### J.R. Simplot Company Characterization of Inserted DNA 1 Appendix 1
Characterization of Inserted DNA

INDEX

Section Page

Index 1

Introduction 2

Results 1 3

Summary 6 2

Materials and M ethods 6 3

Conclusion s 6 6

References 6 8

Cer tification 69

### J.R. Simplot Company Characterization of Inserted DNA 2 INTRODUCTION

E vents were evaluated by DNA gel blot analyses to determine the structure and copy number of the integrated DNA insert (vector map presented in Figure 1 )

. The descriptions of the genetic elements are
shown in Table 1. These studies were carried out as part s of the characterization and biosafety

assessme nt of the events

.

DNA gel blot analysis was performed using DNA extracted from greenhouse

- grown plants and digested
with Eco RV, Hin d III, or Eco RI + S ca I. The digested DNA was run with standard DIG

- labeled molecular weight markers, DIGII and DIGVII, through 0.7% agarose gels in 0.5X TBE buffer and blotted onto Nylon Hybond N+ filters. The DNA insert

- derived probes used were named ASN (from the Asn1/Ppo5 gene silencing cassette), AGP, AGP2 and AGP3 (all from the Agp promoter driving both silencing cassettes),

GBS (from the Gbss promoter driving both silencing cassettes), and PHL (from the R1/PhL promoter silencing cassette) ( Figure 2 ) . To facilitate inter pretations, the original films were divided into separate
segments for each digest with the marker lanes copied and boxed.

### J.R. Simplot Company Characterization of Inserted DNA 3 Figure 1. Vector pSIM1278

The vector backbone region, on the left, starts at position 9,957

- bp and ends at 19,468

- bp (= 9,512

- bp). The backbone DNA consists mainly of bacterial DNA and is intended only to support maintenance of the DNA insert prior to plant transformation.

The DNA insert region, including flanking Border sequences, is on the right from 19,469

- bp to 19,660

- bp and from 1

- bp to
9,956

- bp , which is a total of 10,148

- bp. The DNA insert consists of T

- DNA border

- like sequences and potato genomic DNA which
are intended for stable integration into the plant genome.

### J.R. Simplot Company Characterization of Inserted DNA 4 Table 1. Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border site

Genetic Element Origin Intended Function Genbank

Accession
Number Start

- End

Point in pSIM1278 Reference

1. Left Border ( LB ) site 1 Synthetic Site for secondary cleavage to release single

- stranded DNA insert
fro m pSIM1278 AY566555 2
(bases 1

- 25) 19,469

–

19,493 van Haaren

et al. 1989

2. DNA flanking the LB sequence S. tuberosum var. Ranger

Russet Su pports secondary cleavage at LB AY566555 2
(bases 26

- 187) 19,494

–

19,655

3. KpnI restriction site S. tube rosum Site for connection of DNA insert with LB flanking sequence. AF393847.1 19,656

– 1

4. Promoter for the ADP glucose pyrophosphorylase gene ( pAgp ), 1st

copy S. tuberosum var. Ranger

Russet One of the two convergent

promoters that drives expressi on of an inverted repeat containing fragments of Asn1 and Ppo5, especially in tubers HM363752 2

- 2,261

5. Fragment of the asparagine synthetase

- 1 ( Asn1 ) gene ( 1 st copy antisense orientation) S. tuberosum var. Ranger

Russet Generates with (9) double s tranded

RNA that triggers the degradation of Asn1 transcripts to impair asparagine formation HM363759 2,262

- 2,666 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 Generates with (8) double stranded RNA that triggers the degradation of Ppo5 transcripts to block black spot bruise development HM363754 2,667

- 2,810

7. Spacer

- 1 S. tuberosum var. Ranger

Russet Sequence between the 1st inverted

repeats HM363753 2, 81 7

- 2,973

8. 3’

- untranslated sequence of the polyphenol oxidase

- 5 gene ( Ppo5 ) ( 2 nd copy, in sense orientation) S. verrucosum Generates (6) double stranded RNA that triggers the degradation of Ppo5 transcripts to block black spot bruise development HM3 63754 2,974

- 3,117

9. Fragment of the asparagine synthetase

- 1 ( Asn1 ) gene ( 2 nd copy, in sense orientation) S. tuberosum var. Ranger

Russet Generates with (5) double stranded

RNA that triggers the degradation of Asn1 transcripts to impair asparagine for mation HM363759 3,118

- 3,523 Chawla et
al. 20 12 3

10. 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 One of the two convergent

promoters tha t drives expression of an inverted repeat containing fragments of Asn1 and Ppo5, especially in tubers HM363755 3,5 30

- 4,215

11. pAgp, 2 nd copy S. tuberosum var. Ranger

Russet One of the two convergent

promoters that drives expression of

an inverted re peat containing fragments of the promoters of PhL and R1, especially in tubers HM363752 4,2 32

- 6,491

12. Fragment of promoter for the potato phosphorylase

- L ( pPhL ) gene ( 1 st copy, in antisense orientation) S. tuberosum var. Ranger

Russet Generates wit h (16) double

stranded RNA that triggers the degradation of PhL transcripts to limit the formation of reducing sugars through starch degradation HM363758 6,492

- 7,000

13. Fragment of promoter for the potato R1 gene ( pR1 ) ( 1 st copy, in

antisense orientat ion) S. tuberosum var. Ranger

Russet Generates with (15) double

stranded RNA that triggers the degradation of R1 transcripts to limit the formation of reducing sugars through starch degradation HM363757 7,001

- 7,532

1 4

. Spacer

- 2 S. tubero sum var. Ranger

Russet Sequence between the 2nd inverted

repeat HM363756 7,53 9

- 7,796

### J.R. Simplot Company Characterization of Inserted DNA 5

Table 1 C ontinued . Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border site

Genetic Element Origin Intended Function Genbank

Acc ession
Number Start

- End

Point in pSIM1278 Reference

15. Fragment of promoter for the potato R1 gene (pR1) (2nd copy, in sense orientation) S. tuberosum

var. Ranger

Russet Generates with (13) double

stranded RNA that triggers the degradation of R1 tra nscripts to limit the formation of reducing sugars through starch degradation HM363757 7,797

- 8,328

16. Fragment of promoter for the potato phosphorylase

- L (pPhL) gene (2nd copy, in sense orientation) S. tuberosum var. Ranger

Russet Generates with (1 2) double

stranded RNA containing this sequence will trigger the degradation of PhL transcript to limit the formation of reducing sugars through starch degradation HM363758 8,329

- 8,837

17. pGbss (2nd copy, convergent
orientation relative to the 2nd co py of pAgp) S. tuberosum var. Ranger

Russet 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 HM363755 8,838

- 9,761

18. SacI restriction site S. tuberosum Site for connection of DNA insert with RB flanking sequence. AF143202 9,76 2

– 9,767

19. DNA flanking the RB sequence S. tuberosum var. Ranger

Russet Supports primary cleavage at RB

Like site AY566555 2
(bases 231

- 391) 9,7 71

– 9,931

20. Right Border (RB) sequence 1 Synthetic Site for primary cleavage to release single stranded DNA insert from pSIM1278 AY566555 2
(bases 392

- 416) 9,932

- 9,956 van Haaren
et al. 1989

1 The LB and RB sequences (25

- bp each) were synthetically designed t o 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 9 is referred to as StAst1 in Chawla et al. 2012.



### Figure 2

. DNA Insert Probes

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII 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.

P osition 1 starts from the first Kpn I cutting site in pSIM1278.

Hybridizing bands were based on their approximate sizes and grouped into three different classes ( See summary explaining band interpretation in Table 2 ) . F or clarification purposes, bands in the gels were
read by aligning the top with the marker band (see line marking top of band in example here )

.

### J.R. Simplot Company Characterization of Inserted DNA 6

 Original Bands (OBs) : Plant sequences that hybridize with a DNA insert probe but are not associated
with the inserted D NA. They are i ndicative of hybridizing sequences that are present in both untransformed and transformed plants (shown as “ ”)

.

 Internal Bands (IBs) : Plant sequences that hybridize with a probe, within the ins erted DNA. These DNA fragments with predicted sizes are positioned within the DNA insert and indicative of DNA integrity (shown as “ ”)

. E verything is based on 1 copy of the DNA insert so that the expected
numbers are if we have one copy

.

 Junction Bands (JBs) : Plant sequences that hybridize with a probe and comprise part of both t h e inserted DNA and the insert

- flanking DNA. These s equences compris e both part of the DNA insert and flanking plant DNA

, have unique sizes

, and are in dicative of copy number (shown as “ ”).

Observed and Expect e d Bands : Differences in the number of observed bands (Obs.) compared to expected (Exp.) bands was noted . The data showing more observed bands compared to expected bands
shows us that we have more than one copy or an additional partial copy. If the expected band is missing or smaller than the expected size, the integrated DNA insert has a deletion.

In some cases, Southern blots shown in this appendix will use the same pr obes and enzyme digests as found in other studies (for example, see Appendix 3. Evidence for Stability of the Inserted DNA ). The observations may differ, depending upon the conditions under which the gels were run . Therefore all
bands described in the tabl es representing Southern blot data in this appendix refer to the gels presented within this appendix.

Summary of G el E valuation : In Table 2 , we summarized how gels w ere interpreted to determine original bands, along with internal and junction bands of th e inserted DNA . In addition, in Figures 3

- 6
below , we illustrate the restriction sites and where hybridization occur s on the DNA insert for the various probes , thus summarizing how bands are identified through a visual illustration

. Also included,

is a s ummary of the expected number of original bands for all the varieties ( Table 3 ).

### J.R. Simplot Company Characterization of Inserted DNA 7

Figure 3

. Expected IB and JB of DNA I nsert D igested with V arious E nzymes and H ybridized with A SN
Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

L B = 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.

Figure 4

. Expected IB and JB of DNA I nsert D igested with V arious E nzymes and H ybridized with AGP
Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII 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 fla nking sequence.

### J.R. Simplot Company Characterization of Inserted DNA 8

Figure 5

. Expected IB and JB of DNA I nsert D igested with V arious E nzymes and H ybridized with PHL

P robe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII 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 Characterization of Inserted DNA 9

Figure 6

. Expected IB and JB of DNA I nsert D igested with V arious E nzymes and H ybridized with GBS

P robe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII 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.

### J.R. Simplot Company Characterization of Inserted DNA 10 Table 2

. Band Interpretation in Events

Prob e Restriction

Enzyme Size (bp) Position on

DNA I nsert Band

Type Expected

Bands Notes:

AGP EcoRV Variable Variable OB 3

- 5 Multiple bands relating to variety

2293 2872

- 5165 IB 1 Part of PPO/ASN inverted repeat, 1st GBS promoter and left portion
of the s econd AGP promoter

≥ 1127 LB Flanking

region to

AGP 935 JB 1 Indicates at least a portion of the
left border side of the DNA insert

AGP HindIII Variable Variable OB 3

- 6 Multiple bands relating to variety

4230 2251

- 6481 IB 1 PPO/ASN inverted repeat, 1st GBS promoter and le ft portion of the second AGP promoter

≥ 2443 LB flanking

region to

2251 JB 1 Indicates at least a portion of the

left border side of the DNA insert

AGP EcoRI/ScaI Variable Variable OB 3

- 5 Multiple bands relating to variety

3751 3525

- 72 7 6 IB 1 Compris ing a DNA insert fragment consisting of 1st GBS promoter, 2nd AGP promoter and part of the PHL/R1 inverted repeat

≥ 3717 LB region JB 1 Predicted size indicative of an independent insertion comprising
at least the left border side of the

DNA insert

ASN EcoRV Variable Variable OB 1

- 4 Multiple bands relating to variety
712, 2293 2160

- 2872,

2872

- 5165 IB

- 1,

IB

- 2 2 Indicative of intact ASN1 / PPO5

-

derived inverted repeat and 1st GBS promoter and left portion of the 2nd AGP promoter

ASN HindIII Variable Var iable O B 1

- 3 Multiple bands relating to variety

4230 2251

- 6481 IB 1 Indicative of intact ASN1 / PPO5

- derived inverted repeat as well as 1st GBSs and 2nd AGP promoter Variable = Size and position of Original bands (OB) are variable depending on potato va riety, and position of Junction bands (JB) is variable for each event, depending on the DNA insert integration site.

