Herbicide tolerant soybean plants and methods for identifying same

• US 9,683,242 B2
• Filed: 11/23/2010
• Issued: 06/20/2017
• Est. Priority Date: 11/23/2009
• Status: Active Grant

• Current Assignee: BASF Agricultural Solutions Seed US LLC (BASF SE), MS Technologies LLC
• Original Assignee: Bayer Cropscience NV (Bayer AG), MS Technologies LLC
• Inventors: Welz, Gunter, Mason, Justin Thomas, Eby, Mark Alan, Verhaeghe, Steven, Eby, William H., Lettow, Leslie James, De Beuckeleer, Marc, Habex, Veerle, Ferrulo, Jean-Marc
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/511,631
• Publication Number:
  US 20120304331A1
• Time in Patent Office: 2,401 Days
• Field of Search: None
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8275 Glyphosate
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

The invention provides specific transgenic soybean plants, plant material and seeds, characterized in that these products harbor a stack of specific transformation events at specific locations in the soybean genome. Tools are also provided which allow rapid and unequivocal identification of these events in biological samples.

Claims

1. A method for identifying the simultaneous presence of elite events EE-GM3 and EE-GM2 in biological samples, confirming seed purity in seed samples, or screening seeds for the presence of elite events EE-GM3 and EE-GM2 in samples in seed lots, which method comprises detection of an EE-GM3 specific region with a specific primer pair or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3 and the inserted foreign DNA contiguous therewith, and detecting an EE-GM2 specific region with a specific primer pair or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2 and the inserted foreign DNA contiguous therewith.

2. The method of claim 1, said method comprising amplifying two DNA fragments of between 50 and 1000 bp from a nucleic acid present in said biological samples using a first polymerase chain reaction with at least two primers, one of said primers recognizing the 5′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, the other primer of said primers recognizing a sequence within the foreign DNA of EE-GM3 comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the other primer of said primers recognizing a sequence within the foreign DNA of EE-GM3 comprising the nucleotide sequence of SEQ ID No. 1 or its complement, or comprising the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and using a second polymerase chain reaction with at least two primers, one of said primers recognizing the 5′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, the other primer of said primers recognizing a sequence within the foreign DNA of EE-GM2 comprising the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the other primer of said primers recognizing a sequence within the foreign DNA of EE-GM2 comprising the nucleotide sequence of SEQ ID No. 11 or its complement, whereby said first and second polymerase reaction may be sequential or simultaneous.

3. The method of claim 2,

(a) wherein said primer recognizing the 5′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA of EE-GM3 comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and wherein said primer recognizing the 5′

flanking region of EE-GM2 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or said primer recognizing the 3′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and said primer recognizing a sequence within the foreign DNA of EE-GM2 comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the nucleotide sequence of SEQ ID No. 11 or its complement;

or

(b) wherein said primer recognizing the 5′

flanking region of EE-GM3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA of EE-GM3 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and wherein said primer recognizing the 5′

flanking region of EE-GM2 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or said primer recognizing the 3′

flanking region of EE-GM2 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and said primer recognizing a sequence within the foreign DNA of EE-GM2 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the nucleotide sequence of SEQ ID No. 11 or its complement.

4. The method of claim 3, wherein said EE-GM3 specific primers comprise the sequence of SEQ ID No. 5 and SEQ ID No. 4, respectively, or the sequence of SEQ ID No. 7 and SEQ ID No. 5 respectively, and said EE-GM2 specific primers comprise the sequence of SEQ ID No. 18 and SEQ ID No. 19 respectively.

5. The method of claim 4, which method comprises amplifying an EE-GM3 specific fragment of about 263 or 706 bp using the EE-GM3 PCR Identification Protocol and an EE-GM2 specific fragment of about 151 bp.

11. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with a first specific probe for EE-GM3 and with a second specific probe for EE-GM2.

12. The method of claim 11,

(a) wherein the sequence of said first specific probe has at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of EE-GM3 and part of the sequence of the foreign DNA contiguous therewith and wherein the sequence of said second specific probe has at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of EE-GM2 and part of the sequence of the foreign DNA contiguous therewith;

or

(b) wherein the sequence of said first specific probe has at least 80% sequence identity with SEQ ID No. 2 from nucleotide 1431 to 1472 or SEQ ID No. 3 from nucleotide 220 to 261, or the complement of said sequences and wherein the sequence of said second specific probe has at least 80% sequence identity with SEQ ID No. 14 from nucleotide 301 to 322 or SEQ ID No. 15 from nucleotide 497 to 518, or the complement of said sequences.

