鲢InDel-RFLP标记开发及其在连锁图谱构建中的应用

InDel-RFLPs of silver carp (Hypophthalmichthys molitrix) and their application to constructing the linkage map

基于随机测序的鲢(Hypophthalmichthys molitrix)基因组片段,尝试开发基于InDel的限制性片段长度多态性标记(InDel-RFLP)。结果显示:共133个片段中,65个在一个野生鲢群体(该野生鲢群体采自长江流域荆州段,31个个体)中表现多态性。共检测到163个等位基因,每位点等位基因数从2到4不等,平均2.5个。65个多态性标记中,有35个被添加到鲢SSR-AFLP性别平均连锁图谱上。长约2 kb的片段比长约1 kb的片段更适用于InDel-RFLP标记开发。设计特异引物,扩增基因组片段,用4种限制性内切酶(BsuR I、Hha I、Taq I和Vsp I)之一切成适宜聚丙烯酰胺凝胶电泳分离的小片段,电泳分离、银染分型。该标记系统避免了对InDel精确定位的困难,通过高分辨PAGE分型,可为鲢连锁图谱构建和性状定位提供丰富的标记位点。

In order to detour the limitations faced in the development of SSR markers, 133 randomly sequenced genomic fragments were used to develop InDel based restriction fragment length polymorphism （InDel-RFLP） markers for silver carp （Hypophthalmichthys molitrix）. 65 fragments were polymorphic in a wild silver carp population containing 31 individuals collected from Jingzhou fragment （the middle reach） of Yangtze River. In total, 163 alleles were identified. The allele number per locus varied from 2 to 4 with an average of 2. 5. Thirty five InDel-RFLPs were assigned to the sex average SSRAFLP （amplified fragment length polymorphism） linkage map of silver carp. It was found that ca. 2 kb fragments were more useful to develop InDel-RFLP markers than ca. 1 kb fragments. The fragments were amplified from the genomic DNA and the PCR products were digested by one of the four restriction endonucleases （BsuR I, Hha I, Taq I and Vsp I） into shorter ones which were separated via polyacrylamide gel eleetrophoresis （PAGE） and visualized with silver staining. It a- voided searching for the fine position of InDels and integrated the high resolution of PAGE, which will provide a rich source of molecular markers for linkage map construction and traits mapping of silver carp.