disease-resistance (R) genes include the 

Solanaceae family, Zea mays (maize), Glycine max (soybeans), Oryza sativa L. (rice), and Triticum 

aestivum L. (common wheat) (Bakker et al. 2011). Potato belongs to the Solanaceae family, which 

includes eggplant (Solanum melongena L.), pepper (Capsicum annum L.), and tomato (Solanum 

lycopersicum L.). In addition to these common cultivated crops, the Rpi-vnt1 gene is identical to the Rpiphu1 gene (Rodewald and Trognitz 2013) found in some Solanum phureja species, a potato species 

consisting of many varieties which are currently consumed by indigenous people in South America. The 

prevalence of such resistance genes throughout edible crops suggests that extremely low levels of 

similar proteins are widespread in nature and unlikely to pose risk to human health. 

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2/21/2013

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http://www.agmrc.org/commodities__products/vegetables/fresh-tomatoes-profile/ 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 26

Table 3. Fresh Tomato Varieties with ToMV Resistance Gene

Variety

(

A

Not readily 

available on 

a retail seed 

or plant site; 

B

Rootstock) Reference Description Retail Site Link

Agriset 334

Cornell 

database red round http://www.seedway.com/store/Pages/Product.aspx?pid=6419

Agriset 8279 

F1 (Tami G)

Cornell 

database red grape

http://www.harrisseeds.com/Storefront/p-257-tomato-agriset-8279-grapepelleted.aspx

Apero F1

Cornell 

database

cherry 

plum

http://www.simplyseedsandplants.co.uk/Vegetable-Plants/TomatoApero_177131.htm?ne=AQAAAAUAAAAECFRvbWF0b2Vz

Arbasan F1

Cornell 

database red round http://www.johnnyseeds.com/p-7836-arbason-f1-og.aspx

Atomic 

(German)

Cornell 

database red round http://www.richfarmgarden.com/tomato.html

Bambino

Cornell 

database cherry http://www.territorialseed.com/product/1205/cherry_tomato_seed

Barbarian F1

Cornell 

database large round http://www.harrismoran.com/products/tomato/pdf/barbarian.pdf

Beaufort F1A

Cornell 

database

https://www.paramountseeds.com/seed-varieties-t93/greenhouseseeds/greenhouse-rootstock-tomato.aspx

BHN-589

Cornell 

database

Hybrid 

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

BHN-871

Cornell 

database

hybrid 

round 

yellow http://www.siegers.com/shop/kind.asp?kind_id=BBF

BHN-876

Cornell 

database

goldenorange http://www.seedway.com/store/Pages/Product.aspx?pid=5231P

BHN-961

Cornell 

database

Hybrid 

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

BHN-968

Cornell 

database cherry http://www.johnnyseeds.com/p-8445-bhn-968-f1.aspx

Big Beef F1

Cornell 

database

Hybrid 

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

Bigdena

Cornell 

database beef-type

https://www.paramountseeds.com/seed-varieties-t93/greenhouseseeds/greenhouse-beefsteak-tomato/greenhouse-bigdena-tomato.aspx

Brixmore NCSU red grape

http://www.seedquest.com/vegetables/tomato/expofm/harrismoran/brixmore.ht

m

Camelia EDIS

hydrid 

cherry 

tomato http://www.siegers.com/shop/kind.asp?kind_id=BBD

CarnivalB

Cornell 

database

Celebrity

Cornell 

database

Hybrid 

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

ClermonB

Cornell 

database

Cobra F1

Cornell 

database round red http://www.johnnyseeds.com/p-5711-cobra-f1.aspx

Country 

Taste

Cornell 

database beef-type

https://www.jungseed.com/dp.asp?pID=00205&c=153&p=Country+Taste+Hybrid+

Tomato

Early Goliath

Cornell 

database round red http://www.seedman.com/tomato.htm

Elfin NCSU red grape http://www.reimerseeds.com/elfin-tomato.aspx
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 27