6. A kit for identifying the simultaneous presence of elite event EE-GM3 and elite event EE-GM2 in biological samples, said kit comprising one primer recognizing the 5′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or one primer recognizing the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and one primer recognizing a sequence within the foreign DNA comprising herbicide tolerance genes in EE-GM3, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and wherein said kit further comprises one primer recognizing the 5′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or one primer recognizing the 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and one primer recognizing a sequence within the foreign DNA comprising herbicide tolerance genes in EE-GM2, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the nucleotide sequence of SEQ ID No. 11 or its complement, wherein;

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3;

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions;

or

the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

7. The kit of claim 6,

(a) wherein said primer recognizing said 5′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing the 3′

flanking region of EE-GM3 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within the foreign DNA of EE-GM3 comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and wherein said primer recognizing said 5′

flanking region of EE-GM2 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or said primer recognizing the 3′

flanking region of EE-GM2 comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and said primer recognizing a sequence within the foreign DNA of EE-GM2 comprises 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the nucleotide sequence of SEQ ID No. 11 or its complement;

or

(b) wherein said primer recognizing said 5′

flanking region of EE-GM3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or said primer recognizing said 3′

flanking region of EE-GM3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said primer recognizing a sequence within said foreign DNA of EE-GM3 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 or the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240 or the nucleotide sequence of SEQ ID No. 1 or its complement, or the nucleotide sequence of SEQ ID No. 20 from nucleotide position 1452 to nucleotide position 16638 or its complement, and wherein said primer recognizing said 5′

flanking region of EE-GM2 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or said primer recognizing said 3′

flanking region of EE-GM2 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and said primer recognizing a sequence within said foreign DNA of EE-GM2 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 or the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507 or the nucleotide sequence of SEQ ID No. 11 or its complement;

or

(c) comprising a primer comprising the sequence of SEQ ID No. 4 and a primer comprising the sequence of SEQ ID No. 5 or comprising a primer comprising the sequence of SEQ ID No. 5 and a primer comprising the sequence of SEQ ID No. 7 and further comprising a primer comprising the sequence of SEQ ID No. 18 and a primer comprising the sequence of SEQ ID No. 19.

18. The kit of claim 6, wherein at least one of said primers is labeled.

19. The kit of claim 7, wherein at least one of said primers is labeled.

23. The kit of claim 7, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

24. The kit of claim 7, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

28. The kit of claim 6, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

29. The kit of claim 7, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

32. The kit of claim 6, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

33. The kit of claim 7, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

37. The kit of claim 6, wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

38. The kit of claim 7, wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

8. A set of primer pairs suitable for use in an EE-GM3 and EE-GM2 specific detection, comprising a first primer pair comprising a first primer which-specifically recognizes a sequence within the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, and a second primer which specifically recognizes a sequence within the foreign DNA contiguous with said 5′

or 3′

flanking region in EE-GM3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408, and said foreign DNA contiguous with said 5′

flanking region in EE-GM3 comprising the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1843 and said foreign DNA contiguous with said 3′

flanking region in EE-GM3 comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide 1 to nucleotide 240; and

a second primer pair comprising a third primer which specifically recognizes a sequence within the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM2, and a fourth primer which specifically recognizes a sequence within the foreign DNA contiguous with said 5′

or 3′

flanking region in EE-GM2, said 5′

flanking region comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880, and said foreign DNA contiguous with said 5′

flanking region in EE-GM2 comprising the complement of the nucleotide sequence of SEQ ID No. 14 from nucleotide 312 to nucleotide 810 and said foreign DNA contiguous with said 3′

flanking region in EE-GM2 comprising the nucleotide sequence of SEQ ID No. 15 from nucleotide 1 to nucleotide 507, wherein;

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3;

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions;

or

the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

9. The set of primer pairs of claim 8,

(a) wherein said first primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408 and wherein said third primer comprises a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880;

or

(b) wherein said first primer comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 1451 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 3 from nucleotide 241 to nucleotide 1408 and wherein said third primer comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 14 from nucleotide 1 to nucleotide 311 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 15 from nucleotide 508 to nucleotide 1880.

20. The set of primers of claim 8, wherein at least one of said primers is labeled.

21. The set of primers of claim 9, wherein at least one of said primers is labeled.

25. The set of primers of claim 8, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

26. The set of primers of claim 9, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

30. The set of primers of claim 8, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

31. The set of primers of claim 9, wherein at least one of said primers comprises a nucleotide sequence at their 3′

end spanning the joining region between the plant DNA derived sequences and the foreign DNA sequences, said joining region being at nucleotides 1451-1452 in SEQ ID No 2 or nucleotides 240-241 in SEQ ID No 3, provided that the 17 consecutive nucleotides at the 3′

end are not derived exclusively from either the foreign DNA or plant-derived sequences in SEQ ID Nos. 2 or 3.

