“光合1号”中华绒螯蟹微卫星富集文库的构建与多态性标记的筛选

摘要

A microsatellite-enriched genomic library for selective breeding population of Chinese mitten crab Eriocheir sinensis "Guanghe No.1",was constructed using FIASCO (Fast isolation by AFLP sequences containing repeats) method.One hundred and sixty-eight positive clones were obtained from 225 randomly picked colonies with PCR method,in which 94 (55.95％) containing microsatellite sequence after sequence analysis,the unit number of repetitions in the 5-10 sequence accounted for 34.04％,10-20 accounted for 20.21％,greater than 20 accounted for 45.75％,the maximum number of repetitions of 92 times; 73.40％ were perfect,18.09％ were imperfect and 8.51％ turned out to be compound.Fifty-three pairs of primers designed and synthesized based on microsatellite flanking sequences showed that 31 pairs (58.50％) primers were amplified for specific bands.The selected 14 polymorphism good primer were used to analyze the genetic diversity of the Liaohe wild populations,148 alleles were detected,the number of single locus alleles 7-13,with an average of 10.6,and the observed heterozygosity per locus were varied from 0.214 3 to 0.666 7,and the expected heterozygosity from 0.825 4 to 0.910 1.PIC(Polymorphic information content) per locus was found to be from 0.766 3 to 0.865 4.%采用磁珠富集法(Fast isolation by AFLP sequences containing repeats,FIASCO)构建“光合1号”新品种中华绒螯蟹Eriocheir sinensis基因组微卫星文库.用PCR法对225个单菌落进行鉴定,共获得168个阳性克隆,测序发现有94个含有微卫星座位,其准确率为55.95％,其中单元重复次数为5～10的序列占34.04％,重复次数为10 ～20的序列占20.21％,重复次数大于20的序列占45.75％,重复次数最高达到92次；完美型微卫星座位69个(占73.40％),非完美型微卫星座位17个(占18.09％),混合型微卫星座位8个(占8.51％)；根据微卫星侧翼序列设计并合成53对引物,检测结果显示,有31对(占58.50％)引物能够扩增出特异性条带；选择其中14对多态性良好的引物用于中华绒螯蟹辽河野生群体的遗传多样性分析,共检测到148个等位基因,单个位点等位基因数为7 ～ 13个,平均为10.6个,观测杂合度为0.214 3～0.666 7,期望杂合度为0.825 4～0.910 1,多态性信息含量为0.766 3～0.865 4.