利用FIASCO技术进行波纹巴非蛤微卫星DNA标记分离与筛选的研究

Isolation and Screening of Microsatellite Markers from the Clam Paphia Undulate by Using FIASCO

为揭示波纹巴非蛤种质遗传特性、开发种质库,利用FIASCO(Fast Isolation by AFLP of Sequences Containing Repeats)技术开展了其基因组微卫星标记的分离与筛选研究。基因组DNA经限制性内切酶MseI酶切后与接头连接,用生物素标记的(CA)15或(AAG)7探针与其杂交,然后用磁珠富集、洗脱获得单链目的片段,经PCR扩增后形成双链,最后进行克隆转化,构建微卫星富集文库。挑选克隆用探针引物(CA)15或(AAG)7和载体引物进行PCR筛选,测序得到含有微卫星DNA的序列,根据序列设计和合成微卫星引物,进行引物适用性分析,并分析了湛江群体的遗传结构。结果表明:8对微卫星引物在湛江群体共检测到108个等位基因,每个位点等位基因数为5～19,期望杂合度为0.666～0.926,观测杂合度为0.400～0.882,4个位点(Pun4,Pun5,Pun6,Pun7)显著偏离哈迪-温伯格平衡(P<0.00625);PIC介于0.62～0.92,所有位点均属于高度多态位点(PIC>0.5)。说明FIASCO技术适合于波纹巴非蛤微卫星标记的分离与筛选,筛选得到的8个微卫星位点能用于波纹巴非蛤遗传多样性分析及野生群体与养殖群体的群体结构分析。

In order to reveal the genetic resources and Germplasm repository of the clam Paphia undulate. The present studied reported that isolation and screening of microsatellite markers from the clam Paphia undulate were performed using the method of Fast Isolation by AFLP of Sequences Containing Repeats （FIASCO）. After restriction digests of genomic DNA with Mse I, the digested fragments were ligated with adaptors and then hybridized with 5＇ -biotinylated （CA）15 probe or 5＇ -biotinylated （AAG）7 probe. The tentative microsatellite DNA was isolated by streptavidin-coated magnetic beads from the hybridized mixture. After purification, the isolated microsatellite DNA was amplified using degenerated primer Mse P and then cloned into T-vector. After transforming, the microsatellite- enriched library was constructed. The second PCR screening was performed using the primer of （CA）15 probe or （AAG）7 probe） and T vector＇ s primers. The SSR developed were used for analyzing genetic diversity of Zhanjiang wild population of the species. A total of 108 alleles were detected by the 8 SSR loci. The number of alleles per SSR locus ranged from 5 to 19 in a sample of 39 individuals captured in inshore water of Zhanjiang. Observed and expected heterozygosity per locus variedfrom 0.400 to 0.882 and from 0.666 to 0.926, respectively. Four loci （Pun4, Pun5, Pun6, Pun7） showed significant departure from Hardy - weinberg equilibrium after sequential Bonferroni correction （P〈0.00625）. All loci were high polymorphic loci. This study demonstrated that FIASCO was a useful technique of mierosatellite isolation for the species. 8 mierosatellite markers should provide a tool for assessing genetic diversity and population structure of wild or breeding stocks.