Herbicide tolerant cotton plants and methods for identifying same
View Patent ↗The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific 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.
1. A method for identifying elite event EE-GH3 in biological samples comprising hybridizing a nucleic acid of biological samples with a specific probe that recognizes a part of the 5′ or 3′ flanking region of EE-GH3 and a part of the foreign DNA of EE-GH3 contiguous therewith, said 5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732 and said foreign DNA contiguous with said 5′ flanking region comprises SEQ ID No. 1 from nucleotide 733 to nucleotide 1214, said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 431 to nucleotide 654 and said foreign DNA contiguous with said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430.
2. The method of claim 1 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking region or the 3′ flanking region of EE-GH3 and the sequence of the foreign DNA contiguous therewith.
3. The method of claim 2 , wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 712 to 753 or SEQ ID No. 2 from nucleotide 410 to 451, or the complement of said sequences.
4. A kit for identifying elite event EE-GH3 in biological samples comprising a specific probe, wherein said elite event has a specific region comprising a 5′ flanking region and inserted foreign DNA contiguous therewith and a specific region comprising a 3′ flanking region and inserted foreign DNA contiguous therewith, said 5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732 and said foreign DNA contiguous with said 5′ flanking region comprises SEQ ID No. 1 from nucleotide 733 to nucleotide 1214, said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 431 to nucleotide 654 and said foreign DNA contiguous with said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430, wherein said probe comprises part of the 5′ flanking region and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof, or part of the 3′ flanking region of EE-GH3 and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof.
5. The kit of claim 4 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking region or the 3′ flanking region of EE-GH3 and the sequence of the foreign DNA contiguous therewith.
6. The kit of claim 5 , wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 712 to 753 or SEQ ID No. 2 from nucleotide 410 to 451, or the complement of said sequences.
7. A specific probe for the identification of elite event EE-GH3 in biological samples, wherein said elite event has a specific region comprising a 5′ flanking region and inserted foreign DNA contiguous therewith and a specific region comprising a 3′ flanking region and inserted foreign DNA contiguous therewith, said 5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732 and said foreign DNA contiguous with said 5′ flanking region comprises SEQ ID No. 1 from nucleotide 733 to nucleotide 1214, said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 431 to nucleotide 654 and said foreign DNA contiguous with said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430, wherein said probe comprises part of the 5′ flanking region and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof, or part of the 3′ flanking region of EE-GH3 and part of the sequence of the foreign DNA contiguous therewith, or the complement thereof.
8. The probe of claim 7 , wherein said probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking region or the 3′ flanking region of EE-GH3 and the sequence of the foreign DNA contiguous therewith, or the complement thereof.
9. The probe of claim 8 which has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 712 to 753 or SEQ ID No. 2 from nucleotide 410 to 451, or the complement of said sequences.
10. A specific probe for the identification of elite event EE-GH3 in biological samples, comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID No. 1 from nucleotide 712 to 753 or to SEQ ID No. 2 from nucleotide 410 to 451, or the complement of said sequences.
11. A method for confirming seed purity comprising
(a) hybridizing a nucleic sequence present in said seed samples with a specific probe that recognizes a part of the 5′ or 3′ flanking sequence of EE-GH3 and a part of the foreign DNA of EE-GH3 contiguous therewith, said 5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732 and said foreign DNA contiguous with said 5′ flanking region comprises SEQ ID No. 1 from nucleotide 733 to nucleotide 1214, said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 431 to nucleotide 654 and said foreign DNA contiguous with said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430; and
(b) confirming purity of seed samples.
12. A method for screening seeds for the presence of EE-GH3 comprising
(a) hybridizing a nucleic sequence present in said seed samples with a specific probe that recognizes a part of the 5′ or 3′ flanking sequence of EE-GH3 and a part of the foreign DNA of EE-GH3 contiguous therewith, said 5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732 and said foreign DNA contiguous with said 5′ flanking region comprises SEQ ID No. 1 from nucleotide 733 to nucleotide 1214, said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 431 to nucleotide 654 and said foreign DNA contiguous with said 3′ flanking region comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430; and
(b) screening said seed samples for the presence of elite event EE-GH3.
13. A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH3, said method comprising amplifying DNA fragments of between 100 and 500 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, one of said primers recognizing the 5′ flanking region of EE-GH3, said 5′ flanking region having the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 732, a second of said primers specifically recognizing the 3′ flanking region of EE-GH3, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No. 2 from nucleotide 431 to nucleotide 654, a third of said primers recognizing a sequence within the foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 733 to nucleotide 1214 or the nucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 430.
14. The method according to claim 13 , wherein said first primer comprises the nucleotide sequence of SEQ ID No. 3, said second primer comprises the sequence of SEQ ID No. 10 and said third primer comprises the sequence of SEQ ID No. 11.