Development and application of a high oleic acid allelic mutation of rapeseed bnfad2 gene and its SNP marker primer
A high oleic acid, KASP-1073 technology, applied in the field of in situ mutation, can solve the problems of low throughput, complicated process, high cost, etc., and achieve the effect of accurate and reliable application method and easy operation.
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Embodiment 1
[0051] Example 1. Finding and obtaining a novel allelic mutation based on two copies of BnFAD2 that can be used to indicate the high oleic trait of Brassica napus.
[0052] (1) In our previous research, we used a high oleic acid homozygous strain (FC81, storage date: 2019.12.2, storage number: CCTCC NO: P201923) with an oleic acid content of 87.67% as the female parent, and an oleic acid content of 63.56% % of the homozygous line with medium and low oleic acid (E183, conventional existing varieties) is the male parent, and the F 2 Separate groups. From the above F 2 Genomic DNA was extracted from the fresh and tender leaves of isolated populations and parents, and the specific preparation method was referred to Li Jia et al. ) method, the DNA quality was detected by 1% agarose gel electrophoresis, and the DNA concentration was detected by an ultraviolet spectrophotometer (model: Pharmacia Biotech, Gene QuantII).
[0053] (2) Determination of oleic acid content: the seeds ar...
Embodiment 2
[0070] Example 2. A SNP marker that can be used to detect a new type of high oleic acid allelic mutation in rape BnFAD2 gene was developed.
[0071]The 100bp flanking sequences on both sides were extracted centering on the SNP site in the coding region of the above gene, and combined with the sequence difference between nAFAD2a and BnCFAD2a obtained by cloning, primers were designed to specifically amplify the sequence fragments of the BnAFAD2a and BnCFAD2a genes to exclude the detection of homologous gene sequence pairs generated interference. Multiple primer sets were designed in the experiment, and each set of primers consisted of three primers. Obtained through multiple polymorphism screening and verified by multiple tests in multiple isolated populations, the markers KASP-421 and KASP-1073 have the best amplification effects, which can clearly distinguish the wild type and the high oleic acid mutant in BnAFAD2a and SNP variants of BnCFAD2a. Both pairs of KASP markers co...
Embodiment 3
[0084] Example 3: The establishment of the above-mentioned SNP marker system that can be used to detect a new type of high oleic acid allelic mutation in rapeseed BnFAD2 gene and its application in assisted breeding for high oleic acid traits in Brassica napus.
[0085] Pick one that contains 111 F's 2 F 2 population (♀FC81×♂E183) and a group containing 90 F 2 F 2 Population (♀FC90×♂E121, FC90 and E121 are all conventional varieties, used as a comparison), using the two sets of KASP primers KASP-421 and KASP-1073 designed above, to conduct a primary screening of the isolated population on the LGCSNP line genotyping platform with validation. The specific operation steps are as follows:
[0086] (1) Extract the genomic DNA of the leaves of the material to be tested by conventional methods (CTAB method); use agarose electrophoresis and Nanodrop2100 to detect the quality of the extracted DNA respectively, and the agarose electrophoresis shows that the DNA band is single, and A...
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