J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 1 Appendix 3

Evidence for Stability of the Inserted DNA

INDEX

Section Page

Index 1

In troduction 2

Abbreviations and Definitions 2

Results 3

Discussion 4 5

Materials and Methods 4 6

Conclusion s 49

Reference s 5 0

Certification 5 1J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 2 INTRODUCTION

The stability of the DNA inserts was evaluated in the original transformants and again in propagated

plant material using both DNA gel blot hybridization and a trait evaluation assa y

. These studies were

carried out to ensure that the desired genetic changes in events remained stable over multiple clonal

cycles and expressed the traits consistently over generations. Inconsistent trait expression

generationally might be triggered by r are recombination events or could also be caused by methylation

. Because potatoes are clonally propagated , s tandard assessments for sexually propagated crops we re

not directly applicable . T ubers rather than seeds were used to define subsequent generations since

tubers are what is commercially planted

. Results of DNA blot hybridization demonstrate consistent

bands were present in three generatiuons of plants, indicating stability of the traits

. Further evidence

for stability was obtained by confirming trai t efficacy in three generations of plants ( tuber s) using a

catechol assay for polyphenoly oxidase activity by visually evaluat ing the darkening of tuber flesh

.

In some cases, Southern blots shown in this appendix will use the same probes and enzyme diges ts as

found in other studies (for example, see Appendix 1. Characterization of Inserted DNA ). The

observations may differ, depending upon the conditions under which the gels were run. Therefore all

bands described in the tables representing Southern blot d ata in this appendix refer to the gels

presented within this appendix.

ABBREVIATIONS and DEFINITIONS

G0 In itial generation of tubers utilized from tissue culture

G1 First generation field grown plant (after tissue culture)

G2 Second generation field grow n plant

G3 Third generation field grown plant

wt “wild type”, this term is used to refer to the untransformed control of a given variety

MII Molecular size marker used in lane 1 of each 0.7% agarose gel (approximate range 2.0

- 23.1 kb )

M VII Molecular size m arker used in lane 2 of each 0.7% agarose gel (approximate range 1.2

- 7.7 kb)J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 3 RESULTS

DNA Insert Stability in Ranger Russet Events F10 and F37

The GBS and AGP probes (see Figure 38 in Materials and Methods) were hybridized with DNA of the

Ranger Russet co ntrols and events F10 and F37 and evaluated over 3 generations. Hybridization was

used to visualize three common bands present in the unmodified control Ranger Russet variety (wt) and

the F10 and F37 events. The common bands were visualized on 0.7% agaros e gels with sizes of 7.4 kb,

7.7 kb, and 9.4 kb for the GBS probe and 1.2 kb, 4.9 kb, and 6.2 kb for the AGP probe ( Figures 1 , 2, 3, 4,

5, and 6 )

.

Two additional bands hybridized with the GBS probe were observed in F10 and F37 , one 2. 3 kb band

indicativ e of an internal DNA insert fragment and the other one a junction fragment (7. 1 kb for F10 and

7.7 kb for F37) . These were observed in all G0 , G1

, G2

, and G3 samples , indicating that the insert s of the

original transformant (G0) remained stable into the f irst

, second , and third vegetative generations

( Figure s 1 , 2, and 3 )

. The AGP probe visualized both an IB (internal band) of 2. 3 kb and a JB (junction

band) of 6. 5 kb for F37 in G0 , G1, G2 , and G3 ( Figures 4 , 5, and 6 ). Additionally the AGP probe

hybrid ized with a 2. 6 kb fragment for all F10 samples ( Figures 4 , 5, and 6 ).

Additional evidence for DNA insert stability and black spot bruise efficacy in Ranger Russet events F10

and F 37 was obtained with a catechol assay for Ppo activity

. The results with G2 tubers showed all

Ranger Russet F10 and F37 tubers remained uncolored, indicative of resistance to black spot bruise

( Figure 7 ) through Ppo5 gene silencing.

Overall, hyridization ( Figures 1 , 2, 3, 4, 5, and 6 ) with the GBS and AGP probes show s that ev ents F10

and F3 7 had consistent bands over multiple generations by comparing G0 with G1, G2 and G3 samples

. Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing ( Figure 7) from

the initial generation through G2.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 4

Figure 1

. F10 and F37: DNA Insert S tability in G0 and G1 Samples as V isualized with a GBS P robe

Ranger Russet untransformed controls (wt), F10, and F37 all show consist e ncy with 7. 1 kb, 7.7 kb, and

9.4 kb size bands in the G0 and G1 plants. Additionally, F10 and F37 share a band the size of 2. 3 kb in

both the G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 5

Figure 2

. F 10 and F37: DNA Insert S tability in G2 Samples as V isualized with a GBS P robe

Ranger Russet untransformed controls (wt), F10, and F37 all show consist e ncy with 7. 1 kb, 7.7 kb, and

9.4 kb size bands in the G0 and G2 plants. Additionally, F10 and F37 share a band the size of 2. 3 kb in

both the G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 6

Figure 3

. F10 and F37: DNA Insert S tability in G3 Samples as V isualized with a GBS P robe

Ranger Russet untransformed controls (wt), F10, and F37 all show consist e ncy with 7. 1 kb, 7.7 kb, and

9.4 kb size bands in the G0 and G3 plants. Additionally, F10 and F37 share a band the size of 2. 3 kb in

both the G0 and G3 plants.