### J.R. Simplot Company Characterization of Inserted DNA 11 Table 2 (Continued)

. Band Interpretation in Events

Probe Restriction

Enzyme Size (bp) Position on

DNA insert Band

Type Expected

Bands No tes:

ASN EcoRI/ScaI Variable Variable OB 2

- 4 Variable

≥ 3717 Variable JB 1 Indicative of independent insertion comprising at least the left border
side of the DNA insert

GBS EcoRV Variable Variable OB 2

- 3 Variable

2293 2872

- 5165 IB 1 Comprising part of PPO/ASN inverted repeat, 1st GBS promoter and left po rtion of the second AGP
promoter

≥ 3567 Variable JB 1 I ndicative of independent insertion comprising at least the right border
side of the DNA insert

GBS HindIII Variable Variable OB 1

- 2 Variable

4230 2251

- 6481 IB 1 Comprising intact ASN1 / PPO5

- deriv ed inverted repeat as well as 1st GBSs and 2nd AGP promoter

≥ 2163 Variable JB 1 Indicative of independent insertion comprising at least the right border
side of the DNA insert

GBS EcoRI/ScaI Variable Variable OB 2

- 3 Variable

3751 3525

- 7276 IB 1 Comp rising 1st GBS promoter, 2nd AGP promoter and part of the PHL/R1 inverted repeat

≥ 1903 Variable JB 1 Indicative of independent insertion comprising at least the right border
side of the DNA insert

PHL EcoRV Variable Variable OB 3 Variable

≥ 3567 Var iable JB 1 Indicative of independent insertion comprising at least the right border
side of the DNA insert

PHL HindIII Variable Variable OB 3 Variable

1313 6481

- 7794 IB 1 Comprising part of the PHL/R1 inverted repeat

≥ 2163 Variable JB 1 Indicative o f independent insertion comprising at least the right border
side of the DNA insert

PHL EcoRI/ScaI Variable Variable OB 1

- 5 Variable

3751 3525

- 7276 IB 1 Comprising 1st GBS promoter, 2nd AGP promoter and part of the PHL/R1 inverted repeat

≥ 1903 Varia ble JB 1 Indicative of independent insertion comprising at least the right border
side of the DNA insert Variable = Size and position of Original bands (OB) are variable depending on potato variety, and position of Junction bands (JB) is variable for each event, depending on the DNA 

### insert integration site.

### J.R. Simplot Company Characterization of Inserted DNA 12 Table 3

. Number of Original Bands (OBs) for Conventional (Wild Type, WT) Varieties



### Probe Restriction

Enzyme Ranger

Russet (F ) Russet

Burbank (E) Atlantic (J) G H

AGP EcoRV 3 3 4 4 5

HindIII 4 4 5 3 6
EcoR1/Scal 5 3 4 4 5

A SN EcoRV 4 3 1 2 1

HindIII 3 3 1 1 1
EcoR1/Scal 3 4 2 2 2

P HL EcoRV 3 3 3 3 3

HindIII 3 3 3 3 3
EcoR1/Scal 1 2 5 5 3

GBS EcoRV 3 2 3 2 2

HindIII 2 1 2 1 1
EcoR1/Scal 3 2 3 2 2

### J.R. Simplot Company Characterization of Inserted DNA 13 RESULTS

Copy Number and Integrity: Ranger Russet F10

Plants were molecularly analyzed by Southern blot hybridization using DNA extracted from leaves of greenhouse

- grown plants (see Materials and Methods ). F10 contains a single complete copy of the DNA

insert ( Figure 7 ) . This conclusion was deduced from hybridizations shown in Figures 8

- 1 1 which are
summarized in Table 4 . At the Left Border site, a single JB was visualized when Eco RV, Hin d III and

Eco RI/ Sca I digests were hybridized with the AGP probe. Similarly, hybridization of Eco RI/ Sca I digests
with the ASN probe visualized a single JB . The presence of a single JB at the Right Border side was deduced from hybridizations of Eco RV, Hin dIII, and Eco RI /Sca I di gests with the GBS and PHL probes.

Figure 7

. F10 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Integrity of the DNA insert is indicated by the predicted sizes of IBs visualized in (a) all three digests hybridized with the AGP probe, (b) all three d igests hybridized with the GBS probe, (c) Eco RV and Hin dIII digests with the ASN probe, and (d) Hin dIII and Eco RI/ Sca I digests hybridized with the PHL probe

. Furthermore, JBs are larger than the minimum predicted size from internal restriction site to clea vage site in the left border (>1.1

- kb ) as visualized with Eco RV digest hybridized with the AGP probe, and in the right border (>1.9

- kb ) with Eco RI /ScaI digests hybridized with the GBS probe. Moreover, no other band indicative of any truncation was observed indicating that the integrated copy was complete. Intactness was confirmed by experiments shown in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 14 Figure 8

. Ranger Russet DNA H ybridization with the AGP P robe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with
the AGP probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lan es for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are i ndicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definition s for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 15 Figure 9

. Ranger Russet DNA H ybridization with the ASN P robe

Genomic DNA of Ranger Russet control (RR) and eve nts F10 and F37 were digested and hybridized with
the ASN probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three

enz ymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 16 Figure 10

. Ranger Russet DNA H ybridization with the PHL P robe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with
the PHL probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three

enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction ba nds ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

. Partial digestion of Eco RI site due to overlapping DNA methylation is marked by an arrow.

### J.R. Simplot Company Characterization of Inserted DNA 17 Figure 1 1

. Ranger Russet DNA H ybridization with the GBS P ro be

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with
the GBS probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the bl ot image in kilobase pairs ( k b). Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

. Partial digestion of Eco RI site due to overlapping DNA methylation is marked by a n arrow.

### J.R. Simplot Company Characterization of Inserted DNA 18

Table 4

. Ranger Russet Event F10: DNA F ragments H ybridizing with DNA I nsert

- D erived P robes ( S ummary of obser vations from Figures 8

- 1 1 )

Probe Restriction Enzyme Type of Band Interpretation

OB IB IB JB JB Exp. Obs. Exp. Obs.

AGP Fig ure 8 EcoRV 6. 7

5.0

1.4 2.3 2.3 1.1+ 2.6 1. Copy number at Left Border side Visualization of a single JB in AGP and ASN digests indicates that F10 contains a single DNA insert (at the Left Border side).

2. Copy number at Right Border side
Visualization of a single JB in PHL and GBS digests indicates that F10 contains a single DNA insert (at the Right Border side).

3. Internal DNA insert Integrity Integrity of the DNA insert is indicated by the fact that

the IBs visualized in all DNA digests have nearly the expected sizes (see note 5 below regarding variations)

. See also Fig ure 40 for 0.7

- kb IB.

4. DNA insert Integrity at the extremities The DNA insert is complete because all JBs are greater than the minimum size expected. Closest to JBs are EcoRV fragments with AGP probe (LB) and EcoRI/ScaI fragments with GBS probe (RB).

5. Variation between expected and observed sizes in

DNA insert

. Slight differences were found between
the observed and expected size of a 3.8

- kb internal

band between restriction site s for E coR1 at 3

, 525

- bp
and Sca1 at 7

, 276

- bp

. This band was expected with

the AGP, PHL,and GBS probes with EcoR1 and Sca 1

, but appeared sometimes at 3.6

- kb

. This may be because of differences in gel loading or possibly the influence of plant material resulted i n faster movement within the gel. See further discussion in
section titled “Additional Studies on DNA insert Integrity for all Events.” HindIII 16

8.0

6.5

5.8 4.2 4.2 2.4+ 5. 7

EcoRI/ScaI 4.8

2.7

2.2

1.3

1.0 3.8 3. 6 3.7+ 3.8

ASN Figure 9 EcoRV 8.6

4

. 4

2.5

1.5 2.3,

0.7 2.3,

0.7 # none none

HindIII 3. 2

2.9

2.0 4.2 4.2 none none

EcoRI/ScaI 8.2

7.3

4.8 none none 3.7+ 3. 9

PHL Fig ure 10 EcoRV 22

20

4. 7 none none 3.6+ 7. 2

HindIII 20

8.6

6.0 1.3 1.3 2.2+ 7.0

EcoRI/ScaI 1.5 3.8 3. 6 1.9+ 6. 1

GBS Figure 1 1 EcoRV 8.6

7. 8

7.2 2.3 2.3 3.6+ 6.6

HindIII 1.4

1.3 4.2 4.2 2.2+ 6. 6

EcoRI/ScaI 4.0

3.7

3.4 3.8 3.8 1.9+ 6.1

The definition of OB, IB, and JB can b e found in the Introduction . Bands indicated with the symbol “#” were

visua lized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 19 Copy Number and Integrity: Ranger Russet F37

F37 contains a single complete copy of the DNA insert ( see Figure 1 2

. F37 Insert ) . This conclusion was deduced from hy bridizations shown in Figures 8

- 1 1 which are summarized in Table 5 . The following hybridizations indicated predicted sizes for IBs : Eco RV

, Hin dIII and EcoRI/ScaI digests hybridized with the

AGP and the GBS probes, Eco RV, Hin dIII digests hybridized with th e ASN probe and Hin dIII, Eco RI /Sca I I digests hybridized with the PHL probe. Integrity of the DNA insert is indicated by the fact that the IBs visualized in various DNA digests and probes have predicted sizes.

Figure 1 2

. F37 Insert

RV=EcoRV, R1=Eco R1, Sc=Sca1, Hd=HindIII

Furthermore, the following hybridizations indicate that JBs are larger than the minimum predicted size from internal restriction site to cleavage site in the border: Eco RV

, Hin dIII , and Eco RI /Sca I digests hybridized with the AGP p robe, Eco RI / Sca I digests hybridized with the ASN probe; Eco RV

, Hin dIII , and

Eco RI /Sca I digests hybridized with the PHL and GBS probe. For instance, the JB visualized with the AGP probe in Eco RV digest is larger than the minimum predicted size from restri ction site to cleavage site in the left border (>1.1

- kb) and GBS probe in EcoR I/ Sca I digest is larger than the minimum predicated size (>1.9

– kb) in the right border . Moreover, no other band indicative of any truncation was observed indicating that the inte grated copy was complete.

