J.R. Simplot Company JRS06 Page 1 of 196 

## Title Petition for Determination of Non r egulated S tatus for Innate TM 

## Potatoes wi th Late Blight Resistance, Low Acrylamide Potential , Reduced Black Spot, and

Lowered Reducing Sugars : Russet Burbank E vent W8

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

the article s 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 2 8 , 201 4 J.R . Simplot Company Petition JRS0 6

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 CBIJ.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 i s 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 Figure s

................................ ................................ ................................ ................................ ................ 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 Spo t

................................ 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 Biolog y 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 i nsert

................................ ................................ ............... 57J.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 Su mmary 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

................................ ................................ ................................ ........... 1 09 8.1 Compositional Analysis Results

................................ ................................ ................................ 109 8.2 Glycoalkaloids

................................ ................................ ................................ ........................... 114 8.3 Met hods 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

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

10.0 Trait E fficacy

................................ ................................ ................................ ................................ .. 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 Agronom ic 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 Environ mental 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

................................ ................................ ..... 184J.R. Simplot Company JRS06 Page 7 of 196

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 Reduc ing 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

................................ ................................ ................................ ................................ ... 196J.R. Simplot Company JRS06 Page 8 of 196 List of Tables Table 1. Innate TM 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 backbone 1

................................ . 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, contro l 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 Symptoms 1

................................ ................................ . 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 susce ptibility studies

...................... 177J.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 Hybridizat ion 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 Bu rbank 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 Sout hern 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. South ern 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 tiss ues

................................ ................................ ...... 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 GL N 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 bligh t infection in 2012 are shaded.

................................ ................................ 184J.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 B urbank W8 Using Real

- Time PCRJ.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 GBS 1 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 LysineJ.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 Phe nylalanine PhL Phosphorylase

- L gene P

. infestans P hytophthora 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 ( R pi ) 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 T rue 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 TyrosineJ.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 geneJ.R. Simplot Company JRS06 Page 14 of 196

Summary

The J. R. Simplot Company has advanced plant breeding by employing Simplot’s proprietary Innate TM branded technolog ies to transform plants with plant genomic DNA. In the latest innovation in potato breeding, we added late blight resistance, l ow a crylamide p otential, r educed b lack s pot, and l owered

r educing s ugars into the most popular potato variety, Russet Burbank.

To explain further, we used Innate TM branded t echnolog ies 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 i mmigration. 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 bu t 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

- e ssential free amino acid that is rapidly oxidized to form acrylami de upon frying or baking; and (3 ) susceptib ility to enzymatic browning and discoloration, which happens when polyphenol oxidase leaks out from the damaged plastids of bruised potatoes. In t he 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 s ugars 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 leadi ng 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 b y 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 col d 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 imp act 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 ch ip 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 I nnate TM technolog ies 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 fro m 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 Innate TM technolog ies effectively accelerate the p rocess 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, Innate TM t echnolog ies are particularly attractive because potatoes are notorious for having a high degree of heterozygosity, suffering from inbreeding depression, and limited predominantly to clonal propagat ion

. Historically, t hese inherent factors have significantly hindered and prevented the commercial introduc tion of quality, sought

- after traits into this valuable crop. Traditional breeding techniques result in random geno mic rearrangements and trait segregation, and do not allow for the simultaneous addition of multiple desired traits

.

To illustrate th is 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 Comp any used the techniques of modern biotechnology to improve the quality of the existing Russet Burbank variety by transforming with potato genomic DNA t hat 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 t he VNT1 protein for resistance to late blight. See Table 1 for the OECD unique identifier for W8.

Table 1 . Innate TM 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 composition al 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

c oncerns 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 an d greater than 90% homology discovered between th is 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 ingeste d 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 ofJ.R. Simplot Company JRS06 Page 16 of 196

Plum Pox Virus and Potato Leaf Roll Virus Resistance Ge ne 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 envir onmental 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 l ower 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 co mmercial 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 transfe r range t o 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 p otatoes were grown from true potato seed, the offspring would be so di verse 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 isolate d from commercial production areas, further reducing concerns about cross

pollination with wild species.

The modifications described in this petition were intended to enhance disease resistance and quality, not agronomic characteristics, of potato es . Plan ting, cultivation, management and harvesting techniques were not affect ed by the incorporated traits, with the exception that late blight resistant potatoes should require less fungicide. The InnateTM branded W8 variet y is likely to be planted in areas tha t 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 modification s 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 sp ot 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. T hus
, modified potatoes are unlikely to display enhanced we ediness

. The mechanism of action for resistance proteins like that codedJ.R. Simplot Company JRS06 Page 17 of 196

by Rpi

- vnt1 is highly specific and extremely unlikely to impact insects and other non

- t arget 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 techn ologies 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 Res istance, Low Acrylamide, Reduced Black Spot, and Lowered Reducing Sugars

The Russet Burbank potatoes were transformed using Innate TM 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 Innate TM 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 poss ible 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 phenotyp ic and molecular characterization

.

1 .1 Basis for Determination of Non r egulated Status

Under the authority of the Plant Protection Act and Part 340 of title 7 of the C ode 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 m ore 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 change s in asparagine and reducing sugar levels were associated with a reduced potential to form acrylamide upon frying. The Russet Burbank e vent 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. Include d in the first tra nsformation 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 ev ent W8 using the transformation vector pSIM1678. A reduction i n invertase through gene silencing reduce s the conversion of sucrose to reducing sugars during cold storage, inhibit s formation of sugar related defects such as sugar ends in french fries, and result s 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 determine d by agronomic and phenotypic evaluations, when
compar ed 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 l ate 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 mid

1 800s, 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 mid

twent ieth 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 Europea n 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 s pecies 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; 201 3 ).

Rpi

- v nt1 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, Rpi

v nt1 .1 , Rpi

- v nt1 .2 and Rpi

- v nt1 .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

- v nt1.1 were shown to be re sistant to P. infestans . Of 11 P. infestans isolates tested
, only isolate EC1 from Ecuador was able to overcome Rpi

- v nt1.1 and cause disease on the inoculated plants (Pel 2010). However, Rpi

- v nt1 .1 remains of major importance since it brings resistance a gainst the most dominant late blight isolate in Europe, Blue 13, by responding to the presence of the cognate effector protein AVR

VNT1 (Cooke et al. 2012). Alleles of Rpi

- v nt1 ( Rpi

- v nt1.1, Rpi

- v nt1 .2 and Rpi

- v nt1 .3 ) which differed by only a few nucleotid es 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

v nt1.1 (Pel 2010; Sliwka et al. 201 3 ), suggesting either that this strong resistance gene has been maintained since a common ancestor, due to selection pressure for blight resistance, or that geneticJ.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 Solan um species with 5 genotypes each was screened. Rpi

- v nt1 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

- v nt1 ) 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

- v nt1 (identical genes) in cultivated potatoes (Sliwka et al. 2010; 201 3 ).

Mode of action Expression of Rpi

- v nt1 in wild and cultivated potato confers broad

- spectrum resistance to late blight caused by P. infestans . Resistant pota to 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. infe stans , is recognized by the R

- protein VNT 1 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, VNT 1 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 red uction 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 po tato 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 t he 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 Administ ration 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 f ormation 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 ma y 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 specific ally, 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 Stad ler 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 t he US. The scientific community has since gathered additionalJ.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 hea lth 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 pri marily 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 Gu idance document are consistent with those reported in the Acrylamide Toolbox published by Food Drink Europe (201 1 ).

The introduc tion of InnateTM potatoes with low acrylamide potential would provide potatoes that are largely indistinguishable from existin g varieties. The reduction in asparagine and sugars using Innate TM 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 c ompletion 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 acrylamid e 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 affe cted 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 effectiv eness 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 ; F ood D rink E urope 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 i n 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 multi ple mechanisms. Through the transformation with pSIM1278, we introduced a silencing cassette for theJ.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 af ter 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 convers ion 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 V Inv include improv ed 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 inc luding acrylamide.

The Russet Burbank W8 produce s 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 ma nufacturers 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 dext rose 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 th ose 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 acrylamid e compared with the initial testing at the time of processing. In contrast, the Innate TM 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 w ith Reduced Black Spot

The blackening that occurs after potatoes are bruised affects quality and recovery in processing f rench fries and chips. Potatoes that have been damaged and show b lack 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 f rench fry processing, causing economic loss from trimming or culling potatoes with black spot. Potato es 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. Anothe r 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 19 6

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 tempera ture 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 an d the DNA insert ed into W8 target s 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 Lorbe r th 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 v acuolar 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 V Inv gene through introduction of a gene silencing cassette contained in the pSIM1678 DNA insert . Silencing of V Inv will result in lower levels of reducing sugar throughout the storage period. The V Inv silencing may also allow for lower temperature storage which will reduce yield loss from respiration and disease. There is also evidence that V

Inv 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 a nd 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 lower ing levels of reducin g sugars in cold stored potatoes (Ye et al. 2010 ; Bhaska r et al. 2010). Both research groups rep orted effective reductions in reducing sugars during cold storage with silencing of vacuolar acid invertase. In addition, with less reducing sugar, significant ly 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 fo r 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. WhenJ.R. Simplot Company JRS06 Page 24 of 196

potatoes are low in reducing sugar, colors can be readily developed by adding reducing s ugars , 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 (Dris kill 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 deh ydrated 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 pot atoes 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 potat oes cannot be used for premium f rench 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. I n 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 R esistance, 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 tha t 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 Innate TM technologies. The Russet Burbank represents 2 0.3% 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 ev idence 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

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. Suppression of the Vacular Invertase Gene Prevents Cold

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. Plant Physiology

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Black
, W

. , Mastenbroek
, C

. , Mills
, W

. R

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. ( 1953 )

. A proposal for an international nomenclature of races of Phytophthora infestans and of genes controlling immunity in Solanum demissum derivatives. Euphytica

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.J.R. Simplot Company JRS06 Page 28 of 196

2

. 0 The Biology of Potato

This section is based, in part, on the Organization for Eco nomic Co

- Operation and Development’s (OECD) series on harmonization of regulatory oversight in biotechnology no. 8, Consensus document on the biology of Solanum tuberosum, 1 9 97.

This section describes p otato biology and how it contributes to the effective management of the Innate TM potatoes. All commercial p otato 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 C hile (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 fiel d 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 r apidly 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 i s 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 potat o in 1872. In 20 12 , the United States harvested 46.7 billion lbs of potatoes, enough to make it the world's fo u rth biggest producer (NPC 2013) . Potatoes in the United States are grown in
many state s , although about half of the crop comes from Idaho and Was hington

. 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 potato

producing 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 F eed in the USA

Most h arvested potatoe s are used for food but some are grown for seed for planting

. According to the National Potato Council, t he 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 (p eels, 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. Simplo t Company. Approximately 15,000 tons of processed potato waste and 60,000 tons of raw potato waste areJ.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 G enus 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 thousa nd 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 an d 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 a nd South America ( Hannema n 1994).

2

. 4 Genetics of P otato

The basic chromosome number in the genus Solanum is twelve. S. tuberosum subsp. tuberosum can be diploids (2n= 2x= 24) or tetraploids ( 2 n= 4x= 48). The diploids have a limited range in parts of South America , while the tetrapl oids 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, t he 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 geneti cally modified potatoes, performed in New Zealand, the United Kingdom and Sweden. In each study
, the outcrossing rate was zero whe n receiving plants were separated by more than 20 met er s 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.J.R. Simplot Company JRS06 Page 30 of 196

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) S prouts 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, b ut do not enlarg e . Flowering is initiated at the end of this stag e

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

. 5) Vines turn y ellow 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 c ertified 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 vegetat ive tissues, including roots, leaves, stems, and stolons. Tuber formation begins approximately 30

- 60 days after planting. T ubers 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 . W hen 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

. T he combination of these processes results in the swelling of the subapical part of the stolon. D uring enlargement
, tubers become the largest nutrient sink of the potato plant storing large amounts of carbohydrates (mainly starch) and also significant amoun ts of protein

. F urthermore , potato tubers decrease their general metabolic activity and as such behave as typical storage sinks.

P otato tubers of the Russet Burbank variety are harvested from 140 to 15 0 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. A fter potato vines die back
, the tuber skin s thicken and harden which provides greater protection to tubers during h arvest and blocks entry of pathogen s . Although dormancy is defined as the absence of visible growth, dormant tuber meristems are metabolically active . I n general, rates of many cellular processes such as respiration, transcription, and translation are supp ressed 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 p rimary tuber.

2.6 Seed Propagation

Potato is clonally propagated, which means that tubers rather than seed s 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 t ypical seed bulk

- up program would progress as follows. In the initial year, propagation in tissue culture would produce about 100 plants , and then t hese plants are gr o wn 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, andJ.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 tak e 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 i ntroduction 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 SchematicJ.R. Simplot Company JRS06 Page 33 of 196

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 compri sed 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 Varietie s 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 P 2 O 5 – 330 lb K 2 O 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 seed s are used for planting. Therefore
, pollination to produce seed is not a factor in major commercial potato productio n

. For example , if Innate TM 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 n o t travel much m ore 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 Zea land, the United Kingdom and Sweden demonstrated through genetic testing that outcrossing rate s w ere zero whe n receiving plants were separated by more than 20 meters from genetically modified plants (Conner and Dale 1996). Additionally, p otato pollen grain s 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. Po tato Genebank and on a monogram published by the American Society of Plant Taxonomists entitled Wild Potatoes ( Solanum section P etota; Solanaceae ) of North and Central America (Spooner et al. 2004). The USDA maintains the US Potato Genebank and participat es 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 re cords. 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. stoloniferu m ; 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) determi ned 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 sp ecies
, S. fendleri and S. james i i
, 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 (Bambe rg 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 possi bility 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 organ ic soil of alluvial valleys, sandy fallow fields, grasslands, juniper

- pin yo n 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 4 700 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) indic ate d 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, accordin g 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 s tates with w ild p otato p opulations (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 virt ually 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 hybrid ization, it is unlikely that inter

- species pollination would occur with the Innate TM 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 inJ.R. Simplot Company JRS06 Page 36 of 196

the field would be eliminated by tilling, field p reparations 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, P ythium leak, Verticillium wilt, and powdery scab (Hopkins et al. 2003). P otatoes left to grow in the field are not desired as t hey 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 till age. Soil is disturbed greatly during potato growth and harvest operations, therefore requiring tillage prior to planting the rotation crop. Typically a broad spectrum pre

- emerge nce herbicide is applied in the rotation crop to control a wide variety of w eeds. 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 suc h 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 coloni zer of unmanaged ecosystems. I n 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 bec ome feral

. Furthermore, potatoes generally do not cause toxic effects to other plants or animals including non

target 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 tr opical 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 unlikel y that plants from TPS would persist for the reasons stated above.

2.13 Characterization of the Recipient Potato Cultivar

Russe t 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 vari ety 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

2.1 4 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, p otatoes 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 cross

pollination with wi ld 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 Innate TM 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

- novel

traits/applicants/directive

- 94

- 08/biology

- documents/solanum

- tuberosum

- l

- /eng/1330982063974/1330982145930 (Accessed 23 August 2012).

Conner , A .J., Dale
, P

. J ., (1996)

. Reconsideration o f 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 d iversity. 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 di stribution 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

- Operatio n 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 Pestic ides. 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 Innate TM technolog ies 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 wi th backbone elements to create the vectors for Agrobac terium

mediated 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.

