J.R. Simplot Company Junction Analysis and Event

- Specific Detection 1 Appendix 4
Junction Analysis and Event

- Specific Detection

INDEX

Section Page

Index 1

Introduction 2

Results 3

Materials and Methods 2 9

Conclusions 31

References 3 3

Certification 3 4J.R. Simplot Company Junction Analysis and Event

- Specific Detection 2 INTRODUCTI ON

At least one DNA insert /flanking plant DNA junction was sequenced using either Adapter Ligation

Mediated PCR or Thermal Asymmetric Interlaced PCR. The junction sequence was used to design primers for each event , and these primers were applied for even t

- specific PCR

- based detection method s

. The specificity of the primers was tested on all the events submitted for deregulation to ensure that there was no cross

- reactivity between the detection systems. The method s developed may be used to monitor plants a nd tubers in field and storage as part of quality management programs.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 3 RESULTS
Ranger Russet Event F10

Based on the DNA insert organization of F10 (see Figure 1 below and Appendix 1. Characterization of Inserted DNA ), a fragment was amplified at the R ight Border site. Sequence analysis determined that the DNA insert lacked the first 31 bp upstream from the Right Border cleavage site

. Figure 1. F10 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an even t

specific DNA fragment . Promoter orientation indicated by arrows ( ).

The primers used for line

- specific PCR were F10 1 F, which is derived from the DNA insert , and F10 1 R from the flanking plant DNA ( Figure 1 ). The amplified DNA fragment for the line

- sp ecific PCR had a size of 10 76 bp ( Figure 2 )

. The single DNA insert of F10 had a 31 bp deletion at the Right Border side that did not affect the functional activity of the adjacent silencing cassette.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 4 Figure 2. Ranger Russet F10: Line

- S pecific PCR U sing P rimer P air F10 1F and F10 1R

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), F C = Ranger Russet control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 5 Ranger Russet Event F37

Based on the DNA insert organization of F 37 ( see Figure 3 below and Appendix 1. Characterization of Inserted DNA ), a fragment was amplified at the Right Border site. The junction fragment consists of a truncated Right Border side of the DNA insert (which means that the integrated DNA insert lacks the Right Border and a small adjacent upstream sequence of 13 bp) and is followed by, first, a 13 bp plant

derived ‘stuffer’ DNA and, second, a small fragment (33 bp) of the DNA insert that represents an internal
sequence o f the trailer of the Ppo gene that is present in the silencing cassette of pSIM1278

. Figure 3. F 37 Insert

Arrows below the diagram ( →) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

Two primers, one from the DNA insert and another from the sequenced flanking DNA, F37 A F and F37 A R ( Figure 3 ) , we re used to amplify a 10 91 bp F37

- specific junction fragment ( Figure 4 )

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 6

Figure 4. Ranger Russet F37: Line

- S pecific PCR U sing P rimer P air F37 AF and F37 AR

. Amplified F37

- specific fragment at Right Border side

.

1kb = 1 kb DNA marker lane (Invitrogen ), 100 bp = 100 bp DNA marker lane (invitrogen), F C = Ranger Russet control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 7 Russet Burbank Event E12

The DNA insert organization of E12 is shown in Figure 5 below. A 5 47 bp fragment at the Right Border site lacked the first 92 bp upstream from the Right Border cleavage site. A 609 bp fragment at the Left Border site lacked 17 bp downstream from the Left Border cleavage site ( Figure 6 A and B ).
Figure 5. E12 Insert

Arrows below the diagram ( →) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 8

Figure 6 A

. Russet Burbank E12: Line

- S pecific PCR

. Amplified E12

- specific fragment at the Right Border side , usi ng primer s HY1599 and HY1600

.

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), EC = Russet Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 9

Figure 6 B

. Russet Burbank E12: Line

- S pecific PCR

. Amplified E12

- specific fragment at Left Border side using primer HY1617 and HY1620

.