34. The set of primers of claim 8, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

35. The set of primers of claim 9, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

39. The set of primers of claim 8, wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

40. The set of primers of claim 9, wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.

10. A set of primers, wherein

a first primer comprises at its extreme 3′

end the sequence of SEQ ID No. 5;

a second primer comprises at its extreme 3′

end the sequence of SEQ ID No. 4;

a third primer comprises at its extreme 3′

end the sequence of SEQ ID No. 18; and

a fourth primer comprises at its extreme 3′

end the sequence of SEQ ID No. 19, wherein;

at least one of said primers is labeled;

the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence;

or

at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

22. The set of primers of claim 10, wherein at least one of said primers is labeled.

27. The set of primers of claim 10, wherein the 5′

end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′

or 3′

flanking sequence of foreign DNA sequence.

36. The set of primers of claim 10, wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′

or 3′

flanking region of the elite event or the foreign DNA of the elite event, respectively, and said primer sequence comprises at least one mismatch with said 5′

or 3′

flanking region or said foreign DNA, provided the at least one mismatch still allows specific identification of the elite event with these primers under optimized PCR conditions.

13. A kit for identifying the simultaneous presence of elite event EE-GM3 and elite event EE-GM2 in biological samples, said kit comprising a pair of specific probes for the identification of the simultaneous presence of elite event EE-GM3 and elite event EE-GM2, wherein a first specific probe is capable of hybridizing specifically to a region comprising a part of the 5′

or 3′

flanking region of EE-GM3 and a part of the foreign DNA contiguous therewith, and a second specific probe is capable of hybridizing specifically to a region comprising a part of the 5′

or 3′

flanking region of EE-GM2 and a part of the foreign DNA contiguous therewith.

14. The kit of claim 13,

(a) wherein the sequence of said first specific probe has at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of foreign DNA comprising herbicide tolerance genes in EE-GM3 and part of the sequence of the foreign DNA contiguous therewith and wherein the sequence of said second specific probe has at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of foreign DNA comprising herbicide tolerance genes in EE-GM2 and part of the sequence of the foreign DNA contiguous therewith;

or

(b) wherein the sequence of said first specific probe comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences and wherein the sequence of said second specific probe has at least 80% sequence identity with SEQ ID No. 14 from nucleotide 301 to 322 or SEQ ID No. 15 from nucleotide 497 to 518, or the complement of said sequences.

15. A pair of specific probes for the identification of the simultaneous presence of elite event EE-GM3 and elite event EE-GM2 in biological samples, wherein a first specific probe is capable of hybridizing specifically to a region comprising a part of the 5′

or 3′

flanking region of EE-GM3 and a part of the foreign DNA contiguous therewith, and a second specific probe is capable of hybridizing specifically to a region comprising a part of the 5′

or 3′

flanking region of EE-GM2 and a part of the foreign DNA contiguous therewith.

16. The pair of probes of claim 15,

a) wherein said first probe comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of foreign DNA comprising herbicide tolerance genes in EE-GM3 and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof, and said second probe comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of foreign DNA comprising herbicide tolerance genes in EE-GM2 and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof;

or

b) wherein said first probe has at least 80% sequence identity with SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences and wherein said second probe has at least 80% sequence identity with SEQ ID No. 14 from nucleotide 301 to 322 or SEQ ID No. 15 from nucleotide 497 to 518, or the complement of said sequences;

or

c) wherein said first probe comprises a nucleotide sequence being essentially similar to SEQ ID No. 2 from nucleotide 1441 to 1462 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences, and said second probe comprises a nucleotide sequence being essentially similar to SEQ ID No. 14 from nucleotide 301 to 322 or SEQ ID No. 15 from nucleotide 497 to 518, or the complement of said sequences.

17. A method of detecting the presence of elite events EE-GM3 and EE-GM2 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:

target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising;

a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1452 to nucleotide 1469 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide 223 to nucleotide 240 or its complement;

b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 2 from nucleotide 1434 to nucleotide 1451 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide 241 to nucleotide 258 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;

c) cleaving only the labeled probe within the probe;

target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;

d) recycling of the target nucleic acid sequence by repeating steps (a) to (c); and

e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence, and

f) hybridizing said target nucleic acid sequence to a third nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide 312 to nucleotide 329 or its complement or said third nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 15 from nucleotide 490 to nucleotide 507 or its complement;

g) hybridizing said target nucleic acid sequence to a fourth nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide 294 to nucleotide 311 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No. 15 from nucleotide 508 to nucleotide 525 or its complement, wherein said third and fourth oligonucleotide overlap by at least one nucleotide and wherein either said third or said fourth oligonucleotide is labeled to be said labeled nucleic acid probe;

h) cleaving only the labeled probe within the probe;

target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;

i) recycling of the target nucleic acid sequence by repeating steps (f) to (h); and

j) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.