### M M wt wt wt F10 F10 F10 F37 F37 F37

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2 .3

2.0

1.5 23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.2J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 7

Figure 4

. F 10 and F37: DNA Insert S tability in G0 and G1 Samples as V isualized with an AGP P robe

Ranger Russet untransformed controls (wt), F10, and F37 all show consist e ncy with 1.2 kb, 4.9 kb, and

6. 5 kb size bands in the G0 and G1 plants. F10 and F37 share a band the size of 2. 3 kb in both the G0 and

the G1 plants. F10 also has a band the size of 2.5 kb which is consistent in the G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 8

Figure 5

. F10 and F37: DNA Insert S tability in G2 Samples as V isualized with an AGP P robe

Ranger Russet untransf ormed controls (wt), F10, and F37 all show consistency with 1.2 kb, 4.9 kb, and

6. 5 kb size bands in the G0 and G2 plants. F10 and F37 share a band the size of 2. 3 kb in both the G0 and

the G2 plants. F10 also has a band the size of 2.5 kb which is consis tent in the G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 9

Figure 6

. F10 and F37: DNA Insert S tability in G3 Samples as V isualized with a n AGP P robe

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 1.2 kb, 4.9 kb, and

6. 5 kb size bands in the G0 and G3 plants. F10 and F37 share a band the si ze of 2. 3 kb in both the G0 and

the G3 plants. F10 also has a band the size of 2.5 kb which is consistent in the G0 and G3 plants.



### M M wt wt wt F10 F10 F10 F37 F37 F37

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2 23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.0J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 10 Figure 7

. Assay for Sustained Ppo5 G ene Silencing in G2 Tubers of F10 and F37J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 11 DNA Stability in Russet Burbank Events E12 and E24

Hybridization of DNA of Russet Burbank E12 and E24 plants visualizes two common bands with the GBS

probe (7. 5 and 8. 5 kb) ( Figures 8 , 9, and 10 ) and three with the AGP probe ( 1. 5

, 4. 4 and 5.2 kb) in all

lanes ( Figures 11 , 12, and 13 ) . These bands are indicative of the unmodified genome. Two additional

bands in DNA of all event material, one 2. 3 kb band indicative of an internal DNA insert fragment and

the other o ne representing a DNA insert junction fragment (4. 5 kb for E12 with GBS, ~ 23 kb for E24 with

GBS, 2. 2 kb for E12 with AGP, and ~ 20 kb for E24 with AGP ) , indicate that the insert s of the original

transformant (G0) remained stable into the first

, second , and third vegetative generations G1

, and G2

, and G3 ( Figure s 8 , 9, and 10 ).

Additional evidence for DNA insert stability in events E12 and E24 was obtained with the catechol assay

for Ppo activity , using tubers from the second generation

. As shown in Figure 14

, the G2 tubers did not

brown , except for the untransformed control material

. This demonstrated that the events for each

variety had retained their ability to silence the Ppo5 gene in tubers, after two years of field trials.

Preparation of the field tria l material had taken the events through repeated tissue culture and

vegetative generations without affecting the stability of the inserted genetic material. Note: Slight

differences in flesh color of the event tubers is from natural variation and is not r elated to Ppo silencing.

Overall, hyridization ( Figures 8 , 9, 10, 11, 12, and 13 ) with the GBS and AGP probes show s that events

E12 and E24 had consistent bands over multiple generations by comparing tubers of G0 with G1, G2 and

G3. Also, the visual assa y for PPO with catechol ( Figure 14 ) confirmed stability of Ppo5 gene silencing

from the initial generation through G2.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 13

Fi gure 9

. E12 and E 24: DNA Insert S tability in G2 Samples as V isualized with a GBS P robe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 7. 5 kb and 8. 5 kb

size bands in the G0 and G 2 plants. E12 and E24 share a band the size of 2. 3 kb in both the G0 and the

G 2 plants. E12 has a 4. 5 kb band and E24 has a ~ 23 kb band in the G0 and G 2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 14

Figure 10

. E12 and E24: DNA Insert S tability in G3 Samples as V isualized with a GBS P robe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 7. 5 kb and 8. 5 kb

size bands in the G0 and G3 plants. E12 and E24 share a band the size of 2. 3 kb in both the G0 and the

G3 plants. E12 has a 4. 5 kb band and E24 has a ~ 23 kb band in the G0 and G3 plants.



