基于转录组测序的兴国红鲤微卫星标记筛选  

Development of microsatellite markers in Cyprinus carpio var. singuonensis using next-generation sequencing

作　　者：岳华梅[1] 翟晴[1] 宋明月[1] 叶欢[1] 杨晓鸽[1] 李创举[1] 

机构地区：[1]农业部淡水多样性保护重点实验室,中国水产科学研究院长江水产研究所,武汉430223

出　　处：《淡水渔业》2016年第1期24-28,共5页Freshwater Fisheries

基　　金：国家高技术研究发展计划项目课题一(2011AA100401)

摘　　要：采用Illumina高通量测序技术,对兴国红鲤（Cyprinus carpio var.singuonensis）垂体和性腺等组织进行转录组测序分析,筛选微卫星标记并分析其组成及特征。结果显示：共获得13 652个微卫星标记,对所得位点进行分类,单核苷酸重复类型占47.86%,二、三、四、五、六核苷酸重复类型所占比例分别为34.43%、16.32%、1.25%、0.12%以及0.02%。随机选取30个SSR位点进行PCR验证,有20对可以扩增出清晰稳定的条带。在24个兴国红鲤个体中对上述位点进行多态性分析,结果表明,具有多态性的微卫星引物为9对。不同位点得到的等位基因范围为2~4,平均等位基因数为3.111 1±0.993 8,观测杂合度（Ho）、期望杂合度（He）以及多态信息含量（PIC）平均值分别为0.597 2±0.233 2、0.539 7±0.178 0和0.467 2±0.172 1。9个微卫星位点中,有4个显著偏离哈代-温伯格平衡（Hardy-Weinberg equilibrium,HWE）（P〈0.05）。Illumina高通量测序提供了一种直观、高效开发微卫星标记的方法,所选兴国红鲤群体遗传多样性维持较好。The simple sequence repeats（ SSRs） were detected from the transcriptome of pituitary and gonad in cultured Cyprinus carpio var. singuonensis by high-throughput Illumina sequencing,whose classifications and characterization were analyzed as well. It was shown that a total of 13,652 SSRs were found,which were composed of six repeat units types：mononucleotide repeat units（ 47. 86%）,dinucleotide repeat units（ 34. 43%）,trinucleotide repeat units（ 16. 32%）,tetranucleotide repeat units（ 1. 25%）,pentanucleotide repeat units（ 0. 12%） and hexanucleotide repeats（ 0. 02%）.Twenty out of thirty PCR primers,designed from random SSR sequences,were able to amplify steady and clear sequence bands by PCR. By further PCR amplifications in 24 C. carpio var. singuonensis individuals,nine of the twenty SSRs were proved to be polymorphic. In addition,the number of alleles,heterozygosity scores,and polymorphism information content（ PIC） of the nine SSRs were analyzed. The number of alleles per locus ranged from 2 to 4,with an average of 3. 1111 ± 0. 993 8. The average values of observed heterozygosity（ Ho）,expected heterozygosity（ He） and polymorphism information content（ PIC） were 0. 597 2 ± 0. 233 2、0. 539 7 ± 0. 178 0 and 0. 467 2 ± 0. 172 1,respectively. The χ2test of Hardy-Weinberg equilibrium revealed that genetic disequilibrium was observed in four SSR loci from the nine loci analyzed.These results indicate that Illumina sequencing provides a direct and high-efficient method to develop microsatellite sites,and the Xingguo red carp population analyzed in this study retained fine genetic diversity.

关 键 词：兴国红鲤(Cyprinus carpio var.singuonensis) 高通量测序 微卫星标记 

分 类 号：S917.4[农业科学—水产科学]