磁珠富集法开发蕨麻SSR标记引物

微卫星序列(SSR)因具有分布广、共显性遗传、稳定、多态性丰富等优点而被广泛应用于动植物遗传研究中。利用EcoRⅠ,MseⅠ2 种内切酶酶切蕨麻基因组,酶切片段与用生物素标记的探针(AG)₁₅、(GT)₁₅杂交,然后通过链霉亲和素磁珠富集、含有SSR片段的基因组片段被吸附,再经洗脱、PCR扩增、克隆和测序,完成微卫星序列的收集。共获得有效克隆236个,随机挑选其中80个进行测序,结果显示68条序列(85%)为唯一序列,挑选出其中40条含有SSR位点的序列,用引物设计软件Primer 5 成功设计出 40 对引物,经初步筛选,最终获得多态性丰富,可稳定扩增的SSR引物20对,扩增成功率达50%。对20对引物扩增结果做了初步分析,扩增出多态性位点共338个,平均每对引物扩增出17个,平均有效等位基因数(Ne)、多态性信息含量(PIC)、Nei’s 基因多样性指数(H)和Shannon’s 信息指数(I)平均值分别为1.2440、0.8996、0.1768和0.3078。20对SSR引物为蕨麻遗传变异研究提供了新的分子标记工具。

Development of microsatellite primers in Potentilla anserina by magnetic beads enrichment

Simple sequence repeat (SSR) markers have been used extensively in studies on the heredity of flora and fauna because they are widely distributed throughout the genome,codominant,stable,and polymorphic.The genomic DNA of Potentilla anserina was digested with the restriction enzymes EcoR Ⅰ and Mse Ⅰ and the digested fragments were hybridized with biotinylated (AG)15 and (GT)15 probes.The fragments containing SSR genomic loci were adsorbed onto magnetic beads coated with streptavidin.Then,by elution,PCR amplification,cloning,and sequencing,a collection of SSRs was obtained.A total of 236 effective clones was acquired.Eighty sequences were randomly selected for sequencing,and 68 of them (85％) were found to be sin gle sequences.Forty sequences including SSR loci were selected and 40 primers were developed using primer design soft (Primer 5).Finally,20 pairs of primers with rich polymorphism and stable amplification were obtained through preliminary screening.The success rate of amplification was 50％.A preliminary analysis using the 20 pairs of primers amplified 338 polymorphic sites,with an average of 17 sites per primer pair.The mean values of effective allele number (Ne),polymorphism information content (PIC),Nei's gene diversity (H),and Shannon--Weaver index (I) were 1.2440,0.8996,0.1768,and 0.3078,respectively.These 20 pairs of SSR primers provide novel molecular markers for research on the genetic diversity of P.anserina.