泥蚶34个EST-SSR标记的开发及在格粗饰蚶中的通用性检测

Characterization of 34 polymorphic EST-SSR markers in Tegillarca granosa and their transferability in Anadara craticulata

利用泥蚶转录组高通量测序、拼接获得的大量EST序列开发SSR标记,在1 123条EST序列里筛查到73条含有SSR位点的EST序列,其中54个位点适合设计引物,在位点两侧设计引物并进行PCR扩增。结果显示,46对引物获得稳定扩增的位点,引物在泥蚶奉化群体的多态性检测中发现,有34对引物表现出多态性,共扩增出122个等位基因,各位点的等位基因数Na为2～7个,平均每个位点产生3.59个等位基因,观测杂合度Ho、期望杂合度He、多态信息含量PIC范围分别为0.000～0.600、0.078～0.771、0.106～0.718;Hardy-Weinberg平衡检测显示,13个位点偏离了平衡状态;用Nr和Swiss-Prot蛋白质数据库对含有多态性SSR的EST进行了基因注释,25个SSR位点来自注释基因序列。将34对泥蚶多态性SSR引物在格粗饰蚶中进行了通用性检测,结果有11对成功扩增,8对表现为多态,通用率为23.53%,这些通用引物可用于两种蚶的遗传多样性评价、系统进化分析、比较作图和基因发掘等研究。

EST-SSR is one of important methods to isolate microsatellite loci,which is low cost,high transferability among phylogenetically related species and a probability of being associated with functional regions of the genome.34 polymorphic EST-SSR markers were isolated and characterized in the blood clam,Tegillarca granosa,from the ESTs generated by 454 sequencing.The number of alleles for the 34 SSR markers in 30 individuals of Zhejiang Fenghua population varied from 2 to 7,with an average of 3.59 alleles per locus.The observed heterozygosity ranged from 0.000 to 0.600,while the expected heterozygosity varied from 0.078 to 0.771.Polymorphic information content ranged from 0.106 to 0.718,and 13 SSR loci deviated from Hardy-Weinberg equilibrium.25 EST-SSR identified from annotated genes were expected to be useful for mapping these genes in linkage maps.Interspecific transferability of the 34 markers revealed that 8 were polymorphic in Anadara craticulata,resulting in a transferability rate of 23.53%.To our knowledge,this is the first report on the survey of SSR transferability in blood clams.These novel polymorphic EST-SSR markers should be particularly useful for comparative mapping,gene tagging and QTL mapping among blood clams.