Variety

(

A

Not readily 

available on 

a retail seed 

or plant site; 

B

Rootstock) Reference Description Retail Site Link

Emperador 

F1B

Cornell 

database

Favorita F1

Cornell 

database cherry http://www.johnnyseeds.com/p-7931-favorita-f1.aspx

Fenda

Cornell 

database

large round 

red http://www.harrisseeds.com/Storefront/p-13963-tomato-fenda.aspx

Geronimo F1

Cornell 

database beef-type http://www.johnnyseeds.com/p-7237-geronimo-f1.aspx

Golden Rave 

F1

Cornell 

database

yellow 

baby roma http://www.johnnyseeds.com/p-7054-golden-rave-f1.aspx

Granadero 

F1

Cornell 

database plum http://www.johnnyseeds.com/p-8490-granadero-f1-og.aspx

Grandma's 

gardenB

Cornell 

database

GrowdenaB

Cornell 

database

Halcon

Cornell 

database

large round 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-det.pdf

HermosaB

Cornell 

database

HM 7864 F1B

Cornell 

database

Imperial 

643B

Cornell 

database

Lola

Cornell 

database red round http://www.highmowingseeds.com/organic-seeds-lola-f1-hybrid-tomato.html

Massada F1

Cornell 

database round red http://www.johnnyseeds.com/p-8493-massada-f1.aspx

MaxifortA

Cornell 

database http://www.johnnyseeds.com/p-6895-maxifort-f1.aspx

Mini Charm NCSU red grape http://www.seedman.com/tomato.htm

Montesino 

F1

Cornell 

database

cherry 

grape http://www.highmowingseeds.com/Organic-Seeds-Montesino-F1-Tomato.html

Mt. Glory

Cornell 

database

Hybrid 

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

MurielB

Cornell 

database

Nico

Cornell 

database beef-type http://www.seedway.com/store/Pages/Product.aspx?pid=7400

Orange 

Faruche F1B

Cornell 

database

PalenqueB

Cornell 

database

Palomo F1

Cornell 

database

large oval 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-det.pdf

PanzerB

Cornell 

database

Pegasso F1 

(Pegaso F1?)

Cornell 

database

large oval 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-det.pdf

PilavyBCornell 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 28

Variety

(

A

Not readily 

available on 

a retail seed 

or plant site; 

B

Rootstock) Reference Description Retail Site Link

database

PitenzaB

Cornell 

database

Polbig F1

Cornell 

database large round http://www.seedway.com/store/Pages/Product.aspx?pid=8621

Pony Express

Cornell 

database

large oval 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-det.pdf

Premio F1B

Cornell 

database

Primo Red 

F1

Cornell 

database large round http://www.seedway.com/store/Pages/Product.aspx?pid=5055

Qualit 21B

Cornell 

database

Qualit 23 F1B

Cornell 

database

Red Candy

Cornell 

database red grape http://www.harrismoran.com/products/tomato/pdf/table-fresh-indet.pdf

SahelB

Cornell 

database

Samurai F1

Cornell 

database

large oval 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-indet.pdf

Santa EDIS

Sapho F1B

Cornell 

database

ShirenBEDIS

Solar Fire F1

Cornell 

database

large round 

red

http://www.seedquest.com/vegetables/tomato/expofm/harrismoran/solarfire.ht

m

Sophya F1

Cornell 

database

large round 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-indet.pdf

STM 6701B

Cornell 

database

Sun Gold F1

Cornell 

database

yellow 

cherry http://www.territorialseed.com/product/1152

Sun Sugar

Cornell 

database

hydrid 

cherry 

tomato http://www.siegers.com/shop/kind.asp?kind_id=BBD

Sunoma

Cornell 

database

hybrid 

Roma http://www.siegers.com/shop/kind.asp?kind_id=BBI

Suzanne F1

Cornell 

database

large red 

cherry http://www.highmowingseeds.com/organic-seeds-suzanne-f1-hybrid-tomato.html