T he 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 secon d 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 contain ing 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 medi a containing 3% sucrose and 2 g/l gelzan (co

cultivation medium). Infected explants were transferred, after two days, to M404 (Phytotechnology) medium containing 3% sucrose, 2 g/l gelzan, 30 0 mg/l timentin and 1.2 ml plant protection medium (Phytotechnology) to eliminate Agrobacterium (hormone

- free medium). Evidence that the plan ts were Agrobacterium

- free was obtained by incubating stem and/or leaf fragment s of transformed event s on nutrient broth

- yeast extract ( NBY medium ) for 2 weeks at 28 O C (repeated twice) with no outgrowth . In accordance with 7 CFR Part 340, transformed plant s 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 itse lf 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 ph enotypes, abnormal leaves, or the inability to root due to theJ.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 medi um lacking any synthetic hormones and incubated in a Per cival growth chamber under a 16

- hr photoperiod at 24 o C where they started to form shoots. Many shoots expressed the ipt gene and displayed the cytokinin

overproduction phenotype ; t hese shoots were discarded an d 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 isome rase ( ipt ) marker gene in the plasmid backbone , they were discarded; 2) A bsence 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 "gene

jockeying" tool. Microbiol. M ol 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 Plasmid s 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 P hL genes , whereas the pSIM1678 construct provides the late blight resistance gene ( Rpi

- vnt1 ) and a vacuolar invertase ( VI nv ) silencing cassette. The plasmids, pSIM1278 and pSIM1678, are binary transformation v ector s 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 ), contain ing well

- characterized bacterial origins of replication fro m 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 pSIM167 8 insert consists of the Rpi

- vnt1 expression cassette and a silencing cassette for the plant vacuolar invertase gene, VI nv . The Rpi

- vnt1 gene cassette consists of the VNT1 protein coding region regulated by its native promoter and terminator sequences, whe reas the silencing cassette consists of an inverted repeat of sequence from the potato V Inv gene flanked by opposing plant promoters, pGbss and pAgp.J.R. Simplot Company JRS06 Page 44 of 196

Figure 4

- 1 . pSIM1278 Vector

T he vector backbone region, indicated in grey, starts at position 10,149

- bp and ends at 19, 660

- bp ( 9,51 2

- 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 10,14 8

- 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.J.R. Simplot Company JRS06 Page 45 of 196

Figure 4

- 2. pSIM1678 Vector

T he vector backbone region, indicated in grey, st arts 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 Borde r 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.J.R. Simplot Company JRS06 Page 46 of 196

Table 4

- 1

. Table 4

- 1 Genetic elements of the pSIM1278 and pSIM1678 backbone 1

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. P olyubiquitin 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 isopentenyl

AMP, 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 199 4) 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 Agrobacterium 2

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 ) site 1 Synthetic AY566555 2 (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

. AY566555 2

(bases 1

- 187) 1

– 187 Su pports 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 ( 1 st 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. 20 12 3 ) 6. 3’

- untranslated sequence of the polyphenol oxidase

- 5 gene ( Ppo5 ) ( 1 st 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 ) ( 2 nd 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 ( 2 nd 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 tr anscripts to impair asparagine formation (Chawla et al. 20 12 3 ) 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 granule

bound starch synthase ( pGbss ) gene ( 1 st copy, convergent orientation relative to the 1 st 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, 2 nd 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 ( 1 st 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 ) ( 1 st 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 1 8

. 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 (1 6 ) double stranded RNA that triggers the degradation of R1 transcripts to limit the formation of reducing sugars through starch degradationJ.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 (1 5 ) double stranded RNA that trigger s 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 AY566555 2

(bases 231

- 416 ) 9, 963

–

10, 148 Supports primary cleavage at RB

- Like site

2 4 . Right Border (RB) sequence 1 Synthetic AY566555 2 (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 pSIM16 78, from Left Border site to Right Border
Genetic Element Origin Accession Number Position

(pSIM16 78 ) Intended Function

1. Left Border ( LB ) site 1 Synthetic AY566555 2 (bases 1

- 25) 1

– 25 Site for secondary cleavage to release single

stranded DNA insert from pSIM1 6 78 2. Left Border region sequence including LB S. tuberosum var. Ranger Russet

. AY566555 2

(bases 1

- 187) 1

- 1 87 Su pports 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 p romoter for the late blight resistance gene ( Rpi

vnt1 ) S. venturii FJ423044.1 194

- 902 D rives 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. tuberosum t 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 granule

bound 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

1 5 . SacI restriction site S. tuberosum AF143202 8,896

– 8,901 Site for connection of DNA insert with RB flanking
sequence.

1 6

. Right Border region
sequence including RB S. tuberosum var. Ranger Russet AY566555 2

(bases 231

- 416 ) 8,902

– 9, 0 90 Supports primary cleavage at RB

- Like site

17 . Right Border (RB)
sequence 1 Synthetic AY566555 2 (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 antibiotic

producing 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 transformation s 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 Ph L , 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 ca rried out as part of the characterization and biosafety assessment of the Russet Burbank W 8 event

.
In the following section, we provide a detailed characterization of the genetic inserts associated with transformation of the Russet Burbank variety to prod uce 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 flanki ng 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: Saf ety 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 Insert s

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. How ever, 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 structu ral 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 de termination 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 PC R 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 hybrid izes 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 ca ssette. The G bss 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

- 1 A , 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 pA gp, 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, a nd ScaI, with a map showing the expected fragment sizes provided below the insert map for convenience.

Similarly, Figure 5

- 1 B shows the structure of the original pSIM1678 insert along with Southern probes and a map of commonly used restriction digests. N ote 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 s pecific to the Rpi

- vnt1 promoter and VInv silencing cassette of the pSIM1678 insert,

re s pectively.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, R I =EcoR I , Sc=Sca I , Hd=HindIII, Xb=Xba I, 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.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 en zymes 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 pSIM12 78 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 cont ain 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 molecularJ.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, whe reas 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 rest riction 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 inser t (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 pSIM16 78 To assess the number of integration sites for the pSIM1678 i nsert, 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 corrobor ate 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 s ite 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 t he 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 r oughly 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 iden tified in the flanking sequence , but as described later the structure of this insert was very similar to the input sequence.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 w as 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 a re based upon integration of a single copy of the pSIM1278 DNA insert described in Figure 5

- 1 A

.

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 *Do es 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 locu s. 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 s ection 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 orig inal 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 t o a tandem copy of the longer GBSS promoter separated by r ight b order and a small amount of pPHL sequence (Figure 5

- 4)

.
Although th e structure associated with pSIM1278 is more complicated than anticipated, the duplicated silencing cassettes are still unde r the control of the tissue

- specific promoters and the current structure do es 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) orJ.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 p lant 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 b ands 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 o f 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 polysa ccharides 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 Burba nk 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 m olecular 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 li ne marking top of band in following example).J.R. Simplot Company JRS06 Page 59 of 196

Figure 5

- 4

. Overall s tructure of pSIM1278 DNA Insert with d igest ion and hybridization patternsJ.R. Simplot Company JRS06 Page 60 of 196J.R. Simplot Company JRS06 Page 61 of 196

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 a n 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 colore d 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 comb inations 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 rest riction 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 B lot 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

- 1 5. 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 Flankin g 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 wh en 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 diges ts 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 hybri dized 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 we re 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 existe nce 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 aJ.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, S ca I and BsrG I , bo th cut the insert exclusively in or adjacent to R1, leaving the entire left side of the insert intact. A known restriction site was identifie d in the flanking region for Sca I 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. Southe rn blots probed with an A SN probe identified a single band, as expec ted. 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 Asn 1/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 adja cent 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 con firm 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 describ ed 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 invert ed 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 t he 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 e lucidation 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 i s 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 bandsJ.R. Simplot Company JRS06 Page 63 of 196

ranging from 1.0 to 2.2 kb show n 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 silencin g 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 locati on 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 dis cussed for pSIM1278 were instrumental in elucidating the structure of the insert derived from the pSIM1678 plasmid as both DNA inserts contain A gp and Gbss promoter elements. The data did not suggest the inserted structure deviated from the original pSIM16 78 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 fo r 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 c onsistent 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 pSIM16 78 DNA Insert Digest ion and Hybridization Pattern

The upper figure represents the structure of the insert associated with the pSIM1 6 78 plasmid, including designated restricti on 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 pro be binding sites are indicated by bands, but only the digest/probe combinations necessary to support the model are provided i n this petition. Red boxes denote internal bands (IB) associated with the original pSIM1678 DNA construct. Blue closed boxes indica te 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 Souther n blots is indicated in parenthesis for all junction bands.J.R. Simplot Company JRS06 Page 65 of 196

Figure 5

- 6. Russet Burbank DNA H ybridization with the AGP P robe

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 ban ds are summarized in the table and classified into three groups based on the structure of the DNA inser t: 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 bl ue

. All molecular weights are presented in kilobases

.J.R. Simplot Company JRS06 Page 66 of 196

Figure 5

- 7. Russet Burbank DNA H ybridization with the AGP P robe

Genomic DNA of Russet Burbank control ( WT ) and event W8 were digested with XbaI, Sac1, and PacI and hybridized with the AGP p robe. 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 ban ds ( 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

.J.R. Simplot Company JRS06 Page 67 of 196

Figure 5

- 8. Russet Burbank DNA H ybridization with the ASN P robe

Genomic DNA of Russet Burbank control ( WT ) and event W8 were digested with EcoRV, HindIII, and EcoRI/Scal and hybridized with the ASN p robe. 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.J.R. Simplot Company JRS06 Page 68 of 196

Figure 5

- 9. Russet Burbank DNA H ybridization with the ASN P robe

Genomic DNA of Russet Burbank control ( WT ) and event W8 were digested with XbaI and PacI and hybridized with the ASN probe. Size of th e 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.J.R. Simplot Company JRS06 Page 69 of 196

Figure 5

- 10. Russet Burbank DNA H ybridization with the ASN P robe

Genomic DNA of Russet Bu rbank 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 presente d in kilobases.J.R. Simplot Company JRS06 Page 70 of 196

Figure 5

- 11. Russet Burbank DNA H ybridization with the GBS1 P robe

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 ), intern al 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.J.R. Simplot Company JRS06 Page 71 of 196

Figure 5

- 12. Russet Burbank DNA H ybridization with the GBS1 P robe

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 ban ds 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.J.R. Simplot Company JRS06 Page 72 of 196

Figure 5

- 13. Russet Burbank DNA H ybridization with the R1 P robe

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.J.R. Simplot Company JRS06 Page 73 of 196

Figure 5

- 14. Russet Burbank DNA H ybridization with the R1 P robe

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.J.R. Simplot Company JRS06 Page 74 of 196

Figure 5

- 15. Russet Burbank DNA H ybridization with the G BS2 P robe

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.J.R. Simplot Company JRS06 Page 75 of 196

Figure 5

- 16. Russet Burbank DNA H ybridization with the VNT1 P robe

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.J.R. Simplot Company JRS06 Page 76 of 196

Figure 5

- 17. R usset Burbank DNA H ybridization with the VNT1 P robe

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 ban ds ( IBs ) and the junction bands ( JBs )

. The DNA fragments of pSIM1678 are in blue . All molecular weights are presented in kilobases.J.R. Simplot Company JRS06 Page 77 of 196

Figure 5

- 18. Russet Burbank DNA H ybridization with the INV P robe

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 th ree 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.J.R. Simplot Company JRS06 Page 78 of 196

Figure 5

- 1 9. Russet Burbank DNA H ybridization with the INV P robe

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 ban ds ( IBs ) and the junction bands ( JBs )

. The DNA fragments of pSIM1678 are in blue . All molecular weights are presented in kilobases.J.R. Simplot Company JRS06 Page 79 of 196

5. 2

. 4 Conclusions : Copy number and structure of the DNA Insert s

Genetic and structural characterization of the inser ts 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. T he structure of the DNA derived from transformation of pSIM1278

was complex (Figure 5

- 4) relative to the structure of the original insert. The insert ed 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 do es not negatively impact safety or trait efficacy of the product (See Chapter 9: Safety of Russet Burbank W8 Pota to 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 t he entire T

- DNA left border and 137

- bp of the Rpi

- v nt1 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 Biochemi cal 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 ref erence 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 l ater 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 in dividual 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 undertake n 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

- 2 0 ).J.R. Simplot Company JRS06 Page 80 of 196

Figure 5

- 2 0

. 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 disru pt 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 untransfor med 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 Abs ence 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 develop ed 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) A bsence of the backbone DNA was confirmed with Southern blot hybridiza tion ; 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 o ften 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 (Ko nonov 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 pheno types 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 anyJ.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 pr obes 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 B urbank controls ( WT ) were digested with EcoRI and probed with each of the six probes. As shown in Figure 5

- 2 2 , bands were limited to the positive control (T130) samples in all Southern blots, except for probe 6, which contained a pair of bands common betwe en 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 a ll Southern blots are consistent with a lack of backbone DNA in the W8 genome.

Figure 5

- 2 1

. 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 regio ns 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.J.R. Simplot Company JRS06 Page 82 of 196

Figure 5

- 2 2 . 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 =

a dditional event that was not submitted , W8 = Event W8, T130 = positive control c ontaining backbone DNA . Lane s 1 and 2 are molecular weight markers ( DIG II , and DIGVII, respectively) with sizes indicated next to gel

.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

- 2 1 ). Each of these primer sets successfully amplified sequence corresponding to the gapped regions (Figure 5

- 2 3 , left panel of gels), but the
sequence cor responded 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 an d 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

- 2 1 ). Unlike the internal gap

- s pecific primers, these primers do not amplify Russet Burbank sequence (WT) as shown in Figure 5

- 2 3 ( 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 d id not detect backbone in the W8 samples.

Figure 5

- 2 3

. 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), W T = Control Russet Burbank, W3 = Additional event that was not submitted
, W8 = Submitted Event, PC = Positive control, NC = Negative control

.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

- 2 4 , each junction was tested using two sets of primers. Since t he plasmids have identical right junction s, its presence was tested using a s hared set of PCR primer s (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 ea ch case positive controls amplified as expected (Figure 5

- 2 5 ). 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 withi n the flanking sequences for each insert ( Section 5. 3 )

.

Figure 5

- 2 4

. PCR Primer Sets for Detecti on of Backbone Junction SitesJ.R. Simplot Company JRS06 Page 85 of 196

Figure 5

- 2 5 . Confirmation of the Absence of Backbone DNA Junctions

Detection of Left Border of the DNA insert and flank ing 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

.J.R. Simplot Company JRS06 Page 86 of 196

5. 4.3 Conclusions: Absence of P lasmid Backbone

Collectively, our Southern blot and PCR analyses have shown that the Russet Burbank W8 e vent 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 in sertions are expected to be stable

. T ubers 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 show n 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

. T his 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) usin g 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 (GBS 1 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 geno me 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 GBS 1 . 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 oth er for both probes (Figure 5

- 2 6 ). 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 st ability 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 th ree generations analyzed ( Figure 5

- 2 7 ).J.R. Simplot Company JRS06 Page 87 of 196

Figure 5

- 2 6 . 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.J.R. Simplot Company JRS06 Page 88 of 196

Figure 5

- 2 6 . Southern blot probing of EcoRV digestion (cont.)

Southern blots of genomic DNA (3 μg ) following digestion with EcoRV and probed for GBS 1 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

G2J.R. Simplot Company JRS06 Page 89 of 196

Figure 5

- 2 7 . 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.J.R. Simplot Company JRS06 Page 90 of 196

F igure 5

- 2 7 . 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 Bu rbank 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.J.R. Simplot Company JRS06 Page 91 of 196

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 a ctivity (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 transformati on 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 c onvenient 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 cassett e 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, blac k 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 ins ert derived from pSIM1678 contains an additional expression cassette consisting of the Rpi

vnt1 gene. This gene is expressed under its native potato promoter and provides late blight resistance against P. infestans

. A detailed description and characterizat ion 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 concentration s 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 method s 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 pSIM167 8. 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 se quence 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 compl ex than the original plasmid, but the du plicated 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
met hod 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

Chio u , T

. J

. , Aung , K

. , Lin
, S

. I

. , Wu , C

. C

. , Chiang
, S

. F

. , and Su , C

. I

. (2006)

. Regulat ion 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
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. (2008)

. Unexpected silencing effects from T_DNA tags in Arabidopsis. Trends Plant Sci

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. , Sato , S

. , Kato , T

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. Phosphatidyglycerol is essential for the development of thylakoid membranes in Arabidops is thaliana. Plant Cell Physiol

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- 1464.

Kim
, S

. I

. , Veena, and Gelvin
, S

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. (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
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. Integration of T

- DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J

. 11: 945

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, A

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. , Mendel
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. High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium

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. , Frame , B

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. Generation of backbone

- free, low transgene copy plants by launching T

- DNA from the Agrobacterium chromosome. Plant Physiol

. 152: 1158

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. Cytokinin vectors mediate marker

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. , Ouyang , S

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. , O'Brien
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. 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

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6.0 Characterization and Safety of the VNT1 Protein

The VNT1 protein produced through introduc tion of the Rpi

- vnt1 gene into the Russet Burbank Event W8 has been characterized and assessed fo r safety . A description of VNT1’s origin, function, mode of action, and an assessment for toxi ns and allergen s 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

I dentity 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 flan ked 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 Go verse 2012)

. T he LRR is the least conserved and has b een hypothesized to function in effector recognition.

Using Simplot Innate TM Technologies
, an Rpi

- vnt1 gene (accession: FJ423044.1 ) was transformed into the Russet Burbank potato variet y after insertion into the pSIM1678 plasmid, resulting in late blight resistance. This allele is one of three identified in the wild potato species
, Solanum venturi i , 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

- p rotein and a homolog of the Tm

- 2 2 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 YGV VSIVGMP 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 N DLPIALRPC 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 KKLF LVQGPN ISPISLRVSE RLAKLRISQV LJ.R. Simplot Company JRS06 Page 95 of 196

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 reli es upon recognition of pathogen

- secreted effector proteins by resistance protein receptors (R

- proteins). R

protein 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, f ollowing 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 pathoge n 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 method s 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

- L RR. (B) Quantitative curve showing linearity of western blot data. 

### A BJ.R. Simplot Company JRS06 Page 96 of 196

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 t issues . 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 analys is 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 W 8 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 - 1 or ppb) Russet Burbank

(ng g - 1 or ppb) W8

(ng g - 1 or ppb) Leaf 9 6 0 < 6 0 < 6 0 Tuber 9 30 < 30 < 30 Stem 9 20 < 20 < 20 Root 9 15 < 15 < 15 Flower 9 6 0 < 6 0 < 6 0 VNT1 concentrations were calculated using ng g - 1 dry 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 6 0 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 a ny tissue s 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 assa y 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

c oncentrations 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 f or Bt protein in corn, soybean, cotton, and other crops deregulated by APHIS . In one example, the Cry1Ac protein in Bt soybean samples was expr essed 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 bel ow 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 de tectable 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 de tails). Expression of Rpi

- vnt1 was normalized to a set of endogenous housekeeping genes, α

tubulin and elongation factor 1α, within each sample. Although expression was low in W8 tissue samples, it was considerably higher than in the Russet Burbank control s (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 an alysis. RB (Russet Burbank); W8 (Event W8).

6.4 Assessment of VNT1 for Allergen s and Toxi ns

Using a number of well

- established bioinformatics tools, we performed a comprehensive analysis of
po ssible toxi ns and allergen s 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 homolog ous 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 allergen s or toxi ns

.