Data were confirmed by sequencing one of the junction fragments shown in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 20

Table 5

. Ranger Russet Event F37: DNA F ragments H ybridizing with DNA I nsert

- D erived P robes (S ummary of observat ions from Figures 8

- 1 1 )

Probe Restriction

Enzyme Type of Band Interpretation

OB IB IB JB JB Exp Obs Exp obs

AGP Figure 8 EcoRV 6. 7

5.0

1.4 2.3 2.3 1.1+ 6. 7 (double intensity) 1. Copy number at Left Border side

Visualization o f a single JB in AGP and ASN digests indicates that F37 contains a single DNA insert (at least the Left Border side). The double intensity band at 6. 7
indicates 1 OB and 1 JB are the same

size.

2. Copy number at Right Border side Visualization of a single JB in most PHL and GBS digests indicate that F37 contains a single DNA insert (a t least the Right Border side).

3. Internal DNA insert Integrity Integrity of the DNA insert is indicated by
the fact that the IBs visualized in various DNA digests have nearl y the expected sizes (see note 5 below regarding

variation ). See also Fig ure 40 for 0.7

- kb

IB.

4. DNA insert Integrity at the extremities

The DNA insert is complete because all JBs are greater than the minimum size expected. Closest to JBs are EcoRV fragmen ts with AGP probe (LB) and EcoRI/ScaI fragments with G BS probe (RB).

5. Variation between expected and
observed sizes in DNA insert

. Slight
differences were found between the observed and expected size of a 3.8

- kb

internal band between restriction sites
for EcoR1 at 3

, 525

- bp and Sca1 at 7

, 276

- bp

. This band was expected with the AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared sometimes at

3.6

- kb. This may be because of differences in gel loading or possibly the
influence of plant material resulte d in faster movement within the gel. See
further discussion in section titled
“Additional Studies on DNA insert Integrity for all Events.”

*Partial digestion of Eco RI site due to overlapping methylation ma rked by arrow (Figures 10, 11). HindIII 16

8.0

6

. 5

5.8 4.2 4.2 2.4+ >23

EcoRI/ScaI 4.8

2.7

2.2

1.3

1.0 3.8 3. 6 3.7+ 4.0

ASN Figure 9 EcoRV 8.6

4. 4

2.5

1.5 2.3,

0.7 2.3,

0.7 # none none

HindIII 3. 2

2.9

2.0 4.2 4.2 none none

EcoRI/ScaI 8.2

7.3

4.8 none none 3.7+ 4.2

PHL Figure 10 EcoRV 22

2 0

4. 7 none none 3.6+ 8.2

HindIII 20

8.6

6.0 1.3 1.3 2.2+ >23

EcoRI/ScaI 1.5 3.8 3. 6 1.9+ 2.4, 2.3*

GBS Figure 11 EcoRV 8.6

7. 8

7.2 2.3 2.3 3.6+ 7. 7

HindIII 1.4

1.3 4.2 4.2 2. 2+ >23

EcoRI/ScaI 4.0

3.7

3.4 3.8 3.8 1.9+ 2.4, 2.3*

The definition of OB, IB, and JB can b e found in the Introduction . Bands indicated with the symbol “#” were

visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 21 Copy Number and Integrity: Russet Burbank E12

E12 cont ains a single complete copy of the DNA insert (see Figure 1 3 ). This conclusion was deduced from the hybridizations shown in Figures 1 4

- 1 7 which are summarized in Table 6

.

Figure 1 3

. E12 I nsert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that single JBs with sizes greater than the minimum predicted sizes (from restriction site to border cleavage site) were visualized for both the Right Border side and the Left Border side when the various DNA digest s were hybridized with AGP

, ASN

, PHL
and GBS probes. Intactness of the DNA insert was confirmed by visualization of IBs with predicted sizes. This is also confirmed by the junction study

, which shows both ends of the DNA insert are intact ( Appendix 4. Jun ction Analysis and Event

- Specific Detection )

.

### J.R. Simplot Company Characterization of Inserted DNA 22 Figure 1 4

. Russet Burbank DNA H ybridization with the AGP P robe

Genomic DNA of Russet Burbank control (EC) and events E1 2 and E24 were digested and hybridized with
the AGP probe. The enzymes used a re indicated below each panel. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and J Bs can be found in the Introduction

. Partial digestion of Eco RI site due to overlapping DNA methylation is marked by an arrow. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 23 Figure 1 5

. Russet Burbank DNA H ybridization with the ASN P robe

Genomic DNA of Russet Burbank control (EC) and eve nts E12 and E 24 were digested and hybridized with
the ASN probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three

en zymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , an d the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

. 1. 95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 24 Figure 1 6

. Russet Burbank DNA H ybridization with the PHL P robe

Genomic DNA of Russet Burbank control (EC) and events E12 a nd E 24 were digested and hybridized with
the PHL probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three

enzymes wer e on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the jun ction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 25 Figure 1 7

. Russet Burbank DNA H ybridization with the GBS P robe

Genomic DNA of Russet Burbank control (EC) and events E12 and E 24 wer e digested and hybridized with
the GBS probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three enzymes were on one b lot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction band s ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 26

Table 6

. Russet Burbank Event E12: DNA F ragments H ybridizing with DNA I nsert

- D erived P robes (Summary of observations from Figures 1 4

- 1 7 )

Probe Restr iction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 14 EcoRV 5.2

4.4

1.5 2.3 2.3 1.1+ 2.2 1. Copy number at Left Border side Visualization of a singl e JB with AGP and ASN probes

indicates that E12 contains a single DNA insert (at least the Left Border side).

2. Copy number at Right Border side Visualization of a single JB with PHL and GBS probes indicates that E12 contains a single DNA insert (at least the Right Border side).

3. Inte rnal DNA insert Integrity Integrity of the DNA insert is indicated by the fact that the

IBs visualized in various DNA digests have nearly the expected sizes (see note 5 below regarding vari ation). See also Fig ure 40 for 0.7

- kb IB.

4. DNA insert Integrity at the extremities No unexpected bands were observed, and all JBs have sizes greater than minimum predicted sizes, indicating the
overall intactness of the DNA insert . Closest to JBs are

EcoRV fragments with AGP probe (LB) and EcoRI/ScaI fragments with GB S probe (RB).

5. Variation between expected and observed sizes in DNA
insert Slight differences were found between the observed and expected size of a 3.8

- kb internal band betw een restriction sites for EcoR1 at 3525

- bp and Sca1 at 7276

- bp.

This band was ex pected with the AGP, PHL,and GBS probes
with EcoR1 and Sca1 , but appeared sometimes at 3.4 to

3.6

- kb. This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further dis cussion in section
titled “Additional Studies on DNA insert Integrity for all
Events.” HindIII 8. 4

7.0

6. 6

5.8 4.2 4.2 2.4+ 15

EcoRI/ScaI 4.8

4.0

2.3 3.8 3. 6 3.7+ 5. 3

ASN Figure 15 EcoRV 8.0

4. 4

1.5 2.3

0.7 2.3

0.7 # none none

HindIII 12

3.2

3 .0 4.2 4.2 none none

EcoRI/ScaI 9. 4

4.9

4.0

2.7 none none 3.7+ 5. 8

PHL Figure 16 EcoRV 20.0

15.0

8.0 none none 3.6+ 4. 5

HindIII 18.0

8. 8

6.0 1.3 1.3 2.2+ 5. 5

EcoRI/ScaI 1.6

1.5 3.8 3. 6 1.9+ 3.3

GBS Figure 17 EcoRV 8. 6

7. 6 2.3 2.3 3.6+ 4.5

HindIII 1.3 4.2 4.2 2.2+ 5.2 EcoRI/ScaI 4.2

3. 2 3.8 3. 4 1.9+ 3. 0

The definition of OB, IB, and JB can b e found in the Introduction . Bands indicated with the symbol “#” were

visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 27 Copy Number and Integrity: Russet Burbank E24

E24 contains a single, mostly complete, copy of the DNA insert (see Figure 1 8 ). This conclusion was deduced from the hybridizations shown in Figures 1 4

- 1 7 which are summarized in Table 7

.

F igure 1 8

. E24 In sert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that single JBs were visualized for both the Right Border side and the Left Border side when the various DNA digests were hybridized wi th AGP

, ASN

, PHL and GBS probes.

Visualization of IBs with predicted sizes confirmed that the DNA insert was generally intact. However,
the DNA insert has an approximate 500

- bp deletion at its Left Border side because the associated JBs (3. 2

– kb) are small er than the minimum size expected (3. 7

- kb) when AGP and ASN probes were used for hybridization with Eco RI/ScaI digests. Since the AGP probe, which starts from the 5’ end of AGP promoter, still detected the left side junction signal with lighter intensity, the deletion is within the AGP probe and does not affect efficacy of the silencing constructs

. The R ight Border junction was sequenced
as described in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 28

Table 7

. Russet Burbank Event E24: DNA F r agments H ybridizing with DNA Insert

- D erived P robes ( Summary of obser vations from Figures 1 4

- 1 7 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 14 EcoRV 5.2

4.4

1.5 2.3 2.3 1.1+ 20.0 1. Co py number at Left Border side Visualization of a single JB with AGP and ASN probes indicates that E24 contains a single DNA insert (at least Le ft Border side).

2. Copy number at Right Border side Visualization of a single JB with PHL and GBS probes indicates that E24 contains a single DNA insert (at least the Right Border side).

3. Internal DNA insert Integrity Integrity of the DNA insert is indicated by the fact that the

IBs visualized in various DNA digests have nearly the expected sizes (see note 5 below regarding variation). See also Fig ure 40 for 0.7

- kb IB.

4. DNA insert Integrity at the extremities

The DNA insert suffe rs a small deletion at its Left Border extremity because the JBs are smaller than the minimum size expected ( > 3.7

- kb). The delet ion is upstream from where the AGP probe anneals, which means not greater than a few hundred base pairs, and does not affect the functional activity of the ASN1 / PPO5 gene silencing cassette.

The right side of the DNA insert is intact bec ause all JBs of PHL and GBS are greater than the minimum size expected.

Closest to JBs are EcoRV f ragments with AGP probe (LB) and EcoRI/ScaI fragments with GBS probe (RB).

5. Variation between expected and observed sizes in DNA
insert

. Slight differences were found between the observed and expected size of a 3.8

- kb internal band between

restrictio n sites for EcoR1 at 3525

- bp and Sca1 at 7276

- bp. This band was expected with the AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared sometimes at 3.4 to 3.6

- kb. This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further discussion in section
titled “Additional Studies on DNA insert Integrity for all
Events.” Hind III 8. 4

7.0

6. 6

5.8 4.2 4.2 2.4+ 4.6

EcoRI/ScaI 4.8

4.0

2.3 3.8 3. 6 3.7+ 3.2

ASN Figure 15 EcoRV 8.0

4. 4

1.5 2.3

0.7 2.3

0.7 # none none

HindIII 12

3.2

3.0 4.2 4.2 none none

EcoRI/ScaI 9. 4

4.9

4.0

2.7 none none 3.7+ 3.2

PHL Figure 16 EcoRV 20.0

15.0

8.0 none none 3.6+ 23.0

HindIII 18.0

8. 8

6.0 1.3 1.3 2.2+ 4.7

EcoRI/ScaI 1.6

1.5 3.8 3. 6 1.9+ 2.5

GBS Figure 17 EcoRV 8. 6

7. 6 2.3 2.3 3.6+ 23.0

HindIII 1.3 4.2 4.2 2.2+ 4. 5 EcoRI/ScaI 4.2

3. 2 3.8 3. 4 1.9+ 2.5

The definition of OB, IB , and JB can b e found in the Introduction . Bands indicated with the symbol “#” were

visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 29 Copy Number and Integrity: Atlantic Event J3

J3 contains one nearly intact and one partial copy of the DNA insert , connected reversely at the left border side, with deletions of LB regions and parts of the adjacent AGP promoters. The partial copy
lacks the 3’ end of the central AGP and the rest of the R1/ PHL promoter silencing cassette (see Figure

1 9 ). This conclusion was deduced from Southern hybridizations shown in Figures 20

- 2 3 which are
summarized in Table 8

.