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invi trogen), EC = Russet Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 10 Russet Burbank Event E24

TAIL

- PCR was used to isolate DNA insert junction fragments from event E24 using t he nested, Right Border side

- specific p rimers HY1557, HY1558, HY1576 and HY1577 ( Table 1 )

. Based on the differential shift between the second and third products on agarose gels, E24

- specific PCR products were extracted and cloned. Sequence analysis of the junction fragment demonstrates that E24 carries a full

- length DNA insert (up to the cleavage site within the Right Border)

. T wo primers, one annealing to a sequence within the DNA insert (HY1599) and one annealing to a site in the adjacent plant DNA (HY1601) ( see Appendix 1. Characterization of Inserted DNA , Figure 7 shown below ) , were used to amplify a 5 43 bp E24

- specific DNA fragment ( Figure 8 )

. Figure 7. Insert for E24

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

Sequence analysis demonstrated that t he primers used for line

- specific PCR are the DNA insert specific primer HY1599 and HY1601 which is from flanking DNA

- derived DNA ( Figure 7 )

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 11

Figure 8. Russet Burbank E24: Line

- S pecific PCR U sing P rimer P air HY1599 and HY1601

. Amplified E24

- specific fragment at Right Border side

.

1kb = 1 kb DNA marker lane (Invitrogen), 1 00 bp = 100 bp DNA marker lane (invitrogen), EC = Russet Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 12 Atlantic Event J3

Event J3 contains one nearly intact and one partial copy of the DNA insert , connected head to head at the Left Border site , with deletion s of Left Border region s and parts of the adjacent Agp promoter s ( see Appendix 1. Characterization of Inserted DNA and Figure 9 below)

.
Figure 9. J3 Insert

Arrows below the diagram (→) indicate the approxi mate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

Different combinations of primers at the ends of the inserts were used to amplify DNA fragments, which were then analyzed by sequencing . It was found that one copy had a 430 bp deletion at the L eft Border side while the other copy showed a 251 bp deletion at the L eft Border side

. The resulting structure comprises an inverted repeat carrying two truncated A gp promoters whereby the longer p romoter fragment contains a 179 bp sequence that functions as spacer. To develop a line

- specific PCR method, we designed one primer , HY1768 , that anneals to a n Agp promoter sequence on the one side of the connection and a second primer, HY1772, annealing t o the Agp on the other side ( Figure 9 ). As shown in Figure 10 , these primers could be used to amplify a line specific 464 bp DNA fragment from J3 DNA but not from DNA of untransformed Atlantic , nor events J55 or J78

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 13

Figure 10. Atlantic J3: Line

- S pecifi c PCR U sing P rimer P air HY1768 and HY1772

. Amplified J3

- specific fragment between two DNA insert s at Left Border sides

.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative control PCR reaction (without pl ant DNA).J.R. Simplot Company Junction Analysis and Event

- Speci fic Detection 14 Atlantic Event J55

Both Southern analys es and TAIL PCR indicate d that event J55 ha d two connected copies of the DNA insert . The Right Border region of the first DNA insert is connected to a truncated internal Agp region of the second DNA insert in the reverse orientation ( see Figure 11 below and Appendix 1. Characterization of Inserted DNA )

. Figure 11. J55 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

A short plant

- derived 49 bp stuffer sequence is present between the two DNA insert s. The J55

- specific forward primer JY981 was designed to anneal to the right side of the Gbss promoter of th e first DNA insert and was 29

- nt away from the left side of RB. The J55

- specific reverse primer JY98 2 was targeted to the Agp promoter of the second DNA insert and was 187 nucleotides away from the right side of RB ( Figure 11 ). As shown in Figure 12 , a lin e specific 401 bp DNA fragment was amplified with these primer s in event J55 but neither in untransformed Atlantic nor in J3 or J78.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 15

Figure 12. Atlantic J55: Line

- S pecific PCR U sing P rimer P air JY981 and JY982

. Amplified J55

- specific fragment

.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 16 Atlantic Event J 78

Based on the truncated structure of the DNA insert of J78 (see Figure 13 below and Appendix 1. Charac terization of Inserted DNA ) , a fragment of DNA flanking the Right Border side was sequenced. This sequence was used to design a primer, J78R, that, together with the DNA insert primer J78F ( Figure 13 ) was used to amplify a J78

- specific DNA fragment.
Figure 13. J78 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

As shown in Fig ure 14 , the 513 bp DNA fragment amplified with primer s J78F and J78 R was specific for J78

. Sequ enc ing the junction demonstrated that the DNA insert had a 1583 bp deletion at the Right Border side

