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采用FIASCO(Fast Isolation by AFLP sequences containing repeats)技术和磁珠富集方法开发拟穴青蟹微卫星位点。结果表明,400bp以下长度的序列含微卫星的概率超过400bp以上长度的序列;从二碱基重复类型到五碱基重复类型,完美型微卫星的概率在增加。利用设计的28对引物扩增一个供试群体,其中12对引物能稳定扩增且片段大小基本符合理论长度,10个微卫星位点表现出高度多态性,9个微卫星位点显著偏离Hardy-Weinberg平衡(P<0.05),两组两两位点间存在连锁不平衡现象(P<0.0042,经Bonferroni法校正)。Micro-checker分析揭示其中的5个微卫星位点可能存在无效等位基因。排除混合微卫星位点的引物对以及扩增位点PIc值在0.5以下的引物对,8对引物能用于拟穴青蟹群体遗传学等研究。
The microsatellite loci of Scylla serrata were developed by using FIASCO (Fast Isolation by AFLP sequences containing repeats) and magnetic bead enrichment. The results showed that the sequences with lengths below 400bp contained more than 400bp sequences with the probability of exceeding 400bp. The probability of perfect microsatellites increased from two to five base repeat types. A total of 28 pairs of primers were designed to amplify a test population. Among them, 12 pairs of primers could stably amplify and the size of fragments was basically consistent with the theoretical length. Ten microsatellite loci showed a high degree of polymorphism, and nine microsatellite loci deviated significantly Hardy-Weinberg equilibrium (P <0.05). There was linkage disequilibrium between the two groups (P <0.0042, corrected by Bonferroni method). Micro-checker analysis revealed that five of the microsatellite loci may have invalid alleles. Primer pairs were excluded from the hybrid microsatellite loci and primer pairs with PIc values of 0.5 or less were used for the amplification. Eight pairs of primers were used to study population genetics of Scylla serrata.