Figure 1 9

. J3 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that 2 JBs were visualized with hybridizations for the Left Border side. However, only one IB was observed when the digested DNA was hybridized with the AGP probe , indicating that a truncation occurred within the central AGP promoter in

one copy o f the DNA insert . A dditional evidence for the occurrence of a truncation comes from the fact that only one IB and one JB were visualized from DNA digests hybridized with the PHL probe

, which
means that the truncated DNA insert lacks PHL fragments. Furtherm ore, o nly one IB and two JBs rather
than two IBs and two JBs were visualized with the GBS probe (one IB and one JB per intact DNA insert ).

One 1.6

- kb Eco RV band and one 4.2

- kb Hin dIII band visualized in the hybridization with AGP probe are double intense, suggesting that two copies of the DNA insert can be connected head to head with deletion of Left Borders and part of AGP promoters as shown in Figure 19 (J3 Insert)

. Indeed, 6. 6

- kb bands containing AGP or ASN from both DNA insert copies are found in the Ec oR1/ScaI DNA digestions probed with AGP and ASN , respectively.

This structure comprising two connected DNA insert s was also confirmed by sequence analysis
described in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 30 Figure 20

. Atlantic H ybr idization with the AGP P robe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the

AGP probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are in dicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original ba nds ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 31 Figure 2 1

. Atlantic H ybridization with the ASN P ro be

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the

ASN probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the bl ot image in kilobase pairs ( k b). Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 32 Figure 2 2

. Atlantic H ybridization with the PHL P robe

Genomic DNA of Atl antic control (JC) and events J3, J55 and J78 were digested and hybridized with the

PHL probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 33 Figure 2 3

. Atlantic H ybridization with the GBS P robe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the

GBS probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Lanes for all th ree enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ”

, the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The
definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 34

Table 8

. Atlantic Event J3: DNA F ragments H ybridizing with DNA I nsert

- D erived P robes

( Summary of obser vations from Figures 20

- 2 3 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 20 EcoRV 8.0

5. 2

2. 1

1.4 2.3 2.3 1.1+ 6

. 6
1.6* 1. Copy number at Left Border side

Visualization of two JBs in all DNA digests with the AGP probe
indicates that J3 contains two DNA insert copies (at least at the Left Border side). With the ASN probe, 3 IBs (2.3, 0.7

- double intensity) and 1 unexpected JB (6. 6 ) were visualized in the Eco RV digestion. With the ASN probe in the Hin dIII digestion, we visualized 1 IB (4.2)

and 1 unexpected JB (7. 8 ) containing the ASN inverted repeat, which indicates the presence of two DNA insert copies at the LB side. With the ASN probe and EcoRV, the presence of a junction band indicates

t here is more than 1 c opy.

2. Copy number at Right Border side Visualization of a single JB in all DNA digestions with the PHL probe

indicates that J3 contains one DNA insert at the Right Border side.
The two JBs in all DNA digests probed with GBS (together with one

IB ) indicates that J3 contains three pGbss elements, 2 in the single copy and 1 in the partial copy.

3. Internal DNA insert Integrity Only one IB (no double intensity) was found when the three digestions (EcoRV, HindIII, EcoRI/ScaI) were probed w ith AGP and AGP2 ( Figure 38 , 2.3

- kb), indicating that the second copy of DNA

insert has a truncation in the internal AGP region.
The deletion of the internal AGP includes both Eco RV sites (at 5,165 and 6,390)

. Additional evidence suggests a dele tion of t he internal AGP based on band intensity which indicates only three rather than
four 1.2

- kb bands in an Eco RV digest hybridized with the AGP3 probe ( Figure 3 9 ). Instead of an expected second 2.3

- kb IB in EcoRV digests with the ASN probe, we visualized a 6. 6

- kb fragment, indicating the
deletion of Eco RV in one internal AGP promoter. Only one IB w as shown in HindIII and EcoRI/ScaI digests hybridized with PHL, which indicates the presence of a single copy of DNA

insert . Thus, the second copy lacks the PHL reg ion. Visualization of three bands (1

- IB, 2

- JBs) hybridizing with the GBS probe indic ates J3 contains three pGbss elements, 2 in the single copy and 1 in the partial copy.

4. DNA insert Integrity at the extremities
Both a 1.6

- kb Eco RV band and a 4.2

- kb Hin d III band visualized with AGP are double intense, suggesting that two copies of th e DNA

insert are connected reversely at the LB side. In this case, a 2.2

- kb EcoRV band including LB and partial AGP promoter from both DNA

insert copies will be expected. Ho wever, only a 1.6

- kb band was
observed which me ans a 0.6

- kb deletion from one or both DNA

insert s occurred during the recombination. The partial DNA insert had a deletion at the RB side that extended beyond the first Eco RV site (5,165

- bp ) of the interna l AGP promoter.

One JB band in the EcoRI/ScaI digest that is visualized with the ASN probe confirmed that the two DNA insert s connected at the LB side. When digested with EcoRI/ScaI, we expected double intensity bands of AGP and ASN, with the same size of 6. 6

- kb , and confirmed the

connect ion at the LB. HindIII 16.0

8. 5

7.0

6. 0

2.2 4.2 4.2 2.4+ 7. 8
4.2*

EcoRI/ScaI 3.9

2.9

2.3

1.1 3. 8 3. 7 3.7+ 6. 6 *

2.9 *

ASN Figure 21 EcoRV 1.4 2.3

0.7 2.3

0.7* # none 6. 6

HindIII 3.1 4.2 4.2 none 7. 8

EcoRI/Sca I 8.4

7.4 none none 3.7+ 6. 6 *

PHL Figure 22 EcoRV 18.0

12.0

7 .4 none none 3.6+ 1 2 .0

HindIII 1 8 .0

8. 6

6. 0 1.3 1.3 2.2+ 12.0

EcoRI/ScaI 1.6

1. 5

1. 3

1.1

0.9 3. 8 3.8 1.9+ 3.8

### J.R. Simplot Company Characterization of Inserted DNA 35

Table 8 (Continued). Atlantic Event J3: DNA F ragments H ybridizing wit h DNA Insert

- D erived P robes ( Summary of obser vations from Figures 20

- 23 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

GBS Figure 23 EcoRV 9.2

7. 6

7.0 2.3 2.3 3.6+ 12.0

6. 1 5. Variation between expected and observed sizes in DNA

insert

. Sligh t differences were found between the observed and expected size of a 3.8

- kb internal band between
restriction sites for EcoR1 at 3

, 525

- bp and Sca1 at 7

, 276

- bp. This band was expected with the AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared at 3. 7

- kb with AGP . This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further discussion in section titled
“Additiona l Studies on DNA insert Integrity for all Eve nts.” HindIII 1. 3

1.2 4.2 4.2 2.2+ 12.0

8.4 EcoRI/ScaI 4. 3

3. 8

3. 5 3. 8 3.8 1.9+ 3.8

2. 9

The definition of OB, IB, and JB can be found in the Introduction

. Bands indicated with “*” were double inten sity. Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 36 Copy Number and Integrity: Atlantic Event J55

J55 contains one full copy of the DNA insert , and an additional truncated and linked copy that lacks the R1/ PHL promoter silencing cassette (see Figure 2 4 ). This conclusion was deduced from hybridizations

shown in Figures 20

- 2 3 which are summarized in Table 9

.

Figure 2 4

. J55 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, H d=HindIII

Two observed JBs at the Left Border side in the EcoRV digest hybridized with the AGP probe represented two copies of the DNA insert . Indeed, the presence of 2 copies at the Left Border side is confirmed by
the Eco RI/ Sca l digest hybridized with t he ASN probe. However, only one JB is visualized at the Right Border side, indicating that only one copy of the DNA insert has the Right Border region. A truncation
occurred within the central AGP promoter which removed all sequences at the right of the ce ntral AGP
promoter . Also, about 400

- bp of the central AGP promoter itself (between the Eco RV and Hin dIII sites) is missing because the 2.3

- kb IB visualized when hybridizing an Eco RV digest with the AGP probe is still present. Confirmation for the absence of sequences to the right of the central AGP promoter comes
from the fact that the numbers of IB and JB visualized with the PHL probe are just enough for one intact

DNA insert copy, indicating that the PHL region in the second copy of DNA insert is missing . Since only

one JB and two IB identified in an EcoRV digest with GBS probe, the G bss promoter at the Right Border side is not present in one of the DNA insert s.

Furthermore, a 6

- kb Hin dIII JB was shown in all the hybridizations with the AGP

, ASN

, PHL and GBS probes, indicating that the RB border of the complete DNA insert is reversely connected to the central

AGP promoter of the truncated DNA insert . This structure (see Figure 20 ) is supported by the presence
of 4.5

- kb Sca l

- EcoRI AGP band and a 4.5

- kb Sc a l

- EcoRI GBS band that was double intense because of two copies of GBS promoters . This connection of two DNA insert s is also supported by the fact that a 4.5

- kb band connecting 2 DNA insert s was observed when an Eco RV digest was hybridized with AGP 3 probe ( Figure 39 ). The junction between the two DNA insert s was further confirmed by sequence analyses
described in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 37

Table 9

. Atlantic Event J55: DNA F ragments H ybridizing with DNA I nsert

- D erived P rob es

( Summary of obser vations from Figures 20

- 2 3 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 20 EcoRV 8.0

5. 2

2. 1

1.4 2.3 2.3 * 1.1+ 5.3

2.0 1. Copy number at Left Border side

Visualization of two JB in Eco RV digest with AGP pr obe and Eco RI/ Sca I digest with ASN probe indicates that J55 contains two DNA insert s (at least at the Left Border side).

2. Copy number at Right Border side Visualization of a single JB in with the PHL a nd GBS probes indicates that

J55 contains one DNA insert at the Right Border side.

3. Internal DNA insert Integrity
There are four bands ( IB and JB ) found in all three digests probed with either AGP or ASN, which means that J55 contains two copies of the

DNA insert . However, only one IB (4.2) hybridizing with AGP was
observed in the Hin dIII digestion, indicating that the associated AGP promoter was truncated. The truncated region is about 400

- bp with a breakpoint between Eco RV (6,390) and Hin dIII (6,481) site s in the partial copy because the 2.3

- kb Eco RV IB hybridizing with AGP is still present. The number of bands visualized in all three digests probed with PHL indicates the presence of one intact copy of the DNA insert (thus, the second copy lacks PHL

- hybrid izing sequences). Visualization of three
bands in a ll digests hybridizing with the GBS probe indicates J55 contains three pGbss elements, 2 in the single copy and 1 in the partial

copy.
4. DNA insert Integrity at the extremities In the HindIII digest s, a band was shown that ranged from 5.7 to 6.1 in
the hybridizations pro bed with AGP, ASN, PHL and GBS. These are all the same band but show slight variations with the different probes. This suggests the association of two DNA insert copies, whereby the RB border of the first copy is linked to a truncated pAGP of the second copy.

