Elite event EE-GH7 and methods and kits for identifying such event in biological samples

Patent metadata

Field	Value
Publication number	US-11085050-B2
Application number	US-201716095234-A
Country	US
Kind code	B2
Filing date	Apr 18, 2017
Priority date	Apr 20, 2016
Publication date	Aug 10, 2021
Grant date	Aug 10, 2021

Abstract

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

First claim

The invention claimed is:

1. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1207 to nucleotide 1228, or of SEQ ID No. 1 from nucleotide 8022 to nucleotide 8043, or the complement of said sequences, reference seed comprising said nucleic acid molecule having been deposited at the ATCC under deposit number PTA-122856.

2. A cotton plant, cell, part, tissue, seed or progeny thereof, comprising elite event EE-GH7 in its genome, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856.

3. Cotton genomic DNA obtainable from the cotton plant, cell part, tissue, seed or progeny thereof of claim 2 , said cotton genomic DNA comprising elite event EE-GH7.

4. A seed comprising elite event EE-GH7 deposited at the ATCC under deposit number PTA-122856 or derivatives therefrom, said derivatives comprising elite event EE-GH7.

5. A transgenic cotton plant comprising elite event EE-GH7 in its genome, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, which is tolerant to isoxaflutole and/or glyphosate.

6. The cotton plant, cell, part, tissue, seed or progeny thereof according to claim 2 , further comprising
event T304-40, comprising glufosinate tolerance and the CrylAb gene;
event GHB119 comprising glufosinate tolerance and the Cry2Ae gene; and/or
event COT102 comprising the VIP3A gene.

7. A method for producing a cotton plant or seed comprising elite event EE-GH7, comprising crossing a plant according to claim 2 with another cotton plant, and planting the seed obtained from said cross.

8. A cotton product produced from the cotton plant, cell, part, tissue, seed or progeny thereof of claim 2 , wherein the cotton product comprises cotton genomic DNA comprising elite event EE-GH7.

9. A method for weed control, comprising treating a field in which the cotton seeds of claim 2 are sown with an HPPD inhibitor herbicide, before the cotton plants emerge but after the seeds are sown.

10. A method of weed control, comprising treating the cotton plants of claim 2 with an HPPD inhibitor herbicide after the cotton plants emerge.

11. A method for protecting the emerging cotton plants of claim 2 from competition by weeds, comprising treating a field to be planted with said cotton plants with an HPPD inhibitor herbicide, before the cotton plants are planted or the seeds are sown, followed by planting or sowing of said cotton plants or seeds in said pre-treated field.

12. The method according to claim 9 , further comprising treating the cotton plants with glyphosate.

13. A method for weed control, comprising treating the cotton plants of claim 2 with glyphosate after the cotton plants emerge.

14. A method for identifying elite event EE-GH7 in biological samples, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, which method comprises detection of an EE-GH7 specific region with a specific primer pair which specifically recognizes the 5′ or 3′ flanking region of the foreign DNA comprising herbicide tolerance genes in EE-GH7, and part of the foreign DNA contiguous with said 5′ or 3′ flanking region.

15. A primer pair comprising a first and a second labeled primer, said first primer recognizing the 5′ flanking region of the foreign DNA comprising herbicide tolerance genes in EE-GH7, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1217, or said first primer recognizing the 3′ flanking region of the foreign DNA comprising the herbicide tolerance genes in EE-GH7, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 8033 to nucleotide 9328, and said second primer recognizing a sequence within the foreign DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1218 to nucleotide 8032 or the complement thereof.

16. A specific probe for identifying elite event EE-GH7, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, in biological samples, said specific probe comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GH7 and the sequence of the foreign DNA contiguous therewith, wherein the probe specifically recognizes the nucleotide sequence of SEQ ID NO. 1 from nucleotide 1207 to nucleotide 1228 or of SEQ ID NO. 1 from nucleotide 8022 to nucleotide 8043.

17. A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH7, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, said method comprising amplifying DNA fragments of between 50 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, and the third of said primers recognizing a sequence within the foreign DNA.

18. A method of detecting the presence of elite event EE-GH7, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, in biological samples through hybridization with a 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. 1 from nucleotide 1218 to nucleotide 1235 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 8015 to 8032 or its complement;
b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 1200 to nucleotide 1217 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide 8033 to nucleotide 8050 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.

19. The method according to claim 17 , wherein the two primers which specifically recognizing pre-insertion plant DNA are a primer comprising the nucleotide sequence of SEQ ID No. 11 and a primer comprising the nucleotide sequence of SEQ ID No. 12, and wherein the third of said primers comprises the nucleotide sequence of SEQ ID No. 13.

20. A method for identifying elite event EE-GH7 in biological samples, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-122856, which method comprises detection of an EE-GH7 specific region with a specific probe, said specific probe comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GH7 and the sequence of the foreign DNA contiguous therewith, wherein

Assignees

• BASF Agricultural Solutions Seed US LLC

Inventors

• Jansens Stefan
• Dreesen Rozemarijn
• Aartsen Wendy
• Vanhaelen Jonas
• Moser Hal
• Light Ginger

Classifications

• C12N9/1092

  3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase · CPC title

• C07K2319/08

  containing a chloroplast localisation signal · CPC title

• C12N15/8274

  for herbicide resistance · CPC title

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12Q2600/13

  Plant traits · CPC title