ELITE EVENT EE-GM3 AND METHODS AND KITS FOR IDENTIFYING SUCH EVENT IN BIOLOGICAL SAMPLES

• US 20120304330A1
• Filed: 11/23/2010
• Published: 11/29/2012
• 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 Bioscience NV (Bayer AG), MS Technologies LLC
• Inventors: Mason, Justin Thomas, Lettow, Leslie James, Eby, Mark Alan, Eby, William H., Welz, Günter, Verhaeghe, Steven, De Beuckeleer, Marc, Habex, Veerle, Ferrulo, Jean-Marc
• Granted Patent: US 9,631,202 B2
• Time in Patent Office: Days
• US Class Current: 800/260
• 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
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

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

Claims

1. A method for identifying elite event EE-GM3 in biological samples, which method comprises detection of an EE-GM3 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3.

2. The method of claim 1, said method comprising amplifying a DNA fragment of between 50 and 1000 bp from a nucleic acid present in said biological samples using a 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 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 comprising the nucleotide sequence of SEQ ID No. 1 or its complement, or comprising the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

3. The method of claim 2, wherein said primer recognizing the 5′

flanking region 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 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. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

4. The method of claim 2, wherein said primer recognizing the 5′

flanking region 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 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. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

5. The method of claim 4, wherein said 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. 4, respectively.

6. The method of claim 5, which method comprises amplifying a fragment of about 263 or 706 bp using the EE-GM3 PCR Identification Protocol.

16. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with a specific probe for EE-GM3.

17. The method of claim 16, wherein the sequence of said 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 the sequence of the foreign DNA contiguous therewith.

18. The method of claim 17, wherein the sequence of said 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.

7. A 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, 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. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

8. The kit of claim 7, wherein said primer recognizing the 5′

flanking region 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 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. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

9. The kit of claim 7, wherein said primer recognizing the 5′

flanking region 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 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. 11 from nucleotide position 1452 to nucleotide position 16638 or its complement.

10. The kit of claim 7, 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.

11. A primer suitable for use in an EE-GM3 specific detection, comprising a sequence which, under optimized detection conditions specifically recognizes a sequence within the 5′

or 3′

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 and said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID No 3 from nucleotide 241 to nucleotide 1408.

12. The primer of claim 11, wherein said 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.

13. The primer of claim 11, wherein said 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.

14. A primer comprising at its extreme 3′

end the sequence of SEQ ID No. 5.

15. A primer pair comprising a first primer comprising at its extreme 3′

end the sequence of SEQ ID No. 5 and a second primer comprising at its extreme 3′

end the sequence of SEQ ID No. 4.

19. A kit comprising a specific probe, capable of hybridizing specifically to a specific region of EE-GM3.

20. The kit of claim 19, wherein the sequence of said 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 the sequence of the foreign DNA contiguous therewith.

21. The kit of claim 20, wherein the sequence of said 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.

22. A specific probe for the identification of elite event EE-GM3 in biological samples.

23. The probe of claim 22, which 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 the sequence of the foreign DNA contiguous therewith, or the complement thereof.

24. The probe of claim 23 which 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.

25. A specific probe comprising 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.

26. A method for confirming seed purity, which method comprises detection of an EE-GM3 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, in seed samples.

27. A method for screening seeds for the presence of EE-GM3, which method comprises detection of an EE-GM3 specific region with a specific primer or probe which specifically recognizes the 5′

or 3′

flanking region of foreign DNA comprising herbicide tolerance genes in EE-GM3, in samples of seed lots.

28. A method for deter mining the zygosity status of a plant, plant material or seed comprising elite event EE-GM3, said method comprising amplifying DNA fragments of between 100 and 1000 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plant DNA, such as the a primer comprising the nucleotide sequence of SEQ ID No 5 and a primer comprising the nucleotide sequence of SEQ ID No. 6, the third of said primers recognizing a sequence within the foreign DNA, such as the nucleotide sequence of SEQ ID No. 7.

29. A method of detecting the presence of elite event EE-GM3 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.

30. A transgenic soybean plant, or cells, parts, seed or progeny thereof, each comprising elite event EE-GM3 in its genome, reference seed comprising said event having being deposited at the NCIMB under deposit number NCIMB 41659.

31. The transgenic soybean plant, seed, cells, parts or progeny of claim 30, the genomic DNA of which, when analyzed using the Elite event identification protocol for EE-GM3 with two primers comprising the nucleotide sequence of SEQ ID 4 and SEQ ID 5 respectively, yields a DNA fragment of about 263 bp.

37. A method for producing a soybean plant or seed comprising elite event EE-GM3 comprising crossing a plant according to any one of claims 30 to 36 with another soybean plant, and planting the seed obtained from said cross.