6.5 Conclusions on VNT1 Protein Safety

Biochemical characterizat ion 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 , t he causal agent of late

- blight disease. In fact, the Rpi

- vnt 1 (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 specie s , which shows the breadth and importance of these genes to potato biology ( R odewald and Trognitz 2013)

.
Although we were able to confirm expression of the Rpi

- vnt1 gene in W8 plants, the protei n concentration was below our limit of quantitation in all tissues tested, yet Rpi

- vnt1 conferred late blight resistance (Chapter 10 : Trait Efficacy). A b ioinformatics assessment of possible allergen s and toxi ns 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 transcriptsJ.R. Simplot Company JRS06 Page 99 of 196

Collectively, t he ubiquitous nature of R

- genes and their encoded R

- proteins in Solanum species, including tomato and edible potato es , lack of sequence identity to known toxins or allergens, mode of action not based upon induc ing 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)

. P etition for Determina tion 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 includin g 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). Int eraction 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 Diffe rentiation

. 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 leucine

rich 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 M offett, P. (2008). The Coiled

Coil 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 Agai nst 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. , Swia tek, 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 geneJ.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. Nat ure 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 produ ction 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 dsRN A 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 Innate TM 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 tuber s

. T hese technologies allowed us to specifically silence the target genes in the desired plant tissue with minim al impact on the overall plant biology.

The expression of an inverted repeat is a regulatory s ystem 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 ) gene s (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 inhi bition of a m etabolic 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 t hese 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 cont ain sequence from a total of five different potato genes. The pSIM1278 construct consists of two gene silencing cassettes ( Figure 7

- 1, upper construct ). One cassetteJ.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 inver ted repeat containing sequence from the vacuolar i nvertase ( VInv ) gene ( Figure 7

- 1, lower construct ).

All three silencing cassettes are regulated by the same set of well

- characterized and tissue

- spe cific 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 ha ve shown that transcripts are of variabl e 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 si lencing (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 co nstruct (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 additi onal details of the methods in Appendix C: Allergen and Toxin Assessment for Russet Burbank W8

.J.R. Simplot Company JRS06 Page 104 of 196

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 ca ssette. 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 starc h granules (Lorberth et a l. 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. Northe rn 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.J.R. Simplot Company JRS06 Page 105 of 196

The only tissue other than tubers where some target silencing was ob served 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

- g rown plants f or 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 transcrip ts (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

- g rown 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.J.R. Simplot Company JRS06 Page 106 of 196

Figure 7

- 5. Target Gene Expression in Roots

Northern blot analysis of total RNA (20 μg) isolated from roots of field

- g rown 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 F lowers

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 ea ch sample . W3 = Additional event that was not submitted.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 ta rget 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 phenot ypes 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. Thu s , the RNAi response was both effective and specific and there is no indica tion 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. Plan t Methods

. 3 : 13.

Collinge , S., and Clark , P., 2013. Petition for Determination of Nonregulated Status for Innate TM 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., D edic

- 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

– 11 75.

Llorente, B., Alonso, G.D., Bravo

- Almonacid, F., Rodríguez, V., López, M.G., C arrari, 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.

Lorberth, R., Ritte, G., Willmi tzer, L., and Kossmann, J. (1998). Inhibition of a starch

- granule

– bound 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 ADP

glucose pyrophosphorylase small subunit. Journal of Biological Chemistry

. 269 : 30798

– 30 807.

Petrick, J. S., Brower

- Toland, B., Jackson, A. L., and Kier, L. D. (2013). Safety assessment of foo d and feed from biotechnology

- derived crops employing RNA

- mediated gene regulation to achieve desired traits: a scientific review. Regulatory Toxicology and Pharmacology: RTP

. , 66 : 167

– 1 76.

Pumplin, N., and Voinnet, O. (2013). RNA silencing suppression by plant pathogens: defence, counter

defence and counter

- counter

- defence. Nature Reviews. Microbiology

. 11 : 745

– 7 60.

Tuteja, J.H., Clough, S.J., Chan, W.C., and Vodkin, L.O. (2004). Tissue

- specific gene silencing mediated by a naturally occurring chalcone s ynthase 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

. 21 : 3063

- 3077

.
Visser, R.G., Stolte, A. , and Jacobsen, E. (1991). Expression of a chimaeric granule

- bound starch synthase

- GUS gene in transgenic potato plants. Plant Molecular Biology

. 17 : 691

– 69 9.

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 A ssessment

The compositional assessment study evaluating proximates, vitamins, amino acids, minerals, and glycoalkaloids, was conducted on Russet Burbank E vent 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 de termine if W8 contained equival ent 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 146J.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 E vent 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 with in 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.

 Eve nt W8 tu bers contained, on average, 255.1 mg/100g total aspartic acid as compared to t he 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 tu bers contained, on average, 477.8 mg/100g total glutamic acid as compared to t he control, which contained 310.3 mg/100g (p < 0.0001 ). The mean for Event W8 was within the tolerance interval and combined li terature 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 i nterval 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 contain ed, on average, 72.9 mg/100g total threonine as compared to the control, which contained 65.3 mg/100g (p = 0.002 0 ). 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 ac id, 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 we re 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 Gly c oalkaloids

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 Ev ent 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 i s as safe as con trols 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 f or 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 TI 1

Low TI 1 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 20 13, 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 Type 1 Seed 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 TX278 2 Reference N/A Red Thumb 2 Reference N/A

1 Greenhouse

- grown mini

- tubers were grown from tissue culture plan t lets 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

- G RA, 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 rep licate (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; AOA C 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 lev els 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 Labora tories 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), Magn esium (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 wer e analyzed using the following linear mixed model:

Y ijkl =  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 j th site (random effect),  k(j) denotes the random rep effect (within site), (   ) ik denotes the interaction between the i th treatment and random k th 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 p

values 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 Sunb urst, 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 val ues for each analyteJ.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 c ombined 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 T olerance IntervalJ.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 Used 1 CLR 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 necessaryJ.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 Used 1 CLR (mg/ 100g) Alanine high 53.4 μmol/g Talley et al. 1984 89.1 μmol/g x mol/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol x FW x 1000mg/g x 100g x 0.2 70.0 Aspartic acid high 1476 m g /200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 738.0 Aspartic acid low 677 m g /200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 338.5 Glutamic acid high 1207 m g /200g Rogan et al. 2000 NA mg/200g x 0.5mg/100g 603.5
Glutamic acid low 583 m g /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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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/10 6 μmol 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)J.R. Simplot Company JRS06 Page 120 of 196

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 a nd 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 19 6

8. 6 R eferences

AOAC

. (2005a)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 988.15. A ssociation of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005b)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 967.22. Association of Official Agricultural Chemists International, Gaithersburg, MD, U SA.

AOAC

. (2005c)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 925.09. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005d)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 926.08. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005e)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 923.03

. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005f)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 962.09

. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005g)

. Official Methods of An alysis of AOAC International, 18 th Ed., Method 984.27. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005h)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 985.01. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005i)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 922.06. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005j)

. O fficial Methods of Analysis of AOAC International, 18 th Ed., Method 954.02. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005k)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 944.13. As sociation of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005l)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 960.46. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005m)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 955.04. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005n)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 979.09. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

AOAC

. (2005o)

. Official Methods of Analysis of AOAC International, 18 th Ed., Method 961.15. Association of Offic ial 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, 19 th Edition, Method 997.13. Association of Official Agricultural Chemists International, Gaithersburg, MD, USA.

Atkin
, L

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, A

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. , Williams
, W

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. 15: 141

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Barkholt
, V

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. L

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. 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

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Henderson
, J

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. D

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, 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.

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. J

. , Bookout, J

. T

. , Duncan , D

. R

. , Fuchs
, R

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. , Lavrik
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. , Love , S

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. , 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 derivitizatio n and HPLC. J Chromatogr aphy. 431: 271

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, S

. L

. (1987)

. Potato glycoalkaloids. Acta Hort

. 207: 41

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Talburt
, W

. F

. , and Smith
, O

. (1987)

. Potato processing. Van Nostrand Reinhold, New York.

Talley
, E

. A

. , Toma , R

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. , Orr , P

. H ., (1984)

. Amino a cid composition of freshly harvested and stored potatoes. Am Pot Journal

. 61: 267

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. (1973)

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- 160.J.R. Simplot Company JRS06 Page 123 of 196

9. 0 Safety of Russet Burbank W8 Potato

The rationale for safety of Russet Burbank event W8 is based on the following evidence: 1) p henotype through agronomic studies, 2) c ompositional equivalence, 3) b ioinformatics assessment for toxins and allergens, 4) g lycoalkaloid testing, 5) d ietary risk assessment of VNT1 protein, 6) lo w levels of VNT1 protein, 7) s afety of nucleic acids , 8) l iterature review on gene silencing through RNAi, and 9) p revious regulatory actions for plant incorporated protectants similar to t he 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 at tributes 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 E quivalence

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 literat ure 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 statistic ally equivalent to tubers from the control or within the toleranc e 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 Toxi n Assessment

The bioinformatics approach to assessing toxins and alle r gen s in W8 specifically investigates possible changes related to the DNA inserts and flanking regions. Other evidence that W8 would not contain new alle r gens 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 glycoprote in 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 wo uld avoid them and changes to patatin levels would not be expected to alter that behavior.J.R. Simplot Company JRS06 Page 124 of 196

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 pote ntial 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 compr ehensive bioinformatics analysis of each insertion site was performed to investigate th is possibility (Appendix C : Allergen and Toxi n Assessment ).

BLAST searches indicated that there were no potato transcripts associated with the flanking regions of the i nserts, 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 as sess 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 Univer sity 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 id entity 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 prote in (GI:3201547) from Persea a mericana (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 d atabase 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 allergen ic risk. Collectively
, these data suggest the peptide identified is a false positive an d 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 t o 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 W 8 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 n ative vacuolar invertase gene may actually decrease the allergenic potential of Russet Burbank W 8 potatoes relative to the control.

To assess the potential for these same ORFs to act as toxins, we performed BLAST searches against custom databases containi ng annotated protein toxins found in nature. We did not identify any significant matches (E

- value < 1) for any of the ORFs.J.R. Simplot Company JRS06 Page 125 of 196

In summary, u sing a number of well

- established bioinformatics tools, we performed a comprehensive analysis of potential toxi ns an d allergen s 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 OR Fs 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 : Character ization 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 th an 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 glycoa lkaloids 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 co lor 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 prod uction, 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 ). P eeling potatoes and removing an y 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 i n 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 t otal glycoalkaloids in potato tubers consists of a

- chaconine (solanidine

- glucose

- rhamnose

rhamnose) and a

- solanine (solanidine

- galactose

- glucose

- rhamnose).
Most U.S. commercial potato varieties contain less than 120 parts

- per

- million (ppm) glycoalkaloids w ith 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 th an 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 po tatoes, 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 correla tes 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.J.R. Simplot Company JRS06 Page 126 of 196

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 sign s 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 p otatoes before such symptoms may arise.
Current Industry Practices Mitigate Risk. Notwithstanding the low levels of glycoalkaloids fou nd 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 cove red 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. E vent W8 and the control ha d 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 g ene ( Rpi

- vnt1 ) sequence. Homology of Rpi

- vnt1 with tomato genes identified in the search was >90% with very low E

- values, in dicating 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 (T o MV) resistance genes. In addition, the V NT1 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) / Lycopersic on 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

- vn t1 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

- vnt 1 gene. A lthough a literature search did not yield information about the levels of T o MV resistance protein s in tomatoes or in diets
, T o MV resistant tomato varieties and rootstocks are readily available and grown in the US for fresh market tomato production. G iven the large per capita consumption of fresh marketJ.R. Simplot Company JRS06 Page 127 of 196

tomato es in the United States, consumers have likely been exposed to tomatoes containing homologous T o MV resistant genes and associated proteins in their diets.

Widely consumed plant species that are reported to cont ain 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 pot ato 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 he alth 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 i mmunoblot and q uantitative w estern b lot analys e s, the VNT 1 protein is below 30 ppb in po tatoes 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 propertie s 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 resist ance 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 par t 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 producti on 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 V Inv , all of which result in small RNAs that regulate gene expression. Such s mall RNA (sRNA), including siRNA, miRNA, and piRNA, in plants and animals are generally involved in regulatingJ.R. Simplot Company JRS06 Page 128 of 196

endogenous gene expression, repressing transposons, or targeting invading pathogens for destruction. The sRNA are ubiquitous in nature, including prokaryotes wher e 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 ha d 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 stu dies aimed at reproducing the author’s study

. However, these claims have not been substantiated (Dickinson et al. 2013 ; Snow et al. 2013 ; Witwe r et al. 2013 ; Y. Zhang et al. 2012) , and have been challenged by many experts in the field leading to self

correction 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 (Hein emann et al. 2013) . The concerns of Heinemann and colleagues were thoughtfully considered by fellow scientists associated with the bi

- national
gov ernmental 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 Innate TM potatoes collectively establish these potatoes as safe for human consumption. In fact, t here is no scientific rationale to suggest that sRNA pres ent in GM

- foods are any less safe than those naturally abundant and safely consumed in our current diets.

Stability t hrough Microvesicles or Protein Complexes. Another possible mechanism to increase stability of siRNA would be for the plant to package t hem into microv e sicles 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. Instea d, 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 st rand) are more rapidly turned over. Turchinovich and colleagues found that the vast majority (>97%) of miRNA s 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 (Turchinovi ch et al. 2011)

.J.R. Simplot Company JRS06 Page 129 of 196

In summary, there is evidence that siRNA may be protected by association with RISC complexes w ithin 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 rea lized 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), thi s 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. R NA

- dependent RNA polymerases) that could amplify and sustain a non

- specific
response (Petrick et al. 2013) . In addit ion 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 th at 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 organis m, 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 i s 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 (Fu saro 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 wo uld 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 destroyi ng exogenous dsRNA as a means of protecting against foreign invaders.

Scientific rationale of the safety of orally ingested siRNA(s) derived from Innate TM potatoes. As described previously, the scientific literature does not support a model whereby sRNA present inJ.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 Innate TM potatoes and their use of RNAi:

 The Innate TM potatoes rel y 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 prac tices 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 ) . Innate TM potatoes use RNAi to reduce the accumulation of the precursor asparagine to limit acrylamide potential. Thus, Innate TM
potat oes 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 comm only 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 Innate TM 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 Innate TM potatoes exclusively target plant genes, they are less likely to have adverse o ff

target 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 eff ects greatest.

Numerous physiological barriers have impeded introduction of nucleic acid through oral uptake (O’Neil l 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 t o 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 report ed 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 w ould 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 Innate TM potatoes collectively establish these potatoes as safe for human consumption.J.R. Simplot Company JRS06 Page 131 of 196

9 .9 Previous r egulatory 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 Po x 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 PLR V Resistance Gene, late blight resistance has been pa rt 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 r esistance, 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, l ate 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) Non

occupational 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 fertil ity. 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 t oxicology and the e nvironmental a ssessment. 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 ge neral 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 e nvironmental a ssessment 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 e nvironmental f ate studies were waived because of the lack of toxicity/pathogenicity associated with the active ingredient and lack of significant levels of expression . Requirements for n ontarget 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 t o 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. P otatoes 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. O pen reading frames associated with the DNA inserts, including the late blight resistance gene itself,

w ere 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 in to 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 co n trolling late blight, levels in potato tubers are below our detection limit of 30 ppb. As noted in Section 9.7,

n ucleic 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 P rotein 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 contaminatio n 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 a nd unlikely to pose risk to human health, non

- targets or the environment.

Based on th e foregoing rationale for safety, the J.R. Simplot Company respectfully requests that it should be granted deregulat ed 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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. (2000)

. The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting protein and DNA sequences

. Biotechniques

. 28: 1102

- 1104.

Turchinovich, A., Weiz, L., Langheinz, A., and Burwinkel, B. (2011). Characterization of extracellular circulating microRNA. Nucleic A cids R esearch

. 39 : 7223

– 72 33.

Tuteja, J.H., Clough, S.J., Chan, W., and Vodkin, L.O. (2004). Tissue

- Specific Gene Silencing Mediated by a Naturally Occurring Chalcone S ynthase Gene Cluster in Glycine max . The Plant Cell. 16: 819

– 835.

US EPA

. (2000)

. Biopesticides Registration Action Document. Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf1 gene) (006469) Available from: http://www.epa.gov/opp00001/chem_search/reg_actions/registration/related_PC

- 006469_1

- Apr

00.pdf

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US EPA

. (2010)

. Biopesticides Registration Action Document. Coat Protein Gene of Plum Pox Virus. Available from: http://www.epa.gov/opp00001/chem_search/reg_actions/registration/decision_PC

006354_7

- May

- 10.pdf

.
Van Doorn, W. G., Beers, E. P., Dangl, J. L., Franklin

- Tong, V. E., Gallois, P., Hara

- Ni shimura, I., and Bozhkov, P. V. (2011). Morphological classification of plant cell deaths. Cell D eath and D ifferentiation

. 18 1241

– 124 6.

Van Gelder
, W

. M

. J

. (1990)

. Chemistry, toxicology, and occurrence of steroidal glycoalkaloids: Potential contaminants of the Potato. Chapter 6. Poisonous Plant Contamination of Edible Plants. CRC Boca Raton, F L. AFM Rizk (Ed). p 117

- 156.

Wang, K., Li, H., Yuan, Y., Etheridge, A., Zhou, Y., Huang, D., and Galas, D. (2012). The complex exogenous RNA spectra in human plasma: an interface with human gut biota? PloS one
, 7 : e51009.

Witwer, K.W., McAlexander, M. A , Queen, S.E., and Adams, R.J. (2013). Real

- time quantitative PCR and d roplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: limited evidence for general uptake of dietary plant xenomiRs. RNA B iology

. 10 : 1080

– 108 6.

Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., and Zhang, C.

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– 1 26.

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. A

. (1985)

. Estimation of taste thresholds of three potato glycoalkaloids. J Can Inst Food Sci Technol. 18:337

- 33 9.J.R. Simplot Company JRS06 Page 138 of 196

10. 0 Trait Efficacy

Introduction Russet Burbank Event W8 was created using Innate TM 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, reduc ing acrylamide and lowering levels of reducing sugars

. In the f ollowing discussio n , 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

- formi ng potential. The mean values of asparagine , aspartic acid, glutamine, and glutamic acid for W8 w ere all within the tolerance interval s and combined literature range s , and therefore considered within the normal range for potatoes. A review of the biosynthetic pathway for glutamine and a sparagine 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

- valu e 3 N Range TI 1 CLR 2
Min Max Min M ax M in M ax Asparagine (mg/100g) W8 87.8 <.0001 41 62.1 140

. . . . Asparagine (mg/100g) Burbank 300

. 41 198 469 60.0 48 2 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 5 40

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) ; Shep he rd 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

.

1 0. 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 pro moters 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 br own 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 Activ i ty 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, espec ially 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 ea ch of W8 and Russet Burbank control were used for the assay wh ich 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 Express ion , 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 Inverta se 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 year

round 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 proces s 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 stor age 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. A t harvest and all storage time points and temperatures, W8 tubers contained lower levels of reducing sugars compared with the control ( Table s 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 tuberJ.R. Simplot Company JRS06 Page 141 of 196

Table 10

- 2. Potato Sugar Levels at Harvest and After Storage at 46 ° F
Timing Variety LS Mean P

- value 1 N Range Tolerance Interval Min Max Min Max Fructose + Glucose (mg/100g)

Fresh W8 38.4 0.0002 41 9. 68 106 1.0 0 424

Control 146

. 41 14.0 406

Month 3 W8 122 0.0056 9 54.1 210 1.0 0 996

Control 483

. 9 298 598

Month 6 W8 116 <.0001 9 20.8 310 1.0 0 640

Control 261

. 9 153 459

Month 9 W8 106 0.032 9 79.7 160 1.0 0 648

Control 224

. 9 105 372 Sucrose (mg/100g) Fresh W8 395 <.0001 41 161 775 1.0 0 512

Control 241

. 41 113 558 Month 3 W8 651 <.0001 9 520 738 1.0 0 1125

Control 148

. 9 56.2 228 Month 6 W8 202 0.0021 9 177 229 1.0 0 345

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

Timing 1 Variety LSMean P

- value 3 N Range TI 2

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 15 1 <.0001 3 102 18 8 183 1586

Control 754

. 3 703 788 Sucrose (mg/100g)

Month 6 W8 96 3 <.0001 3 945 986 1.0 0 661

Burbank 18 2

. 3 138 206

Month 9 W8 64 5 <.0001 3 598 714 1.0 0 32 5

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 w er e within the tolerance interval and combined literature range s, 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 toJ.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

Re duced asparagine levels in W8 ( Table 1 0

- 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 product s because they ar e reactants in the formation of acrylamid e . In order to demonstrate the efficacy of reducing acrylamide, field

- grown tubers of W8 and the c ontrol 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% l ess acrylamide than the control (Table 10

- 4) . When potatoes were stored throughout nine months at 46 ̊F, acrylamide levels in W8 were 78 to 8 3.7 % lower than control Russet Burbank ( Table 10

- 4 )

. Acrylamide le vels 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. Th ese significantly lower acrylamide levels in W8 were expected from silencing the Asn1 and V Inv 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

- value 3 Percent Reduction 2 N Range (ppb) TI 1 (ppb)

Min Max Min Max

Fresh Acrylamide W8 75.3 <.0001 85.0 41 32.7 185 10.0 1035 Burbank 50 3

. . 41 229 971

Month 3 Acrylamide W8 86.1 <.0001 80.9 9 74.5 94.3 10.0 59 9 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 10.0 104 7 Burbank 52 8

. . 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

Timing 1 Compound Variety LSMean (ppb) P

- value 4 Percent Reduction 3 N Range (ppb) TI 2 (ppb)

Min Max Min Max

Month 6 Acrylamide W8 20 3 <.0001 86.2 3 199 207 1155 179 2 Burbank 1473

. . 3 1450 1500

Month 9 Acrylamide W8 212 <.0001 90.8 3 201 234 761 383 9 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 sile nced. 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 pre sented 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.J.R. Simplot Company JRS06 Page 144 of 196

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 show s that W8 ha s 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 associ ated with reduced black spot through silencing of the Ppo5 gene (Chapter 7: Characterization of Gene Silencing and Target Gene Express ion
, 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 Tuber s

. Conversion of L

- DOPA to dopachrome was monitored over time by measuring A 474nm . Δ A 474nm ·min - 1 , which was converted to units of μmol·min - 1 ·mg dw tuber - 1

.

10. 1. 5 Late Blight Resi s tance

T he P otato L ate B light R esistance G ene (also known as Rpi

- vnt1 ) has been added to W8, successfully conferring late blight resistance. To demonstrate the efficacy of late blight resistanc e, stud ies were conducted by inoculating both foliage and tubers.

Foliar Late Blight Test Results At the end of the trial period, t h e last rating from each site is summ a ri zed in Table 10

- 6 to show the

e ffect of t he potato late b light 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

- va lue 2 CVR 1

Control 58.3 <.0001 18. 8

- 100 W8 0.50

. . 1 CVR = Conventional variety range. The range of mean values of conventional varietie s. 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 b light resistance gene confers resistance in W8 and is efficacious in the foliage.

Tuber Late Blight Results Tuber infection rate determin ed by percent infection is summari zed 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 tuberJ.R. Simplot Company JRS06 Page 146 of 196

Table 10

- 7 . Percent Tuber Late Blight Infection Isolate Variety Mean Percent Late Blight Tuber Infection P

- value 1
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

suc h 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 stor age 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 t hrough the catechol assay. In addition, the enzymatic assay showed a 90% reduction in PPO activity in tubers from W8 compared to the control.

T ests with late blight inoculum on tubers and foliage support the conclusion that the P otato L ate B light R esista nce G ene 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 reduc tions 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 Method s for Trait Efficacy and Composition

10. 3 .1 Analytical Methods for Free Amino Acids, Sugars, Acrylamide

Sample Source, Preparation, and Analysis Tuber s were analyzed at harvest and after 3, 6, and 9 months of storage to determine the eff icacy of the traits leading to decreased reducing sugars and acrylamide at harvest and throughout storage at normal and cold temperatu res. 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 andJ.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 st andard 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 Bar kholt 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 T echnical 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 analy sis wa s performed by using SAS 9.3.

Data were analyzed using the following linear mixed model:

Y ij =  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 f or 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 bet ween W8 and the control were considered important for trait efficacy. For substantial equivalence, mean values were first compared wi th tolerance intervals calculated f rom 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 commun ication)
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 indirec t 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 th is 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 PPO

dependent 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 Design 1 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 Res istance 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 w ere used to produce the crop. The entire trial was treated with the same agronomic inputs and pesticide and fertilizer ap plications 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 Used 1 CLR (mg/100g)

Asparagine low 11.8 mmol/kg Shepherd et al. 2010 132.1 mmol/kg x mol/10 3 mmol 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 high 2 2968.0 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 74.2J.R. Simplot Company JRS06 Page 149 of 196

Table 10

- 10 . Late Blight Study Information Year Study Site State Site County Material Tested Trial Design 1

2012 Late Blight

- Tubers WA Grant W8, Control RCB, 3 Reps

2013 Late Blight

- Foliar MI Ionia W8, Control
, TX278 2 , Bintje 2

, Nicolet 2 , Golden Sunburst 2 RCB, 4 Reps

2013 Late Blight

- Foliar PA Centre W8, Control
, TX278 2 , Bintje 2

, Nicolet 2 , Golden Sunburst 2 RCB, 4 Reps

2013 Late Blight

- Foliar ID Boundary W8, Control
, TX278 2 , Bintje 2

, Nicolet 2 , Golden Sunburst 2 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

- 12x10 4 sporangia/mL. Details on inoculation at each site are listed in Table 10

- 1 1 . After inoculation, plots were irrigated regularly to induce a humid environment and facilitate spread of disease (Personal Communication with Joe Coom bs, Michigan State University).

Table 10

- 1 1 . 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 e ffect of the p otato l ate b light r esistance g ene on foliar late blight resist a nce 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 perfor med by using SAS 9.3.

Data were analyzed using the following linear mixed model:

Y ijkl =  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 j th site (random effect),  k(j) denotes the random rep effect (within site), (   ) ik denotes the interaction between the i th treatment and random k th 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 random ly 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 min 1 at 15 o C in dry air (30% relative humidity) for four hours prior to ino culation. 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 diame ter) 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 - 5 sporangia per plug. Negative control tubers were inoculated with cold (4 o C) sterile dist illed H 2 O. 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 10 o C 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 wasJ.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:

Y ij =  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 N 2 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 centrifugatio n @ 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 A 474 nm at 8 sec intervals over a 10 min time co urse. The Δ A 474nm ·min - 1 was converted to moles·min - 1 using ε = 3700 M - 1 ·cm - 1 for 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 A 474nm . Δ A 474nm ·min - 1 the n 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 HEP ES

- 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 de salted 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 pro tein 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 Na

phosphate, 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 - 1 protein.J.R. Simplot Company JRS06 Page 152 of 196

10. 4 R eferences

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.

Behbahani
, I

. , Miller
, S

. A

. , and Okeeffe
, D

. H

. (1993). A Comparison of Mushroom Tyrosinase Dopaquinone and Dopachrome Assays Using Diode

- Array Spectrophotometry: Dopachrome Formation vs Ascorbate

- linked Dopaquinone Reduction. Microchemical Journal

. 47: 251

– 260.

Bhaskar
, P

. B

. , Wu , L

. , Busse , J

. S

. , Whi tty , 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.

Brummell
, D

. A

. , Chen
, R

. K

. Y

. , Harris
, J

. C

. , Zhang
, H

. , Hamiaux
, C

. , Kralicek
, A

. V

. , and McKenzie
, M

. J

. (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: 35 19

- 3534.

Davies
, A

. M

. C

. (1977)

. The free amino acids of tubers of potato varieties grown in England and Ireland. Potato Res

. 20: 9

- 21.

Dionex Technical Note 20. Analysis of Carbohydrates by High

- Performance Anion

- Exchange Chromatography with Pulsed Amperometric Detection (HPAEC

- PAD).

FDA

. (2003)

. United States Food and Drug Administration, Center for Food Safety and Applied Nutrition of Plant & Dairy Foods and Beverages. Detection and Quantification of Acrylamide in Foods

. FDA, 2003. Accessed Online : 23 May 2011.

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.

Lilla
, Z

. , Sullivan
, D

. , Ellefson
, W

. , Welton
, K

. , and Crowley
, R

. (2005)

. Determination of “net carbohydrates” using high

- performance anion exchange chromatography. J of AOAC International

. 88: 714

- 719.

Lisinska
, G

. , and 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.

Musser, S

. M

. (2003). Detection and Quantitation of Acrylamide in Foods. U.S. Department of Health and Human Services, Food and Drug Administration. Accessed Online: February 2003.

Schuster
, R

. (1988)

. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivitization and HPLC. J Chromatogr

. 431: 2 71

- 284.J.R. Simplot Company JRS06 Page 153 of 196

Shepherd
, L

. V

. T

. , Bradshaw
, J

. E

. , Dale
, M

. F

. B

. , McNicol
, J

. W

. , Pont , S

. D

. A

. , Mottram , D

. S

. , and Davies
, H

. V

. (2010)

. Variation in acrylamide producing potential in potato: Segregation of the trait in a breeding population. Food Chem

. 123: 568

- 573.

Zrenner , R

. , Shuler
, K

. , and Sonnewald
, U

. (1996)

. Soluble acid invertase determines the hexose

- to

sucrose ratio in cold

- stored potato tubers. Planta 198: 246

- 252.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 ha s 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 incor porated new traits a nd maintains all the benefits of the conventional Russet Burbank parent variet y . 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

- li ke 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
p otato 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 input s 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 Potato es 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 P 2 O 5 - 330 lb K 2 O/

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 s ites 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

Design 1 Rows x

Planted Tubers/ Rep Seed Type Sticky Traps Deployed Regional Specifics

2012 12

- 066

- 102n ID Canyon W8, Control RCB, 3 reps 4x20 Mini

tubers Y

Typical for Southwest Idaho, an important

potato

- growing region in the Northwest that produces about 120 million cwt/year, mainly for the f rench 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 Mini

tubers N

2013 13

- 079

- 106n ID Canyon W8, Control, Nicolet, Bintje, TX278, Golden Sunburst RCB, 4 reps 2x20 NFT Mini

tubers Y

2013 13

- 072

- 112n ID Minidoka W8, Control, Nicolet, Bintje, TX278, Golden Sunburst RCB, 4 reps 6x20 NFT Mini

tubers Y Typical for Southern Idaho, an important potato

growing region that produces about 120 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. The growing season is relatively sho rt because of cooler temperatures.

2012 12

- 066

- 102n WA Grant W8, Control RCB, 3 reps 4x20 Mini

tubers N Typical for Washington, a state in the Northwest

that produces about 85 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. Ideal growing conditions give rise to very high yields per acre. 2013 13

- 072

- 112n WA Grant W8, Control, Nicolet, Bintje, TX278, Golden Sunburst RCB, 4 reps 6x20 NFT Mini

tubers Y

2013 13

- 072

- 112n ID Jerome W8, Control, Nicolet, Bintje, TX278, Golden Sunburst RCB, 4 reps 2x20 NFT Mini

tubers Y Typical for Southern Idaho, an important potato

growing region that produces about 120 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. The growing season is relatively short because o f cooler temperatures.

2013 13

- 072

- 112n WA Adams W8, Control, Nicolet, Bintje, TX278, Golden Sunburst RCB, 4 reps 2x20 NFT Mini

tubers N Typical for Washington, a state in the Northwest

that produces about 85 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. Ideal growing conditions give rise to very high yields per acre.

1 RCB=Randomized Complete Block designJ.R. Simplot Company JRS06 Page 156 of 196

Table 11

- 2 continued. Field Trial Locations

Year USDA Notification # Site

State Site County Material

Tested 1 Trial

Design 1 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 Mini

tubers Y Typical for North Dakota, a Midwest state that

produces about 20 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. The Red River Valley is the 3 rd 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 Mini

tubers N Typical for Minnesota, a Midwest state that

produces about 20 million cwt/year for the f rench fry and chip industry, with harvests in the F all. 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 Mini

tubers N 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 e valuations 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 W 8 and the control for final emergence
, stems per plant, or vine desiccation. For early emergence, W 8 was lower than the control ( 39.5 vs 6 1

. 5 %)
, for plant vigor, W 8 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 w as s horter than the contr ol, t hese characteristics do not indicate i ncreased 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

. T here 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, spe cific 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 inv ertase silencing. None of these differences in size or quality would indicate increased plant pest potential. Characteristic Variety N Mean P

- Value 1 SD 2 90% CI 3 CVR 4

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 46.9J.R. Simplot Company JRS06 Page 158 of 196

Table 11

- 4 . Yield and Grading Characteristics Characteristic Variety N Mean P

- Value 1 SD 2 90% CI 3 CVR 4
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 = str essor 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 observ ations 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 stresso rs. 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 trial s with inoculated strains of late blight confirm ed 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 W 8 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

Differences 1 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 w ere 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 w ere 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 w ere 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

Differences 1 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 w ere 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 observation s, 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 observation s, 1 at Canyon County, ID and 1 at Minidoka County, ID. HS values in W8 w ere within the CVR

. Nutrient Imbalance (NI) 6 1 NI in W8 was higher during 1 observation at Adams County, WI

. NI values in W8 w ere 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 Table s 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 betw een 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 bot h 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 arthropodJ.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 insertion s of the traits do not have an impact on abundance within the potato agro

- ecosystem.

Table 1 1

- 6

. Beneficial Arthropod Abundance in Field Trials
Arthropod Variety N Average Abundance P

- value 1 SD 2 90% 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 0.81 00 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 1 1

- 7

. Pest Arthropod Abundance in Field Trials
Arthropod Variety N Average Abundance P

- value 1 SD 2 90% C onfidence 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 t hese agronomic trials confirmed that W8 had an equivalent phenotype compared to the control when grown a t 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 variet y.

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 compare d 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 coul d 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 expect ed

r esult 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 potent ial 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. I t can be conclude d 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

Thi s summary includes agronomic evaluation s of W8 and the Russet Burbank control at geographical ly 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 summariz ed 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 commerc ially

- 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 Type 1 Seed 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 C SS Farms

- Colorado City, CO W8 Test pSIM1278+pSIM1678 Golden Sunburst Reference N/A Bintje Reference N/A Nicolet Reference N/A TX278 2 Reference N/A Red Thumb 2 Reference 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

T he experiments were established in a randomized complete block design (RCB D ). The RCB D is typical for the evaluation of new potato varieties and event s. In 2012, e ach 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 wi th 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 trial s, greenhouse grown mini

- tubers for W8 and the control were planted

. S eed 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 th e smallest pieces were planted in Grant County, WA

.

