基于转录组平台的蛤仔微卫星标记筛选

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 基于转录组平台的蛤仔微卫星标记筛选 DOI: 10.5846/stxb201305151071 作者: 作者单位: 大连海洋大学 水产与生命学院,大连海洋大学 水产与生命学院,大连海洋大学 水产与生命学院,大连海洋大学 水产与生命学院,大连海洋大学 水产与生命学院,大连海洋大学 水产与生命学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家863计划(2012AA10A410-2); 现代农业产业技术体系建设专项资金资助(CARS-48). Development of microsatellite markers in Ruditapes philippinarum using next- generation sequencing Author: Affiliation: Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian,Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian,Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian,;China,Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian,Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian,Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University,Dalian Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:以菲律宾蛤仔转录组测序所得拼接序列为基础,采用MISA软件进行微卫星分析,对其中的145个微卫星位点进行引物设计,得到具有清晰扩增条带的微卫星位点58个。对大连庄河野生蛤仔群体的扩增结果表明,18个位点显示单态性,40个位点表现为多态性。该群体40个多态性微卫星位点得到的等位基因数在2-6之间,平均等位基因数为3.4250±0.9718,观测杂合度和期望杂合度分别在0.0000-1.0000和0.0615-0.7996之间,平均值分别为0.2727±0.2272和0.4739±0.1902,群体平均Nei指数为0.4664±0.1872。多态信息含量(PIC)在0.0586-0.7529之间,平均值为0.4148±0.1707,其中16个微卫星位点的PIC值大于0.5,为高度多态性,15个位点0.25 < PIC < 0.5,为中度多态性,其余9个为低度多态性。经Sequential Bonferroni校正的Hardy-Weinberg平衡检验,有10个位点尚未偏离平衡。基于转录组平台筛选微卫星标记的方法,在很大程度上推动了DNA分子标记的开发。研究开发的微卫星标记可用于蛤仔群体遗传学、遗传连锁图谱构建及其他相关研究,为蛤仔分子标记辅助育种及群体种质保护等工作提供技术支持。 Abstract:Ruditapes philippinarum has a high growth rate, short culture cycle, and is highly adaptable. Because of these traits, it is one of China's four major cultured shellfishes, and one of the world's major cultured shellfishes. Microsatellites known as simple sequence repeats are widely used to assess genetic diversity in farmed aquatic species populations, construct molecular genetic maps, and carry out gynogenesis, gene mapping, gene cloning, and paternity tests. These molecular markers have high stability and polymorphism, are site-specific and easily detected, and exhibit codominant inheritance and transferability of SSR primers. At present, the sustainable culture of Ruditapes philippinarum is threatened by having a single breeding method and difficulties with disease prevention, control, and treatment. We developed a series of microsatellite markers using a transcriptome-based platform to provide a foundation for genetic research in Ruditapes philippinarum. These markers may also be used for Ruditapes marker-assisted breeding. Genetic diversity measures the degree of variability of biological genetic information. DNA is the primary carrier of genetic information, so the diversity of DNA directly reflects the degree of genetic variation. The genetic diversity of a population can be represented by the number of alleles, heterozygosity scores, and polymorphism information content (PIC). We sequenced a large number of ESTs and screened 145 potential microsatellites of trinucleotide repeats using MISA software. We successfully obtained clear, reproducible bands for 58 microsatellite loci. These were amplified in 32 wild clam individuals sampled from Zhuanghe, Dalian, Liaoning. A single allele was detected at 18 loci and another 40 were polymorphic (number of alleles per locus ranged from 2 to 6, with an average of 3.4250±0.9718). The observed and expected heterozygosity was 0.000-1.000 (0.2727±0.2272) and 0.0615-0.7996 (0.4739±0.1902), respectively. The average of the Nei index was 0.4664±0.1872. The polymorphism information content (PIC) of all loci ranged from 0.0586 to 0.7529 (0.4148±0.1707). Among these, 16 loci had a PIC of > 0.5, so were classified as highly polymorphic. An additional 15 loci were moderately polymorphic with PICs ranging from 0.25 to 0.5. The PIC of 9 loci was < 0.25, meaning that these were classified as low polymorphic loci. Using a test of the Hardy-Weinberg principle (χ2 test) and sequential Bonferroni calibration, all except 10 loci had deviated equilibrium. Eight loci had a core sequence of TTG, 6 had a core sequence of TGG, and 5 each had a core sequence of TGT or ATC. These four core sequences accounted for 41.38% of the 58 SSR screened loci. Based on this, we infer that TTG, TGG, TGT, and ATC are relatively abundant copy categories of the trinucleotide repeat sequences. Our results provide a reference for subsequent repetitive sequence screening and further understanding the characteristics of the Ruditapes genome. Microsatellite marker development by transcriptome sequencing proved to be efficient and feasible in R. philippinarum . Further in-depth analysis based on transcriptome analysis will likely yield more microsatellite sites which are associated with functional genes, thereby providing more molecular markers for Ruditapes artificial marker-assisted breeding. These polymorphic markers may also be used in future studies of population genetics, linkage mapping and assisted breeding in R. philippinarum. 参考文献 相似文献 引证文献