The second DNA insert is in the reverse orientation. Evidence for this structure comes from the presence of (1) a 4.5

- kb Sca l

- Eco RI band hybridizing with AGP, (2) a double

- intense 4.5

- kb Sca l/ Eco RI GBS

hybridizing band, and (3) a double

- intense 6.0

- kb HindIII GBS band. Furthermore, a 4.5

- kb band is visualized when Eco RV digest is hybridized with the AGP3 probe ( Figure 39 ).

5. Variation between expected and observed sizes in DN A insert

. Slight differences were found between the observed and expected size of a

3.8

- kb internal band between restriction sites for EcoR1 at 3

, 525

- bp and

Sca1 at 7

, 276

- bp. This band was expected with the AGP, PHL,and GBS probes with EcoR1 and Sca1 , bu t appeared at 3. 7

- kb with AGP . This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further
discussion in section titled “Additional Studies on DNA insert Integrity
f or all Events.” HindIII 16.0

8. 5

7.0

6. 0

2.2 4.2 4.2 2.4+ 6.7

5.8

3.4

EcoRI/ScaI 3.9

2.9

2.3

1.1 3.8 3. 7 3.7+ 5.0

4.5

3.8

ASN Figure 21 EcoRV 1.4 2.3

0.7 2.3 *

0.7 # * none none

HindIII 3.1 4.2 4.2 None 5.8 EcoRI/ScaI 8.4

7.4 None none 3.7+ 5. 3

4.0

PHL Figure 22 EcoRV 18.0

12.0

7 .4 None none 3.6+ 4. 4

HindIII 1 8 .0

8. 6

6. 0 1.3 1.3 2.2+ 5. 7

EcoRI/ScaI 1. 6

1.5

1. 3

1.1

0.9 3.8 3.8 1.9+ 4. 8

GBS Figure 23 EcoRV 9.2

7. 6

7.0 2.3 2.3 * 3.6+ 4. 4

HindIII 1. 3

1.2 4.2 4.2 2.2+ 6.1 *

EcoRI/Sca 4. 3

3. 8

3. 5 3.8 3.8 1.9+ 4.5*

The definition of OB, IB, and JB can be found in the Introduction . Bands indicated with “*” were double intensity. Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7

- k b IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 38 Copy Number and Integrity: Atlantic Event J78

J78 contains a single truncated DNA insert (see Figure 2 5 ). This conclusion was deduced from hybridizations shown in Figures 1 6

- 1 9 which are summarized in Table 10

.

Figure 2 5

. J78 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Visualization of a single JB when Eco RI, Hin dII and Eco RI / Sca l digests are hybridized with the AGP probe demonstrates that J78 contains a single copy of the DNA insert at the Left Bor der side. However, there is
a truncation at the Left Border side because the JB is 200

- bp shorter than the predicted minimum size
when Eco RI/ Sca l digests are hybridized with AGP or ASN probe. Using GBS as a probe, only one IB was visualized while the expe cted JB band on R ight Border side was not found in all three digestions, indicating that the G bss promoter adjacent to the Right Border was deleted. PHL hybridization further proved that the whole or partial PHL fragment next to G bss promoter was also miss ing in both Hin dIII
and Eco RI/ Sca l digestions. Indeed, the junction sequence analysis demonstrated that 486

- bp of PHL was deleted as described in Appendix 4. Junction Analysis and Event

- Specific Detection

.

### J.R. Simplot Company Characterization of Inserted DNA 39

Table 10

. Atlantic Event J78: DNA F ragments H yb ridizing with DNA Insert

- D erived P robes

(Summary of observations from Figures 20

- 2 3 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB Exp. IB
Obs. JB Exp. JB
Obs.

AGP Figure 20 EcoRV 8.0

5. 2

2. 1

1.4 2.3 2.3 1.1+ 12 .0 1. Copy number at Left Border side Visualization of a single JB in the all digestions with AGP probe and EcoR/ScaI with ASN probe indicates that J78 contains one copy of DNA insert (at least the Left Border side).

2. Copy number at Right Border side J78 contains a single DN A insert without the Right Border because only internal PHL and GBS bands were visualized.

3. Internal DNA insert Inte gr ity Integrity of the DNA insert is indicated by the fact that the IBs visualized in various DNA digests have nearly the expected sizes (se e note 5 below regarding variation). See also Fig ure
40 for 0.7

- kb IB.

4. DNA insert Integrity at the extre mities

JB bands are about 200

- bp shorter than the expected sizes when genomic DNA was digested with either Hin dIII or

Eco R1/ Sca I and probed with eithe r AGP or ASN, indicating that there is a truncation at the Left Border region. In addition, J78
suffers a deletion of about 486

- bp comprising PHL and GBS

hybridizing sequences on the right border side

.

5. Variation between expected and observed sizes in DN A
insert

. Slight differences were found between the observed and expected size of a 3.8

- kb internal band between

re striction sites for EcoR1 at 3

, 525

- bp and Sca1 at 7

, 276

- bp. This band was expected with the AGP, PHL,and GBS probes
with EcoR1 and Sca1 , bu t appeared at 3. 7

- kb with AGP . This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further discussion in section titled “Additional

Studies on DNA insert Integrity f or all Events.” HindIII 16.0

8. 5

7.0

6. 0

2.2 4.2 4.2 2.4+ 2. 1

EcoRI/ScaI 3.9

2.9

2.3

1.1 3.8 3. 7 3.7+ 3. 5

ASN Figure 2 1 EcoRV 1.4 2.3

0.7 2.3

0.7 # None none

HindIII 3.1 4.2 4.2 None none

EcoRI/ScaI 8.4

7.4 None none 3.7+ 3. 5

PHL Figure 2 2 EcoRV 18.0

12.0

7 .4 None none 3.6+ 3.0

HindIII 18.0

8. 6

6. 0 1.3 1.3 2.2+ none

EcoRI/ScaI 1. 6

1.5

1. 3

1.1

0.9 3.8 3.8 1.9+ none

GBS Figure 2 3 EcoRV 9.2

7. 6

7.0 2.3 2.3 3.6+ none

HindIII 1. 3

1.2 4.2 4.2 2.2+ none

EcoRI/ScaI 4. 3

3. 8

3. 5 3.8 3.8 1.9+ none

The definition of OB, IB, and JB can be found in the Introduction . Bands indicated with the symbol “#” were

visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 40 Copy Number and Integri ty: G Event G11

The hybridization data for event G11 are shown in Figures 2 7

- 30 and summarized in Table 1 1

. In summary, a single JB in AGP and ASN hybridizations indicated that G11 contains a single DNA insert with
an intact Left Border (See Figure 2 6 )

. No JB was observed in DNA digests hybridized with the PHL probe
and Eco RV and Hin dIII digests hybridized with the GBS probe. This indicated that G11 does not contain an insert with a Right Border. Observation of IBs with sizes that are different from the p redicted sizes for

Eco RI/ Sca I digests when hybridized with AGP , PHL and GBS probes confirmed that the Right Border side
of the DNA insert was missing. The truncation occurred on the Right Border side, upstream from the Sca I site at position 7

, 276

- bp. A jun ction study described in Appendix 4. Junction Analysis and Event

- Specific

Detection showed that the actual truncation site was at position 6

, 618

- bp within the DNA insert . A summary of the deduced insert for G11 is shown below. Event G11 does not have the p PHL

- pR1 inverted repeat DNA. The second AGP promoter is immediately upstream from the DNA insert /plant DNA junction

.

Figure 2 6

. G11 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

### J.R. Simplot Company Characterization of Inserted DNA 41 Figure 2 7

. G Event DNA H ybridization with the AGP P robe

Eco RV Hin dIII Eco RI/ Sac I

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the AGP probe. The

re striction enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot origi nally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) a re indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 42 Figure 2 8

. G DNA H ybridization with the ASN P robe

Eco RV Hin dIII Eco RI/ Sac I

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the ASN probe. The enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers ar e indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 43 Figure 2 9

. G DN A H ybridization with the PHL P robe

Eco RV Hin dIII Eco RI/ Sac I

Genomic DNA of G control (GC) and event G11 was digested a nd hybridized with the PHL probe. The enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all

three en zymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , an d the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

. Observations are
summarized in Table 8

.

### J.R. Simplot Company Characterization of Inserted DNA 44 Figure 30

. G DNA hybridization with the GBS probe

Eco RV Hin dIII Eco RI/ Sac I

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the GBS probe. The enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segme nts (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for O Bs

, IBs and JBs can be found in the Introduction

. 1.95

1.88 1.95

1.88 1.95

1.88

### J.R. Simplot Company Characterization of Inserted DNA 45 Table 1 1

. G Event G11: DNA F ragments H ybridizing with DNA Insert

- D erived P robes

( Summary of observations from Figures 2 7

- 30 )

Probe Restriction

Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB Exp. Obs. Exp. Obs.

AGP Figure 27 EcoRV 8. 4

5. 3

2. 1

1.5 2.3 2.3 1.1+ 5.3 * 1. Copy number at Left Border side Visualization of a single JB in each of Eco RV digested AGP hybridizations and Eco RI/ Sca I digested ASN hybridization indicates
that G11 con tains a single DNA insert (containing the Left Borde r side). Although 2 bands were observed in Eco RI/ Sca I digested AGP hybridization, the absence of an IB in this digest indicates one of the
bands is the truncated IB.

*The double intensity band at about 5.3

- kb appears to include both
the 5.3

- kb OB and the 5.3

- kb JB

2. Copy number at Right Border side

No JB was o bserved in each DNA digest with PHL hybridization and

Eco RV

, Hin dII I , or EcoRI/ScaI

. Digests with GBS hybridization indicated
that G11 does not con tain the Right Border DNA insert . The missing IB
from the Eco RI/ Sca I d ige st with GBS hybridization and the visible 5. 3

- kb JB is indicative of the truncated IB and confirms there is only one copy of the Gbss fragment in the DNA insert at the Left Border si de of

the construct.

3. Internal DNA insert Integrity

IBs with expected sizes observed in Eco RV or Hin dIII digests with AGP and GBS hybridizations indicated the integrity of the DNA insert to the

second Hin dIII site (6

, 481

- bp position on pSIM1278). The absen ce of
IBs for the Eco RI/ Sca I d igest with AGP and GBS hybridizations indicated that the Right Border side of the DNA insert was truncated at a point before the first Sca I site (upstream from 7

, 276

- bp).