Sweet 

Chelsea F1

Cornell 

database

large red 

cherry http://www.reimerseeds.com/sweet-chelsea-tomato.aspx

Sweet Gold 

Cornell 

database

yellow 

cherry http://www.tradewindsfruit.com/vegetables/sweet_gold_tomato.htm

Sweet 

Hearts

Cornell 

database red grape http://www.reimerseeds.com/sweet-hearts-tomato.aspx

Sweet 

Million 

Cornell 

database red cherry http://www.reimerseeds.com/sweet-million-tomato.aspx

Sweet Cornell 
Dietary Exposure to Rpi-vnt1.1, J.R. Simplot Co. Page 29

Variety

(

A

Not readily 

available on 

a retail seed 

or plant site; 

B

Rootstock) Reference Description Retail Site Link

OrangeBdatabase

Sweet Treats 

F1

Cornell 

database red cherry http://www.reimerseeds.com/sweet-treats-tomato.aspx

Toronjina F1

Cornell 

database

orange 

cherry http://www.highmowingseeds.com/Organic-Toronjina-F1-Tomato-Seeds.html

Trigress EDIS

orange 

cherry http://www.highmowingseeds.com/Organic-Toronjina-F1-Tomato-Seeds.html

Trinity F1

Cornell 

database

large round 

red http://www.harrismoran.com/products/tomato/pdf/table-fresh-det.pdf

Yellow Mini

Cornell 

database

yellow 

cherry http://www.johnnyseeds.com/p-7117-yellow-mini-f1.aspx
1 of 9

Appendix E: Detection of Russet Burbank W8 Using Real-Time PCR

JR Simplot Regulatory Plant Sciences

Stephanie McInerney and Jingsong Ye

Date

March 28, 2014

Contents

1. Description of the methods ........................................................................ 2

2. Equipment / reagents .................................................................................. 3

3. Procedure ...................................................................................................... 3

4. Testing of the detection methods .............................................................. 6

5. Conclusions .................................................................................................... 8

6. References ..................................................................................................... 9
2 of 9

1. Description of the Methods

General Description

This report describes a Real-Time TaqMan® Polymerase Chain Reaction (PCR) method for the detection 

of Russet Burbank Event W8 developed by the J.R. Simplot Company. Testing was performed using DNA 

extracted from freeze-dried tuber and leaf samples. The method is event-specific and able to reliably 

detect 0.2% event positive potato or leaf material in a sample with a 95% confidence interval.

 

Primers and Probes

Event-specific primers were designed to amplify the junction between a W8-specific DNA insert and the 

adjoining flanking sequences. These event-specific primers amplify a region consisting of 99 bp (47 bp 

construct, 52 bp flanking region) where a target-specific fluorescent probe allows real-time detection 

and quantitation of product.

The fluorescent probe is labeled at both ends with a 6-FAM (6-carboxyfluorescein) moiety at the 5 ́ end 

and a TAMRA (tetramethylrhodamine) moiety at the 3 ́ end. Fluorescence by 6-FAM is quenched by the 

presence of TAMRA on the same oligonucleotide. During PCR any probe annealed to the target strand 

being amplified will be cleaved by the 5'-to-3' nuclease activity of Taq DNA polymerase, which prevents 

quenching by TAMRA and leads to fluorescence. The probe is designed as a locked nucleic acid (LNA) to 

increase thermal stability and increased specificity (6). 

Primers and a dual labeled (TAM/FAM) probe specific to Elongation Factor 1α (EF1α) from Solanum 

tuberosum were used as an endogenous control in the assays. Elongation Factor 1α was chosen based 

on its stability during various biotic and abiotic stress studies in potato using Real-Time PCR (5). The 

oligonucleotides used in the procedure are summarized in the Table 1. 

Table 1. Sequences of primers and probes.