For the 2013 field trial evaluations, minitubers were produced using nutrient film technology ( NFT ) mini

tubers for W8 and the control

. This seed was grown at CSS Farms in Colorado City, Colorado. P la nts 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

Timing 1 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=excell ent 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 i n 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. 2 Grading 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 f rench f ried p otatoes Percent of tubers with fry strips which, when compared with the Munsell Color Cha rt for f rench f ried p otatoes , 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 f rench f ried p otatoes Percent of tubers with fry strips which ha s 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 f rench f ried p otatoes.

Fry 0 , Fry 1, Fry 2, Fry 3, Fry 4 Grading Color rating of fried strips based on Munsell Color Chart for f rench f ried p otatoes Percent of tubers with fry strips which, when the predominate color of the darkest side is compared with the Munsell Color Chart for f rench f ried p otatoes , 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 i nternal 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 mad e during the crop senescence stage (about 100

- 140 days after planting).
2 Data on tubers <4oz. w ere 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 yi eld.J.R. Simplot Company JRS06 Page 165 of 196

Early emergence ( at approximately 50% emergence ) and final emergence ( at complete emergence )

w ere 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 w ere evaluated by counting the num b er 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 pr incipal 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

- sys tematically 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 Symptoms 1 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 R olling 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 n ot 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 season s 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 ve ctoring 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. T he 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 s ix

- ounce s produce optimal f rench fries and allow the f rench fry processor to meet most customer specifications at the highest potential recovery.J.R. Simplot Company JRS06 Page 167 of 196

T he 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.

Hi gh 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 w as 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 f rench f ried p otatoes , 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 w hich, when the predominate color of the darkest side is compared with the Munsell Color Chart for f rench f ried p otatoes 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 w as determined by counting the tota l 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 bro wn 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 art hropod abundance data was performed by Simplot using SAS 9.3. All data were analyzed using the following linear mixed model:

Y ijkl =  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 j th site (random effect),  k(j) denotes the random rep effect (within site), (   ) ik denotes the interaction between the i th treatment and random k th 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 p

values 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 rang e (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 plan t 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 we re 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) Monitor ing 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 o f 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 commi ts 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 guide lines 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. Simplo t 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, manyJ.R. Simplot Company JRS06 Page 169 of 196

of the regulated fiel d 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 minim izes 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 h ave not found any potatoes to persist in test p lots 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 consi sted of small plots that are carefully monitored by expert researchers and represent a well

- controlled environment.

11. 4 Disease S usceptibility Assessment

A s 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 P otato L ate B light R esistance G ene ( Rpi

- vnt1 ) which
express es the VNT1 protein

. Th is protein
, with 891 amino acid s , induces resistance to late blight with a broad spectrum against P. infestan s isolates (Foster et al. 2009)

. Also, r esearchers 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 e vidence 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 th ese stud ies was to evaluate the susceptibility of W8 to common foliar, stem, stolon, and tuber diseases in order to determine if W8 has alter ed 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 t ested for resistance to Rhizoctonia solani. Additionally, field grown tubers were tested for resistance to R solani ( black scurf )
, Fusarium sambucinum (dry rot), P hytophthora erythroseptica (pink rot), Pythium ultimum (pythium leak), Pectobacterium carotov ora (soft rot), and Streptomyces scabies (common scab )

. Details of each disease specificity trial are listed in Table 11

- 1 1

.J.R. Simplot Company JRS06 Page 170 of 196

Table 11

- 1 1

. 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 Design 1 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, TX278 2

, Bintje 2 , Nicolet 2

, Golden Sunburst 2 RCB,

4 reps 2X5 NFT

Mini

tubers 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 R hizoctonia solani 13

- 079

- 108n MI Ionia W8, Burbank Control, TX278 2

, Bintje 2 , Nicolet 2

, Golden Sunburst 2 RCB,

4 reps 1X10 NFT

Mini

tubers 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, Pi nk Rot, Pythium Leak, Soft Rot

- Tubers 12

- 066

- 102n WA Grant W8, Burbank Control RCB,

3 reps 4x20 Greenho

use

grown Mini

tubers Typical for Washington, a state in the Northwest that produces about 85 million cwt/year, mainly for the f rench fry industry, with harvests in Fall. Ideal growing conditions give rise to very high yields per acre.

2013 Common Scab 13

- 079

- 108n MI Montca l m W8, Burbank Control, TX278 2

, Bintje 2 , Nicolet 2

, Golden Sunburst 2 RCB,

4 reps 1X5 NFT

Mini

tubers 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

, R hizoctonia solani
, Fusarium sambucinum
, P hytophthora erythroseptica
, Pythium ultimum
, and Pectobacterium
carotovora were not significantly different between W8 and the control ( Table 11

- 1 2 ) . 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 m ean percent coverage of tuber s and mean severity of lesion type

. W8 had less scab than the control as measured by mean percent coverage and lesion s were less severe than the control ( Table 11

- 1 2 ). Altered response to S. scabies was not expected in W8, but would be considered a positiv e 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 con trol, 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 i n susceptibility compared to the control with the exception of a reduction in tuber coverage and severity of common scab cause d 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 link ed to altered diseas e 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 meaning ful 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 en hanced weediness or plant pest potential.J.R. Simplot Company JRS06 Page 172 of 196

Table 1 1

- 1 2

. Disease Susceptibility Assessment Results Pathogen Test and Data Variety Mean Value P

- Value CVR 1

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 Weig ht) Control 57.4

.

. . W8 47.9 0.1715 Common Scab ( Mean Percent Coverage of Tuber ) Control 2.65

. 2.48 4.58 W8 1.9 0 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 applicableJ.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 pr event ed from movement into the plots from adjacent plots inoculated with P

. infestans with weekly fungicide applications that pr event 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 Aug ust and untreated controls reached about 15% foliar infection by September 10

. B . cinerea developed slowly durin g Aug ust 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 h arvested 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 c ounting 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 ye ar 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 Sta te University)

.

Fusarium sambucinum in Tuber s 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 tube rs 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 15 o C in dry air (30% relative humidity) for four hours prior to inocu lation. Tubers were injected with 20 μl of a conidial suspension (approximately 10 6 conidia/ml as determined by a hem o cytometer) 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 atJ.R. Simplot Company JRS06 Page 174 of 196

10 o C 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 evaluat ed 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 Pack ard Co., Houston, TX) for image processing . S canner 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 d etermine the area of the lesion. The lesion length and width (mm) were measured to calculate the dimension and area (mm 2 ) of the lesions, and calibrated to convert image pixels to a unit of measurement using the ruler within the image. The measurement “fil l’ 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 protoco l (Personal Communication with Dr. Willie Kirk, Michiga n 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 H 2 O 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 15 o C in dry ai r (30% relative humidity) for four hours prior to inoculation. Tubers were immersed in a water/oospore/sporangial suspension (approximately 10 4 propagules/ml) for 24 hours; control tubers were immersed in distilled water for a corresponding period. After i noculation, 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 10 o C and 95% hu midity 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 Kir k, Michigan State University)

.

Pectobacterium carotovora in Tubers Samples of W8 and the c ontrol 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 inJ.R. Simplot Company JRS06 Page 175 of 196

distilled H 2 O to remove soil. The tubers were then surface

- sterilized by soaking in 2% sodium hypochlorite solu tion for 4 hours. Tubers were dried in a controlled environment with forced air ventilation at 5950 l min - 1 at 15 o C in dry air (30% relative humidity) for four hours prior to inoculation. The washed, surface

- sterilized tubers were inoculated by a sub

- peri dermal injection of a bacterial suspension of 1x10 10 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 (Mo del I

- 36LLVL, Geneva Scientific, LLC, PO Box 408, Fontana, WI)]. The chambers were set at 10 o C and 95% humidity and the sample tubers were incubated for 40 days until evaluation (Personal Communication with Dr. Willie Kirk, Michigan State University)

.

Tu bers 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 i ncubation, 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 t rial 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 grow n every year in this field and organic matter is added to promote disease development. The t rial 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

- 1 3 (Personal Communication with Joe Coombs, Michigan State University)

.

Table 1 1

- 1 3 . 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 >50J.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:

Y ij =  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 m eaningful 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 bi ologically 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 control s 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 regula tions by submitting the field test reports for each notification ( Table 11

- 1 4 ).J.R. Simplot Company JRS06 Page 177 of 196

Table 11

- 1 4. 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

- 10 1 n 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

T he 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 Russe t Burbank.

Results of t he 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 cont rols. 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 po tential.

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. sca bies

. 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 link ed 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. I t 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 R eferences

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 Potato es. 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 resist ance. 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 characterizati on, compositional assessment, VNT1 protein evaluation, and bioinformatics screening for toxi ns and allergen s. Throughout these studies, the concept of familiarity of potato was considered. F amiliarity 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). Th is 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.

I nformation on W8 has been reviewed to determine the pot ential risk to the environment using the following five criteria: (1) p otential 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) pote ntial 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, inc reased plant vigor, increased tuber set, delayed senescence, or other key agronomic characteristics associated with weediness or survival outside of cultivation. Additionally, all field s ites 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, parti cularly 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 agro nomic assessment show no differences between W8 and its conventional control that would alter survivability, and thus it is unlikely that W8 possesse s increased potential to become a weed of agriculture or to be invasive of natural habitats

.

12.2 Potentia l for gene flow to sexually compatible plants

Gene flow from Russet Burbank Event W8 is nonexistent because the variety is sterile. Generally, t he 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 unli kely; 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 po tatoes”, 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 sin ce 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 19 94; 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 f rom 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 R io 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 i n 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). L ove 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
gene rations, is impossible or at least highly improbable (Love 1994).

T he 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 incompati ble 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 variet y 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

me ters, 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 wou ld 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 produ ction 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 a gronomic performance section show that in multi

- year and multi

site 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

- t arget 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 l ate 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 b ioinformatics assessment showed no new toxins or allergens that coul d 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, t he r eduction of acrylamide levels in food products made with these potato e s 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 t raits, 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 p otato variet y 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 u nlikely 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 submissio n, was assessed by compar ing W8 with the Russet Burbank control variety. F amiliarity of potato was considered d uring evaluation of the data, including agronomic performance, trait efficacy, specificity, compositional assessment, toxicology, and allergenic ity

.

I nformation on W8 has been reviewed to determine the potential risk to the environment using the following five criteria: (1) p otential to become a weed of agriculture or to be invasive of natural habitats, (2) potential for gene flow to sexually com patible plants, (3) potential to become a plant pest, (4) potential impact on non

- target species including humans, and (5) potential impact on biodiversity.

Regulated f ield trials over multiple years with W8 did not provide any evidence for increased plan t 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 spec ies. F ield 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

. T he introduction of W8 would not result in a signif icant expansion of planted acres or a change in the areas where potatoes would normally be grown . T herefore, the potential to impact insects and other nontarget organisms, weed or disease susceptibility, endangered species
, or biodiversity is negligible fr om 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 intr oduction 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

- novel

traits/applicants/directive

- 94

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- tuberosum

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- /eng/1330982063974/1330982 145930 (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, Koe hler 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

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. (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
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. Wild potatoes ( Solanum section Petota : Solanaceae ) of North and Central America. Syst Bot Monogr

. 68: 1

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. (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

Russe t Burbank Event W8 has multiple traits that provide benefits to potato pro cessors, growers and

consumers including late blight resistance, low acrylamide potential, reduced black spot, and lowered reducing sugars. The se benefits were added to Russet Burban k potatoes through tools of modern biotechnology such as gene silencing using po tato genomic DNA and in the case of late blight resistance,
genes were obtained from a wild potato species. Integrating these traits simultaneously through conventional breedin g 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. C ommercially 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 1 3

- 1 . Areas of late blight infect ion 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 19 6

and still incur yield losses of 20% to 70%. In terms of pes ticide 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 fungic ide 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 savi ngs 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 sub ject 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. E vidence suggest s that tuber damage is far more likely to oc cur if the pl ant 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. T he loss of entire crops due to late blight is not unco mmon 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 reducti on 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.” A n 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 resis tant 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 (201 1 ).

The introduction of I nnateTM potatoes with low acrylamide potential would provide potatoes that are largely indistinguishable from existing varieties. The reduction in asparagine and sugars using Innate TM 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 potent ial

.
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 78J.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 a n 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 f rench fries and chips. Potatoes that have been damaged and show b lack spot must be trimmed or could be

re jected 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 f rench fry processing, causing economic loss from trimming or culling potatoes with black spot. Potatoes may develop black

s pots from pressure bruising which result s 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 i ndicates 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 su gar 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 shrink age due to lower respiration r ates.

With introduction of pSIM1278 into Russet Burbank potatoes (Collinge and Clark 2013), we found silencing of the promoters for R1 and Ph L to result in slight reductions in reducing sugars at the time of harvest or after one month of storage. The si lencing cassette contained in pSIM1678 results in silencing of VInv in W8 , which result s in lower levels of reducing sugar s throughout the storage period. V Inv silencing may also allow for lower temperature storage which will reduce yield loss from respir ation 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 h ave low reducing sugars at the time of harvest that remain low throughout their storage life (Driskill et al. 2007). There is also evidence that V Inv 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 byJ.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 . Addition ally, 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 201 2 was 467 mil lion 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 potatoe s, 4% other frozen products, and 1% canned (NPC 2013). Annual per capita potato use in the US in 201 2 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 201 3 fiscal year (Ju ly 201 2

- June 201 3 ) 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 92 6,553 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 . F inally, 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 201 2 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
te sting 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 w ill be filed after experiments are completed under the EUPs. Simplot has initiate d 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
event s 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 201 4 ). 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 acciden tal 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
p otatoes. 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

- p ollination 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 B urbank 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 o f 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) soy beans 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/data

products/adoption

- of

- genetically

- engineered

- crops

- in

- the

- us/recent

- trends

- in

- ge

adoption.aspx#.UnGtr_ns_cA )

. These two crops provide significant quantities of biotechno l o gy

- 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 adoptio n 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 Potato es 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 co mplete 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 d eveloped thatJ.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 effecti ve 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 progr am 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 outline d 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. T he 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 a cceptance 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 preser ved” with respect to the well

controlled 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 identi ty 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 t he 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 req uirements 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

Th is voluntary guideline was adopted because, according to BIO: “asynch ronous 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 giv en 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 shoul d 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 temp eratures, 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.

Knowle dge 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 pr ogram 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. 1 1 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 )

. P etition for Determination of Nonregulated Status for Innate TM 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 G enet

. 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 N ovember 2013).

F ood D rink E urope

. ( 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 F3 44/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/Thor nton.pdf (Accessed 31 May 2011).

USDA ERS

. (201 4 )

. 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

- pulses

outlook/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

1 4

. 0 Conclusions : Determination of Non r egulated 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 Innate TM techn ologies , 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 outcro ssing. 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 e nvironmental 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 acrylamid e. Significant reductions in reducing sugars and acrylamide were observed throughout long term storage

.
Collectively, t he ubiquitous nature of resistance genes like Rpi

- v nt1 and their encoded resistance proteins such as VNT1 in Solanum species, includi ng potato es , lack of toxins or allergens, mode of action not based upon inducing pest toxicity, and extremely low expression levels provides confidence i n the safe use of potato es 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 exp ected 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 t he W8 potatoes are unlikely to pose a plant pest risk and respectfully submit that these plants should not be classified as “ r egulated artic les” 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. a nd Kerwin Bradley, respectively.J.R. Simplot Company JRS06 Page 195 of 196

1 6.0 Statement of G rounds U nfavorable

JR Simplot is not aware of any information indicating that Innate TM 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 222 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

................................ ................................ ................................ ................................ ...... 223 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 Structure s

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 condition s and used as a background control. After one to two months of growth, leaf ma terials were taken from W8 and control plants for genomic DNA isolation.

DNA Isolatio n. 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 o C until ready to proc ess. 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 re suspended in 2 ml extraction bu ffer containing 200 μg RNase A. After incubating the resuspended DNA at 65 0 C 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 Bro mide) and 800 μl of 5% Sarcosyl, it was mi xed with an equal volume of chloroform : isoamyl alcohol (24:1)
, vortex ed 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, a nd 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
μ g/ml RNase) and incubated at 55 o

- 65 o C for 30 min f ollowed 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. C ontrol and event DNA were always extracted with the same method for a ny 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 37 o C. Digested DNA was c oncentrated by ethanol precipitation (40 μl of 3M NaOAc, pH5.3 and 1 ml ethanol) at

- 80 o C 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 Tris

HCl, 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 T aq enzyme and buffer (Fisher BioReagents) according to Roche’s DIG labeling instructi ons. A standard 50 μ l reaction consisted of 5 μl of 10 x 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 ampli fication 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

s lower than control PCR product). The probe was denatured before use by incubating the probe at 100 o C 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 L eft B order and 162

- bp flanking sequence. RB = Right Border like region containing 25

- bp R ight B order and 161

- bp flanking sequence.

Hybridization. The nylon membrane carryin g transferred DNA was prehybridized in 40 ml pre

- warmed DIG Easy Hybridization solution (Roche) at 42 o C 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 prehybr idization buffer with a fresh amount of the same preheated solution, now containing 25

- 50 μl denatured DIG labeled probe, and continuing the incubation at 42 o C, 20

- 25 rpm for about 16 hrs. The hybridization solution could be store at

- 20 o C and reused up t o 3 times. The reused hybridization solution was heated at 68 o C for 10 minutes before use.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 wash ed twice in washing solution I f or 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, 60 0 C) was added immediately. The membrane was washed twice in washing solution II at 60

- 63 o C for 20 min each at 2 5

- 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 blo cked 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 equil ibrated 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 dar k. Depending on the experiment
, multiple exposures w ere 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 det ermine 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 l abeled 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 dig est 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 t he 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. Simi larly, 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 expec ted

– 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 fragmen t 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 Bur bank 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 f rom potato plants have a tendency to alter the migration of higher molecular weight bands.7 of 22

Figure A

- 3

. The primers and PCR fragments to confirm 375 3

- bp EcoRI /Scal fragment of P

- DNA.

