菜豆EST-SSR分子标记开发及部分种质分子身份证构建

Development of EST-SSR Marker System for Phaseolus vulgaris and Construction of Molecular ID for Some Important Germplasms

为开发菜豆高效新型分子标记,从分子水平解析菜豆重要种质的遗传多样性及亲缘关系,基于菜豆转录组数据,设计EST-SSR引物,建立EST-SSR标记系统.利用PCR筛选出多态性引物,结合毛细管电泳,分析81份菜豆种质的亲缘关系,并构建分子身份证.通过MISA软件,在菜豆转录组数据85276条非冗余序列中,共挖掘出11649个EST-SSR位点.菜豆转录组SSR标记主要重复单元为1~3个碱基,其中单碱基为主要类型,占总量的56.7%;其次是三碱基重复,占总量的21.1%.从128对EST-SSR引物中筛选出18对多态性高、重复性好的引物进行PCR扩增.供试种质的多态性信息量(PIC)为0.50~0.95,平均为0.88;遗传相似性系数为0.15~0.65,以0.35为阈值可将81份种质分为3大类,其中贵州种质主要分布于第Ⅰ,Ⅲ类,而省外品种仅分布在第Ⅱ类.利用核心引物TDc9和TDc66可以有效区分81份菜豆种质,并构建其分子身份证.研究开发的EST-SSR标记系统,可用于菜豆种质的鉴定及亲缘关系分析.

To develop an efficient marker system for Phaseolus vulgaris as well as to elucidate the phylogenetic relationships of important germplasms,in the present work,EST-SSR primers were designed based on the transcriptome data,subsequently,an EST-SSR marker system was established.Using PCR and capillary electrophoresis with selected polymorphic primers,the 81 Phaseolus vulgaris germplasms,most of which originated in Guizhou Province,were fingerprinted and genetically identified.With MISA software,a total of 11649 EST-SSR loci were identified from 85276 unigene sequences in the transcriptome data of Phaseolus vulgaris.The main repetitive units of SSR markers in the transcriptome of Phaseolus vulgaris are 1 to 3 bases,with single base repeats being the main type,accounting for 56.7%of the total,followed by triple base repeats,accounting for 21.1%of the total.Totally,18 pairs of EST-SSR primers with higher polymorphism and better repeatability were selected from 128 primer pairs.The polymorphic information content(PIC)of the tested germplasms were ranged from 0.50 to 0.95,with an average value of 0.88.The genetic similarity coefficient(DICE)varied from 0.15 to 0.65.When DICE is 0.35,the 81 germplasms can be divided into three categories,among which,the germplasms from Guizhou were mainly distributed in classⅠandⅢ,and the cultivars from the other provinces were only distributed in classⅡ.The core primers TDc9 and TDc66 might efficiently differentiate the 81 Phaseolus vulgaris germplasms,thereby were employed to construct the molecular ID.Conclusively,the established EST-SSR marker system may be effective for identifying Phaseolus vulgaris germplasms and elucidating genetic diversity.