芸薹属SSR标记e-PCR与云南省芥菜型油菜种质遗传多样性分析

Genetic Diversity Analysis of Brassica by SSR Marker e-PCR and Brassica juncea Germplasm in Yunnan Province

云南芥菜型油菜种质资源丰富,为整理前人搜集的275份芥菜型油菜种质材料亲缘关系与评估电子PCR技术(e-PCR)的实用性,本研究利用搜集的17713对公开发表的芸薹属作物SSR标记在芥菜型油菜参考基因组和cDNA序列中进行e-PCR分析后挑选引物进行群体PCR分析。e-PCR分析结果显示8016对预期有扩增产物,共计扩增37272个位点,平均每对引物扩增4.65个位点;其中336对引物在基因组与cDNA序列均预期扩增2个位点。从336对引物中随机挑选合成20对预期扩增位点覆盖全基因组的SSR标记组合进行群体分析,PCR电泳结果共检测出63个条带,每对引物多态性片段为2~5个,平均每对引物可扩增3.15个条带;标记PIC值变化范围0.03~0.69,平均PIC值0.45,结果符合e-PCR分析预期。群体遗传聚类分析结果表明,云南芥菜型油菜的亲缘关系并不完全与地域性相关,少数来源不同地区的材料也可聚成一类;275份材料可在遗传相似系数为0.70处被划分为八大类群,95份原始记录信息缺失的种质材料大多位于第Ⅷ大类。本研究同时从分子标记层面理清了275份芥菜型油菜种质材料亲缘关系,验证了e-PCR分析结果的有效性,并绘制扩增芥菜型油菜基因内部序列的SSR标记图谱,这将为芥菜型油菜种质资源评价,保护及后续育种利用提供有力的理论依据与技术支持。

In order to analyse the relationship of the 275 mustard rapeseed germplasm materials collected by previous studies,and evaluate the practicability of electronic PCR(e-PCR)technology,in this study,17713 published SSR markers of Brassica crop were collected for e-PCR analysis using the reference genome and cDNA sequence of Brassica juncea,and then primers were selected for population PCR analysis.e-PCR analysis results showed that a total of 37272 sites were amplified expected by 8016 SSR markers,and each pair of primers was supposed to amplify 4.65 loci on average.Among them,336 SSR could be expected to amplify two loci in both genome and cDNA sequence.Among the 336 SSR,20 SSR markers with expected amplification sites covering the whole genome were randomly selected and synthesized for population analysis.A total of 63 bands were detected by electrophoresis,and 2 to 5 polymorphic fragments were detected in each SSR,with an average of 3.15.The PIC values of 20 SSR ranged from 0.03 to 0.69,with an average PIC value of 0.45.The results were in line with the expectations of EPCR analysis.Population genetic clustering analysis results show that the phylogenetic relationship of B.juncea in Yunnan was not completely related to the region,and a few materials from different regions could be clustered into one group.The 275 material can be divided into eight groups in the genetic similarity coefficient of 0.70.And most of the 95 germplasm materials with missing original record information were located in the VIl category.In this study,the genetic relationships of 275 B.juncea germplasm materials were identified by molecular markers,and verified the validity of e-PCR analysis results,and drew the SSR marker map of the internal locus of the amplified genes,which will provide a strong theoretical basis and technical support for the subsequent germplasm resources evaluation and breeding utilization.