Confirmation of a 375 3

- 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 ( I nvitrogen), PC = positive control, WT = untransformed recipient variety (“wild type”), NC = negative control. Event W3 not included in this petition.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 Sunshin e 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/m 2 /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

- 80 o C until ready to process. 10 ml extraction buffer (0.35 M Sorbi tol, 0.1 M Tri s

- 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 resu spended DNA at 65 o C 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 Bro mide) and 800 μl of 5% Sarcosyl, it was mixed with an equal volume of chloroform : isoamyl alcohol (24:1)
, vortex ed 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 al ternative 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
μ g/ml RNase A ) and incubated at 55

- 65 o C for 30 min followed by centrifug ation 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 rins ed 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. C ontrol and event DNA samples were always ext racted 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 t hen depurinated by submersion in 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 / 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 Eco RI 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 vector s pro vided positive controls.9 of 22

Table A

- 1

. Backbone Probes for pSIM1278 and pSIM1678 1

Backbone Probe Size (bp) pSIM1278 Coordinates Forward Primer (5  ́ to 3  ́ ) Reverse Primer (5  ́ to 3  ́ )

1 1,47 4 10,149

- 11,622 ACTAGTTGTGAATAAGTCGCTGTG ATCGGAATCGACTAACAGAACAT 2 1,51 5 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 83 2 18,827

- 19,658 GCTCTAATATAGCGCATTTCAAG GCTTCCAGCCAGCCAACAGCTC 1 Numbering system based on pSIM1278, but backbone sequences identical for pSIM1678.

Figure A

- 4. Probes for backbone testing and PCR primers for G ap A and G ap B 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 Ga p 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 probe s w er e achieved using Hotmaster T aq enzyme and buffer (Fisher BioReagents) according to Roche’s DIG labeling protocol . A standard 5 0 μl reaction consisted of 5 μl of 10X Hotmaster Taq Buffer, 2

- 5 μl of 10 μ M forward primer, 2

- 5 μl of 10 μM reverse primer, 5 μl of DIG labeled dNTP s (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 dNTP s instead of DIG labeled dNTP s 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 100 o C for 5 min, and then quenched on ice fo r 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 42 o C 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 42 o C for abo ut 16 hrs. The probe

- containing hybridization solution was store d (

- 20 o C ) and reused up to 3 times.10 of 22

The reused hybridization solution was heated at 68 o C for 10 minutes before use. The hybridization solution was removed and replaced by 100 ml washi ng solution I (2X SSC/0.1% SDS). The membrane was wash ed 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, 60 0 C) was added immediately . The membrane was washed twice in washing solution II at 60

- 63 o C 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 incubat ed in 1X Blocking solution (Roche) for 0.5

- 3 h our s 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 memb rane 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. T he membrane was wrapped in plastic film and exposed to the Z

- ray film in the dark. Depending on the experiment multiple
exposures w ere taken from 30 sec to 30 min

. F ilms 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 dNTP s, 0.6 μl of 10 μM forward primer, 0.6 μl of 10 μM reverse prime r, 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 follow ed 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 indica tive of (1) junctions between DNA insert border regions and flanking backbone DNA or (2) regions entirely within th e 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 (s ee Figure A

- 5 and Table A

- 2 )

. The primer pair JY718

- JY719 amplified a 58 7

- bp fragment comp rising the junction at the Left Border region and flanking backbone of pSIM1278 , and primers JY 1029

- JY 1030 were used to amplify a 503

- bp backbone fragment flanking t he 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 a nd flanking backbone of pSIM1678, and primers JY719

- Y180 were used to amplify a 307

- bp fragm ent also spanning the Lef t 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 pSIM167811 of 22

Table A

- 2 . Primer sequences and PCR products for detection of b ackbone adjacent to left and right borders of pSIM1278 and pSIM1678

PCR Primers for D etecting B ackbone A djacent to Left Border of pSIM1278

Name Sequence (5 ́ to 3 ́) Location in pSIM1278 Product length (bp) Backbone ( bp )

JY719 GAGCTGTTGGCTGGCTGGAAG 19 ,637

- 1 , 9 657 ( backbone )

587 24 JY718 GTTGGAAATCAATTATCACTGAG 541

- 563 ( AGP promoter )

JY 1029 CCGTTCTTCCGAATAGCATC 19 ,507

- 19 ,526 (backbone)

503 154 JY 1030 CGGGTTATCGGTTCTTAACG 330

- 349 (AGP promoter)

JY749 GAAGCCGACTGCACTATAGCAG 10 ,342

- 10 ,363 ( backbone )

PCR Primers for D etecting B ackbone A djacent 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 )

377 41 JY726 GATCTCAAACAAACATACACAGCG 10,166

- 10,189 ( backbone )

JY915 ACTAGTTGTGAATAAGTCGCTGTG 10,149

- 10,172 ( backbone )

215 215 JY749 GAAGCCGACTGCACTATAGCAG 10 ,342

- 10 ,363 ( backbone )

PCR Primers for D etecting B ackbone A djacent to Left Border of pSIM1 6 78

Name Sequence (5 ́ to 3 ́) Location in pSIM1 6 78 Product length (bp) Backbone ( bp )

Y 179 AAAGTCTGCCGCCTTACAAC 18,481

- 18,500 ( backbone )

405 bp 122 Y 180 GGCGTTCGGTTCGATATTTA 264

- 283 (vnt1 promoter )

JY719 GAGCTGTTGGCTGGCTGGAAG 18,579

- 18,599 ( backbone )

307 bp 24 Y 180 GGCGTTCGGTTCGATATTTA 264

- 283 (vnt1 promoter )

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 F igure 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 G ap A is potato ubi3 terminator ( Garbarino and Belknap , 1995 ) and the G ap B is part of potato ubi7 promoter ( G arbarino 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 G ap A and G ap 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 G a p A and G ap 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 G ap A and G ap 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)

609 Y280 AGTTGTTTATATTTCGGGTGGA 16,592

- 16,613 (backbone)

Y281 GATCCACCTCCACGTAGACGGAG 17,668

- 17,690 (UGP7 promoter)

1,179 Y282 GAAATGCGCTATATTAGAGCATA 18,824

- 18,846 (UGP7 promoter)

Y283 TATCGCGGTCGTCGTCTTTC 17,604

- 17,623 ( ipt gene in backbone)

1,264 Y284 GGTCATGTTCATGATAAACTTG 18,846

- 18,867 (UGP7 promoter)

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 store d 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 resus pended DNA was incubated at 65 o C 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 vol ume of isopropyl alcohol, washed with 70% ethanol, air dried, and dissolved in 400

- 700 μl 1X Tris/EDTA buffer (TE). DNA con centration 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 Eco RV and hybridized with two probes (GBS and AGP) or digested with XbaI and hybridized with t wo 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 concen trated by ethanol precipitation (40 μl of 3M NaOAc, pH5.3 and 1 ml ethanol) at

- 80 o C for 10 min followed by a wash with 70% ethanol. The DNA pellet was dissolved in 20 μl13 of 22

1X TE followed by addition of 2 μl DNA gel loading buffer, which consists of 40% suc rose and 0.35% Orange G (Sigma) in water.

Figure A

- 6 . P robes U sed for the Southern Blot Stability A nalysis

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, Cal ifornia), and then depurin ated 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 1 5 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 equili brated 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 Chap ter 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

. G reenhouse 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. vent urii was used as a positive control for VNT1 expression and is resistant to late blight. W8 express es 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 liqu id N 2 , and stored at

- 80 °C.

Expression Vectors, Primers, Chemicals, and other Reagents

. pMAL expression vectors and restriction enzymes were purchased from New England B ioLabs (NEB) (Ipswich, MA); pCR

- Blunt

- TOPO TA cloning kit and PCR primers from Life Te chnologies (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,14 of 22

reagents, and laboratory supplies were purchased from Sigma

- Aldrich (St. Louis, MI), Fisher Scientific (Pittsburg, PA), or Research Products Inter national (Mt. Prospect, IL). Construction of E. coli Expression Vectors for Full

- length Rpi

- vnt1 and Rpi

- vnt1 Fragments

. The pMAL

c5x and pMAL

- p5x vectors (NEB) were mod ified 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  ́

- GCTGATTTATAACAA AGATCT GCTG

- 3  ́ (BglII restriction site underlined) and reverse primer: 5  ́

- A GAATTC C GATCGAGACC CCGAGGTTGTTGTTATTGTTAT

- 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 restric tion 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 B glII 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 Rpi

vnt1 (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 s trategy 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  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- CTTAACATGC CTGCAG TCACCACAAT

- 3  ́ Rpi

- vnt1 (1

- 891)

- 3’ For 5  ́

- ATTGTGGTGA CTGCAG GCATGTTAAG

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 3  ́ Rpi

- vnt1 (1

- 125) For 5  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- T GAATTC TTA ATTGGATTGTTGAATTTTTGG

- 3  ́ Rpi

- vnt1 (126

- 544) For 5  ́

- A GGTCTCCATCG AGGGAAGG AAG TTCATTTGTTGCCTTAAGAC

- 3  ́

Rev 5  ́

- T GAATTC TTA CCTAGCAACATTGCTAGG

- 3  ́ Rpi

- vnt1 (545

- 891) For 5  ́

- A GGTCTCCATCG AGGGAAGG GTG CGAAGGATTACATTCTAC

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 3  ́ Rpi

- vnt1 (1

- 544) For 5  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- T GAATTC TTA CCTAGCAACATTGCTAGG

- 3  ́ Rpi

- vnt1 (126

- 891) For 5  ́

- A GGTCTCCATCG AGGGAAGG AAG TTCATTTGTTGCCTTAAGAC

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 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 BL2115 of 22

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 l evels of VNT1 were determined using multiple induction temperatures (17°C and 37 °C) and times (2

- 8 hr s . and overnight). Full

- length VNT1 (1

- 891) was maximally expresse d by overnight incubation at 37 °C. VNT1

- LRR (545

- 891) was maximally express ed from 2 hr. incubations at 37 °C. Cell cultures we re 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 contai ning 1 mM EDTA, 500 mM NaCl, 10 % glycerol and Complete Protease Inhibitor Cocktail Tablets (Roche) and l ysozyme (2 mg/ml). The cells were sonicated for 2 min. using a Branson Probe Sonifier (Branson; Danbury, CT). Extrac ts 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 wa s 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

- i nch pots with Sunshine Professional Growing Mix LC1 (Sun Gro Horticulture; Vancouver, Canada). The pl ants 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 prepare d 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 dilut ion). Cells were pelleted by centrifugation and resuspended in 10 mM Tris

- HCl (pH 7.5) buffer containing 10 mM MgCl 2 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 morta r and pestle, and stored at

- 80 o C.

Protein Extraction from Potato Tissues

. A combination of trichloroacetic acid (TCA)/acetone washes together with ph enol 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 2 0% TCA/acetone followed by 20% TCA/H 2 O. 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 R abbits by Anaspec (Fremont, CA) with two animals per antigen. Seve ral 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 collect ed 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 wer e affinity16 of 22

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 r aised 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. F ourteen days following the boost, serum was collected and processed from each rabbit. Characterization of all antisera was done by SDS

PAGE/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 mi n. 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 buf fer 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 fo r 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

- IgG

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. e ach. 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 E SI ionization source was used for peptide separation and analysis. Digested samples of purified VNT1

- LRR were sepa rated by reverse d

- 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% Bu ffer B (Buffer A = 0.1% formic a cid 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, N 2 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 S tudies

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 e xtracted from 1 g of tuber, and root tissue using Plant RNA reagent (Invitrogen, Carlsbad, California), whereas Trizol reagent (Invitrogen) was used to extract RNA from17 of 22

leaves, flowers and stems. The concentration of iso lated RNA w as measured spectrophotometrical ly 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 tota l RNA was denatured in RNA loading dye for 10 min at 65 0 C foll owed 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 bro mide and 2% formaldehyde) . The gel was run at 80

- 85 volts for 2

- 3 hrs an d then photographed using the gel documentation system from Alpha Innotech (Santa Clara, California). The gel was soaked twice in 10 X SSC for 15 min to remove the formaldehyde. RNA was transferred from to a positively charged nylon membrane (Roche, Indiana polis) by capillary blot ting with 10X SSC for 16

- 18 hrs and stabilized by UV cross

- linking (UVP, Upland, California). The RNA

- containing membrane was stored at 4 0 C 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 18 S rRNA. The primers used for PCR amplification and the length s of probes are listed in Table A

- 5

. A typical 50 μl labeling reaction consisted of 5 μl Ho tMaster 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 dH 2 O 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 100 0 C ) 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 42 O C (Amerex Inst ruments). The hybridization solution was replaced by a mix of 40 ml fresh pre

warmed hybridization solution and 25

- 50μl of denatured DIG

- labeled probe, and the membrane was incubated in this mix for 3

- 16 hrs at 42 O C. The hybridization solution was store d at

- 20 0 C and reused. The reused hybridization solution was heated at 68 0 C for 10 minutes before use.18 of 22

Table A

- 5 . Primers and P robes U sed for Northern A nalysis of P otato G enes Probe

name Hybridization

target Primer sequences Probe size (bp) ASN1 Asn1 transcript 5  ́

- GGTTGATGACTGATGTCCCCTTTG

- 3  ́ 5  ́

- TAGTTAGCTCCTTATTGTGAGCTC

- 3  ́ 1115

PPO 1 Ppo5 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 untrans lated 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 o ven 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 60 0 C for 20 min each at 25

- 30 rpm. This was fo llowed by a wash for 20 min with washing solution III (0.1X SSC/ 0.1% SDS) at 65 0 C 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 i ncubated in 1X Blocking solution (Roche, Indianapolis) for 3 0 min

- 3 hrs on a shaker. The blocked membrane was incubated with DIG antibody solution (1:10,000 dilution of Anti

- DIG

alkaline phosphate conjugate with 1X Blocking solution, Roche, Indianapolis) f or 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 w ere 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) w ere collected from W8 and untransformed Russet Burbank plants and used for RNA isolation.

RNA Isolation

. RNA was isolated as above and then treated with D N ase I (Qiagen). Approximately 20 μg RNA was added to 10 μl RDD Buffer (Qiagen), 4.0 μl D N ase 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) accord ing to the manufacturer’s instructions.
cDNA Synthe s is. cDNA and negative controls (No

- RT) were made by combining 16 μl 5 X iScript reverse transcription reaction mix or No

- RT Control Mix (Bio Rad), 3 μ g RNA and DEPC water to 80 μl on ice. Reactions were then incubated at 25 o C for 5 minutes, 42 o C for 30 minutes , and 85 o C for 5 minutes and cDNA concentration was measured with the Qubit ssDNA Assay Kit (Invitrogen).19 of 22

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 1 α and α

- Tubulin
, in addition to primers for Rpi

- v nt 1 ( Table A

- 6 ) . RT

- qPCR reactions included 10 ng of cDNA, 10 μl of 2 X SSO Advanced S YBR 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 6 0 seconds. Reactions were then heated to 95.0°C for 10 seconds and a melt ing curve analysis was performed from 65.0°C to 95.0°C, increasing in i ncrem ents 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 wer e appropriate. Expression was normalized to the endogeno us reference genes.

Table A

- 6 . Primers U sed 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 S equence A nalysis

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 T ris

- 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 ( Eco RI, Hin dIII, Bam HI, Ase I/ Nde I, 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 specific20 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 we re 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.

The rmal asymmetric interlaced (TAIL) PCR. This method was used as an alternative to adapter ligation

mediated PCR. Three series of reactions using AD (Arbitrary Degenerate) and SP (nested sequence

specific) primers were carried out to obtain DNA insert juncti on 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 Taq TM (TAKARA BI O 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 secon d 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 ne gatively 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 sequ ences 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, biot inylated 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.