4. DNA insert Integrity at the extremities

No IB was visu alized in the digest hybr idized with the GBS probe and EcoRI/ScaI, indicating that the DNA insert for G11 was truncated between the HindIII site at 6,481

- bp and the Scal site at 7

, 276

- bp. No

JB was observed when PHL probes for the right border side of the DNA

insert were used with all three digests. This indicated that a portion of
the DNA insert was missing from the right border side of the DNA

insert , including the PHL element. The absence of the IB in the

Eco RI/ Sca I digests probed with PHL suggests tha t no intact PHL sequences are present in the truncated insert. HindIII 7.0

6. 7

6.1 4.2 4.2 2.4+ None

EcoRI/ScaI 4. 2

3.0

2. 5

1.2 3.8 none 3.7+ 5. 9

5.5

ASN Figure 28 EcoRV 2 .5

1.5 2.3

0.7 2.3

0.7 # None None

HindIII 3. 1 4.2 4.2 None None

Ec oRI/ScaI 7. 6

7.0 none none 3.7+ 5. 5

PHL Figure 29 EcoRV 20.0

15.0

8.0 none none 2.2+ none

HindIII 15.0

8. 6

5.8 1.3 none 3.6+ none

EcoRI/ScaI 1.7

1.6

1.5

1.3

1.2 3.8 none 1.9+ none

GBS Figure 30 EcoRV 8. 6

7.0 2.3 2.3 3.6+ none

HindIII 1.3 4.2 4 .2 2.2+ none EcoRI/ScaI 4.2

3. 4 3.8 none 1.9+ 5. 3

The definition of OB, IB, and JB can be found in the Introduction . Bands indicated with “*” were double intensity. Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 46 Copy Number and Integrity: H Event H37

The hybridization data for event H37 are shown in Figures 3 2

- 3 5 and summarized in Table 1 2

. Event H37 contains one complete DNA insert, two truncated inserts, and one additional small fragment only comprising a part of the AGP promoter (see Figure 3 1 below). To simplify explanations, these copies have been labeled as copy number 1, 2, 3, and 4 respectively.

Figure 3 1

. H37 Insert s

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd =HindIII Diagram (4) is a small fragment containing part of the AGP promoter.

At least one copy of the DNA insert is complete as indicated by the IBs and JBs in all the DNA digests that have the expected sizes. Two extra JBs (one was expected, 3 total) were observed when EcoRV digests were hybridized with the AGP probe, indicating two more DNA insert copies are in H37. One of the copies is truncated at the first A sn 1 fragment downstream from the Left Border. This is indicated by only

### J.R. Simplot Company Characterization of Inserted DNA 47

one copy of IB s ( 0. 7

- kb , 2.3

- kb) visualized when the EcoRV digests were hybridized with the ASN probe ( F igure s 3 3 and 3 9 ) and the observation of JBs with Eco RV and Hin dIII when probed with ASN

.

H37 possesses a third partial DNA insert containing the Right Border side. This is indicated by the two

JBs and two IBs observed in the Hin dIII digest with the PHL probe. Only one IB (3.7

- kb ) was observed
when Eco R1/ Sca l digests were hybridized with GBS and AGP probes, which indicated that this third DNA insert was missing the Left B order region and started at the central GBS near the Eco R1 site, extending to the Right Border.

In addition, a very faint band with a size of about 4.2

- kb, indicating the presence a small portion of part
of an AGP promoter, was detected in H37 with the A GP 3 probe ( Figure 3 4 ). This fragment was not

visualized with the AGP or AGP 2 probe, indicating that the insert itself is smaller than 1.0

- kb and has been depicted as copy number 4. A similar ~4.2

- kb band was identified in event G9, which is not part of
thi s submission.

In summary , the deduced inserts for event H37 are shown above . This event is believed to have four
inserts. One is a full

- length DNA insert . The second is a shortened insert with only one AGP promoter and a portion of one ASN 1 fragment. The third insert is also a shortened DNA insert containing only the second inverted repeat with the p PHL and pR1 fragment inverted repeats and the Right Border. The fourth insert consists of a part of the AGP promoter only.

### J.R. Simplot Company Characterization of Inserted DNA 48 Figure 3 2

. H H ybridization with th e AGP P robe

Genomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the AGP probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The origina l bands ( OBs ) are indicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 49 Figure 3 3

. H H ybridization with the ASN P robe

Ge nomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the ASN probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs )

are i ndicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 50 Figure 3 4

. H H ybridization with the PHL P robe

Geno mic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the PHL probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pa irs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs )

are ind icated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 51 Figure 3 5

. H H ybridization with the GBS P robe

Ge nomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the GBS probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b) in gray and black, respectively. Lanes for all three enzymes were on one blot originally, and were separated into three segments (one for each digest) with the marker lanes copied and boxed for better illustration. The original bands ( OBs )

are i ndicated by “ ” , the internal bands ( IBs ) are indicated by “ ” , and the junction bands ( JBs ) are indicated by “ ” . The definitions for OBs

, IBs and JBs can be found in the Introduction

.

### J.R. Simplot Company Characterization of Inserted DNA 52 Table 1 2

. H Event H37: DNA F ragments H ybridizing with DNA Inser t

- D erived P robes

( Summary of obser vations from Figures 3 2

- 3 5 )

Probe Restriction Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 32 EcoRV 8.0

7.2

5. 4

2. 2

1. 5 2.3 2.3 1.1+ 3. 8

3.2

2. 2 1. Copy number at Left Bo rder side

H 37 showed three JB bands with the Eco RV and Hin dIII digests using the AGP probe. This indicated three copies of the insert. However,

the EcoR I/ Sca I digest with the AGP probe gave only two JB bands, which suggested that one of the three copies w as truncated at the
left border side.

2. Copy number at Right Border side H37 contains two copies of the DNA insert extending to the Right Border side as viewed with the PHL and GBS enzymes. However, it
contains three rather than two JBs with Eco RV. The th ird JB (in bold

, EcoRV with GBS ) is smaller than the minimum size expected, and was generated by truncation of one of the two DNA insert s at a site within the centrally

- located pGbss.

3. 3. Internal DNA insert Integrity

4. The first DNA insert cop y ( DNA insert 1) is intact because nearly all IBs

visualized in various DNA digests have expected sizes (see comment 5 below) . The second copy ( DNA insert 2) contains the first AGP promoter and is truncated within the Asn1

- derived sequence,
because the e xpe cted IBs for a second complete copy were not found. Copy number 3, DNA insert 3, is truncated as it misses the Left Border side, and lacks additional IBs (2.3, 0.7) associated with the ASN fragments when digested with Eco RV and hybridized with the ASN pr obe.

5.

6. 4. DNA insert Integrity at the extremities

DNA insert 1 is complete because the JBs are greater than the minimum size expected. Evidence for the structure of DNA insert 2
were the JBs found with Eco R V and Hin dIII digests with the ASN probe when n one are expected. DNA insert 2 is truncated at Asn1 as indicated by visualization of a JB band that was unexpected and smaller in size (2.4) with the ASN probe in the Eco RI / ScaI digest. The total size of this copy should be smaller than 2.4

- kb since

Eco R I /Sca I would cut outside of the proposed DNA insert 2. DNA

insert 3 is missing the left side as indicated by a smaller than expected band (3.2

- kb , in bold) in the Eco RV digestion with GBS probe. Also, DNA insert 3 has one IB band plus 3 JB bands in other digestions with Eco RI/ Sca I, and both GBS and PHL probes. These 3

- JBs that hybridize with the PHL and GBS probes are found instead of the expected IBs, indicating a truncation to the right of the EcoR1 site at 3,525

- bp

.

5. Variation between expected and observed sizes in DNA insert

. Slight differences were found between the observed and expected
size of a 3.8

- kb internal band between restriction sites for EcoR1 at

3

, 525

- bp and Sca1 at 7

, 276

- bp. This band was expected with the AGP, PHL,and GBS probes wi th EcoR1 and Sca1, but appeared

sometimes at 3. 7

- kb. This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further discussion in section titled

“ Additional Studies on DNA insert Integrity for all Events.” HindIII 12.0

8. 6

7.0

6.6

6.1

5.8 4.2 4.2 2.4+ 20

5. 8

3.3

EcoRI/ScaI 4. 9

4. 3

3.0

1.3

1.1 3. 8 3. 7 3.7+ 5. 8

3. 5 *

ASN Figure 33 EcoRV 1.4 2.3

0.7 2.3

0.7 # none 3. 3

HindIII 3.4 4.2 4.2 none 6.4 EcoRI/ScaI 9

. 4

8.6 none none 3.7+ 3.7

2.4 (weak)

PHL Figure 34 EcoRV 20.0

14.0

8.2 none none 3.6+ 4. 7*

HindIII 16.0

9.2

6. 5 1.3 1.3* 2.2+ 6. 5

3. 3

EcoRI/ScaI 2.4

1.8

1.7 3.8 3. 7 1.9+ 5. 8

2.4

2.1

GBS Figure 35 EcoRV 1 2

8.4 2.3 2.3 3.6+ 4. 7 *

3.2

Hi ndIII 0.9 4.2 4.2 2.2+ 6. 5

6.1

3. 3 EcoRI/ScaI 4.3

3.6 3.8 3.8 1.9+ 3. 5

2.4

2.1

The definition of OB, IB, and JB can be found in the Introduction . Bands indicated with “*” were double intensity. Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV digestion).

### J.R. Simplot Company Characterization of Inserted DNA 53 Copy Number and Integrity: H Event H50

The hybridization data for event H50 are shown in Figures 3 2

- 3 5 and summarized in Table 1 3

. Event H50 contains one complete DNA in sert as well as one truncated copy (see Figure 3 6 below).

Figure 3 6 . H50 Insert s

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

The presence of two JBs at the left border side was observed in all the digests hybridized with the AGP probe and in the Eco RI/ S ca I digest hybridized with the ASN probe. These results suggested two inserts with a Left Border side. However, all the digests probed with GBS indicated the presence of a single copy containing a Right Border side. One of the inserts is complete, because all the expected IB s were present and because the JBs identified were greater than the minimum size expected. However, no I Bs
were observed in the other DNA insert copy when HindIII and EcoR1/Scal digests were hybridized with PHL probe, indicating that pa rt of p PHL/ p R1 invert ed repeat was deleted. This analysis was confirmed by sequencing the junction fragments (see Appendix 4. Junction Analysis and Event

- Specific Detection ).

### J.R. Simplot Company Characterization of Inserted DNA 54 Table 1 3 . H Event H50: DNA fragments Hybridizing with DNA Insert

- Derived P robe s

( Summary of obser vations from Figures 3 2

- 3 5 )

Probe Restriction Enzyme Type of Band (kb) Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP Figure 32 EcoRV 8.0

7.2

5. 4

2.2

1. 5 2.3 2.3* 1.1+ 4.0

1.9 1. Copy number at Left Border side

Visuali zation of two JB s in three DNA digests with the AGP probe and EcoRI/ScaI with the ASN probe indicated that H50 contained two copies of the DNA insert comprising the Left Border side

.

2. Copy number at Right Border side Visualization of a single JB in most d igests with the PHL and GBS probes indicated that H50 contained at least one DNA insert comprising the Right Border side. The two JB bands observed with the HindIII digestion and PHL probe indicated a second insert copy
with a truncation on the RB side.

3. I nternal DNA insert Integrity *The double intensity IB bands observed with the AGP, ASN and GBS probes indicated the presence of two copies of the DNA insert

. However , only single intensity IB band s w ere observed with the PHL probe. This suggests that at l east one copy of the DNA insert is complete as indicated by the IBs having the expected sizes. The
second DNA insert copy has a complete cassette on the LB side (see

doubl e intensity IBs with AGP, ASN and GBS probes and EcoRVand HindIII) and truncation on the RB side (seen with the PHL probe).

