Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof

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.