### M M wt wt wt E12 E12 E12 E24 E24 E24

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2 23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.0

0.7J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 16

Figure 12

. E12 and E 24: DNA Insert S tability i n G2 Samples as V isualized with an AGP P robe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 1. 5 , 4. 4 , and 5.2 kb

kb size bands in the G0 and G2 plants. E12 and E24 share a band the size of 2. 3 kb in both the G0 and

t he G2 plants. E12 has a 2. 2 kb band (slightly below the 2.2 kb band) and E24 has a ~2 0 kb band in the

G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 17

Figure 13

. E12 and E24: DNA Insert S tability in Samples as V isualized with an AGP P robe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 1. 5 , 4. 4 , and 5.2 kb

(very light bands) size bands in the G0 and G3 plants. E12 and E24 share a band the size of 2. 3 kb in both

the G0 and the G3 plants. E12 has a 2. 2 kb band (slightly b elow the 2.2 kb band) and E24 has a ~2 0 kb

band in the G0 and G3 plants.



### M M wt wt wt E12 E12 E12 E24 E24 E24

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3

2. 8

2.3

2.0

1.5 23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 18 Figure 14

. Assay for Sustained Ppo5 G ene Silencing in G2 Tubers of E12 and E24J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 19 DNA Insert Stability in Events J3, J55 and J78

Hybridization of DNA of Atlantic, J3, J55 and J78 plants visualizes three common bands with the GBS

probe (7. 0 , 7.8, and 8.6 kb ) and four with the AGP probe (1.4 , 2.0, 5.0, and 7.2 kb ) in all lanes ( Figures

15 , 16, 17, 18, 19, and 20 )

. These bands are indicative of DNA fragments of the unmodified genome.

Three additional bands in DNA of J3, one 2. 3 kb band indicative of an internal DNA insert fragment and

two others representing DNA insert junction fragments (5. 8 and ~1 2 kb with GBS and 1.6 and 5.7 kb

with AGP) , indicate that the insert s of the original transformant (G0) remained stable into the first

, second, and third vegetative generations ( Figures 15 , 16, 17, 18, 19, and 20 )

. Similarly, J55 contains the

internal 2. 3 kb internal band and additional bands (2.3 and 4.1 kb with GBS and 1.95, 2.3 and 4.8 k b with

AGP) that are identical in G0, G1, G2

, and G3 s amples ( Figures 15 , 16, 17, 18, 19, and 20 )

. Event J78 also

contains the internal 2. 3 kb DNA insert fragment in all generations and a junction fragment that only

hybridizes with the AGP probe (~9 kb) ( F igures 18, 19, and 20 ) . There is no junction fragment with the

GBS probe because the J78 insert had a DNA insert deletion of the Gbss promoter at the Left Border site

( as described in the Characterization of Inserted DNA study ).

The Atlantic intrangenic G2

- tubers could not be assayed for Ppo activity because cut tuber surfaces of

this untransformed variety do not develop a brown precipitant when exposed to catechol. It is possible

that they do not produce the enzyme that catalyzes the oxidation of polypheno ls. Instead, a dditional

evidence for DNA insert stability in events J3, J55 and J78 was obtained with a PCR test using primers

HD121F and HD121R (see Figure 39 in Materials and Methods ) , which demonstrated that all Samples

analyzed contained the same ampli fied 0.8 kb product (part of the A sn 1/ Ppo5 gene) and showing

stability of the DNA insert in G2 Samples ( Figure s 21 and 22 )

. Although G0 Samples were not available

for this test, the presence of part of the A sn 1/ Ppo5 gene indicates insertion stability in G2 Samples

.

Overall, hybridization ( Figures 1 5, 16, 17, 18, 19, and 20 ) with the GBS and AGP probes show s that

events J3, J55, and J78 had consistent bands over multiple generations by comparing s amples of G 0 with

G1, G2 and G3. Also, PCR testing ( Figure s 21 and 22 ) showed amplification of the same 788 bp DNA

insert fragment in all G2 events, that was not found in the control Atlantic samples , indicating that the

DNA insert was stable through at least the second generation

.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 20

Figure 15

. Atlantic J3, J55, J78: DNA Insert S tability in G0 and G1 Samples as V isualized with a GBS

P robe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size

bands in the G0 and G1 plants. J3 and J78 share a band the size o f 2. 3 kb in both the G0 and the G1

plants. Additionally, J3 has 5. 8 kb and ~1 2 kb bands in the G0 and G1 plants. J55 has bands of 2.3 kb and