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 17

Figure 14. Atlantic J78: Line

- S pecific PCR U sing P rimer P air J78F and J78R

. Amplified J78

- specific fragment at Right Border side.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 18 G event G11

Based on the truncated structure of the DNA insert of G11 (see Figure 15 below and Appendix 1. Characterization of Insert ed DNA ), the sequence of DNA flanking the Right Border side was determined. Two primers, one annealing to a sequence within the DNA insert (HY1680) and the other one annealing to flanking plant DNA (HY1683) ( Figure 15 ), were used to amplify a 734 bp G11

- s pecifc junction fragment ( Figure 16 ). Sequence analysis of the fragment demonstrated that the DNA insert lacked 3 , 317 bp at the Right Border side , which means that it contained a full Asn1 / Ppo5 gene silencing cassette and lacked the R1/PhL promoter silenci ng cassette

. Figure 15. Insert for G11

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 19

Figure 16. G11: Line

- S pecific PCR U sing P r imer P air HY1680 and HY1683

. Amplified G11

- specific fragment ( 734 bp ) at Right Border side.

100 bp = 100 bp DNA marker lane (invitrogen), GC = G control variety (untransformed), G9 = G event, not included in this submission, NC = negative control PCR reaction (without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 20 H event H37

Based on the DNA insert organization of H37 (see Figure 17 below and Appendix 1. Characterization of Inserted DNA ), a Right Border junction fragment was isolated using the nested primers (HY 1557 , HY1558, and HY1 576 ) by TAIL

- PCR. Sequencing of the fragment made it possible to design two primers, one annealing to a sequence within the DNA insert (JY939) and the other one annealing to a flanking plant DNA sequence (JY938) ( Figure 17 )

. Figure 17

. H37 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

specific DNA fragment . Promoter orientation indicated by arrows ( ).

The event

- specific primers were used to amplify the junction . As demonstrated in Figure 18 , the 553 bp DNA frag ment was amplified with H37 DNA but not with DNA from either the conventional variety H or the event H50

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 21

Figure 18. H37: Line

- S pecific PCR U sing P rimer P air JY938 and JY939

. Amplified H37

- specific fragment ( 553 bp ) at Right Border side

.

100 bp = 10 0 bp ladder (Invitrogen) , H C = H Control , H74 = H event not included in this submission , NC = negative control ( PCR reaction without plant DNA ).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 22 H event H50

The DNA insert organization of H50 is shown in Figure 19 below and Appendix 1. Characterization of Inserted DNA . A junction fragment was isolated using TAIL

- PCR with t he nested primers HY 1562 , HY15 66 , and HY15 67 ( Table 2 )

.

Figure 19. Insert for H50

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an event

sp ecific DNA fragment . Promoter orientation indicated by arrows ( ).

Based on the size difference between the first and second PCR products on agarose gels, specific PCR products were cloned and sequenced. The H50 Left Border junction specific primers JY979 and JY980

were used to amplify a line specific 507

- bp junction fragment in the H50 event but not in the conventional variety H n or H37 ( Figure 20 )

.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 23

Figure 20. H 50 : Line

- S pecific PCR U sing P rimer P air JY979 and JY980

. Amplified H 50

- specific fragment

a t Left Border side.

M = 100 bp ladder (Invitrogen) , H C = H Control , H74 = event not included in this submission , NC = negative control (PCR reaction without plant DNA).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 24 Line

- Specificity of PCR

- Based Detection Methods.

As shown in Figure 21 , the 10 event

- specific PCR methods do not cross react with the other events in this deregulation submission.

Figure 2 1. Line

- S pecific PCR Detection Methods

M = 100

- bp ladder from invitrogen; EWT = Russet Burbank C ontrol; FWT = Ranger Russet C ontrol; JWT = Atlantic C ontrol; GWT = G C ontrol; HWT = H C ontrol . NC = negative control (PCR reaction without plant DNA). Test lines as indicated in the text (F10, F37).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 25 Figure 21 (Continued). Line

- S pecific PCR Detection Methods

M = 100 bp lad der from invitrogen; EWT = Russet Burbank C ontrol; FWT = Ranger Russet C ontrol; JWT = Atlantic C ontrol; GWT = G C ontrol; HWT = H C ontrol

. NC = negative control (PCR reaction without plant DNA). Test lines as indicated in the text ( E12, E24).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 26 Figure 21 (C ontinued). Line

