【作者】 许新； 王江勇； 区又君；

【Author】 XU Xin WANG Jiang-yong OU You-jun South China Sea Fisheries Research Institute,CAFS,Guangzhou510300,China

【机构】 中国水产科学研究院南海水产研究所；

【摘要】 杂色鲍是我国南方重要的海水养殖贝类。为构建杂色鲍的遗传连锁图谱,构建了其肌肉和内脏等全组织的cDNA文库,测序共获得了8016条EST序列,经拼接和去掉重复序列后获得Unigene4781条。用SSR Hunter软件,从Unigene中查到173个重复序列,隶属于161个EST,含微卫星的EST数占总EST总数的3.37%。其中双碱基重复序列110个,占约63.6%,三碱基重复序列47对,占约51.8%,其他类型共占约9.2%。选取含有丰富侧翼序列的ESTs,从中设计93对引物,有78对可以扩增,占合成总引物的83.9%,其中有25对引物扩增的片段超出预期的片段。用深圳,汕尾等3个群体中挑选5个个体经聚丙烯凝胶电泳检测,多态性引物19对。经深圳九孔鲍群体(36个个体)来检测,19个位点等位基因数范围2-18个,平均等位基因7.55个每位点。标记转换性测试另外两个养殖品种,除几个位点的少数部分个体不能得到扩增,所以微点都可以在其它两个养殖品种扩增。选取10对引物在三个杂色鲍养殖群体通过聚丙烯酰胺凝胶电泳来检测SSR引物的群体遗传多样性的应用。经检测,10对引物都可用于杂色鲍群体遗传多样性分析。多态性EST-SSR的筛选将为杂色鲍的分子遗传学研究,种质鉴定和野生资源保护等提供可靠的工具。更多还原

【Abstract】 Haliotis diversicolor is an important marine aquaculture shellfish in South China.A complementary DNA(cDNA) library was constructed from H.diversicolor muscle and visceral.A total of 8016 expressed sequence tag(ESTs) were obtained by sequencing clones from the cDNA library,and after stitching and remove the repeat sequences we were obtained 4781 Unigene and screened for simple repeat sequences(SSRs) with at least 5 repeats for di-,tri-,tetra and penta-,and 4 repeats for hexanucleotide repeats with the software of SSR Hunter,a total number of 173 SSRs were found.the number of the di-nucleotide repeats and the tri-nucleotide repeats are 110(about 63.6%) and 47(27.2%).Primers were designed for 93 SSR-containing ESTs with good and sufficient flanking sequences.Polymerase chain reaction(PCR) amplification was successful for 78(83.9%)primer pairs,including 25(32.1%) pairs that amplified fragments longer than expected sizes,probably due to introns.53 pairs that produced fragments were screened for polymorphism in five animals from three populations via polyacrylamide gels,and 19(35.8%) of them were polymorphic.Nignteen locus were examined in the Shenzhen H.diversicolor population via polyacrylamide gels,in order to test the numer of alleles.The number of alleles per locus varied from 2 to 18, with an average of 7.55 alleles per locus.Marker transferability was tested on two other H.diversicolor farmed species,and all loci gave successful amplifications in both farmed species.But several markers failed to express to a few individuals of other twe population,resepectly.Ten prime pairs selected from the nineteen polymorphic primers were examined by testing for amplification of the SSR loci in three H.diversicolor populations(Shanwei×50%Japan(36 abalone individuals),Huidong×japan(36) and Shenzhen(36) via polyacrylamide gels,in order to test the adaptability of microsatellite primers and genetic diversity of three populations.After testing we found that the 10 pairs of primers can be applied to detect genetic diversity of Haliotis diversicolor.The results obtained in this study suggest that ESTs are valuable resources for the development of SSR markers in the H.diversicolor,and H.diversicolor EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection,and evolutionary studies.更多还原