拟穴青蟹(CA)_n微卫星DNA多态性引物筛选  

Screening for polymorphic primer pairs of(CA)_n microsatellite from Scylla paramamosain(mud crab) genome

作　　者：宋忠魁[1,2] 聂振平[1] 王芳宇[3] 

机构地区：[1]广西海洋研究所广西海洋生物技术重点实验室,广西北海536000 [2]钦州学院化学与生物系,广西钦州535000 [3]衡阳师范学院生命科学系,湖南衡阳421008

出　　处：《海洋科学》2013年第10期98-103,共6页Marine Sciences

基　　金：广西自然科学基金项目(0991275);广西科学研究与技术开发计划项目(11107012-11);广西海洋生物技术重点实验室主任基金项目(0801)

摘　　要：利用FIASCO(Fast Isolation by AFLP Sequences Containing repeats)技术建立拟穴青蟹Scylla paramamosain基因组文库,并与生物素标记的(CA)15寡核苷酸探针杂交,联合磁珠富集法构建拟穴青蟹微卫星富集文库。测序194个阳性菌落,分析其中的150条序列,结果表明:两碱基重复类型占90%以上,其中重复拷贝数在30以上的占27.45%;含微卫星座位189个,其中完美型146个、非完美型28个和复合型15个。设计125对引物扩增一个拟穴青蟹野生群体(含20个个体),其中的19对引物能稳定扩增且片段大小基本符合理论长度。遗传变异分析表明,17个位点表现出高度多态性,16个位点显著偏离Hardy-Weinberg平衡(P<0.05),4组两两位点间存在连锁不平衡现象(P<0.0026,经Bonferroni法校正),7个微卫星位点可能存在无效等位基因。若排除混合微卫星位点的引物对以及扩增位点PIC(polymorphism information content)值在0.5以下的引物对,则13对引物能用于拟穴青蟹群体遗传学等研究。Genomic library of Scylla paramamosain （mud crab） was first constructed by FIASCO （Fast Isolation by AFLP Sequences Containing repeats） technique and then was hybridized to （CA）15 biotin-labeled probe. The magnetic bead enrichment method was employed to capture the target fragments for microsatellite DNA enrichment library. A total of 194 positive bacterial colonies were sequenced, among of which 150 sequences were investigated. As a result, dinucleotide repeat preponderated over 90%, whose repeat copy more than 30 times accounted for 27.54%. 189 microsatellite sites were found, containing 146 perfect type, 28 imperfect type, and 15 compound type. The polymorphisms of 125 primer pairs were estimated through amplifying a solo wild population of Scylla paramamosain （containing 20 individuals）. 19 loci showed stable amplification, whose products conformed to theory size basically. Genetic varaiability analysis suggested 17 highly polymorphic loci. 16 loci were deviated from Hardy-Weinberg equilibrium significantly （P〈0.05）. Significant linkage disequilibrium was found in four pairwise loci （P〈0.0026, corrected by Bonferroni method） while null alleles were detected at seven loci. Regardless of the loci of microsatellite mixture （containing two microsatellite sites or over） plus some loci with PIC value lower than 0.5, 13 primer pairs proved to be useful for the population genetic analysis.

关 键 词：FIASCO(Fast Isolation by AFLP Sequences Containing repeats)技术 磁珠富集法 拟穴青蟹Scylla paramamosain 微卫星 

分 类 号：Q953.5[生物学—动物学] S917.4[农业科学—水产科学]