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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。研究亮点:首次利用多碱基(三、四核苷酸重复)探针筛选出微卫星分子标记结合同位素探针进行两次筛选,是开发鲢微卫星分子标记过程中新的尝试与探索。在与其他利用二碱基核苷酸重复探针开发的鲢微卫星分子标记的对比中发现,四核苷酸重复的微卫星比二、三核苷酸重复微卫星具有更高的筛选效率和多态性。
Three and four nucleotide repeat microsatellite molecular markers of silver carp (Hypophthalmichtys molitrix) were screened by magnetic bead enrichment. The genomic DNA of large silver carp was extracted and digested with restriction endonuclease Sau3AI and sucrose density gradient centrifugation to collect the fragments of 400-900 bp in length. The full-length PCR library of silver carp was constructed and the biotin-labeled microsatellite probes (TGA ) 8, (CAG) 8 and (AGAT) 6, the magnetic beads were enriched in DNA fragments containing microsatellite sequences to obtain single-stranded sequences containing microsatellites; these sequences were amplified by PCR and ligated into pMD18-T vector , Transformed into competent Escherichia coli DH5α to obtain a microsatellite library, and then screened by γ-32P radioisotope probe for secondary hybridization to obtain 1 758 positive clones. Among 348 sequences sequenced, A total of 280 microsatellite loci were found, of which 167 were perfect (59.64%), 28 (10%) were imperfect, and 85 (30.36%) were mixed. Primer 3.0 software designed 60 pairs of primers and used the wild silver carp population of Pearl River to detect the polymorphic loci. Statistical software analysis showed that 22 of them had high screening efficiency and polymorphism and could be used as tools for genetic analysis such as silver carp breeding evaluation. Research Highlights: The first screening of microsatellite molecular markers with isotopic probes using multi-base (tri- and tetranucleotide repeat) probes for the first time is a new attempt in the development of microsatellite molecular markers for silver carp. Compared with other silver carp microsatellite markers developed by using two-nucleotide repeat probes, it was found that tetracycline-repeat microsatellites have higher screening efficiency than two-and three-nucleotide repeat microsatellites Polymorphism.