富集文库—菌落原位杂交法筛选栉孔扇贝的微卫星标记

Isolation and characterization of microsatellite markers for Zhikong scallop by screening SSR-enriched library

应用微卫星富集文库——菌落原位杂交法，筛选得到了40个栉孔扇贝的微卫星标记。用固定了（AG）15和（AC）15探针的尼龙膜（HybondN^＋）捕捉含有微卫星DNA的片段，经洗脱、PCR扩增和TA克隆，构建栉孔扇贝的微卫星富集文库。利用ECL试剂盒（Amersham公司）标记的（AG）15和（AC）15探针进行菌落原位杂交筛选微卫星富集文库，阳性克隆经测序获得微卫星DNA。富集文库中1200个重组克隆经过菌落原位杂交后，532个（44．3％）为阳性克隆。任意挑选100个克隆测序，结果显示所有的克隆都至少含有一个微卫星位点。利用软件设计了65对特异性PCR引物，40对能扩增出清晰的带谱；利用48个栉孔扇贝个体评价微卫星位点，分析表明37个位点具有多态性。不同的位点获得的等位基因数目为2～14个不等，37个多态性位点共获得258个等位基因，平均每个位点获得7．0个等位基因。观测杂合度（H0）、期望杂合度（HP）及多态性信息含量值（P配）的范围分别为0．1000-1．0000、0．1197-0．9831和0．1172-0．9782。结果表明，富集文库一菌落原位杂交法适合大规模筛选目标物种的微卫星标记。

In this study, 40 microsatellite markers were isolated and characterized for Zhikong scallop （Chlamys farreri） using the method of screening SSR-enriched library. DNA fragments containing microsatellites were captured by a piece of nylon membrane （Hybond N^＋ ） bound with the probe combinations of （AG）15 and （AC）15. The nylon membrane was washed three times in 2 × SSC, 1% SDS at 62 ℃, two times for 15 min with the final wash for 30 min. The captured DNA was eluted in 0. 1 × TE buffer and used to construct an SSR enrichment library. After transformation, the clones were regularly re-arrayed in a new agar plate with the density of about 300 clones per plate and screened with （AG）15 and （AC）15 probes labelled by the ECL system （Amersham）. A total of 1200 clones derived from the enrichment library were screened and 532 clones gave the positive response. One hundred clones were randomly selected for sequencing and the results showed that all of the clones contain at least one microsatellite. Sixty-five primer pairs were designed using the software Primer Premier 5.0, of which 40 pairs can be amplified scorable PCR products. The polymorphisms of these scorable loci were assessed using 48 Chlarnys farreri individuals, and the results showed that 37 loci were polymorphic. The number of alleles per polymorphic locus ranged from 2 to 14 with an average of 7.0 alleles per locus, and the values of Ho, He and PIC varied from 0. 1000 to 1.0000, 0. 1197 to 0.9831 and 0. 1172 to 0.9782, respectively. The results indicated that the method of screening SSR-enriched library is efficient and suitable to isolate a large amount of microsatellite markers for the target species of interest.