- S pecific PCR Detection Methods

M = 100 bp ladder from invitrogen; EWT = Russet Burbank C ontrol; FWT = Ranger Russet C ontrol; JWT = Atlantic C ontrol; GWT = G C ontrol; HWT = H C ontrol. NC = negative control (PCR reaction without plant DNA ). Test lines as indicated in the text (J3, J55).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 27 Figure 21 (Continued). Line

- S pecific PCR Detection Methods

M = 100 bp ladder from invitrogen; EWT = Russet Burbank C ontrol; FWT = Ranger Russet C ontrol; JWT = Atlantic C ontrol; GWT = G C ontrol; HWT = H C ontrol. NC = negative control (PCR reaction without plant DNA). Test lines as indicated in the text ( J78, G11).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 28 Figure 2 1 ( C ontinued)

. Line

- S pecific PCR Detection Methods

M =

100bp ladder from invitrogen; EWT = Russet Burbank C ont rol; FWT = Ranger Russet C ontrol; JWT = Atlantic C ontrol; GWT = G C ontrol; HWT = H C ontrol. NC = negative control (PCR reaction without plant DNA). Test lines as indicated in the text (H37, H50).J.R. Simplot Company Junction Analysis and Event

- Specific Detection 29 MATERIALS AND METHODS

Potato material. Leaves from greenho use
- grown events and untransformed plants were used to extract DNA and isolate and characterize the DNA insert junctions.

Adapter ligation

- mediated PCR Junction fragments were amplified by PCR using digested DNA ligated with adapter primers AP1 and AP2 as described by O’Malley et al

. ( 2007 ) and detailed below. The amplified product containing a DNA insert primer binding site was selected by a secondary PCR reaction. The sequences of DNA insert primers used to analyze RB and LB junctions are shown in Table s 1 and 2 , respectively. Plant DNA was isolated using either the CTAB method or the Qiagen Plant DNAeasy kit: 200

- 500 ng DNA was digested for 3

- 5 hours with a restriction enzyme for which an adapter had been designed ( Eco RI, Hin dIII, Bam HI, Ase I/ Nde I) in a total volume of 20 μl. The digested DNA (3 μl) was ligated with its respective adapter (1 μl) in a mixture containing 0.5 μl T4 DNA ligase, 0.3 μl ATP, 1.0 μl 10X ligation buffer, and 4.2 μl water. The primary PCR was carried out using the first DNA inser t specific primer and AP1 with Hot Master Taq polymerase (Fisher BioReagents) under the following amplification conditions: 1 cycle of 3 min at 95°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 60°C, 2.5 min at 68°C, and finishing with 10 min at 68°C . A 1 μl aliquot of the primary PCR product was used for secondary reactions ( 1 cycle of 3 min at 95°C; 35 cycles of 30 sec at 94°C, 30 sec at 62°C, 2.5 min at 68°C; 1 cycle of 10 min at 68°C) with the second DNA insert specific primer and AP2. This proto col was performed at the same time for non

- transformed control plants and the events. Products of the secondary PCR were run on 1% agarose in TAE buffer. Bands unique for the event s were gel

- extracted using a Qiagen QIAquick Gel Extraction kit, cloned int o pGEM

- T Easy vector (Promega, Madison WI), and sequenced. Primers specific for DNA insert

- flanking DNA were used, together with a DNA insert primer, to amplify junction fragments.

T able 1. DNA Insert P rimers U sed to Analyze RB J unction F ragments Primer Name Primer position in pSIM1278

PHL

- GBSS 8822

- 8844 GBSS

- PR 9756

- 9777 HY1669 1675

- 1698 ; 5905

- 5928 HY1670 6479

- 6504 HY1671 6516

- 6541 HY1557 8824

- 8848 HY1558 4011

- 4035; 9320

- 9344 HY1576 9751

- 9777 HY1577 9846

- 9869 RB3R

- T1a 76 96

- 7719 RB3L

- T1b 7736

- 7759 RB3R

- T1d 7866

- 7889J.R. Simplot Company Junction Analysis and Event

- Specific Detection 30 Table 2

. DNA Insert Primers Used to A nalyze LB J unc tion F ragments Primer Name Primer position in pSIM1278

HY1562 460

- 485; 4690

- 4715 (reverse) HY1566 19643

- 7 (reverse) HY1567 19590

- 19614 (rever se)

