利用SSR荧光标记构建山楂种质分子身份证

Using the Fluorescent Labeled SSR Markers to Establish Molecular Identity of Hawthorn

以91份山楂种质资源为材料,使用SSR标记对其进行分子身份证构建,并进行遗传多样性分析。从山楂基因组数据库中开发并设计了96对引物,筛选出了12对多态性好的引物,利用SSR荧光标记毛细管电泳技术检测扩增条带的分子量,并分析参试样品的扩增带型。采用数字与字母组合的方式,依据12对引物的多态性信息含量由高到低排列引物扩增结果,生成各材料的指纹编码。基于非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)对91份山楂资源进行聚类分析。结果表明,12对SSR引物从91个山楂种质资源样品中共扩增到67个等位基因,其平均等位基因数、有效等位基因数、观测杂合度、期望杂合度分别为5.583、2.730、0.490、0.593,Shannon’s信息指数平均值为1.195,多态性信息含量介于0.300~0.715之间,平均为0.551。91份山楂资源的遗传相似系数范围为0.25~0.98,平均值为0.529,在遗传相似系数0.46处时可分为5大类群。根据引物的多态性信息含量高低选择引物组合,构建了山楂种质区分需要的最少引物组合。

In present research,simple sequence repeats(SSR)markers were employed to analyze the genetic diversity and to establish the molecular identity of 91 hawthorn accessions.Ninety-six pairs of SSR primers were developed from the hawthorn genome database,and 12 pairs with good polymorphism were selected.The screened primers were labeled with four kinds of fluorescence and used for PCR amplification.The PCR products of the 91 samples were analyzed by capillary electrophoresis.The GeneMarker analysis software was used to determine the sizes of the amplified fragments and the genotypes.The individual digits and uppercase English letters were used to mark each band to encode a fingerprint.According to the polymorphic information content(PIC)of 12 primers,the order of these primer pairs were sorted from high to low,and molecular identities of 91 hawthorn accessions were established.Cluster analysis of 91 hawthorn accessions were performed using the Unweighted pair-group method with arithmetic means(UPGMA).The results showed that a total of 67 alleles were detected from 12 pairs of SSR primers,with an average of 5.583 alleles per each primers and an average of 2.730 effective alleles.The mean values of observed heterozygosity(Ho),expected heterozygosity(He),Shannon’s information index(I),and polymorphic information content(PIC)were 0.490,0.593,1.195,and 0.551(range 0.300–0.715),respectively.The genetic similarity coefficient of 91 hawthorn accessions ranged from 0.25 to 0.98 with an average of 0.529,and the samples could be classified into five groups at the genetic similarity coefficient of 0.46.The molecular identities of the 91 hawthorn accessions were constructed through the selection of primers according to its PIC,these accessions could be distinguished by the least specific primers.This study could provide reliable basis for the identification,evaluation,utilization,and genetic relationship analysis of hawthorn germplasm resources.