基于测序分型方法开发日本沼虾微卫星分子标记

Development of microsatellite markers in Macrobrachium nipponense based on sequencing genotyping

利用无参考基因组SSR开发方法筛选微卫星序列并结合测序分型方法对分离的微卫星序列进行基因分型。利用两个样本的全基因组测序数据,通过ebwt2InDel软件从这两个样本的测序数据中寻找包含InDel标记的序列。基于包含InDel标记的序列,利用MISA软件搜索包含SSR的序列并删除非SSR型InDel标记的序列,最终得到包含完美型SSR的序列111条。利用测序分型软件SSRgenotyper对30尾日本沼虾(Macrobrachium nipponense)测序样本进行多态性分析,111条含SSR的序列中,共有19个SSR可以成功分型,分型成功率为17.12%。分型成功的19个SSR位点均为3碱基重复4~6次,其中以ATC/ATG为单位的重复序列占比最多,为6个(31.58%)。19个SSR中,低度信息含量的标记2个,中度信息含量的标记17个。本研究制备的19个微卫星标记可用于评估日本沼虾种群的遗传参数,为日本沼虾种质资源的开发提供有用的遗传标记。

The microsatellite sequences were obtained using a simple sequence repeats(SSR)development method without needing a reference genome and were genotyped through sequencing.The ebwt2InDel software was utilized to identify Insertion/Deletion(InDel)-containing sequences from the whole genome sequencing data of two samples.Using MISA software,111 perfect SSR-containing sequences were obtained from the InDel-containing sequences by eliminating non-SSR-containing sequences.The SSRgenotyper software was then used to analyze the polymorphism of 30 M.nipponrnse samples,successfully genotyping 19 out of the 111 SSR-containing sequences with a genotyping success rate of 17.12%.Successful genotyping was achieved at 19 SSR loci,all exhibiting 3-base repeats ranging from 4 to 6.Among them,the highest proportion of repetitive sequences was observed with the ATC/ATG unit,accounting for six loci(31.58%).Two of the 19 SSR markers had low polymorphism information content,and 17 had moderate polymorphism information content.These 19 microsatellite markers helped to evaluate the genetic parameters of freshwater shrimp populations and develop freshwater shrimp germplasm resources.