Name Oligonucleotide DNA Sequence (5' to 3')

W8 forward primer Not shown

W8 reverse primer Not shown

W8 probe Not shown

Elongation Factor 1α forward primer CTCTCGACCAGATTAATGA

Elongation Factor 1α reverse primer GTTCCAATACCACCAATC

Elongation Factor 1α probe CCAA*CAG*ACA*AGC*CAC

*Locked-Nucleic Acid (LNA)

The following sections include a list of all equipment and reagents (Tables 2, 3), an outline of the 

procedure, parameters for the PCR reaction (Table 4) and cycling protocol (Tables 5 and 6).
3 of 9

2. Equipment and Reagents

Table 2. Equipment.

Equipment Number/Specification

Table top microcentrifuge Thermo Scientific #15002536

qPCR Thermal Cycler BioRad CFX96 Real-Time System 

w/C1000 Touch Thermal Cycler

qPCR Microseal Adhesive Sealers BioRad #MSB-1001

Hard-Shell 96 well PCR plates BioRad #HSP9601

Table 3. Reagents.

Chemicals/Reagents Number/Specification

TaqMan® Universal PCR Master Mix Applied Biosystems 4304437

SSO Advanced SYBR Green Supermix BioRad 172-5274

Nuclease-free water E&K Scientific #NUPW-1000

Illustra Nucleon Phytopure Genomic

DNA Extraction Kit

GE RPN8511

Chloroform Sigma-Aldrich C2432-500ML

Ethanol Sigma-Aldrich E7023-500ML

2-Propanol Sigma-Aldrich I9516-500ML

QIAquick PCR Purification Kit Qiagen 28104

3. Procedure

General instructions and precautions

 The procedures require experience with working under sterile conditions.

 The quality and the concentration of DNA need to be checked before performing the PCR assay.

 All PCR reagents and equipment used must be DNase-free, DNA-free, and sterilized prior to use.

 Filter pipet tips should be used and pipettes should be checked regularly for precision.

 Keep the working area clean. Use only powder-free gloves and change them frequently.

 All reagents and DNA should be kept on ice.

DNA Extraction Protocol using the GE Illustra Nucleon PhytoPure Kit

1. Add about 200 mg of starting pre-ground material to a 2 ml tube.

2. Add 1.2 mls Reagent 1 and vortex to mix thoroughly.

3. Add 400 μl of Reagent 2 and invert to mix well.
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4. Incubate at 65°C with occasional mixing for 30 minutes.

5. Add RNase A to a concentration of 20 μg/ml and incubate at 37°C for 30 minutes.

6. Put samples on ice for 20 minutes.

7. Centrifuge samples at 21,000 x g for 5 minutes at room temperature. 

8. Transfer supernatant to a fresh tube. 

9. Add 1 ml of chloroform (previously chilled at -20°C).

10. Place samples on a shaker at room temp for 10 minutes.

11. Centrifuge samples at 21,000 x g for 10 minutes. 

12. Transfer upper phase to a clean tube.

13. Add 400μl of PhytoPure DNA extraction resin (make sure resin is fully resuspended by 

shaking right before use.)

14. Place samples on a shaker at room temp for 10 minutes.

15. Centrifuge samples at 1,300 x g for 10 minutes. 

16. Transfer upper phase to a clean tube.

17. Add an equal volume of cold isopropanol (previously chilled at -20°C).

18. Invert tube until DNA precipitates (can go overnight at -20°C).

19. Centrifuge at 21,000 x g for 5 minutes to pellet DNA.

20. Wash pellet with cold 70% ethanol (previously chilled at -20°C).

21. Centrifuge pellet at 21,000 x g for 5 minutes.

22. Air-dry pellet for 10 minutes.

23. Resuspend DNA in 200 μl TE buffer.

24. Optional: Further clean DNA using the Qiagen QIAquick PCR Purification Kit column and 

elute DNA twice with 50 μl TE. 

25. Measure DNA concentration.

Note: Protocol can be scaled-up to increase DNA yield.

