紫云英SSR分子标记的开发及在品种鉴别中的应用

Development of Microsatellite Markers for Astragalus sinicus L.and Its Application for Variety Identification

利用生物素标记的(AG)15、(CT)15、(AC)15、(GT)15探针及链霉素亲和磁珠,从紫云英的基因组中富集微卫星(SSR)序列。在用富集片段构建插入文库的950个转化子中,经PCR检测及测序共得到127个SSR序列,微卫星序列的富集效率达15.8%。除去重复或无效的序列,得到33个序列用于引物设计。对征集的9个紫云英品种进行多态性分析,有6对引物扩增出明显且稳定的多态性位点18个。在紫云英的品种水平上,这些SSR位点产生的多态率为50%～100%,有效等位基因数为1.19～1.64,Nei氏遗传多样性为0.13～0.38,Shannon多态信息指数为0.22～0.56;利用这6对引物可将参试品种完全区分开,证明这些SSR位点可用于紫云英品种的指纹鉴别。根据SSR位点的相似性系数,将9个紫云英品种主要聚成两个类群,与传统按生育期的紫云英品种划分结果无必然的联系。

Astragalus sinicus L.is a traditional green manure used in rice field in China.To identify Astragalus sinicus L.varieties in agricultural production,we developed SSR molecular markers by using biotinylated（AG）15,（CT）15,（AC）15,（GT）15 probes and streptavidin coated magnetic beads,the SSR sequences were enriched from A.sinicus genome.Of the 950 transformants selected from the library constructed by obtained fragments,127 clones were confirmed as SSR insert by PCR identification and final sequencing,with a SSR enrichment efficiency of 15.8%.By deleting the duplicated and unavailable sequences,33 SSR loci were used for primer design.After nine A.sinicus varieties were sampled for analysis of the genetic diversity based on these SSR primers,only six SSR primer pairs amplified a total of 18 stable and repeatable polymorphic bands within tested plant samples.At the level of variety population of A.sinicus,based on these SSR loci we calculated the percentage of polymorphic band ranging from 50% to 100%,effective number of alleles ranging from 1.19 to 1.64,Nei＇s gene diversity ranging from 0.13 to 0.38,and Shannon＇s information index ranging from 0.22 to 0.56;the selected six primer pairs could completely distinguish the tested plant samples,and the application of these SSR loci for fingerprinting identification of variety was proved to be feasible to A.sinicus.Based on similar coefficient generated from six SSR loci,we clustered nine tested varieties of A.sinicus mainly into two groups,which had no certain linkage to the traditional classification of variety according to its growth period.The study opened up a prospect of using SSR molecular marker for genetic analysis,variety identification,and protection of intelligent property in commercial breeding of A.sinicus.