4.1 kb in the G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 21

Figure 16

. Atlantic J3, J55, J78: DNA Insert S tability in G2 Samples as V isualize d with a GBS P robe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size

bands in the G0 and G 2 plants. J3 and J78 share a band the size of 2. 3 kb in both the G0 and the G 2

plants. Additionally, J3 has 5. 8 kb and ~1 2 kb bands in the G0 and G 2 plants. J55 has bands of 2.3 kb and

4.1 kb in the G0 and G 2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 22

Figure 17

. Atlantic J3, J55, J78: DNA Insert S tability in G3 Samples as V isualized with a GBS P robe 1

1 Tubers w ere unavailable for this test so leaves from third generation plant s were used

. Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size

bands in the G0 and G3 plants. J3 and J78 share a band the size of 2. 3 kb in both the G0 and the G 3

plants. Additionally, J3 has 5. 8 kb and ~1 2 kb bands in the G0 and G 3 plants. J55 has bands of 2.3 kb and

4.1 kb in the G0 and G 3 plants.



### M M J3 J3 J3 J55 J55 J55 J78 J78 J78 WT WT WT

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2

1.0

0.7

0.5 23.1

9.4

8.6

7.4

6. 6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 23

Figure 1 8

. Atlantic J3, J55, J78: DNA Insert S tability in G0 and G1 Samples as V isu alized with an AGP

P robe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb,

and 7.2 kb size bands in the G0 and G 1 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G1 plants. J3

and J78 share a band the size of 2. 3 kb in both the G0 and the G 1 plants. Additionally, J 55 has bands the

size of 1.95 , 2.3, and 4.8 kb in G0 and G1 plants. J78 has a band the size of ~9 kb in G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 24 Figure 1 9

. Atlantic J3, J55, J78: DNA Insert S tabil ity in G2 Samples as V isua lized with an AGP

P robe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb,

and 7.2 kb size bands in the G0 and G2 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G2 p lants. J3

and J78 share a band the size of 2. 3 kb in both the G0 and the G2 plants. Additionally, J55 has bands the

size of 1.95, 2.3, and 4.8 kb in G0 and G2 plants. J78 has a band the size of ~9 kb in G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 25

Figure 20

. Atlanti c J3, J55, J78: DNA Insert S tability in G3 Samples as V isualized with an AGP P robe 1

1 Tubers were unavailable for this test so leaves from third generation plant s were used

. Atlantic untransformed controls (wt), J3, J55, and J 78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb,

and 7.2 kb size bands in the G0 and G2 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G2 plants. J3

and J78 share a band the size of 2.2 kb in both the G0 and the G2 plants. Additionally, J55 has bands the

size of 1.95, 2.3, and 4.8 kb in G0 and G2 plants. J78 has a band the size of ~9 kb in G0 and G2 plants.



### M M J3 J3 J3 J55 J55 J55 J78 J78 J78 WT WT WT

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2

1.0

0.7

0.5 23.1

9.4

8.6

7.4

6. 6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 26

Figure 2 1

. PCR

- B ased C onfirmation of I nsert S tability in G2 T ubers of Events J3, J55 and J78

The first lane of each gel contains At lantic controls labeled JC, followed by a 1 k b DNA ladder

(Invitrogen)

. The subsequent 8 lanes contain DNA from non

- systematically chosen G2 tubers from

Hancock, Parma, Cody, and Aberdeen. Headings above each gel correspond to the site location where

the tubers were grown. A consistent band of 788 b p of DNA insert was amplified from the G2 tubers,

but not controls, indicating stable integration of the DNA insert

.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 27

Figure 2 2

. PCR

- B ased C onfirmation of I nsert Stability in G2 T ubers of Events J3, J55 and J7 8

The first lane of each gel contains Atlantic controls labeled JC, followed by a 1 kb DNA ladder

(Invitrogen). The subsequent 8 lanes contain DNA from non

- systematically chosen G2 tubers from

Othello, Lakeview, and Larimore. Headings above each gel correspond to the site location where the

tubers were grown. A consistent band of 788 bp of DNA insert was amplified from the G2 tubers, but

not controls, indicating stable integration of the DNA insert

.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 28 DNA Insert Stability in Event G1 1

Hybridizati on of DNA of the G variety and G1 1 plants visualizes two common bands with the GBS probe

(8.2 and ~12 kb) and four with the AGP probe (1.45, 2.1, 5.3, and 8.6 kb) in all lanes. These bands are

indicative of DNA fragments of the unmodified genome. One addit ional internal DNA insert band of 2.2

kb that hybridizes with both the GBS and AGP probes indicate s that the insert of the original

transformant (G0) remained stable into the first , second, and third vegetative generations G1

, G2, and

G3 ( Figure s 2 3 , 24, 25 , 26, 27, and 2 8 ). An additional 5.2 kb band only hybridizing with the AGP probe

represent ed a DNA insert junction fragment and confirm ed the insert stability.