PCR set up

An example reaction mix is provided in Table 4 and the cycling protocol is described in Tables 5, 6.

Table 4. PCR Reaction Mix

Reagents Stock concentration Amount per reaction Final concentration 

Nuclease-free water Up to 20 μl

TaqMan® Universal PCR 

Master Mix or SSO 

Advanced SYBR Green 

Supermix (melt curves)

2X 10 μl 1X

Forward primer 10 μM 1.0 μl 0.6 μM

Reverse primer 10 μM 1.0 μl 0.6 μM

Probe 10 μM 0.4 μl 0.2 μM

DNA* 195 pg to 200 ng

Total volume 20 μl

*20 ng were used for specificity testing and a serial dilution from 200 ng down to 195 pg was used for sensitivity tests.
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Table 5. Probe Cycling Parameters

Probe Cycling Parameters:

1. Enzyme Treatment (UNG) 50.0°C for 2 minutes

2. Initial Denaturation 95.0°C for 10 minutes

3. Denaturation 95.0°C for 15 seconds

4. Annealing & Extension 58.0°C for 60 seconds

Plate Read

Go to #3, 39 more times

Table 6. SYBR Green Cycling Parameters

SYBR Green Cycling Parameters:

1. Initial Denaturation 98.0°C for 2 minutes

2. Denaturation 98.0°C for 5 seconds

3. Annealing 50.0°C for 15 seconds

4. Extension 56.0°C for 60 seconds

Plate Read

Go to #2, 39 more times

5. Melt Curve Denaturation 95.0°C for 10 seconds

6. Melt Curve Melt Curve from 65.0°C to 95.0°C (ramp 0.5°C every 5 seconds)

Plate Read

Data analysis

The quantification threshold for calculating the quantification cycle (Cq) value was set at 150 Relative 

Florescence Units or at a value above the baseline, but sufficiently low to be within the exponential 

growth region of the amplification curve (1). The point at which samples crossed this line was reported 

as the Cq value for the samples. For sensitivity testing, the Cq and standard deviation were determined. 

For specificity testing, if the Cq value was less than 40 the sample was considered event positive. The 

percentage of false positives and false negatives was determined.
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4. Testing of the detection methods

The aim of the testing was to verify that the Real-Time PCR assay described above detected W8-

specifically, using potato leaf and tuber DNA at a level of 0.2% with a 95% confidence interval. 

Efficiency

The qPCR efficiency (E value) was determined for each primer pair using leaf and tuber DNA, which 

describes how much of the target is produced with each cycle. An efficiency of 100% indicates target 

doubling with each cycle, whereas acceptable values are between 95-110% with a corresponding slope 

between 3.1 and 3.6 (2, 3). The R2coefficient, a measure of linearity of the standard curve, was also 

determined and should be ≥0.98 (3). The W8 primers and probe were shown to fall within the range of

appropriate reaction efficiency values (Table 7).

Table 7: Summary of PCR efficiency values.

Sample Ave R2Ave E value Ave Slope

W8 0.993 98.1% -3.37

A separate assay was performed using the intercalating dye, SYBR Green, to allow the generation of 

melt curves at the end of the protocol, which allowed for analysis of primer specificity. The eventspecific primer pair demonstrated a single peak for each sample (Figure 1).

Figure 1. Melting curves for primer pairs. 

Sensitivity 

It was our goal to detect 0.2% of W8 in a sample of leaves or tubers. Towards this end Real-Time PCR 

assays with event-specific primers were performed on Russet Burbank leaf and tuber DNA samples 

spiked with varying amounts of W8 genomic DNA (2.5%, 0.63%, 0.16%, and 0.04% and 0.01%) in a total 

of 200 ng control Russet Burbank DNA. The absolute copy number for each sample was determined by 

dividing the sample DNA weight, in nanograms, by the published average value for a single copy of the 

potato genome (1.8 pg) (1). All samples were analyzed in triplicate.

