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利用454焦磷酸测序技术开发棕点石斑鱼微卫星标记并将其应用于相关物种

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species.

作者信息

Kang J-H, Yang S G, Moon T S, Park J-Y, Choi T-J

机构信息

Biotechnology Research Division, National Fisheries Research and Development Institute, Busan, Republic of Korea.

出版信息

Genet Mol Res. 2013 Nov 13;12(4):5485-93. doi: 10.4238/2013.November.13.1.

Abstract

The kelp or longtooth grouper (Epinephelus bruneus), which inhabits Eastern Asia, is the most economically important of 11 grouper species that inhabit the Southern Sea near Jeju Island in Korea. This species is listed as vulnerable by the International Union for the Conservation of Nature and Natural Resources because of a rapid decrease in its resources. We developed microsatellite markers for E. bruneus using the pyrosequencing technique for applications in resource management and aquaculture. In addition, we tested the cross-species transferability of the microsatellite markers in four species belonging to the Epinephelus genus. Among 66,452 simple sequence repeats, 64 loci containing more than eight CA or TG repeats were randomly selected for primer synthesis; 45 primer sets (75.0%) produced polymerase chain reaction (PCR) products of 100-300 bp and were selected as candidates. After primary testing with four E. bruneus fish, 28 polymorphic loci were selected as the final microsatellite markers, and 23 sets showing clear amplification of polymorphic loci were used to analyze 71 fish. These loci have allele numbers ranging from 2 to 23. Null alleles were detected at three loci, and three loci showed an excess of homozygotes in the Hardy-Weinberg equilibrium test. Of the three species used for cross-species transfer of these markers, Epinephelus moara showed the highest transferability (92.9%) and polymorphism (67.9%), followed by Epinephelus fuscoguttatus (75.0 and 67.9%, respectively) and Epinephelus septemfasciatus (57.1 and 46.4%, respectively). These results suggested that these microsatellite loci should be valuable tools for population genetic studies of the species Epinephelus.

摘要

褐点石斑鱼(Epinephelus bruneus)栖息于东亚地区,是韩国济州岛附近南海海域11种石斑鱼中经济价值最高的物种。由于其资源量迅速减少,该物种被国际自然及自然资源保护联盟列为易危物种。我们利用焦磷酸测序技术开发了用于褐点石斑鱼的微卫星标记,以应用于资源管理和水产养殖。此外,我们还测试了这些微卫星标记在石斑鱼属四个物种中的跨物种转移性。在66,452个简单序列重复中,随机选择了64个含有超过8个CA或TG重复的位点用于引物合成;45个引物组(75.0%)产生了100 - 300 bp的聚合酶链反应(PCR)产物,并被选为候选引物。在用4条褐点石斑鱼进行初步测试后,选择了28个多态性位点作为最终的微卫星标记,并使用23组显示多态性位点清晰扩增的引物来分析71条鱼。这些位点的等位基因数范围为2至23。在三个位点检测到无效等位基因,并且在哈迪 - 温伯格平衡测试中,有三个位点显示纯合子过剩。在用于这些标记跨物种转移的三个物种中,云纹石斑鱼(Epinephelus moara)显示出最高的转移性(92.9%)和多态性(67.9%),其次是棕点石斑鱼(Epinephelus fuscoguttatus)(分别为75.0%和67.9%)以及七带石斑鱼(Epinephelus septemfasciatus)(分别为57.1%和46.4%)。这些结果表明,这些微卫星位点应是石斑鱼属物种群体遗传研究的有价值工具。

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