东北雅罗鱼微卫星分子标记的筛选及特征分析  

Isolation and Characterization of Microsatellite from Genome of Leuciscus waleckii Dybowski

作　　者：刘金亮[1,2] 常玉梅[1] 徐丽华[1,2] 刘春雷[1,2] 梁利群[1] 韩国苍 高玉奎 柳玉海 

机构地区：[1]中国水产科学院黑龙江水产研究所,黑龙江哈尔滨150070 [2]上海海洋大学水产与生命学院,上海201306 [3]赤峰市克什克腾旗达里诺日渔场,内蒙古赤峰024000

出　　处：《华北农学报》2011年第1期87-93,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家"863"计划项目(2007AA10Z186);水产分子育种共性技术的建立与应用项目(200903045)

摘　　要：通过磁珠富集法筛选东北雅罗鱼的微卫星分子标记。采用限制性内切酶Sau 3AI对东北雅罗鱼基因组DNA进行酶切,选取400～900 bp的片段进行PCR全基因组扩增,并利用生物素标记的(CAG)8、(TGA)8、(AGAT)6、(GATT)6 4个探针进行微卫星片段的富集。将得到的片段与T载体连接后转入DH5α大肠杆菌中,然后以SaulA为引物对长出的菌落进行PCR扩增,挑取扩增后片段大小符合条件的菌落送生物公司测序。结果表明:1 416个阳性克隆中有1 047个克隆含有重复次数大于5次的微卫星序列,完美型占67.99%,非完美型为15.31%;混合性标记占16.70%。从中选出160个微卫星序列设计105对引物并合成,经过筛选,56对引物可扩增出清晰条带,其中23对具有多态性。选取18对多态性引物对达里湖东北雅罗鱼群体进行扩增,结果显示等位基因数为3～11,多态信息含量(PIC)为0.207 7～0.882 0,67%(12对/18对)的位点处于高度多态水平(PIC>0.5)。Magnetic beads enriched method was used to isolate microsatellite DNA from Leuciscus waleckii Dybowski genome.L.waleckii genomic DNA was extracted and then digested with restriction enzyme Sau 3A I.Targeted segments of 400-900 base pairs 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（CAG）8,（TGA）8,（AGAT）6 and（GATT）6.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 DH 5α,and finally a microsatellite library was obtained.One thousand,four hundred and sixteen positive colonies were obtain through twice screens by colony PCR.Sequencing of these positive colonies confirmed that 1 047 contained microsatellite loci（number of repeats ≥5）.Among these sequences,737 repeat motifs（about 67.99%）were perfect,166 repeat motifs（about 15.31%）were imperfect,and 181 repeat motifs（about 16.70%）were compound.We design 105 pairs of primers from 160 microsatellite sequence and compose them.As a result,56 pairs were screened and used successfully to amplify special fragment,among which 23 pairs were polymorphism.Using 18 pairs of these microsatellite markers,we analyzed genetic structures of one L.waleckii population sampled from Dali Lake.The result showed that the alleles were ranged from 3-11,while the polymorphic information content（PIC）were 0.207 7-0.882 0.The statistic results indicated that 67%loci（12/18）were in high polymorphic level.

关 键 词：东北雅罗鱼 微卫星 磁珠富集 

分 类 号：Q78[生物学—分子生物学]