刺参(Apostichopus japonicus)EST序列中微卫星分布分析及其标记的筛选

利用微卫星查找软件对现已公布的6632条刺参ESTs的数据库中2—6个碱基重复单元组成的简单序列重复进行筛选,进而对其微卫星的丰度和分布进行比较研究。共发现微卫星序列416个,占整个ESTs数据库的6.48%;其中含双碱基重复序列146个,数量最多,占ESTs数据库中发现微卫星序列总数的65.86%;三、四、五、六碱基重复序列分别占微卫星序列总数的26.68%、1.68%、0.24%、5.53%。对含有SSR位点符合微卫星引物设计的EST序列利用Primer软件结合人工方法设计合成引物21对,其中13对有扩增产物且均为多态位点。扩增产物变性聚丙烯酰胺胶电泳,统计每个微卫星标记等位基因数目,计算等位基因频率、观测杂合度、期望杂合度等统计学指标的评价。结果表明,在13个微卫星位点上,等位基因的数目从3到8不等,等位基因长度从175—382bp,观测杂合度和期望杂合度分别为0.158—0.650和0.198—0.841,所筛选微卫星标记可于遗传分析。

ISOLATION OF MICROSATELLITE MARKERS FROM APOSTICHOPUS JAPONICUS ESTs

Frequency and density of 2-6bp simple sequence repeats(SSRs) were analyzed from Apostichopus ja-ponicus EST(expressed sequence tag) database comprising 6632 sequences. The result shows that 416 microsatellite se-quences(taking 6.48%) were obtained. The dinucleotide repeats motifs appeared to be the most abundant type in A. ja-ponicus ESTs(taking 65.86%), followed by trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeats at 26.68%, 1.68%, 0.24%, and 5.53%, respectively. 21 primer pairs were designed with Primer software and man-ual-searching method from EST sequences containing microsatellites. 13 primers had polymorphic PCR products in Chi-nese and Russian sea cucumber samples by electrophoresis analysis. In the 13 microsatellite loci, the alleles numbers dis-tributed from 3 to 8, and the range of these alleles were between 175 to 382bp. The observed heterozygosity and expected heterozygosity distributed from 0.158-0.650 and 0.198-0.841, respectively, which showed that these 13 microsatellite loci were suitable for genetic analysis.