Granted Patent B2

US 12,252,721 · App. 18/504,940 · Granted Mar 18, 2025

Inventors: Yunxing C. Cui (Carmel, IN); Thomas Hoffman (Zionsville, IN); Ning Zhou (Zionsville, IN); Stephen N. Novak (Westfield, IN); Julissa Colon (Cidra, PR); Dawn M. Parkhurst (Avon, IN); Sandra G. Toledo (West Lafayette, IN); Terry R. Wright (Westfield, IN); Sean M. Russell (Carmel, IN); Bruce Held (Ames, IA); Vaithilingam Sekar (Ames, IA)

Assignees: CORTEVA AGRISCIENCE LLC; M.S. TECHNOLOGIES, L.L.C.

C12N9/93C12N9/1092C12N15/8241C12N15/8274C12N15/8275C12Q1/6895C12Y205/01019C12Y604/01002A01N39/04C12Q2600/13C12Q2600/156C12Q2600/16

Abstract

Claims (27)

1. A method for determining event zygosity of a soybean plant comprising soybean Event pDAB8264.44.06.1, as present in seed deposited under ATCC Accession number PTA-11336, said Event pDAB8264.44.06.1 comprising a transgene construct, said transgene construct being flanked by a 5′ flanking soybean genomic DNA and a 3′ flanking soybean genomic DNA, said method comprising:

obtaining a DNA sample comprising genomic DNA from said soybean plant;

producing a contacted sample by contacting said DNA sample with

a. a first event primer and a second event primer, wherein said first event primer specifically binds said transgene construct, said second event primer specifically binds said 5′ flanking soybean genomic DNA or said 3′ flanking soybean genomic DNA, and wherein said first event primer and said second event primer produce an event amplicon when subjected to PCR conditions;

b. a reference forward primer and a reference reverse primer that produce a reference amplicon from an endogenous soybean reference gene when subjected to PCR conditions;

c. a florescent event probe that is labeled with a fluorescent dye and a quencher and hybridizes with said event amplicon;

d. a florescent reference probe that is labeled with a fluorescent dye and a quencher and hybridizes with said reference amplicon;

subjecting said contacted sample to fluorescence-based endpoint PCR conditions in the presence of a thermostable polymerase, dNTPs, said fluorescent event probe and said fluorescent reference probe;

quantitating said florescent event probe that hybridized to said event amplicon;

quantitating said florescent reference probe that hybridized to said reference amplicon;

comparing amounts of hybridized florescent event probe to amounts of hybridized florescent reference probe; and

determining zygosity of said Event pDAB8264.44.06.1 by comparing florescence ratios of said hybridized fluorescent event probe and said hybridized fluorescent reference probe.

2. The method of claim 1 , wherein said amplicons consist of about 50 to about 150 nucleotide residues.

3. The method of claim 1 , wherein said reference gene is an endogenous soybean GMFL01-25-J19 gene, as disclosed in GenBank accession number: AK286292.1.

4. The method of claim 1 , wherein said first event primer binds an insert sequence of SEQ ID NO: 27, and said second event primer binds SEQ ID NO: 1 or SEQ ID NO: 2, or the complements thereof.

5. The method of claim 1 , wherein said method is used for breeding introgression of the Event pDAB8264.44.06.1 into a soybean line said method comprising crossing a first parental soybean plant comprising soybean Event pDAB8264.44.06.1, as present in seed deposited under ATCC Accession number PTA-11336, with said soybean line thereby producing a plurality of progeny plants; then selecting a progeny plant that comprises Event pDAB8264.44.06.1.

6. The method of claim 5 , wherein said soybean line lacks said Event pDAB8264.44.06.1.

7. The method of claim 1 , wherein said reference gene comprises or hybridizes to a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

8. The method of claim 1 , wherein said reference primers are selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 24, and said reference probe comprises SEQ ID NO: 26.

9. The method of claim 1 , wherein said fluorescent event probe comprises FAM as said fluorescent dye at the 5′ end of said fluorescent event probe and an MOB quencher on the 3′ end of said fluorescent event probe.

10. The method of claim 1 , wherein said fluorescent reference probe is labeled with HEX at the 5′ end of said fluorescent reference probe and a Black Hole Quencher 1 (BHQ1) at the 3′ end of said fluorescent reference probe.

11. The method of claim 1 , wherein said fluorescent event probe comprises SEQ ID NO: 20.

12. The method of claim 1 , wherein said event primers are selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 22.

13. The method of claim 1 , wherein results of said method are read directly in a plate reader.

14. The method of claim 1 , wherein said DNA sample is obtained from a soybean plant in a field.

15. A kit for performing the method of claim 1 , said kit comprising said first event primer, said second event primer, said reference forward primer, said reference reverse primer, said event probe, and said reference probe.

16. The kit of claim 15 , wherein said first event primer consists of SEQ ID NO: 18 and said second event primer consists of SEQ ID NO: 19, said reference forward primer consists of SEQ ID NO: 24 and said reference reverse primer consists of SEQ ID NO: 25, said event probe consists of SEQ ID NO: 20, and said reference probe consists of SEQ ID NO: 26.

Assignments (3)

Continuity (10)

References Cited (148)