4. DNA insert Integrity at the extremities
A total of four IB and JB bands with the AGP and ASN probes an d
three IB and JB bands with the PHL and GBS probes were found in the H50 event digests. One of the inserts is com plete because the

JBs are greater than the minimum size expected. The second insert was truncated (between the HindIII site at 6,481 and the Sca1 site at 7,276) and the deleted region included the pPHL and pR1 inverted repeat, Gbss promoter and Right Borde r.

5. Variation between expected and observed sizes in DNA insert

. Slight differences were found between the observed and expected
size of a 3.8

- kb internal band between restriction sites for EcoR1 at

3

, 525

- bp and Sca1 at 7

, 276

- bp. This band was expected w ith the AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared

sometimes at 3. 7

- kb. This may be because of differences in gel loading or possibly the influence of plant material resulted in faster movement within the gel. See further discussion in sect ion titled
“Additional Studies on DNA insert Integrity for all Events.” In the EcoR1/Sca1 digest we expected two JBs that were the same size, however there was slight variation with one of the band sizes specifically between the AGP and ASN probes. Hi ndIII 12.0

8. 6

7.0

6.6

6.1

5.8 4.2 4.2* 2.4+ 4. 7

3. 6

EcoRI/ScaI 4. 9

4. 3 3.0

1.3

1.1 3. 8 3. 7 3.7+ 5. 7

4. 3 *

ASN Figure 33 EcoRV 1. 4 2.3,

0.7 2.3*

0.7 # * none none

HindIII 3. 4 4.2 4.2* none none

EcoRI/ScaI 9

. 4

8.6 none none 3.7+ 6. 1

4. 2

PHL Figur e 34 EcoRV 20.0

14 .0

8.2 none none 3.6+ 6. 9

HindIII 16.0

9.2

6. 5 1.3 1.3 2.2+ 5. 7

4. 3

EcoRI/ScaI 2.4

1.8

1.7 3. 8 3. 7 1.9+ 3.7

GBS Figure 35 EcoRV 12.0

8.4 2.3 2.3* 3

. 6+ 6.9

HindIII 0.9 4.2 4.2* 2.2+ 5. 7

EcoRI/ScaI 4.3

3.6 3. 8 3. 8 1.9+ 4. 3

3.7

The definition of OB, IB, and JB can be found in the Introduction . Bands indicated with “*” were double intensity. Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7

- kb IB with the ASN probe in EcoRV dige stion).



### Additional S tudies on DNA Insert I ntegrity for A ll Event s

### J.R. Simplot Company Characterization of Inserted DNA 55

In some cases, minor differences were observed between expected sizes of internal bands and what we observed. In multiple events, an internal band was observed of about 3.6

- kb in siz e where we knew the expected size to be 3,751 or about 3.8

- kb. This band occurs between restriction sites for EcoR1 at

3

, 525

- bp and Sca1 at 7

, 276

- bp and was expected to be visualized with the AGP, PHL, and GBS probes
with EcoR1 and Sca1 digests. The dif ference of about 0.15

- kb between observed and expected sizes most likely was related to small differences in gel loading or the influence of plant tissues that would not be part of the marker lanes. The largest difference between expected and observed val ues was found for events E12 and E24. The same sequence of DNA insert containing the second AGP promoter (expected size was 3.8

- kb with EcoR1 and ScaI) was studied using the Hin dIII digests. For example, expected bands with the AGP, ASN, and GBS probes o f 4.2

- kb between 2,251 and 6,481

- bp all include the AGP promoter region. For all three probes, the observed bands matched the size of the expected
bands for both events E12 and E24 (Tables 6 and 7)

. In addition, the 1.3

- kb band between 6,481 and
7,794

- bp restriction sites for HindIII , was observed with the PHL probe in both E12 and E24. Thus, additional probes and restriction digests support the observation that the DNA insert in the region of the second APG promoter was intact.

To investigate the poss ibility of a truncation, we conducted studies with probes that would anneal to the AGP promoter in that region. Further evidence for the structure of the various inserts was obtained by performing additional studies using a new probe that was designed to anneal to the downstream part
of the AGP promoter. This 402

- bp probe, AGP 2, was amplified with primers HY1659 and HY1660 ( Figure
37 and Table 1 4 in Materials and Methods ) . As shown in Figure 3 8

, hybridization with AGP 2 visualized an expected 2.3

- kb band in DNA of all events, indicat ing that no AGP promoter residing within the DNA

insert was truncated in this region. A 999

- bp probe, AGP 3 ( Figure 3 9 )

, was amplified with primer HY1661

and HY2662 ( Figure 37 and Table 1 4 in Materials and Methods ) and specificall y targeted the AGP promoter region between the two EcoR V digests with an expected size of 1

, 225

- bp . As shown in Figure

3 9

, the J55 and H37 events each had extra hybridizing DNA fragments in addition to the 1

, 225

- bp fragment. This indicated that an AGP pr omoter residing within one of the DNA insert s of these events was truncated at this range.

A short electrophoresis run of Eco RV digests probed with ASN confirmed the presence of the relatively
small internal Eco RV fragments with an expected size of 0.7

- kb ( Figure 40 ). The presence of this band provides additional proof of the integrity of the DNA insert s.

Even more evidence exists for the completeness of the DNA insert in the region of the AGP promoter (3,525 to 7,276

- bp) in the gels used for stability t esting (See Appendix 3, Evidence for stability of the

inserted DNA ). For events F10 and F37 ( Evidence for stability of the inserted DNA : Figures 4, 5, and 6 ), the gel shows the 2.3

- kb section of DNA insert that hybridizes with the AGP probe between the E coR V restriction sites at 2,872 and 5,165

- bp. This same 2.3

- kb band is visualized for events E12 and E24 (Evidence for stability of the inserted DNA : Figures 11, 12, and 13 ), J3, J5, and J78 ( Figures 18, 19, and

20 ), G11 ( Figures 25, 26, and 27 ) and H37 and H50 ( Figures 33, 34, and 35 ). Thus, the stability studies provide additional evidence that the DNA insert is intact in this region.

### J.R. Simplot Company Characterization of Inserted DNA 56

Figure 37

. Expected IB and JB of DNA Insert D igested with V arious E nzymes and H ybridized with (A)

AGP 2 and (B) AGP3 p ro be s

.

(A)

(B)

RV=EcoRV 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 Characterization of Inserted DNA 57 Figure 3 8 . Hybridization of EcoRV D igests with AGP2 P robe

Genomic DNA was digested with EcoRV and hybridized with the AGP 2 probe. Samples were loaded following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlant ic), G (G) and H (H). EC, FC, JC, GC and HC are wild type control samples for each variety. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Events G9 and E67 are not part
of this sub mission.

### J.R. Simplot Company Characterization of Inserted DNA 58 Figure 3 9

. Hybridization of Eco R V D igests with AGP3 P robe

Genomic DNA was digested with EcoRV and hybridized with the AGP 3 probe. Samples were loaded following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlantic), G (G) a nd H (H). EC, FC, JC, GC and HC are conventional variety control samples for each variety. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). A boxed segment of the blot is shown again be low the blot with different brightness and contrast to highlight the

4.2

- kb bands in J55, G9, and H37. Event s G9 and E67 are not part of this submission.

### J.R. Simplot Company Characterization of Inserted DNA 59

Figure 40

. Visualization of an E xpected 0.7

- kb Band in A ll E vents when Hybridized with the ASN P robe

(in addition to previously observed bands in shorter runs)

Genomic DNA was digested with EcoRV and hybridized with the ASN probe. Samples were loaded following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlantic), G (G) and H (H). EC , FC, JC, GC and HC are wild type control samples for each variety. Size of the DigII and Dig VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs ( k b). Events G9 and E67 are not part
of this submission.

### J.R. Simplot Company Characterization of Inserted DNA 60 PCR Confirmati on of EcoR1/Scal Fragments in E, F J, and H lines

Minor differences were observed in some cases between expected sizes of internal bands and what we actually observed. The description and figures below clarify why we observed the minor differences.

When the plant genomic DNA is digested with EcoR1 and Scal a 3751

- bp internal fragment of the DNA

insert is expected in all events except G11. In addition to Southern blot analysis, a PCR approach was used to further ensure the completeness of the fragment. As shown in Figure 41 , four primers were designed to amplify two fragments: (A) a 1653

- bp fragment including EcoR1 site (3525) and EcoRV site (5165); (B) a 2125

- bp fragment from EcoRV site (5165) to Scal site (7276). Since the component targeted by a forward primer was different from one targeted by a reversed primer, no fragments were expected to be amplified from untransformed parent controls. The pSIM1278 plasmid DNA was also used as a positive control to amplify the fragment A and B. The results were show n in Figure 42 . Both fragment A and fragment B were amplified from transformed events but not from untransformed controls. The sizes of the two fragments are the same as the expected and the positive controls. We can conclude that the

3751

- bp internal frag ment of DNA insert exists in events E12, E24, F10, F37, J3, J55, J78, H37, and H50.

Figure 41. The primers and PCR fragments to confirm 3751

- bp EcoR1/Scal fragment of DNA insert

### J.R. Simplot Company Characterization of Inserted DNA 61

Figure 42. Confirmation of a 3751

- bp internal fragment of DNA inser t in E, F, J, and H lines

Bands on the ethidium bromide

- stained gels are indicative for the presence of fragment A and B . Data for all events, except for G11 , are shown. M = one kb DNA marker lane (invitrogen), PC = positive control, wt = untransformed recipient variety (“wild type”, the negative control).

### J.R. Simplot Company Characterization of Inserted DNA 62 SUMMARY

Event s F10, F37, E12, and E24 contain a single DNA insert comprising all sequences needed for expression of both the Asn1 / Ppo5 gene silencing cassette and R1/PhL promoter silencing cassett e.

Events J78 and G11 contain a single DNA insert comprising the sequences needed for expression of the

Asn1 / Ppo5 gene silencing cassette. These two events do not contain all sequences required for expression of the R1/PhL promoter silencing cassette.

E vents J3, J55, and H50 contain 2 copies of the DNA insert , one of which is full and the other one lacking at least part of the R1/PhL promoter silencing cassette.

Event H37 contains one full copy of the DNA insert and three additional DNA insert fragments

.

### J.R. Simplot Company Characterization of Inserted DNA 63 MATERIALS AND ME T HODS

Plant M aterial. Plants used for DNA gel blot analysis were grown for two months in Sunshine mix

- 1 (www.sungro.com) in two

- gallon pots in a greenhouse that was controlled for temperature (18°C minimum/27°C maximum) and light (16

- h photoperiod with an intensity of ~1500 μmol/m2/s).

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a mortar using liquid nitrogen. The ground tissue was transferred to a pre

- cooled 15 ml conical tub e with

a pre

- cooled spatula and stored at

- 80 o 

### 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 1 5 min at room temperature. The pellet was thoroughly re

- suspended in 2 ml extraction buffer containing 200 μg RNase A. After incubating the resuspended DNA at 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 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 preci pitated 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 runni ng 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 p H 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 followed by centrifugation at 3,000 rpm for 15 min. DNA was extracted twice with equal volumes of chloroform: isoamyl alcohol (24:1) by shaking for 10 min and centrifug ed 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 dried in air and dissolved in TE buffer. The quality and concentration of DNA was determined spectrop hotometrically and by running a sample on a 0.8% agarose gel. C ontrol and event DNA was always extracted with the same method for a specific comparison.

