湖北钉螺微卫星锚定PCR产物序列分析

目的 分析4个种群湖北钉螺中微卫星序列及其两侧序列的特点.方法 以(CA)8RY为引物对湖北钉螺基因组DNA进行微卫星锚定PCR(SSR-PCR)扩增,对全部159个扩增产物进行T克隆并测定其中82个片段的核苷酸序列.结果 SSR-PCR的扩增产物是湖北钉螺基因组DNA上的散在区域,不是微卫星序列,但扩增产物中含有微卫星序列,测序的82个片段中36个克隆片段含有微卫星序列.微卫星序列其侧翼序列有一定的保守性,同一个微卫星的侧翼序列多数情况下是相同的.(GA/CT)n、(TTAGGG/CCCTAA)n两类微卫星在4个钉螺种群中均有发现,(CAA)n仅在福建福清种群中发现,(TCTCTG)n仅在安徽贵池种群中发现,(GAA～TTC)n、(CAA/TTG)n、(CAT)n三种微卫星序列仅在四川普格种群中发现.结论 SSR-PCR扩增的不是微卫星,其结果的分析应当类似于随机扩增多态性DNA.因此SSR-PCR不能十分有效体现微卫星作为分子标记的优势,应当根据微卫星的侧翼序列设计针对微卫星的引物,对微卫星进行PCR扩增并进行分析.

Sequencing on products of Oncomelania hupensis through simple sequence repeat anchored polymerase chain reaction amplification

Objective To analyze the sequence of microsatellite and the flanking sequence from four populations of Oncomelania hupensis. Methods We cloned 159 SSR-PCR amplification products of a commonly used primer, (CA)8RY, using O. hupensis genomie DNA as template, and sequenced 82 products Results The sequences obtained were novel O. hupensis genomic sequences but not repeat simple sequence. It was observed that 36 out of 82 clones contained microsatellites between priming sites.The flanking sequences of certain microsatellite were invariant. Both (GA/CT). and (TTAGGG/CCCAA)n were found in four populations of O. hupensis. However, (CAA)n were found only in O. hupensis from Fuqing,Fujian province and (TCTCTG), were found only in O. hupensis from Guichi,Anhui province and (GAA/TTC)n, (CAA/TTG)n, (CAT), were found only in O.hupensis from Puge,Sichuan province. Conclusion The results obtained by SSR-PCR should not be interpreted as the amplification of microsatellite loci, and analytical rules similar to those for Random Amplified Polymorphic DNA should be used. SSR-PCR could not make the most of the priority of microsatellite. It seems better to amplify the microsatellites with the primers designed on the basis of the flanking sequence.