鹅基因组微卫星富集文库的构建与分析

为分离鹅(Anser cygnoides)微卫星序列片段,提取鹅基因组DNA,用Hae III和Rsa I内切酶消化并连接接头,再用接头特异引物进行PCR扩增。扩增产物与生物素标记的(AC)12探针杂交,杂交复合物用链霉亲和素包裹磁珠进行结合,得到单链DNA目标片段。再经PCR扩增,连接pMD19-T载体,转化入感受态大肠杆菌,得到微卫星富集小插入片段DNA文库。用Colony-PCR法筛选获得318个阳性克隆,并进行测序分析。结果表明,所测的318个序列有242个含微卫星序列,197个为有效微卫星序列,其中完全型(perfect)占60.9%,非完全型(imperfect)20.8%,混合型(compound)18.2%。(CA)n重复最为常见。文章为鹅种资源遗传多样性、分子进化、遗传图谱的构建及重要经济性状基因座定位等研究奠定了基础。

Construction and Analysis of Microsatellite Enriched Library from Goose Genome

For isolating microsatellite repeat sequence of goose(Anser cygnoides),genomic DNA of goose was extracted and digested with restriction enzyme Hae III and Rsa I.Adapters were ligated to the end of restriction fragments.Ligated products were amplified by PCR with primers complementary to adapters.The PCR products were hybridized with biotin-labeled Oligo(CA)12 probes,hybridized complex was fished by magnetic beads coated with streptavidin.Then selected fragments were amplified by using adapter specific primers and cloned into pMD19-T vectors and transformed into E.coli hosts,SSR enriched genomic DNA library was constructed.318 clones were selected by colony-PCR method and then sequenced.197 distinct microsatellite loci were detected.Of these sequences,120(60.9 %) were perfect repeat,41(20.8 %) imperfect and 36(18.2 %) compound repeat.(CA)n was the most common repeat.This research laid the foundation for the study of genetic diversity,molecular evolution,genetic linkage mapping and QTLs location of the main economic traits of goose.