利用FIASCO(Fast Isolation by AFLP Sequences Containing repeats)技术建立拟穴青蟹Scylla paramamosain基因组文库,并与生物素标记的(CA)15寡核苷酸探针杂交,联合磁珠富集法构建拟穴青蟹微卫星富集文库。测序194个阳性菌落,分析其中的150条序列,结果表明:两碱基重复类型占90%以上,其中重复拷贝数在30以上的占27.45%;含微卫星座位189个,其中完美型146个、非完美型28个和复合型15个。设计125对引物扩增一个拟穴青蟹野生群体(含20个个体),其中的19对引物能稳定扩增且片段大小基本符合理论长度。遗传变异分析表明,17个位点表现出高度多态性,16个位点显著偏离Hardy-Weinberg平衡(P<0.05),4组两两位点间存在连锁不平衡现象(P<0.0026,经Bonferroni法校正),7个微卫星位点可能存在无效等位基因。若排除混合微卫星位点的引物对以及扩增位点PIC(polymorphism information content)值在0.5以下的引物对,则13对引物能用于拟穴青蟹群体遗传学等研究。
[Objective] The aim was to explore a new sampling approach to obtain genomic DNA of marine shellfish,as well as to provide reference for the molecular biology research on precious shellfish.[Method] Meretrix meretrix,Atrina pectinata,Perna viridis,Crassostrea hongkongensis and Scapharca kagoshimensis were used as experimental materials and the genomic DNA of adductor muscle was taken as reference to extract the genomic DNA of shell cavity fluids with the conventional phenol-chloroform method.And then biophotometer,agarose gel electrophoresis,amplification and sequencing of the target fragments were used to examine the quality of genomic DNA.At the same time,the phylogenic tree was constructed to verify the reality of source contributions.[Result] The quality of genomic DNA of shell cavity fluids extracted by the phenol-chloroform method was better than the genomic of adductor muscle.The genomic DNA extracted by this method showed less content of protein,polyphenol and pigment,which could completely meet the demands of amplification and sequencing of the target fragments.Through the phylogenic tree,it was verified that the source contributions of shell cavity fluids were not come from foreign pollutions.[Conclusion] It is completely feasible to obtain the genomic DNA from shell cavity fluids,which could be applied in the target fragments amplification.