Additional evidence for DNA insert stability in event G1 1 was obtained with the catechol assay f or Ppo

activity

. As shown in Figure 2 9 , all event tubers remained uncolored, whereas the untransformed

control material developed a deep brown color

. This demonstrated that the G1 1 event had retained

their ability to silence the Ppo5 gene in tubers, after two years of field trials. Preparation of the field trial

material had taken the events through repeated tissue culture and vegetative generations without

affecting the stability of the inserted genetic material.

Overall, hyridization ( Figures 23, 24, 25, 26, 27, and 28 ) with the GBS and AGP probes show s that event

G11 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and G3.

Also, the visual assay for PPO with catechol ( Figure 29 ) confirmed stability of Ppo5 gene silen cing from

the initial generation through G2.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 29

Figure 2 3

. G1 1: DNA Insert S tability in G0 and G1 Samples V isualized with a GBS P robe 1

1 GC = control G ; G1

- 1 and G1

- 2 refer to leaves from 2 independent G1 plants

G variety untransformed co ntrols (wt) and G 11 show consistency with 8.2 kb and ~12 kb size bands in

the G0 and G 1 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 30

Figure 2 4

. G1 1: DNA Insert S tability in G2 Samples as V isualized with a GBS P robe 1

1 GC = G C ontrol

G variety untransformed controls (wt) and G11 show consistency with 8.2 kb and ~12 kb size bands in

the G0 and G 2 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G2 plants. 23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48 6.1

1.95

1.88 

### M M GC GC GC G11 G11 G 11

II VII G0 G2 G2 G0 G2 G2J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 31 Figure 2 5

. G11: DNA Insert S tability in G3 Sam ples as V isualized with a GBS P robe

1 GC = control G

G variety untransformed controls (wt) and G11 show consistency with 8.2 kb and ~12 kb size bands in

the G0 and G 3 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G3 plants.



### M M wt wt wt G11 G11 G11

II VII G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5 23.1

9.4

8.6

7.4

6. 6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 32

Figure 2 6

. G1 1: DNA Insert S tability in G0 and G1 Samples V isualized with an AGP P robe 1

1 GC = control G

, G1

- 1 and G1

- 2 refer to samples from 2 independent G1 plants

G variety untransformed controls (wt) and G11 show co nsistency with 1.45 kb, 2.1 kb, 5.3 kb, and 8.6 kb

size bands in the G0 and G 1 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0

and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 33 Figure 2 7

. G1 1: DNA Insert S tability in G2 Samples as V isualized with an AGP P robe 1

1 GC = control G

G 2

- 1 and G 2

- 2 refer to samples from 2 independent G 2 plants

G variety untransform ed controls (wt) and G11 show consistency with 1.45 kb, 2.1 kb, 5.3 kb, and 8.6 kb

size bands in the G0 and G 2 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0

and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 34 Figure 28. G1 1: DNA Insert S tability in G3 Samples as V isualized with an AGP P robe

G variety untransformed controls (wt) and G11 show consistency with 1.45 kb, 2.1 kb, 5.3 k b, and 8.6 kb

size bands in the G0 and G 3 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0

and G3 plants. 

### M M wt wt wt G11 G11 G11

II VII G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2 23.1

9.4

8.6

7.4

6. 6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 35 Figure 2 9

. Assay for Sustained Ppo5 G ene Silencing in G2 Tubers of Event G1 1J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 36 DNA Insert Stability in Events H37 and H50

Hybridization of DNA of the H variety as well as H37 and H50 plants visualizes two common bands with

the GBS probe (7.8 and 10.0 kb) and five with the AGP probe (1.4, 2.1, 5.4, 6.7 , and 8.0 ) in all lanes.

These bands are indicative of DNA fragment s of the unmodified genome. Three additional GBS

hybridizing bands in DNA of H37, one 2. 3 kb band indicative of an internal DNA insert fragment and two

others representing DNA insert junction fragments (3.0 and 4.5 kb) , indicate that the insert s of the

ori ginal transformant s (G0) remained stable into the first

, second , and third vegetative generations G1

,

G2 , and G3 ( Figure s 30 , 31, 32, 33, 34, and 3 5 ). Similarly, there are three additional bands consistently

visualized with the AGP probe in H37 DNA (2.0 , 3 .2 and 3.6 kb).

Event H50 also contains the 2.3 kb internal DNA insert fragment in all generations. Additionally, it carries

a single junction fragment hybridiz ing with the GBS probe (6.5 kb) and two with the AGP probe (1.9 and

3.8 kb) , as was expected ba sed on the DNA insert organization of this event

.

Additional evidence for DNA insert stability in events H37 and H50 was obtained with the catechol assay

for Ppo activity

. As shown in Figure s 3 6 and 3 7 , all event tubers remained uncolored, compared with the

dark

- colored untransformed contr ol material. This demonstrated that the se events for this variety had

retained their ability to silence the Ppo5 gene in G2 tubers, after two years of field trials. Preparation of

the field trial material had taken the e vents through repeated tissue culture and vegetative generations

without affecting the stability of the inserted genetic material.

