METHOD TO DETERMINE ZYGOSITY OF THE FAD3 GENE IN CANOLA USING END-POINT TAQMAN PCR
The subject disclosure relates in part to endpoint TaqMan® PCR assays for the detection and high throughput zygosity analysis of the fad-3c gene in canola. The subject disclosure further relates, in part, to the use of wild type DNA as a reference for use in determining zygosity. These and other related procedures can be used to uniquely identify the zygosity and variety of canola lines comprising the subject gene. The subject disclosure also provides related kits for determining zygosity from a sample of a canola plant or seed, for example.
1 - 16 . (canceled)
17 . A method for determining zygosity of a canola plant comprising a fad-3c gene, said method comprising:
obtaining a sample of genomic DNA from a canola plant;
hybridizing the sample of genomic DNA with a first primer, a second primer, a first probe, and a second probe, wherein said first primer and said second primer comprise SEQ ID NO: 2 and SEQ ID NO: 3, and said first probe and said second probe comprise SEQ ID NO: 5 and SEQ ID NO: 4, wherein each of said first probe and said second probe is labeled with a fluorescent dye and a quencher;
measuring florescence of said first probe, said second probe, or a combination thereof; and
determining zygosity of said canola plant.
18 . The method of claim 17 , wherein the sample of genomic DNA comprises a mutated fad-3c sequence having a single nucleotide polymorphism, wherein said single nucleotide polymorphism consists of a G-to-A polymorphism.
19 . The method of claim 18 , wherein the sample of genomic DNA further comprises a wild-type fad-3c sequence.
20 . The method of claim 17 , wherein said method is used for breeding introgression verification of cross-bred canola plants.
21 . The method of claim 17 , wherein said first probe comprises VIC as said fluorescent dye at the 5′ end of said first probe and a MGB quencher on the 3′ end of said first probe.
22 . The method of claim 17 , wherein said second probe is labeled with FAM at the 5′ end of said second probe and a MGB quencher at the 3′ end of said second probe.
23 . The method of claim 17 , wherein measuring florescence comprises measuring and analyzing fluorescence directly in a plate reader.
24 . The method of claim 17 , wherein said DNA sample is obtained from a canola plant in a field.
25 . The method of claim 17 , wherein said first probe and said second probe hybridizes to the sample of genomic DNA for a period of time and at a temperature of 50-70 degrees Celsius.
26 . The method of claim 25 , further comprising increasing said temperature after the period of time.
27 . The method of claim 26 , wherein said florescence produced by each of said first probe and said second probe during the step of increasing said temperature is measured by increments.
28 . The method of claim 17 , wherein said first probe hybridizes to a region of a wild-type fad-3c sequence and said second probe hybridizes to a region of a mutated fad-3c sequence having a single nucleotide polymorphism (SNP).
29 . A kit for performing the method of claim 17 , said kit comprising said first primer, said second primer, said first probe, and said second probe.
30 . The kit of claim 29 , wherein said first primer is SEQ ID NO:2, said second primer is SEQ ID NO:3, said first probe is SEQ ID NO:5, and said second probe is SEQ ID NO:4.