用等位基因特异PCR检测普通小麦(Triticum aestivum L.)的单核苷酸多态性

Assaying Single Nucleotide Polymorphism in Wheat (Triticum aestivum L.) with Allele-Specific PCR

【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 hexaploid wheat cultivars （Triticum aestivum L.）, Opata85 and W7984, their 111 recombinant inbred lines （RILs）, and three diploid relative-species, T. urartu, Ae. Speltoides and Ae. Tauschii were used as plant materials to study the method of assaying single nucleotide polymorphism with allele-specific PCR in wheat. [Method] Two SNPs were discovered on B genome by aligning the TaDREB1 genes in two hexaploid wheat cultivars and three diploid relative-species. To type these SNPs, allele-specific primers and their complementary primers were designed using the SNPs as their 3＇-end. In addition, we studied the effect of the mismatched bases at the 3＇-end of the allele-specific primers on PCR and the optimum PCR system. [Result] There were distinct effects of the mismatched bases at 3＇-end different sites of the allele-specific primers on allele-specific PCR, so did different types of mismatched bases. Moreover, the concentrations of Mg^2＋, dNTP and Taq DNA polymerase in allele-specific PCR were higher than that in conventional PCR. [ Conclusion] It is feasible to assay SNPs by allele-specific PCR in hexaploid wheat, as long as proper mismatched bases were introduced at 3＇-end proper sites of the allele-specific primers and the PCR system was optimized reasonably.