黄鳝EST-SSR标记的开发及其通用性检测

Development and Characterization of EST-SSR Markers from Monopterus albus and Their Transferability to Other Speices

利用SSRrepeat软件对NCBI数据库中黄鳝肠道EST序列进行挖掘、验证,分析微卫星(SSR)在这些序列中的总体分布特点,并开发黄鳝EST-SSR引物。结果显示,在黄鳝肠道的814条EST序列中共搜索到51个SSR位点,分布在43条EST中,出现频率为6.27%。这些EST-SSR的平均长度为18.80 bp。其中,三核苷酸重复为主导类型,占总SSR的49.02%,其次为二核苷酸重复,占29.41%。四、五、六碱基重复序列分别占微卫星总数的3.92%、11.76%、5.88%。多态性引物验证结果表明,在设计合成的21对EST-SSR引物中,12对在黄鳝中获得有效扩增。其中7对引物显示出多态性,共扩增出29个等位基因;每个位点等位基因数为2~7个(平均4.14),观测杂合度(Ho)为0~0.857 1,期望杂合度(He)为0.135 1~0.822 7,多态信息含量(PIC)为0.123 8~0.781 1。在鲤形目和鲈形目中进行的跨物种扩增结果表明,12对引物中有7对引物在5个不同物种间得到成功扩增,5对引物在个别物种间表现出多态。研究获得的EST-SSR引物,可用于黄鳝的种资资源评价及遗传结构研究。

The abundance and distribution of the microsatellites were analyzed from a total of 814 intestinal EST sequences of Monopterus albus in the NCBI database using the SSRrepeat software.The results showed that 51 SSR loci were found in these ESTs with a frequency of 6.27%.The most abundant EST-SSR types were trinucleotide repeats,accounting for 49.02% of the total SSRs,followed by dinucleotide,pentanucleotide,hexanucleotide and tetranucleotide,accounting for 29.41%,11.76%,5.88% and 3.92%,respectively. A total of 21 primers were designed according to the most appropriate flanking sequences in these SSRs,of which 12 were successfully amplified in 30 individuals and 7 polymorphic microsatellite markers were screened out.A total of29 alleles were identified from 7 polymorphic SSR loci,ranging from 2 to 7,with an average of 4.14 alleles/locus.The values of the observed and expected heterozygosity ranged from 0 to 0. 857 1 and from 0. 135 1 to0.822 7,respectively.The transferability of the 12 EST-SSR primers showed that 7 primers were successfully amplified in five different species belonging to order Cypriniformes and Perciformes,of which 5 primers showed polymorphism.These markers will be useful for the conservation studies and population structure assessment for these species.