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

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

specific) primers were carried out to obtain DNA inse rt junction fragments. The standard 20 μl reaction mixture consisted of 2 μl 10X PCR buffer, 0.4 μl 10 mM dNTP, 0.4 μl 10 μM SP1, 5 μl 8

- 16 μM AD, 1 μl template, with either 0.1 μl HotMaster Taq polymerase (extension temperature 68 °C) or TaKaRa Ex Taq TM ( TAKARA BIO Inc). A 1 μl aliquot of 50ng/ μl leaf DNA was used as template for the first PCR; 1 μl of a 1:50 dilution of the first reaction was used as template for the second PCR; and 1 μl of 1:50 dilution of the second reaction was used for the third PCR. The second and third reactions were run on a gel, and bands with sizes that changed between the second and third reactions were cloned into pGEM

- T Easy vector (Promega, Madison WI) and sequenced. Primers specific for DNA insert

- flanking DNA were used, toge ther with DNA insert

- specific primers to clone junction fragments that were sequenced to confirm their identity.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 31 CONCLUSIONS

At least one junction where the DNA insert meets the flanking plant DNA was sequenced for each of the 10 events and those sequen ce s were used to design event

- specific PCR

- based detection methods (see summary of primers for each event in Table 3 )

. The specificity of the primers was tested on all the events to ensure that there was no cross

- reactivity with other events

. The methods may be used to monitor plants and tubers in field and storage as part of quality management programs and can be developed further to provide for routine testing in food.

T able 3. P rimers U sed for Event

- specific PCR

Event Ranger Russet F10 : Primers F101F and F101R can be used to amplify an F10

- specific DNA fragment of 1076 bp , and thus results in a line specific test method for event F10

. Event Ranger Russet F37 : Primers F37AF and F37AR ca n be used to amplify an F37

- specific DNA fragment of 1091 bp , and thus results in a line specific test method for event F37.

Event Russet Burbank E12 : Primers HY1 617 and HY16 20 can be used to amplify an E12

- specific DNA fragment of 609

- bp at the Left Borde r , and primers HY1599 and HY1600 can be used to amplify a 547 bp fragment at the Right Border, and thus either set of primers could resul t in a line specific test method for event E12.

Event Russet Burbank E24 : Primers HY1599 and HY1601 can be used to ampl ify an E24

- specific DNA fragment of 543 bp , and thus results in a line specific test method for event E24.

Event Atlantic J3 : Primers HY1768 and HY1772 can be used to amplify a J3

- specific DNA fragment of 46 4 bp , and thus results in a line specific test me thod for event J3.

Event Atlantic J55 : Primers JY981 and JY982 can be used to amplify a J55

- specific DNA fragment of 401 bp , and thus results in a line specific test method for event J55. Event Forward Primer Reverse Primer PCR Product (bp)
F10 F101F F101R 1076 F37 F37AF F37AR 10 91 E12 (Left Border) HY1617 HY1620 609 E12 (Right Border) HY1599 HY1600 547 E24 HY1599 HY1601 543 J3 HY1768 HY1772 464 J55 JY981 JY982 401 J78 J78F J78R 513 G11 HY1680 HY1683 734 H37 JY939 JY93 8 553 H50 JY979 JY980 507J.R. Simplot Company Junction Analysis and Event

- Specific Detection 32

Event Atlantic J78 : Primers J78F and J78R can be used to amplify a J 78

- specific DNA fragment of 513 bp , and thus results in a line specific test method for event J78

.
Event G11 : Primers HY1680 and HY1683 can be used to amplify a G11

- specific DNA fragment of 734 bp , and thus provide an event

- specific test method for G11.

Ev ent H37 : Primers JY938 and JY939 can be used to amplify an H37

- specific DNA fragment of 553 bp , and thus provide an event

- specific test method for event H37.

Event H50 : Primers JY979 and JY980 can be used to amplify an H50

- specific DNA fragment of 507 bp , and thus provide an event

- specific test method for event H50.J.R. Simplot Company Junction Analysis and Event

- Specific Detection 33 REFERENCES
O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation

- mediated PCR method for high

- throughput mapping of T

- DNA inserts in the Arabidopsis gen ome. Nature Protocols, 2:2910

- 2917.