扁吻鱼微卫星分子标记的筛选及特征分析

Isolation and Characterization of Microsatellite from Genome of Aspiorhynchus laticeps

通过磁珠富集法筛选扁吻鱼的微卫星分子标记。利用限制性内切酶Sau3AI对扁吻鱼的基因组DNA进行酶切,并选取片段大小在250～750 bp间的片段,利用PCR技术进行全基因组扩增,采用生物素标记的(CA)8探针对微卫星片段进行富集。富集后的片段与T载体连接后利用DH5α大肠杆菌进行转化,然后以Sau3AI为引物对菌落进行PCR扩增,扩增后选片段大小符合条件的菌落挑取至测序板,进行测序。结果表明:PCR筛选共获得563个阳性克隆,其中测序192个阳性克隆后发现有53个微卫星序列;序列分析表明,完美型占67.92%,非完美型为22.64%;混合性标记占9.43%。根据测序结果设计微卫星引物24对,经PCR扩增筛选,琼脂糖凝胶电泳结果显示,其中22对引物可扩增出清晰的条带,其中具有多态性的13对。利用13对多态性引物对扁吻鱼野生群体进行扩增,PCR扩增结果显示,等位基因数为3～6,多态信息含量(PIC)为0.625 3,12个位点处于高度多态水平(PIC>0.5)。

Magnetic beads enriched method was used to isolate microsatellite DNA from Aspiorhynchus laticeps genome.Aspiorhynchus laticeps genomic DNA was extracted and then digested with restriction enzyme Sau3AI.Targeted segments of 250-750 bp were collected by centrifugation of sucrose density gradient and ligated to adaptors.Then the purified ligated DNA was hybridized with Biotin-labeled simple sequence repeats probes（CA）8,（TGA）8.After capture of target fragments by magnetic beads and PCR amplification,the selected DNA s were cloned into the pMD18-T vector and transformed into competent Escherichia coli DH5α,and finally a microsatellite library was obtained.563 positive colonies were obtain through twice screens by colony PCR.Sequencing of these positive colonies confirmed that 53 contained microsatellite loci（number of repeats ≥5）.In these sequences,36 repeat motifs（about 67.92%） were perfect,12 repeat motifs（about 22.64%） were imperfect,and 5 repeat motifs（about 9.43%） were compound.We design 24 pairs of primers from 50 microsatellite sequence and compose them.As a result,56 pairs were screened and used successfully to amplify special fragment,among which 24 pairs were polymorphism.Using 24 pairs of these microsatellite markers,we analyzed genetic structures of one Aspiorhynchus laticeps population.The result showed that the alleles were ranged from 3-6,while the polymorphic information content（PIC） were 0.625 3.Twelve of the loci were in high polymorphic level.