Overall, hyridization ( Figures 30, 31, 32, 33, 34, and 35 ) with the GBS and AGP probes show s that events

H37 and H50 had con sistent bands over multiple generations by comparing samples of G0 with G1, G2

and G3. Also, the visual assay for PPO with catechol ( Figure s 36 and 37 ) confirmed stability of Ppo5

gene silencing from the initial generation through G2.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 37

Figure 30

. H37 and H 50 : DNA Insert S tability in G0 and G1 Samples V isualized with a GBS P robe

MII M VII WT WT WT H37 H37 * H37 H50 H50 H50

G0 G1 G1 G0 G1 G1 G0 G1 G1

* Background smear in the lane marked “H37 G1 ” at 1.7 kb is not a band.

H variety untransformed controls (wt) , H37, and H50 show consistency with 7.8 kb and 10.0 kb size

bands in the G0 and G 1 plants. H37 and H50 have one additional band of 2. 3 kb in size in G0 and G1

plants. Additionally, H3 7 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a

size of 6.5 kb in G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 38

Figure 3 1

. H37 and H 50 : DNA Insert S tability in G0 and G2 Samples V isualized with a GBS P robe

MII M VII WT WT WT H37 H37 H 37 H50 H50 H50

G0 G2 G2 G0 G2 G2 G0 G2 G 2

H variety untransformed controls (wt) , H37, and H50 show consistency with 7.8 kb and 10.0 kb size

bands in the G0 and G 2 plants. H37 and H50 have one addit ional band of 2. 3 kb in size in G0 and G2

plants. Additionally, H37 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a

size of 6.5 kb in G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 39

Figure 3 2

. H37 and H50: DNA Insert S tability in G3 Samples as V is ualized with a GBS P robe

H variety untransformed controls (wt) , H37, and H50 show consistency with 7.8 kb and 10.0 kb size

bands in the G0 and G 3 plants. H37 and H50 have one additional band of 2. 3 kb in size in G0 and G3

pl ants. Additionally, H37 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a

size of 6.5 kb in G0 and G3 plants.



### M M wt wt wt H37 H37 H37 H50 H50 H50

II VII G 0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5 23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 40

Figure 3 3

. H37 and H50 : DNA Insert S tability in G0 and G1 Samples V isualized with an AGP P robe

MII M VII WT WT WT H37 H37 H37 H50 H50 H50

G0 G1 G1 G0 G1 G1 G0 G1 G 1

H variety untransformed controls (wt) , H37, and H50 show consistency with 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb siz e bands in the G0 and G 1 plants. H37 contains three additional bands of 2.0 kb, 3. 2 kb,

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G1 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 41

Figure 3 4

. H37 and H 50 : DNA Insert S tability in G0 and G2 Samples V isualized with an AGP P robe

MII M VII WT WT WT H37 H37 H37 H50 H50 H50

G0 G2 G2 G0 G2 G2 G0 G2 G 2

H variety untransformed controls (wt) , H37, and H50 show consistency w ith 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb size bands in the G0 and G 2 plants. H37 contains three additional bands of 2.0 kb, 3. 2 kb,

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G2 plants.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 42

Fig ure 3 5

. H37 and H50: DNA Insert S tability in G3 Samples as V isualized with an AGP P robe

H variety untransformed controls (wt) , H37, and H50 show consistency with 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb size bands in the G0 and G 3 plants. H37 contains three additional bands of 2.0 kb, 3. 2 kb,

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G3 plants.



### M M wt wt wt H37 H37 H37 H50 H50 H50

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2 23.1

9.4

8.6

7.4

6.6

6.1

4.9

3.6

1.9 4.6J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 43

Figures 3 6

. Assay for Sustained Ppo5 G ene Silencing in G2 Tubers of Events H37 and H5 0J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 44

It should be mentioned here that this assessment did identify insert instability in another event

, which is

not part of this petition . The G2 tubers of this event displa yed instability at a low frequency ( Figure 3 7 )

. Of the 50 tubers tested, one t uber develop ed some color when subjected to catechol. This color

development is probably indicative of a partial reversion to Ppo5 gene expression. Importantly, this

indicates that the visual catechol test is effective for detecting gene instability in pot ato events

transformed with this construct. Given the demonstrated sensitivity of the test to identify low

frequencies of instability, it will be used to monitor trait stability in commercial potato seed (except for

Atlantic, where the PCR

- based stability test will be used).

