一种大豆SNP分型新方法

单核苷酸多态性(SNP)在大豆基因组中分布广泛,SNP分型是大豆SNP遗传作图,关联分析、分子标记辅助选择等研究的重要技术.本文以Rhg4基因开发的5个SNP为例,介绍了一种大豆SNP分型的新方法-片段长度差异等位基因特异性PCR(FLDAS-PCR).采用AS-PCR原理,针对某个SNP位点设计2条相差4-5bp的特异引物和1个公用引物,PCR产物约100bp或150bp,2种等位基因型PCR产物相差4-5bp,在2个特异引物3'端第3和4碱基位置分别人为引入错配碱基来提高PCR特异性,通过6%变性聚丙烯酰胺凝胶电泳可将纯合和杂合基因型检测出来.探讨了特异引物浓度和退火温度对Rhg4-1592扩增效果的影响,研究表明,可通过调整特异引物浓度和退火温度优化扩增效果.FLDAS-PCR对18份种质分型结果与PCR产物克隆测序法一致,表明本研究建立的FLDAS-PCR法是一种简便、快捷、新型的大豆SNP分型方法.

A METHOD OF SNP GENOTYPING IN SOYBEAN

Single nucleotide polymorphism(SNP) is a genome-wide mutation type for organisms.SNP genotyping is an important technique for SNP genetic mapping,association analysis,and molecular assistant selection and so on.We introduced a fragment length discrepant allele specific PCR(FLDAS-PCR) method for SNP genotyping in soybean,using 5 SNPs in Rhg4 as examples.For a SNP loci,we designed 2 allele specific primers which had 4-5bp length difference and 3'-end complementary to each of bi-allelic SNPs,and 1 common primer,as a result PCR products of different alleles had 4-5bp difference in length,which could be separated by 6% denature polyacrylamide gel.In order to improve PCR specificity and data accuracy,an additional base mismatch was introduced at the 3rd or 4th nucleotide closest to the 3'-end of each of the specific primers.We also investigated the influence of annealing temperature and primer concentration on PCR amplification,Rhg4-1592 as an example,and found that lower concentration of the specific primer would reduce its annealing competition in PCR reaction,and behaved more sensitive to annealing temperature,so we could optimize PCR by increasing or reducing specific primer concentration.The genotypes of SNPs by the FLDAS-PCR agreed with the results by PCR cloning and sequencing for 18 soybean genotypes,which approved that the method was effective in SNP genotyping in soybean.