Bioinformatic s analysis

. Illumina data was processed by splitting samples by barcode a nd trimming for quality. The reads were aligned with CLC Genomics Workbench to Sanger sequenced PCR products to21 of 22

confirm support for novel junctions in the candidate transgenic line, compared with other transgenic lines prepared at the same time. Independen t 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

.
G arbarino JE, Oosumi T , and Belk nap 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, Ni ko la o s Georgelis, Roshani Shakya, Kelsey Wood , Alexi Balmuth and Jingsong Ye 3/28/2014A 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 ReceptorA Detection Method for the Solanum venturii R

- Protein VNT1, J.R. Simplot Co. Page 3

IN TRODUCTION

Plants utilize a two

- part innate immune response to detect and combat in vading 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 (PAMP s ) that bind trans

- membrane pattern recognition receptors (PRRs) at the cell surface (Jones and Dangl, 2006 ; Monaghan and Zipfel, 2012)

. Pathogen recognition initiate s mitogen

- activated protein kinase (MAPK) signaling cascades that mediate physiological changes such as calcium influx, oxidative burst , increase d phytoalexin production , and other immune respon ses (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, s uccessful pathogens overcome PTI by secret ing effector proteins that suppress the PAMP

triggered immune response (Jones and Dangl, 200 6)

. A secondary layer of defense relies upon recognition of pathogen effector proteins either directly or indirectly by r e s istance p rotein receptors (R

- proteins)

. R

protein mediated immunity , known as Effector Triggered I mmunity (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. infest ans 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 ra pid evolution (Haas et al., 2009)

. The effectors secreted by the pathogen are recognized by potato R

- proteins in resistant plants. Th e 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 regula ted (Spoel and Dong, 2012) . R

- protein expression levels are extremely low and an auto

- inhibitory mecha nism blocks signaling except in the presence of pathogen effectors (Ayliffe et al., 1999; Tan et al., 2 007) . The p otato genome encodes for at least 430 R genes, many of these share > 8 0 % sequence identi t y (Jupe et al., 2012)

. R genes are mainly found in gene cluster s 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), h a ve demonstrated a mechanism for regulating R

- protein activity (Mo ffett 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 initiat ion 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 t he 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 processingA 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 e vidence for safe

use (Bushey, 2014)

. VNT1 is a n 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 Innate TM 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 Innate TM potato lines and demonstrate that the VNT1 pr otein 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 Healthca re Life Sciences (Piscataway, NJ); and Amicon Centrifugal Filter Unit s 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 int roduce a BsaI restriction cut site at the multiple cloning site (Fig. 1 A ). A BsaI

- containing PCR fragment with 5  ́

- BglII and 3  ́

- EcoRI restriction sites was amplified from the pMAL vector template using forward primer: 5  ́

- GCTGATTTATAACAA AGATCT GCTG

- 3  ́ (BglII restriction site underlined) and reverse primer: 5  ́

- A GAATTC C GATCGAGACC CCGAGGTTGTTGTTATTGTTAT

- 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 intro duced 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 ligat ion into the pMAL

- c5x and pMAL

- p5 x 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). T he 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 ligat ion into the pMAL

- BsaI vectors using BsaI and EcoRI restriction enzymes. Full

- length Rpi

- vnt1 contains an internal BsaI cut site and was cloned usin g a 3

- piece ligation strategy (Fig. 1 D 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 protein s. 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 s upplemented with ampicillin (100 μg/ml) to an O.D. 600 = 0.5. VNT1 expression was induced by adding 1 mM i sopropyl

- 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 m aximally expressed by overnightA Detection Method for the Solanum venturii R

- Protein VNT1, J.R. Simplot Co. Page 5

Figure 1. pMAL Vector M odification 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 Bs aI, 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  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- CTTAACATGC CTGCAG TCACCACAAT

- 3  ́ Rpi

- vnt1 (1

- 891)

- 3’ For 5  ́

- ATTGTGGTGA CTGCAG GCATGTTAAG

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 3  ́ Rpi

- vnt1 (1

- 125) For 5  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- T GAATTC TTA ATTGGATTGTTGAATTTTTGG

- 3  ́ Rpi

- vnt1 (126

- 544) For 5  ́

- A GGTCTCCATCG AGGGAAGG AAG TTCATTTGTTGCCTTAAGAC

- 3  ́

Rev 5  ́

- T GAATTC TTA CCTAGCAACATTGCTAGG

- 3  ́ Rpi

- vnt1 (545

- 891) For 5  ́

- A GGTCTCCATCG AGGGAAGG GTG CGAAGGATTACATTCTAC

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 3  ́ Rpi

- vnt1 (1

- 544) For 5  ́

- A GGTCTCCATCG AGGGAAGG ATG AATTATTGTGTTTACAAGACT

- 3  ́

Rev 5  ́

- T GAATTC TTA CCTAGCAACATTGCTAGG

- 3  ́ Rpi

- vnt1 (126

- 891) For 5  ́

- A GGTCTCCATCG AGGGAAGG AAG TTCATTTGTTGCCTTAAGAC

- 3  ́

Rev 5  ́

- T GAATTC TTA TAGTACCTGTGATATTCTCA

- 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. incubation s 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

- V NT 1 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). S eedlings were transferred to 4

- inc h 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 MgCl 2 and washed once. C ulture s 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 usi ng a syringe. Leaves were harvested 2

- 3 days post

infiltration, ground in liquid nitrogen using a mortar and pestle, and stored at

- 80 o C.

Protein I dentification of MBP

- VNT1 (LRR) and GroL from E. coli

— An Agilent 1200 HPLC coupled to an Agilent 6320 Ion

- T rap M ass S pectrometer 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 P eptide 3.6 μm

- C 18

- 2 50 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 35 0  C, N 2 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 Antibod ies

— Polyclonal antibodies were raised in New Zealand W hite rabbits by Anaspec (Fremont, CA) w ith 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. P eptides were conjugated to keyhole limpet hemocyanin:bovine serum albumin ( 7.0:1.5 (w/w) ) before injection

. A standard 70

- day protocol was ext ended and final bleeds were collected 100 days post

- immunization ( initial + 6 boosts ; peptide antibodies 5361

- 5366). A 28

- day “ speedy ” protocol was followed f or antibodies 5467

- 5472. F inal bleed s were collected 28 post

- immunization (initial + 4 boosts ) . Th e 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 antibod y s against VNT1 used p urified V NT1

- LRR as antigen. A ntibodies were raised in New Zealand White rabbits (specific

pathogen free) by Pierce Biotechnology (Rockland, IL) using the company’s s tandard 90

- day protocol, with two animals per antigen. V NT1

- LRR was run on an SDS

- polyacrylamide gel electrophoresis (PA GE) 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 d ays following the boost
, serum was collected and processed from each rabbit. Characterization of all antisera was done by SDS

PAGE /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

- denatur ing 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

- Al drich). S ample s 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 o C for 5 min. The protein samples were aliquoted and stored at

- 20 o C. A lternatively , 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 o C for 10 min

. and centr ifuged 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 o C for 5 min. The protein samples were aliquoted and stored at

- 20 o C. A TCA/acetone and phenol extr action was also performed. T issue materials were washed several times in acetone followed by additional washes in 10% TCA/acetone and 10% TCA/H 2 O. 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 ex tracted 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 o C 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

— P roteins 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 S DS

- pol y acrylamide 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 . F or anti

- MBP a 1:10,000 (v/v) , and for anti

- VNT1 a 1:1000 (v/v) dilution w as used . The membrane was incubated with primary antibody for 30 min. an d 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

. SuperSig nal ® West F emto Maximum Substrate (Thermo Scientific) was added and after 5 min. b lots 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 V NT 1 protein fragments tested for expression in E. coli were full

length 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.m su.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

— H igh expression levels of VNT1 (1

- 891) were observed with 6 hr

. to overnight ind uction s at 37 °C. The full

- length V NT 1 (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 ). S eve ral 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 ea rly void volumes indicating that they formed a complex in solution while the small molecular weight bands were successfully separated away. A ttempts t o separate the 80 and 60 kDa proteins using ion

- exchange chromatography, hydrophobic interacting chromatog raphy, 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 b y 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 t he sensitivity and specificity of the antibody

. FHSSKLPFGVWESKIL had the highest specificity of all antibodies tested and was used in further analyses for detecti o n of V NT 1 in Innate TM lines.

Limit of Detection and Quantitation for VNT 1

— 9 pg was the lowest amount of V NT1

- 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 toA Detection Method for the Solanum venturii R

- Protein VNT1, J.R. Simplot Co. Page 9

one copy V NT 1

- LRR. The concentration of VNT1

- LRR was used to estimate the concentration of f ull

length 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 6 0 ppb in leaf, 30 ppb i n tuber, 2 0 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 Innate TM Line s

— VNT1 was not detected in crude protein extracts from Innate TM W8 tissues using the FHSSSKLPFGVWESKIL antibody (Fig. 8 ) . The W8 line is resist ant to l ate b light in leaf inoculation studies (Fig. 8 ) , however
, based on the measured LOQ for VNT1 the amount of VNT1 protein expressed in Innat e TM lines is low er 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 detect s low picogram levels of purified VNT1 protein , but was

u nsuccessful at detect ing VNT1 protein expressed in Simplot Innate TM transformed potato line W8

. Results of q uantitative Western Blot analys e s 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 th at the late blight resistance of Innate TM lines is independent of high R

- protein expression. Innate TM lines appear otherwise phenotypically equivalent to u ntransformed 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. Lan e 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 MATE RIALS 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

a mylose (to p) and gel filtration column s (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 a mylose column (top). L ane 1: MW marker; Lane 2: empty vector; Lane 3: f raction from a mylose 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 suc cessfully removed. A 60 kDa protein, GroL ( E. coli chaperone) co

- elutes with the VNT1

- LRR domain . B oth 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. Colle cted 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

Fig ure 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 demonstrat e the high sensitivity of the FHSSSKLPFGVWESKIL antibody for VNT1. Lane 1: VNT1

- LRR, Lane 2: full

length 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

- 89 1). 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

Fig ure 6. L imit 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 b etween 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 Innate TM W8 Tissues

. Western Blot analysis using antibody FHSSSKLPFGVWESKIL demonstrate s 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

w ere spiked into root extracts

. LOQ values are shown in ppb

. A d etached l eaf a ssay 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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## Appendix C : Allergen and Toxin Assessment for Russet Burbank W8

Bioinformatics E valuation of the Ins ert s and Flanking Sequences

AUTHOR s

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 14J.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

................................ ................................ ................................ ................................ ...................... 1 2

ORF detection

................................ ................................ ................................ ................................ ......... 12

Database and software resources

................................ ................................ ................................ .......... 12 Allergenicity database searches

................................ ................................ ................................ .............. 12

Toxin database searches

................................ ................................ ................................ ......................... 13 Native gene disruption

................................ ................................ ................................ ............................ 13

Refere nces

................................ ................................ ................................ ................................ .................. 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 introduc e a n unexpected toxin or al lergen through expression of a novel open reading frame (ORF). This report describes our analysis of possible native gene disruption, toxicity, and allergen icity for the Russet Burbank W8 event using the standard bioinformatics techniques summarized in Tab le D

- 1.

Disruption of native genes and expression of unexpected toxins or allergens are all highly unlikely events, particularly in Innate TM 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 s table 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 double

stranded hairpins that are processed by the RNA interference pathway into small interfering RNAs, preventing them from being translated. The pSIM1678 plasmid also cont ains 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 allerge nicity and toxicity pot ential 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

ana lyses. 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 in dication of gene disruption.J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 4



### Stop

- to

- stop ORF I dentification 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 insert ion locus and
108 with the pSIM1678 insertion locus when a start codon was not required. These ORFs were converted in to 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 ORFJ.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 5



### A llergenicity E valuation
A llergenicity 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 kn own 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 pSIM127 8 insert and one ORF in our pSIM1678 insert because the sequence is derived from an open reading frame contained within the GBS

S promoter , which is native to the potato and coopted to drive expression of the silencing

constructs cont ained in the insert. It is highly unlikely that this polypeptide is responsible for the allergenicity of the endoch itinase protein given that it represents only one of 319 distinct 8

- mers present in the full

- length, allergenic endochitinase protein

. As further confirmation, we v erified 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

s equence is present in both potato and numerous human proteins. Collectively
, these data suggest the peptide identified is a false positive an d 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 o nly found in tomatoes, but is also found in other Solanum species, such as potatoes. Th e presence of this partial ORF was by design as the potato vacuol ar invertase ( VInv ) gene was the target of one of our silencing cassettes. Thus, the VInv silencing cass ette 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
invertas e 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 M inor 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 simil arity 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. Th e algorithm only identified one poten tial 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

M inor allergen beta

fructofuranosidase 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 QSRHRJ.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 toxi n

- 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 homolo gy between our ORFs and known protein toxins

. We took advantage of two repositories of annotated toxin s for this analysis. The first database (mvirDB) consists of known bacterial toxins , virulence factors, and antibiotic resistance genes, which is a public ly

available repository maintained as a combination of at least eight independent database sources ( Zhou
, 200 6)

. A second BLAST

- compatible database was created to supplement the bacterial toxin database by

extr acting all eukaryotic proteins annotated as to xins 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 ar e 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 prot eins with significant homology when the toxin databases were queried with the ORF sequences (Table D

- 5) . Thus, no toxin

- related safety conce rns were identified for any of the Innate TM potato event s

.
Native gene disruptio n
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 redu ndant 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 techni ques (The Potato Genome Sequencing Con sortium). 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 organiz ation 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 pl ant with up to three fun ctional 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 change s in phenotype. In the ev ent that a native gene is disrupted by an Innate TM 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.

I n 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_sea rches.shtml )

.
Neither the blastn nor blastx search es 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 e xpressed transcript disrupted by the pSIM1278 integration

event

. Similarly, v isu al 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 2J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 9

p SIM16 78 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 a nd 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 DM 1

- 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 c andidates for native gene disruption. Collectively, we found no evidence of native gene disruptions associa ted with the insertion of pSIM16 78 in W8 for further consideration

. Figure D

- 3

. W8 pSIM1678 insertion on chromosome 10

The sequences for the left (L1

- L6) and right junction s (R1

- R6) were analyzed as part of the analysis described above and are summarized in more detail in Table s D

- 5 and D

- 6 . As shown, no potential toxins or allergens were identified associated with the ORFs specifically covering the junction regions

.J.R. Simplot Company Bioinformatics Evaluation of Allergenicity and Toxicity for Event W8 10

Table D

- 5

. Summary of toxi city and allergenicity of pSIM12 78 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 *EPISNRDSRFVSYSI K LIFGQFISVNLIFDWELREVIWSHVVPDFSS* 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 co mprehensive analysis on the loci including each of the inserts in Russet Burbank W 8 . 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 ev idence of allergens or toxins associated with inserts that pose a safety concern. If anything, our Russet Burbank W 8 event is predicted to have reduced allergen potential due to silencing of the vacuolar invertase protein, which is listed as a mild allerge n 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 , a nd (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 (OR Fs) 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 id entify 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 t he i nsert 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

. Th e results were converted int o FASTA

- formatted file s 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 B LAST

compatible databases were generated and/or downloaded to support the toxin analysis described herein. The most recent NCBI non

- redundant (nr) protein data base (August 26, 2013 post date ) was downloaded using the update_blastdb.pl script provided with the B LAST application suite. Searches against this database are comprehensive, but non

- specific and produce data that can be challenging to associate with puta tive 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
resis tance genes (Zhou et al., 2006) that was downloaded from the Law rence 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 sou rces, including Tox

- Prot, SCORPION, PRINTS, VFDB, TVFac, ARGO, and VIDA. A second eukaryote

- specific (t axid: 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 U niprot technical support at the SIB Swiss Institute of Bioinformatics:

http://www.uniprot.org/uniprot/ ?query=taxonomy%3a 2759 +keyword%3atoxin&force=yes&format=fas ta&include=yes

Thi s toxin dataset was converted into a B LAST

- compatible database ( uniprot_toxin) using the makeblastdb.exe utility provided in the B LAST 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 t he insert and flanking regions w ere 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 theJ.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 c omprehensive set of ORFs predicte d by the ORF Finder application was used as input for B LAST (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 biological ly significan t (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 potentia l homology between the ORFs and the sequences in the toxin

- specific databases, we analyzed our BLAST searches using a higher E

value threshold of 1.0. However, since the E

- value calculation i s 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 10 1 1 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 .0 1

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, t he sequences identified as the left and right flanking regions were used as input for a BLAST ( blastn ) search agai nst the NCBI potato genome to identify the likely integration site and any genes associated with th e 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 databas e (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 : S 165

- 186.

The Potato Genome Sequencing Consorti um, “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 microb ial database of protein toxins, virulence factors and antibiotic resistance genes for bio

- defence applications ” , Nucleic Acid Research, 200 7 , 35:D391

- D394.

B LAST help : http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=ProgSelectionGu ideDietary 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 2 9Dietary 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 Tomat oes with ToMV Resistance Gene

................................ ................................ ....... 21
Conclusions

................................ ................................ ................................ ................................ ................. 21

Literature Cited

................................ ................................ ................................ ................................ ........... 22Dietary 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 T o MV Resistance Gene 26



## List of Figures

Figure 1. Rpi

- vnt1.1 Gene Sequence (FJ4230441.1) 7Dietary 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 geneDietary 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 Rpi

vnt1, 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 provid es 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 mid

twent ieth 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 design ated 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 progr ams ( 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 breed ing , 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, Rpi

vnt1 .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 p hysical 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

- v nt1.1 were shown to be resistant to P. infestans . Of 11 P. infestans isolates tested
, only isolate EC1 from Ecuador was able to overcome Rpi

- v nt1.1 and cause disease on the inoculated plants (Pel 2010). However, Rpi

- vnt1 .1 remains of major importance since it brings resi stance 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

- v nt1.1, Rpi

- vnt1 .2 and Rpi

- vnt1 .3 ) which differedDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 6

by only a few nucleotid es 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

- v nt1.1 (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 Solan um species with 5 genotypes each was screened. Rpi

- v nt1 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
po tatoes (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 (effec tor 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 si gnal 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 Pr oteins

– 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
p lant 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 Solanac eae family, which includes eggplant ( Solanum melongena L.), pepper ( Capsicum annum L.), and tomato ( Solanum lycopersicum L.).