Figure 3 7

. Assay for Ppo5 G ene Silencing in G2 Tubers of a G Event That is N ot C onsidered for

D eregulationJ.R. Simplot Company Evidence for Stab ility of the Inserted DNA 45 DISCUSSION

Given the demonstrated DNA insert stability in the 10 vegetatively propagated events described in this

petitio n, it is unlikely for instability to arise during subsequent cycles of vegetative propagation

. Continued trait efficacy c ould be readily confirmed by performing the catechol assay for Ppo activity on

non

- systematically chosen tubers. This assay is indicati ve of not only black spot bruise tolerance but also

low asparagine formation because Ppo5 and Asn1 gene silencing is mediated by the same silencing

cassette. In the unlikely case that a specific line would display trait instability, individual tubers would

revert back, either partially or fully, to the phenotype of their untransformed counterparts. Such

reversion events will not be associated with the expression of phenotypes associated with pests or

noxious weeds. For the Atlantic events, the PCR detectio n assay can be used instead of the catechol

assay.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 46 MATERIALS and METHODS

Materials. DNA insert stability was demonstrated in the originally

- transformed material (G0) by

extracting and evaluating DNA from leaves of plants that had been propagated in vitr o and never

planted in soil. For generation

- 1 ( G1 ) analyses, two propagated plants from each event and one plant

from each control were planted in the greenhouse; one of the tubers harvested from each plant was

planted to obtain leaves from G1 plants that were used to isolate DNA and evaluate the G1 generation.

Tubers from this generation were planted again, and leaves of the resulting G2 plants allowed a

characterization of that generation. For event F37, G2 tubers rather than leaves were used for the

anal yses. For all G3 samples, the genomic DNA was extracted from tubers.

In summary, all G0, G1, and G2 DNA was from leaves except that G2 material from F37 was from tubers,

and G3 material all came from tubers. Extracted DNA from leaves or tubers were dige sted with Eco RV

and hybridized with two probes (GBS and AGP).

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a

mortar using liquid nitrogen. The ground tissue was transferred to a pre

- cooled 15 ml conical tube with

a pre

- cooled spatula and stored at

- 80 ° C until ready to process. 10 ml extraction buffer (0.35 M Sorbitol,

0.1 M Tris

- HCl, pH8.0, 0.05M EDTA) was added to, and mixed with, the powder, and the resulting

suspension was centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was thoroughly

re

- suspended in 2 ml extraction buffer containing 200 μg RNase A. After incubating the resuspended

DNA at 65 o C for 20 min with 2 ml of nuclear lysis buffer (0.2 M Tris

- HCl pH7.5, 0.005 M EDTA pH 8.0 and

20 mg/ml CTAB Hexadecyl Trimetyl Ammonium Bro mide) and 800 μl of 5% Sarcosyl, it was mixed with

an equal volume of chloroform: isoamyl alcohol (24:1)

, vortex ed for about 1 min, and centrifuged at

3000 rpm for 5 min at room temperature. The DNA was precipitated with an equal volume of isopropyl

alcohol, washed with 70% ethanol, air dried, and dissolved in 400

- 700 μl 1X Tris/EDTA buffer (TE). DNA

concentration was measured using a spectrophotometer at an OD of 260 nm, whereas quality was

confirmed by running the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE) for 30

- 40 min at 80

volts. In some cases, an alternative method was used to isolate DNA. The ground tissue from 0.7 g young

leaves was mixed with 7 ml CTAB buffer (2% CTAB, 1.4 M NaCl , 0.1 M pH 8.0 Tris

- HCI, 20 mM pH8.0

EDTA, 100 μ g/ml RNase) and incubated at 55 o

- 65 o C for 30 min followed by centrifugation at 3,000 rpm

for 15 min. DNA was extracted twice with equal volumes of chloroform: isoamyl alcohol (24:1) by

shaking for 10 min and centrifuged at 3000 rpm for 5 min at room temperature. The DNA was

precipitated with equal volumes of ethanol and rinsed with 70% ethanol. The resulting pellet was dried

in air and dissolved in TE buffer. The quality and concentration of DNA was determined

spectrophotometrically and by running a sample on a 0.8% agarose gel. It should be mentioned here

that control and event DNA w ere always extracted with the same method for a specific comparison. For

event F37, G2 tubers rather than leaves were used for DN A extraction using the same protocol.

DNA Gel (Southern) Blot Analysis. The probes used in the Southern blot analysis are represented in

Figure 3 8

. 10 μg of DNA was digested overnight in 400 μl final volume with at least 5 μl (10 units/μl)

restriction en zyme (Invitrogen) at 37 ° 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.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 47 Figure 3 8

. P robes U sed for the Southern A nalysis

Membrane Preparation. Digested DNA was loaded on a large 0.7% agarose gel (170 ml ) containing 0.5X

TBE buffer and 3

- 5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 hrs. The gel was

photographed using a gel documentation system from Alpha Innotech (Santa Clara, California), and then

depurinated by submerging it into 0.25 N HCl for 20 min. After subsequent denaturing in 0.5 M NaOH

/1.5 M NaCl for 2 X 15 min and neutralization in 1.5 M NaCl and 0.5 M Tris

- HCl, pH7.5, for 2X15 min on a

shaker at room temperature and equilibrated with 10X SSC for 10 min. The transfer of DNA to the nylon

membrane was carried out using 10X SSC according to a standard capillary transfer method (Sambrook

and Russel l 2001).