DNA Gel (Southern) Blot Analysis. 10 μg of plant DNA was digested overnight in 400 μl final volume

wit h at least 5 μl (10 units/μl) restriction enzyme (Invitrogen) at 37 o 

### C. Digested DNA was concentrated 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

- ED TA ( TBE ) buffer and 3

- 5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 hrs. The gel was photographed using a gel documentation system from Alpha Innotech (Santa Clara, California), and then depurinated by submerging it into 0.25 N HCl for 20 mi n. After subsequent denaturing 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 mem brane was carried out using 10X SSC according to a standard capillary
transfer method

.

Probe Preparation. The labeling of the PCR

- derived probe was achieved using Hotmaster taq enzyme and buffer (Fisher BioReagents) according to Roche’s DIG labeling ins tructions. A standard 50 μl reaction consisted of 5 μl 10 x Hotmaster Taq Buffer, 2

- 5 μl 10 uM forward primer, 2

- 5 μl 10 μM reverse primer, 5 μl DIG labeled dNTP (Roche), 10 ng plasmid template, 0.75 μl Hotmaster Taq polymerase, and

### J.R. Simplot Company Characterization of Inserted DNA 64

water. The PCR amplifi cation 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 slo wer 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 4 3

.

Figure 4 3

. DNA Insert Probes

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII 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 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 bottle in a standard hybridization oven (Amerex Instruments Inc.) at 20

- 25 rpm. Hybridization was carried out by replacing the prehybridization
buff er 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 to 3 times. The reused hybridization solution was heated

at 68 o 

### C for 10 minutes before use.

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 for 10 min at r oom 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. Thi s was followed by a brief rinse with 2X SSC to remove SDS. The membrane was rinsed with 150 ml of 1X DIG Washing Solution (Roche) in a tray for 2 min and incubated in 1X Blocking solution (Roche) for 0.5

- 3 hrs on a low

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

- DIG

- alkaline phosphate conjugate with 1X Blocking solution) for 30 min on a shaker. The membrane was washed twice (15 min each) with 1X DIG Washing Solution (Roche) and equilibrated with 1 X detection buffer. The detection reaction was
carried out with 2 ml CDP

- Star solution (1:100 diluted stock of CDP

- Star with 1X detection buffer) for 5 min. The membrane was wrapped in a plastic film and exposed to the Z

- ray film in the dark. Depending on the experiment

, multiple exposures 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.

### J.R. Simplot Company Characterization of Inserted DNA 65

Explanation of Bands Visualized by DNA Gel Blot Ana lysis

. DNA gel blot analysis was performed using DNA extracted from greenhouse

- grown plants and digested with the following restriction enzymes: (1)

Eco RV, (2) Hin d III, and (3) Eco RI + Sca I. The positions of the restriction sites and probes employed are
sh own in Figure 4 3 . The digested DNA was run with standard DIG

- labeled molecular weight markers, DIGII and DIGVII, through 0.7% agarose gels in 0.5X TBE buffer and blotted onto Nylon Hybond N+ filters.
The four DNA insert

- derived probes used were AGP

, PHL

, A SN , and GBS (see Table 1 4 ). A dditional probes used were AGP 2 and AGP 3. Each of the original films from hybridization experiments contained, apart from the 2 markers, three DNA digests of the untransformed control and the events for one variety. To facilita te interpretations, the original films were separated into three separate segments (one for each digest) with the marker lanes copied and boxed.

When reading the bands sizes in DNA gel blots, such observations are subject to interpretation by the

reviewe r and slight variation could occur between the expected and observed sizes

. The observed sizes could be influenced by many factors including the sample matrix or slight differences in gel running

conditions. Therefore

, other information was considered

, inc luding the expected sizes and observations from other gels and digests when reporting the sizes of the bands.

Table 1 4 . Molecular P robes

Probe (bp) Primer

name Primer sequence Genetic
Elements Position in

pSIM1278 Size of hybridizing bands

Eco RV Hin dIII Eco RI

- Sca I

AGP (511) AGP 3F

AGP 3R CCAAAACTGAAAGTGGGAAACC

ACAACAACCCCCTCCTTGTG AGP promoter fragment 17

- 527

4,247

- 4,757 OB : several

IB : 2,293

JB : 1,127+ OB : several

IB : 4230

JB : 2443+ OB : several

IB : 3751

JB : 3717+

ASN (549) HD076F2

HD076R2 GAAC TCTTTATCCAGAAATG

TAGTCTCTATTGAATCTGCT ASN 1 and PPO 5 gene fragments 2,262

- 2,810

2,974

- 3,523 OB : several

IB : 712, 2293

JB : none OB : several

IB : 4230

JB : none OB : several

IB : none
JB : 3717+

PHL (436) PhoLF

PhoLR AAAGTGTGAAGGGTATTTTTGTAAAC

CGAATGAGAGTGATAAGAG AGTGAGG PHL promoter fragment 6,518

- 6,953

8,376

- 8 ,811 OB : several

IB : none
JB : 3567+ OB : several

IB : 1313

JB : 2163+ OB : several

IB : 3751

JB : 1903+

GBS (546) HD077F

HD077R TGTGGTCTACAAAAAGGGGAAT

GGTAGCGGTAGGAGGGAGTT G bss promoter fragment 3,536

- 4,081

8,845

- 9,390 OB : several

IB : 2293

JB : 3567+ OB : several

IB : 4230

JB : 2163+ OB : several

IB : 3751

JB : 1903+

AGP 2 (402) HY1659

HY1660 ACAAGGAGGGGGTTGTTGTACT

CTTGATGTAAACACCGAATACGA AGP promoter fragment 509

- 910

4,739

- 5,140 OB: several

IB: 2,293 JB: 1,127+

AGP 3 (999) HY1661

HY1662 TAAATTGGACGGACGATGAAAT

CGGAATTTA CGAGAGGACCATA AGP promoter fragment 1,130

- 2,128

5,360

- 6,358 OB: several

IB: 1, 225, 1,225

JB: none



### PCR Confirmation of EcoR1/Scal Fragments in E, F, J, and H lines. The genomic DNA was isolated from the leaves of events E12, E24, F10, F37, J3, J55, J78, H37, and H50. The primers used for the confirmation of EcoR1/Scal fragments in E, F, J, and H lines were described in Figure 41

. The PCR was carried out in 30 μl of PCR reaction mixture consist ing of 3 μl of 10X PCR buffer, 0.6 μl of 10 mM dNTP, 0.6 μl of 10 μM forward primer, 0.6 μl of 10 μM reverse primer, 100 ng of genomic DNA template, and 0.2 μl of HotMaster Taq polymerase (Fis her BioReagents). The PCR amplification conditions were as follows: 1 cycle of 3 min at 95 °C followed by 40 cycles of 30 sec at 94 °C, 30 sec at 55°C, 2 min at 68 °C, and finishing with 10 min at 68 °C.

### J.R. Simplot Company Characterization of Inserted DNA 66 CONCLUSION S

Event Ranger Russet F10 contains a sin gle copy of the DNA insert . As shown in Appendix 6. Field
Performance and Tuber Evaluations and Appendix 9. Compositional Analyses

, this insertion is not linked to any unexpected event

- specific agronomic or biochemical changes.

Event Ranger Russet F37 co ntains a single copy of the DNA insert

. T his insertion is not linked to any unexpected event

- specific agronomic or biochemical changes.

Event Russet Burbank E12 contains a single copy of the DNA insert

. T his insertion is not linked to any unexpected event

- specific agronomic or biochemical changes.

Event Russet Burbank E24 contains a single copy of the DNA insert with the exception of a 0.5

- kb deletion at the Left Border side. This deletion does not affect construct efficacy. T his insertion is not
linked t o any unexpected event

- specific agronomic or biochemical changes.

Event Atlantic J3 contains one nearly intact copy of DNA insert with a small deletion at the Left Border site. Attached to this first copy is a second copy in a head

- to

- head orientation, wh ereby the second copy
also had a small deletion at the Left Border site and a much larger deletion, comprising the R1/PhL
promoter silencing cassette, at the Right Border site. This insert arrangement resulted in a junction between part of the Agp promoter and flanking plant DNA that is unlikely to result in expression of a new open reading frame because the partial Agp promoter lacks the TATA

- box, CCAAT

- box, and additional 5’ elements needed to induce transcription. T he insertions are not linked to any un expected

event

- specific agronomic or biochemical changes.

Event Atlantic J55 contains one full copy of the DNA insert and an additional truncated copy lacking the R1/ PhL promoter silencing cassette. The two copies integrated at the same site as an inverte d repeat.

T he insertions are not linked to any unexpected event

- specific agronomic or biochemical changes.

Event Atlantic J78 contains a single truncated DNA insert . The insert has a small deletion at the Left Border that did not affect efficacy of the as sociated Asn1/Ppo5 gene silencing cassette. A larger deletion
occurred at the Right Border site that included one of the promoters associated with the R1/ PhL
promoter silencing cassette , and loss of efficacy of that cassette. T his insertion is not linked t o any unexpected event

- specific agronomic or biochemical changes.

Event G G11 contains a single DNA insert insert with a large deletion at the Right Border site that includes the R1/ PhL silencing cassette and results in a junction between the Agp promoter and flanking plant DNA. This flanking DNA does not contain known genes or new ORFs in the vicinity of the Agp promoter (as deduced by BLAST analysis of a 2.5

- kb flanking DNA fragment, see Appendix 4. Junction Analysis and Event

- Specific Detection , indicating that it is unlikely for the insert to have altered the expression pattern of one or more plant genes. T his insertion is not linked to any unexpected event

specific agronomic or biochemical changes.

Event H H37 contains one complete DNA insert insert and three DNA insert fragment s. One of the

inserted fragmen ts consists of a small part of the Agp promoter of the DNA insert . This particular insertion may have resulted in the operable fusion of a promoter and open reading frame. However, if
such a fusion did occur, it neither affected plant agronomics nor tuber biochemistry. The second truncated fragment lacks the Asn1/Ppo5 gene silencing cassette except for the Gbss promoter. The

### J.R. Simplot Company Characterization of Inserted DNA 67

remaining Gbss promoter is not functional ly active because it lacks the 5’ sequences carrying CCAAT and

TATA boxes. The fourth small fragment consists of a small part of the Agp promoter that is, for the same reasons, not functionally active. The insertions are not linked to any unexpected event

- specific agronomic or bioc hemical changes.

Event H H50 contains one full copy of the DNA insert and an additional truncated copy lacking the R1/ PhL promoter silencing cassette. The junction created by insertion of the truncated DNA insert
results in fusion of the Agp promoter with flanking plant DNA. It is possible that this fusion could result in the expression of an open reading frame. However, if such expression did occur, it neither affected plant agronomics nor tuber biochemistry. The insertions are not linked to any unexpecte d event

- specific agronomic or biochemical changes.

### J.R. Simplot Company Characterization of Inserted DNA 68 R EFERENCES

Chawla R, Shakya R, Rommens CM (2012) Tuber specific silencing of asparagine synthetase

- 1 reduces the acrylamide

- forming potential of potatoes grown in the field without affecting tuber shap e and yield. Plant Biotechnol J, 10: 913

- 924.

Van Haaren MJJ, Sedee NJA, de Boer HA, Schilperoort RA, Hooykaas PJJ (1989) Mutational analysis of the
conserved domains of a T

- region border repeat of Agrobacterium tumefaciens. Plant Mol Bio,

13: 523

- 531.