鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析*

采用磁珠富集法结合放射性同位素杂交法分离出鲤鱼(Cyprinus carpio)三、四核苷酸重复的微卫星阳性克隆1248个。对其中的384个克隆进行测序分析，共得到微卫星序列325个，其中完美型244个（75.08%），非完美型59个（18.15%），混合型22个（6.77%）。依据得到的微卫星序列，用Primer 3软件在线设计并合成引物145对，检测结果显示71.43%（80对/112对）的引物在野生个体间表现出多态性，等位基因数在3~12之间，多态信息含量在0.2109~0.8607之间，80%的位点处于高度多态水平。对群体的遗传结构评估结果显示，四核苷酸重复的微卫星标记在黑龙江鲤野生群体表现出的多态性水平（PIC=0.7740）高于三核苷酸重复（PIC=0.5161）和双核苷酸重复（PIC=0.49），适用于群体间遗传差异分析和遗传连锁图谱的构建。

Isolation and Characterization of Polymorphic Tri-and Tetranucleotide Repeat Microsatellite Loci in Common Carp(Cyprinus carpio)

Enrichment by magnetic beads is a simple and efficiency method for rapid isolation of microsatellite DNA.In the experiment,four libraries rich in GAT,CAT,AGAC and AGAT mierosatellites respectively were constructed from the common carp(Cyprinus carpio)genome with this method.One thousand two hundred and forty-eight positive clones were obtained through the second screening by radioactive labeling hybridization,including 167 motifs with GAT/CTA,189 with CAT/GTA,684 with AGAC/TCTG and 208 with AGAT/TCTA.Three hundred and twenty-five microsatellite sequences were isolated from 384 positive clones.There were 244(75.08%)perfect motifs,59(18.15%)imperfect motifs and 22(6.77%)compound motifs in these sequences.One hundred and forty-five pairs of primers were designed according to these sequences with software Primer 3 and synthesized.And results showed that 112 pairs of primers(77.24%)successfully produced specific products and 80 primers(71.43%)showed polymorphism in Heilongjiang River wild common carp population.Fifty polymorphic loci were used to analyze genetic structure of the wild population.The number of alleles ranged from 3 to 12.The polymorphism information content(PIC)of these loci ranged from 0.2109to 0.8607.The statistic results indicated that 80%loci(40/50)were in high pelymorphic level and suited to do analysis of population genetic diversity and genetic mapping.The evaluated results revealed that the polymorphic level of tetranucleotide repeat microsatellite(PIC=0.7740)was higher than that of trinucleotide repeat(PIC=0.5161)or dinucleotide repeat(PIC=0.49).