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【目的】以2份六倍体小麦Opata85和W7984及其重组近交系(RIL)的111个株系和3份小麦二倍体野生近缘种为材料,研究用等位基因特异PCR检测普通小麦中单核苷酸多态性的方法。【方法】利用直接测序的方法检测2份六倍体小麦和3份小麦二倍体野生近缘种TaDREB1基因的DNA序列,在B基因组上发现了2个SNPs。以其为3′端,设计等位基因特异引物及其互补引物,对SNP进行分型,同时研究了特异引物3′端碱基错配对等位基因特异PCR的影响,优化了PCR反应体系。【结果】等位基因特异引物3′端不同位置的碱基错配及不同类型的碱基错配对PCR结果影响较大;在等位基因特异PCR中,Mg2+、dNTP及TaqDNA聚合酶的用量均大于普通PCR。【结论】只要在等位基因特异引物3′端加上合适的错配碱基,并且优化其PCR反应体系,用等位基因特异PCR方法检测六倍体小麦中的单核苷酸多态性是可行的。
【Objective】 Two hundred and six hexaploid wheat lines Opta185 and W7984 and their recombinant inbred lines (RIL) were used in this study. Allele-specific PCR was used to detect common Method for Single Nucleotide Polymorphism in Wheat. 【Method】 The DNA sequence of TaDREB1 gene in two hexaploid wheat and three diploid wheat relatives was detected by direct sequencing. Two SNPs were found in the B genome. The SNP was genotyped at the 3 ’end by designing the allele-specific primers and its complementary primers. At the same time, the effect of 3’ base mismatch on allele-specific PCR was studied and the PCR reaction system was optimized. 【Result】 The results showed that the mismatch of bases and mismatches of base mismatches at 3 ’end of allele-specific primers had a great influence on PCR results. In allele-specific PCR, the amount of Mg2 +, dNTP and Taq DNA polymerase Greater than normal PCR. 【Conclusion】 As long as a suitable mismatch base is added to the 3 ’end of the allele-specific primer and the PCR reaction system is optimized, the allele-specific PCR method is used to detect single nucleotide polymorphisms in hexaploid wheat It works.