Elongation Factor 1α W8
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The mean Cq values and associated standard deviations were calculated and are summarized in Table 8. 

Event-specific primers were able to amplify DNA below the required 0.2% level with standard deviations 

below the recommended threshold of 0.35 for samples containing 0.04% or more of W8 DNA in both 

leaf and tuber (4). Some PCR inhibition was seen in the 2.5% W8 DNA sample in freeze-dried tuber. 

Therefore the Limit of Detection (LOD) for this method is 0.04%.

Table 8. Statistics for samples containing W8 DNA in the range 0.01 – 2.5%. 

Copy Number of 

W8 Genomic DNA 

 

% W8 

Leaf Tuber 

Mean Cq Cq St Dev Mean Cq Cq St Dev 

2778 2.5% 29.29 0.06 33.58 0.10

694 0.63% 30.61 0.14 33.97 0.08

174 0.16% 32.55 0.07 35.50 0.11

43 0.04% 34.10 0.18 37.88 0.21

11 0.01% 37.52 0.55 39.60 1.07

Specificity

The specificity of the primer pair was tested by using DNA extracted from six independent sources of 

potato leaf or tuber (F10, E12, J3, J55, and Russet Burbank) along with the W8 event. Assays were run 

for 40 cycles with 20 ng of genomic DNA. Those samples which crossed the threshold (150 Relative 

Florescence Units) by 40 cycles were considered positive and those that did not were considered 

negative. qPCRs were performed using the W8 specific primers and probe. Each plate included 18 W8-

positive samples and 54 W8 event-negative samples. Endogenous control primers to Elongation Factor 

1α were used as positive controls for each DNA sample and no template controls were included for each 

primer and probe used. Three repetitions of each PCR were performed. No false negatives or positives 

were detected for the W8-specific primers and probes as shown in Table 9 with an acceptable standard 

deviation (of less than 0.35) in W8 event-positive samples (4).

All samples spiked with W8 DNA were detected using this method and single-peak melt curves 

demonstrated primer specificity (Figure 1). Therefore, this method allows for the reliable and sensitive 

detection of W8 containing DNA within the required 95% confidence interval. 

Table 9. Percent of false positives and false negatives during specificity testing. 

Event –specific 

Primers

% False 

Positive

% False 

Negative Ave Cq Cq Std Dev

W8 Leaf DNA 0.00% 0.00% 30.07 0.1273

 W8 Tuber DNA 0.00% 0.00% 29.04 0.0423
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5. Conclusions

1. The Real-Time PCR method, developed to exclusively detect the W8 event, demonstrated 

appropriate efficiencies with an LOD of 0.04%. 

2. Our results showed that the detection methods are event-specific and do not cross-react with 

other samples from other transformation events with a 95% confidence interval.

3. The event-specific detection method can be used to reliably detect 0.2% of W8 DNA within a 

mixed sample of either leaves or tubers.
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6. References

1. Arumuganathan K, Earle ED (1991). Nuclear content of some important plant species. 

Plant Molecular Biology Reporter. 9:208-218.

2. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, 

Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009). The MIQE Guidelines: 

Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin 

Chem. 55:611-622.

3. European Network of GMO Laboratories (ENGL), (2008). Definition of Minimum 

Performance Requirements for Analytical methods of GMO Testing, http://gmocrl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Anaytical_methods.pdf.

4. D’haene B, Hellemans J. (2010). The Importance of Quality Control During qPCR Data 

Analysis. Int’l Drug Discovery. Aug/Sept: 18-31.

5. Nicot N, Hausman, JF, Hoffmann L, Evers D (2005). Housekeeping gene selection for realtime RT-PCR normalization in potato during biotic and abiotic stress. Journal of 

Experimental Botany. 56(421):2907-2914.

6. Petersen M, Wengel J, (2003). LNA: A versatile tool for therapeutics and genomics. 

Trends in Biotechnology. 21:74-81.