PCR

- b ased s tability t est. For Atlantic, insert stability was evaluated with PCR using tuber

- extracted DNA

as template and the primer set HD121F (5’

- 

### CCTCTCGTAAATTCCGACAAA , at position 2113

- 2133 of

the DNA insert , which is the 3’ end of AGP promoter) and HD121R (5’

- TCTCAACTTGCGAACCTGAC

, at

position 2882

- 2901 of the spacer in the first inverted repeat ) to amplify a 789

- bp DNA inser t fragment

containing part of the A sn 1/P po5 gene

. Figure 39 below shows the location of the amplified fragment

on the DNA insert

.

Figure 39. PCR Primer P ositions for Confirmation of Stability in Atlantic Events

Catechol a ssay for Ppo activity. The second stability assessment of the events was based on their black

spot bruise susceptibility. The events including Ranger Russet and Russet Burbank were planted in plots

with three replications in the spring for the 2 nd year field trials (2010) in Parma, Larimore, Aberdeen, and

Othello ( Table 1) . They were grown and harvested using standard commercial production practices. For

each event, twelve or thirteen tubers from each site (four or five from each rep) for a total of 50 G 2

-

tubers from all four sites were non

- systematically selected from harvested tubers (see the following

Table 1 ). Similarly, G and H plants were planted in Rhinelander

, WI and 50 G2

- tubers were non

systematically selected from harvested tubers ( Table 1 ). O ne slice of each tuber was then assayed for

the absence of the functional PPO enzyme by adding 1.0 mL of 25 mM catechol in 50 mM MOPS buffer,

pH 6.5, onto each freshly cut tuber slice surface and reviewing for color development.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 48

Table 1. Number of Tubers from Each Location Assayed for PPO Activity with Catechol

Location Event

F10 Event

F37 Event

E12 Event

E24 Event

G11 Event

H37 Event

H50

Parma, ID 13 13 13 13 * * *

Larimore, ND 12 12 12 12 * * *

Aberdeen, ID 13 12 12 12 * * *

Othello, WA 12 13 13 13 * * *

Rhinelander, WI * * * * 50 50 50

Total Number of Tubers 50 50 50 50 50 50 50

* None from this locationJ.R. Simplot Company Evidence for Stab ility of the Inserted DNA 49 CONCLUSION S

Overall Conclusion: Our data provides evidence that the genetic insertion s remain stable

, which was

confirmed in the genotyp e and phenotype of the 10 events that are the subject of this submission

, meaning that their inserts are stable in the original transformant s as well as in G1

, G2 , and G3 plant

materials . Because potato is a vegetatively propagated crop, material from tis sue culture and tubers

w ere used in stability testing.

Ranger Russet Events F10 and F37: Hy b ridization with the GBS and AGP probes shows that events F10

and F37 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and

G3 . Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the

initial generation through G2.

Russet Burbank Events E12 and E24: Hy b ridization with the GBS and AGP probes show s that events E12

and E24 had consistent bands over multiple generations by comparing sample s of G0 with G1, G2 and

G3. Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the

initial generation through G2.

Atlantic Events J3, J55, and J78: Hybridizatio n with the GBS and AGP probes shows that events J3, J55,

and J78 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and

G3. Also, PCR testing showed amplification of the same 788 bp DNA insert fragment in all G2 events,

that was not found in the control Atlantic samples, indicating that the DNA insert was stable through at

least the second generation.

G Event G11: H y b ridization with the GBS and AGP probes shows that event G11 had consistent bands

over multiple generatio ns by comparing samples of G0 with G1, G2 and G3. Also, the vis ual assay for

PPO with catechol confirmed stability of Ppo5 gene silencing from the initial generation through G2.

H Events H37 and H50: H y b ridization with the GBS and AGP probes show s that events H37 and H50 had

consistent bands over multiple generations by comparing samples of G0 with G1, G2 and G3. Also, the

visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the initial generation

through G2.

Th us, th e sta bility of the inserts has been confirmed and trait stability monitoring will remain part of the

commercial strategy for release of planting material for these events. Importantly, the stability of the

inserts will not alter the pest characteristics of thes e events compared to conventional potato varieties

and will not alter the environmental impact of potato production compared to conventional varieties

.J.R. Simplot Company Evidence for Stab ility of the Inserted DNA 50 REFERENCE S

Sambrook J, Russell DW (2001) Southern Hybridization. In: Molecular Cloning: A Laboratory Manual. (Ed.

Argentine J) Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY (pp. 6.33

- 6.38).