Th e 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 R

proteins 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 g enes 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 ge ne 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 orDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 7

amino acid sequences with similarity to the Rpi

- vnt1.1 gen e 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

- rich

repeat domain (NB

- LRR) (Bakker et al. 2010; Jupe et al. 2012), additional BLAST analyses were conducted to identify edible plant species that contain disease resistan ce 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 Voss en,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="MNYCVYKTWAVDSYFPFLILTFRKKKFNEKLKEMA EILLTAVIN

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 actcgagtc a ttatgatgat gtctcacgac caaatcaaat
61 caaagttaaa taaatatcga accgaacgcc cactctgtat gagtatggca aaagattttg
121 agagaatcaa gttgcataaa agcctaattt tcatggaaca tacaaattga gtctcataat
181 agcccaaact cacagccatg aacccaaatt gggtaaagtt ttgcaagacg ttca tcaaac
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 tacgcg tagt 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 ggtgatg tgg aggatctatt agatgagttt cttccaaaaa ttcaacaatc
1081 caataagttc atttgttgcc ttaagacggt ttcttttgcc gatgagtttg ctatggagat
1141 tgagaagata aaaagaagag ttgctgatat tgaccgtgta aggacaactt acagcatcac
1201 agatacaagt aacaataatg atgattgcat tccattggac cg gagaagat tgttccttca
1261 tgctgatgaa acagaggtca tcggtctgga agatgacttc aatacactac aagccaaatt
1321 acttgatcat gatttgcctt atggagttgt ttcaatagtt ggcatgcccg gtttgggaaa
1381 aacaactctt gccaagaaac tttataggca tgtctgtcat caatttgagt gttcgggact

144 1 ggtctatgtt tcacaacagc caagggcggg agaaatctta catgacatag ccaaacaagt
1501 tggactgacg gaagaggaaa ggaaagaaaa cttggagaac aacctacgat cactcttgaa
1561 aataaaaagg tatgttattc tcttagatga catttgggat gttgaaattt gggatgatct
1621 aaaacttgtc cttcctgaat gtga ttcaaa 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 gatttgg ttt caagaaactt gattcaagtt gccaaaagga catatgatgg
2221 aagaatttca agttgtcgca tacatgactt gttacatagt ttgtgtgtgg acttggctaa
2281 ggaaagtaac ttctttcaca cggagcacaa tgcatttggt gatcctagca atgttgctagDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 10 2341 ggtgcgaagg attacattct actctgatga taatgccatg aa tgagttct tccatttaaa
2401 tcctaagcct atgaagcttc gttcactttt ctgtttcaca aaagaccgtt gcatattttc
2461 tcaaatggct catcttaact tcaaattatt gcaagtgttg gttgtagtca tgtctcaaaa
2521 gggttatcag catgttactt tccccaaaaa aattgggaac atgagttgcc tacgttatgt

258 1 gcgattggag ggggcaatta gagtaaaatt gccaaatagt attgtcaagc tcaaatgtct
2641 agagaccctg gatatatttc atagctctag taaacttcct tttggtgttt gggagtctaa
2701 aatattgaga catctttgtt acacagaaga atgttactgt gtctcttttg caagtccatt
2761 ttgccgaatc atgcctccta ataa tctaca 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 tcacagg tac tataaataat tatttacgtt taatatccat gattttttta
3421 aatttgtatt tagttcatca actaaatatt ccatgtctaa taaattgcag ggatgccttt
3481 gaaaatgatt ctgtgttgga gagaatcttc tgatgcctgt tggtattata atactaataa
3541 taagagaaaa agtttgatta ctgtttcaag ttaattgctt gt gatttgta aaaacaaatt
3601 acttttatat ttctctttgt tttattttat gtttatttat ctttaattaa tggagtaata
3661 aaataaaaat cttattttca atagaaaaaa gtagacctta tttgtggtgc atgtatggta
3721 tctttttgaa atttttgata tatttgctct ttgattcgaa tttcttgctt atatgatgat

378 1 ttgcataaat ataaaatatt atacaaatac ctatgggttg gaaaatatag aaatatgcca
3841 atcaaatgta tacaaaaatc attaatagat agaatcgtaa aagatataca aatgagaaat
3901 gcttgactaa gaagcttcgt gcaacctctc acactgagca caatgcattt ggtgatctcg
3961 gcactattgc tgttacttgt aaga ctacgt 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 cttttcaaagDietary 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 mega blast 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 Rpi

vnt1.1 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, t he “nr” database option was selected for databases against which to search. Due to constraints of computational cost, the “nr” database collection is no longer non

redundant; however, this was not a nticipated 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 includi ng EST (expressed sequence tags), STS (sequence

tagged 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 t he 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 Tm

2(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,

BlastnDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 13

Solanum lycopersicum cultivar LA0656 disease susceptible protein tm

2 (tm

- 2) gene, partial cds > gb[FJ817599.1] Solanum lycopersicum cultivar Peto 95

- 43 LA3528 disease susceptible protein tm

2(tm

- 2) gene, partial cds Solanum venturii late blight resistance ( Rpi

- vnt1.2 ) gene,

c omplete 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 (Tm

2nv) gene, 5’ UTR and partial cds Tomato HM027885.1 90%, 90% 6e

- 08,

6e

- 08 Discontiguous,

Blastn

S olanum 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, BlastnDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 14

Lycopersico n esculentum ToMV

resistance locus, Tm

- 2 resistance allele, complete cds Tomato AF536200.1 86%, 85%, 85% 0.0,

0.0,
0.0 Megablast
,

Discontiguous, Blastn

Lycopersicon esculentum ToMV

resistance locus, Tm

2^2 resistance allel e, complete cds Tomato AF536201.1 86%, 85%, 85% 0.0,

0.0,
0.0 Megablast
,

Discontiguous, Blastn

Lycopersicon esculentum ToMV

resistance 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 (Tm

2nv ) 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 BlastnDietary 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 T m

2 ( T m

- 2) gene, partial cds > gb [FJ817607.1] Solanum lycopersicum cultivar Peto 95

- 43 LA3528 disease susceptible protein T m

2 ( T m

- 2) gene, partial cds Tomato FJ817601.1 76%, 76% 6e

- 141,

6e

- 141 Discontiguous,

BlastnDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 16

Solanum lycopersicum cultivar Anahu LA0655 disease susceptible protein T m

- 2( T m

- 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 Tm

2^2 (Tm

- 2) gene, Tm

2^2 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 InternationalDietary 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

- values 1 , 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 wheth er 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 ove rall homology despite the maximum identity value of 77%.
Protein Homology A protein BLAST search was perfor med 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 tomat o and potato, as indicated by very low e

- values and relatively high maximum identity values.

Table 2

. Protein Sequences with Significant Homology to Rpi

- v nt1.1 (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 Sol anum 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 oth er entries in the general BLAST output, which contained many hits, most of which were not edible Sola num. 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 i s 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, di s contiguous megablast, and blastn searches were performed for Rpi

- vnt1 .1 (F J423044.1 ) 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 GenBan k. A search of the database for genes from eggplant or aubergine u sing the three search methods did not reveal any homolog ous genes to Rpi

- vnt1.1 (FJ423044.1). T he search performed for genes from Dutch eggplant returned a gene for an N B S

- coding re s istance protein

- like protein RGA3 () with an e

- value of 2e

- 173 in the mega blast search
, and 0.0 in the discontiguous megablast and blastn search es. Maximum identity value s for these gene s
were 88% in all three searches

. Finally, t he search for homologous genes from pepper returned one entry for a homol o gous 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 m embers, 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 pimpinellifol ium (curra nt 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 ( f lax ). No signif icant 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 fr om flax

. Specific BLAST searches against the genomes of th e 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 homolo gous sequences w ere returned in these searches, overall results do not provide evidenceDietary 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 rela tively favorable maximum identity values, the overall homology between Rpi

- vnt1.1 ( FJ423044.1) and genes within these three commonly consumed organisms is quite low.



## T o MV 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 T o MV resistance proteins were reported in food and therefore could be used to ass ess 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

m osaic 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 cita tions. 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 expressio n levels of resistance protein were found. T he 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 fo r 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 se arch did not yield any papers providing information about the lev els of ToMV resistance protein s in tomatoes or in the diets. Some publications do point to the similarity between T o MV and Rpi

- V NT 1 , confirming the findings of the BLAST search results prese nted above.Dietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 20



## Tomato Varieties with T o MV Resistance Gene

Database Search In addition to searching for literature that would provide levels of the T o MV resistance protein, a search was conducted of potato and tomato varieties that contain the virus resist ance. Databases containing tomato or potato varieties and details on pathogen resistance of each vari ety were located using the Google search engine. Both tomato and potato variety databases were identified; however, potato varieties did not appear to di splay the trait of T o MV resistance, so the potato variety search was abandoned. Tomato variety databases and listings which contained information on T o MV resistance of each particular variety included a publication by North Carolina State University (Janua ry 2010 revision, http://ipm.ncsu.edu/Production_Guides/Tomatoes/AG

- 405Web.pdf ), a table compiled by the Cornell Department of Plant Pathology (dated April 201 2, http://vegeta blem donline.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 T o MV resistant tomato varieties for pla nting, 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 T o MV resistant tomato were found in the databases and publications that were searched. Of the 78 tomato varieties identified as T o MV resistant tomato varieties, all are considered f resh market tomatoes and fifty five of them are available on the various retail web sites (see Table 3)

. Many of the T o M V resistant tomato varieties are recommended by the university agriculture extension offices to commerci al producers and individual gardeners as being suitable varieties to grow in their areas. The Seminis’s Cheong Gang tomato rootstock is T o MV resistant

a nd recommended for grafting purposes for fresh market growers.

The U.S. tomato processi ng industry, com prised primarily of tomato pastes, sauces, and canned tomato products, is distinctly separate from the fresh

- market industry. Nearly 100 percent of pro cessing tomatoes are produced and sold under contract between growers and processing firms. Approximatel y 34 percent of the world’s processed tomatoes are grown using Heinz proprietary tomato seeds. None of these processed varieties appear to have been br ed for T o MV resistance as the breeding focus has beenDietary 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 25 3, has been bred for T o MV resistance but has only recently been introduced commercially and is not considered part of the 2012 top 50 Calif ornia processed tomato list.



### Consumption of Tomatoes with T o MV 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 ma jor 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 Unite d 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 i ncluding 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 tom atoes 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 (T o MV) resistanc e gene s. In addition, the V NT1 protein (ACJ66594.1 ) is greater than 75% homologous to Tm

- 2 To MV proteins from Solanum lycopers icum (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 h uman 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 gene s 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 histor y of dietary exposure to the protein product of the Rpi

- vnt1.1 gene

. Several searches to determine the level s of T o MV resistance proteins in food and existing dietary exposure to tomato varieties with the T o MV resistance gene were carried out. A literature search did not yield any papers providing infor mation about the levels of ToMV resistance protein s in toma toes or in the diets . However, T o MV 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 T o MV resistant tomatoes in their diet. Since the Rpi

vnt1.1 gene is substantially similar to the T o MV resistance gene (approximately 90% homologous), it isDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 22

reasonable to conclude that US consumers have di etary exposure to proteins that a re 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 ( Solanu m 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 Solanum phureja species, a potato species consisting of many varieties which are currently con sumed 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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- 25 2000: 38

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, Puddephat I ( 1999 ) Deployment of disease resistance genes by plant transformation: a “mix and match” approach. Trends Plant Sci. 4:71

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- Hampton M, Vallad GE, Dittmar PJ, Webb SE, Smith HA, Olson SM (2013) Chapter 12. Tomato Production. Document HS739, Horticultural Sciences Department, University of Florida /IFAS Extension, August 2013. http://edis.ifas.ufl.edu/pdffiles/cv/cv13700.pdf (Accessed 8 Oct 2013).

Sliwka J, Jakuczun H, Lebecka R, Marczewski W, Gebhardt C and Zimnoch

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, M., and Zimnoch

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- phu1 during incompatible interactions with Phytophthora infestans

. Plant Pathology 62: 1072

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- like protein encoded by the rice disease resistance gene, Xa21. Science, Dec 15;270(5243):1804

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Tan MY, Hutten RC, C elis C, Park T

- H, Niks RE, Visser RG and van Eck HJ ( 2008 ) The RPi

- mcd1 locus from Solanum microdontum involved in resistance to Phytophthora infestans , causing a delay in infection, maps on potato chromosome 4 in a cluster of NBS

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- bearing Solanum species. BMC Plant Biol. 18;11:116.

Wastie R ( 1991 ) Breeding for resistance. In Phytophthora infestans , the Cause of Late Blight of Potato, ed. D Ingram, P Williams, pp. 193

– 224. London: Acad emic

Weber H and Pfitzner AJ ( 1998 ) Tm

- 2(2) Resistance in Tomato Requires Recognition of the Carboxy Terminus of the Movement Protein of Tomato Mosaic Virus. Mol Plant Microbe Interact . June; 11(6):
498

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- 8781.Dietary Exposure to Rpi

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U.S. Department of Agriculture, Economic Research Service (ERS), 2012. http://www.ers.usda.gov/topics/crops/vegetables

- pulses/tomatoes.aspx ; original accessed date 2/21/2013

U.S. Department of Agriculture, Economic Research Service (ERS), 2008. 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 T o MV Resistance Gene Variety ( A Not readily available on a retail see d 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

- grape

pelleted.aspx

Apero F1 Cornell

database cherry

plum http://www.simplyseedsandplants.co.uk/Vegetable

- Plants/Tomato

Apero_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 F1 A Cornell

database https://www.paramountseeds.com/seed

- varieties

- t93/greenhouse

seeds/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 golden

orange 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/greenhouse

seeds/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

Carnival B Cornell

database

Celebrity Cornell

database Hybrid

round red http://www.siegers.com/shop/kind.asp?kind_id=BBB

Clermon B 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.aspxDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 27

Variety ( A Not readily available on a retail see d or plant site ; B Rootstock ) Reference Description Retail Site Link Emperador F1 B 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 garden B Cornell

database

Growdena B Cornell

database

Halcon Cornell

database large round

red http://www.harrismoran.com/products/tomato/pdf/table

- fresh

- det.pdf

Hermosa B Cornell

database

HM 7864 F1 B Cornell

database Imperial 643 B 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

Maxifort A 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

Muriel B Cornell

database

Nico Cornell

database beef

- type http://www.seedway.com/store/Pages/Product.aspx?pid=7400 Orange Faruche F1 B Cornell

database

Palenque B Cornell

database

Palomo F1 Cornell

database large oval

red http://www.harrismoran.com/products/tomato/pdf/table

- fresh

- det.pdf

Panzer B Cornell

database Pegasso F1 (Pegaso F1?) Cornell

database large oval

red http://www.harrismoran.com/products/tomato/pdf/table

- fresh

- det.pdf Pilavy B CornellDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 28

Variety ( A Not readily available on a retail see d or plant site ; B Rootstock ) Reference Description Retail Site Link

database

Pitenza B 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 F1 B Cornell

database Primo Red F1 Cornell

database large round http://www.seedway.com/store/Pages/Product.aspx?pid=5055

Qualit 21 B Cornell

database

Qualit 23 F1 B Cornell

database

Red Candy Cornell

database red grape http://www.harrismoran.com/products/tomato/pdf/table

- fresh

- indet.pdf

Sahel B Cornell

database

Samurai F1 Cornell

database large oval

red http://www.harrismoran.com/products/tomato/pdf/table

- fresh

- indet.pdf

Santa EDIS

Sapho F1 B Cornell

database
Shiren B EDIS

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 6701 B 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 CornellDietary Exposure to Rpi

- vnt1.1 , J.R. Simplot Co. Page 29

Variety ( A Not readily available on a retail see d or plant site ; B Rootstock ) Reference Description Retail Site Link Orange B database

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.aspx1 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 2 8 , 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 ..................................................................................................... 92 of 9 

## 1. Description of the M ethods

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. T he 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 s ame 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 a n 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 Oligonucleoti de 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 CCA A *CA G *AC A *AG C *CAC *Locked

- Nucleic Acid (LNA)

The following sections include a list of all equipment and reagents (Table s 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

. R eagents. Chemicals/Reagents Number/Specification TaqMan® Universal PCR Master Mix Applied Biosystems 4304437 SSO Advanced SYBR Green Supermix BioRad 172

- 527 4 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

 Th e 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

- f ree, 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.4 of 9

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 res in is fully re suspended 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 μ l TE buffer.
24. Optional: Further c lean DNA using the Qiagen QIAquick PCR Purification Kit column and elute DNA twice with 50 μ l 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 Table s 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) 

### 2 X 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.5 of 9

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 5 8 .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 R elative
F lorescence U nits or at a value above the baseline, but sufficiently low to be within the exponenti al growth region of the amplification curve (1) . The point at which samples crossed this line was reported as the Cq value for the sample s

. 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

.6 of 9 

## 4. Testing of the detection methods The aim of the testing was to verify that the Real

- Time PCR assay described above detected W8

- specific ally
, using potato leaf and tuber DNA at a level of 0.2% with a 95% confidence interval.



### Efficiency
The q PCR efficiency (E value ) was determi ned 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 R 2 c oefficient, a measure of linearity of the standard curve, was als o determined and should be ≥0.9 8 (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 R 2 Ave E value Ave Slope
W8 0.99 3 98.1 %

- 3. 37

A separate assay was performed using t he 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 event

specific primer pair demonstrated a single peak for eac h sample (Figure 1).

Figure 1

. Melt ing 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 %, 0.1 6 %, and 0.0 4 % and 0.01%) in a total of 200 ng control Russet Burbank DNA. The absolute copy nu mber for each sample was determined by dividing the sample DNA weight, in nanogra ms, 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 W87 of 9

T he mean Cq values and associated standard dev iations 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 le af 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 0.1 6 % 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 ( F 10, E12, J3, J55, and Russet Burbank ) along with the W8 event. Assays were run for 40 cycles with 20 ng of genomic DNA. Those samp les 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 includ ed 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 perf ormed. 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 positi ves 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 0.04238 of 9 

## 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 9 5% 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 tuber s

.9 of 9 

## 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 An alytical methods of GMO Te sting, http://gmo

crl.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, H ausman, JF, Hoffmann L, Evers D (2005). Housekeeping gene selection for real

time 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.