鲢三、四核苷酸重复微卫星标记的筛选及其特征分析

Development and characteristics of tri- and tetra-nucleotide microsatellites for silver carp (Hypophthalmichtys molitrix)

摘要:

通过磁珠富集法筛选鲢(Hypophthalmichtys molitrix)的三、四核苷酸重复微卫星分子标记。提取鲢血液大片段基因组DNA，用限制性内切酶Sau3AI进行酶切，蔗糖密度梯度离心收集400~900 bp长度的片段，构建鲢全基因组PCR文库，用生物素标记的微卫星探针(TGA)₈、(CAG)₈以及(AGAT)₆进行筛选，磁珠富集含有微卫星序列的DNA片段，获得含有微卫星的单链序列；将这些序列通过PCR扩增，连接pMD 18-T载体，转入感受态大肠杆菌(Escherichia coli)DH5α，获得微卫星文库；再用γ-³²P标记的放射性同位素探针进行二次杂交筛选，获得1 758个阳性克隆，选择测序348个克隆序列中，共含有280个微卫星座位，其中完美型167个(59.64%)，非完美型28个(10%)，混合型85个(30.36%)。Primer 3.0软件设计60对引物，并用珠江水系的野生鲢群体对引物进行多态性位点检测。统计软件分析显示其中22对具有较高的筛选效率和多态性，可作为鲢种质评价等遗传分析的工具。

Abstract:

It was a study to isolate the tri- and tetra-nucleotide repeat microsatellite from sliver Carp (Hypophthalmichtys molitrix) with magnetic beads enriched method. Sliver carp (Hypophthalmichtys molitrix) genomic DNA was extracted from blood and digested with restriction Sau 3A I. The segments of 400-900 bp were collected by centrifugation of sucrose density gradient and then a whole genome PCR library was created. The genomic DNA was hybridized with biotinlabeled microsatellite probes (TGA)₈，(CAG)₈，(AGAT)₈,and the singlestranded DNA containing the selected microsatellite DNA was obtained. Those selected DNAs were amplified with PCR, cloned into pMD 18-T vector, transformed into the competent Escherichia coli DH5α，and the microsatellite library was finally obtained. The second screen was performed with radiolabeled probes. 1758 positive clones were obtained and 348 microsatellite clones contained 248 microsatellite sequences. There were 167 (59.64%) perfect motifs, 28 (10%) imperfect motifs, 85 (30.36%) compound motifs. 60 pairs of primers were designed with the software Primer 3.0 and detected with the wild population of sliver Carp ( Hypophthalmichtys molitrix ) from the Pearl River. The results showed that 22 pairs of primers could be used to amplify special fragments and a genetic tool to evaluate germplasm resources of sliver Carp (Hypophthalmichtys molitrix) .