32. Seed comprising elite event EE-GM3 deposited at the NCIMB under deposit number NCIMB 41659 or derivatives therefrom.

33. A soybean plant, plant part, cell or tissue, or seed comprising elite event EE-GM3 obtainable from the seed of claim 32.

34. A soybean plant, or seed, cells or tissues thereof, each comprising elite event EE-GM3 in its genome, obtainable by propagation of and/or breeding with a soybean plant grown from the seed deposited at the NCIMB under deposit number NCIMB 41659.

35. A soybean seed comprising elite event EE-GM3, reference seed comprising said event having been deposited at the NCIMB under deposit number NCIMB 41659.

36. A transgenic soybean plant, cell or tissue, comprising elite event EE-GM3, obtainable from the seed of claim 35.

38. Soybean genomic DNA comprising elite event EE-GM3.

39. An isolated nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 2 from nucleotide 1441 to nucleotide 1452 or SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences.

40. The isolated nucleic acid molecule of claim 40 comprising a nucleotide sequence essentially similar to SEQ ID No. 2 from nucleotide 1431 to nucleotide 1462 or SEQ ID No. 3 from nucleotide 220 to 261, or the complement of said sequences.

41. A soybean plant, comprising in its genome in order the following nucleotide sequences:

a) the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to 1451;

b) the nucleotide sequence of the complement of the nucleotide sequence of SEQ ID 1 from nucleotide 6760 to nucleotide 6958;

c) the nucleotide sequence of SEQ ID 1 from nucleotide 6874 to nucleotide 7298;

d) the nucleotide sequence of SEQ ID 1 from nucleotide 7 to nucleotide 7291;

e) the nucleotide sequence of SEQ ID 1 from nucleotide 12 to nucleotide 7265;

f) the nucleotide sequence of SEQ ID 3 from nucleotide 217 to nucleotide 240; and

g) the nucleotide sequence of SEQ ID No 3 from nucleotide 241 to nucleotide 1408.

42. A DNA molecule comprising in order the following nucleotide sequences:

a) the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to 1451;

b) the nucleotide sequence of the complement of the nucleotide sequence of SEQ ID 1 from nucleotide 6760 to nucleotide 6958;

c) the nucleotide sequence of SEQ ID 1 from nucleotide 6874 to nucleotide 7298;

d) the nucleotide sequence of SEQ ID 1 from nucleotide 7 to nucleotide 7291;

e) the nucleotide sequence of SEQ ID 1 from nucleotide 12 to nucleotide 7265;

f) the nucleotide sequence of SEQ ID 3 from nucleotide 217 to nucleotide 240; and

g) the nucleotide sequence of SEQ ID No 3 from nucleotide 241 to nucleotide 1408.

43. A transgenic soybean plant, plant cell, tissue, or seed, comprising in its genome event EE-GM3 comprising a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 2 from nucleotide 1441 to nucleotide 1.462 and a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 3 from nucleotide 230 to 251, or the complement of said sequences.

44. A soybean plant, plant cell, tissue, or seed, comprising in its genome event EE-GM3 comprising a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 2 and a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 3, or the complement of said sequences.

45. A soybean plant, cell, tissue or seed, comprising EE-GM3 and comprising in the genome of its cells a nucleic acid sequence with at least 80%, 90%, 95% or 100% sequence identity to SEQ ID No. 2 from nucleotide 1431 to 1472 and a nucleic acid sequence with at least 80%, 90%, 95% or 100% sequence identity to SEQ ID No. 3 from nucleotide 220 to 261, or the complement of said sequences.

46. A soybean plant, plant cell, tissue, or seed, comprising in its genome a nucleic acid molecule comprising a nucleotide sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID No 1 or the complement thereof, or at least 99% sequence identity to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or the complement thereof, or at least 99% sequence identity to the nucleotide sequence of SEQ ID No. 11 or the complement thereof.

47. A soybean plant, plant cell, tissue, or seed, comprising in its genome a nucleic acid molecule hybridizing to the nucleotide sequence of SEQ ID No 1 or the complement thereof, or hybridizing to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or the complement thereof, or hybridizing to the nucleotide sequence of SEQ ID No. 11 or the complement thereof.

48. An isolated nucleic acid molecule comprising a nucleotide sequence with at least 99% sequence identity to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or the complement thereof, or a nucleotide sequence with at least 99% sequence identity to SEQ ID No. 11 or the complement thereof.

49. An isolated nucleic acid molecule comprising a nucleotide sequence hybridizing to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1452 to nucleotide position 16638 or the complement thereof, or hybridizing to the nucleotide sequence of SEQ ID No. 11 or the complement thereof.