生物技术通报 ›› 2014, Vol. 0 ›› Issue (6): 139-144.

• 研究报告 • 上一篇    下一篇

三角帆蚌四核苷酸重复微卫星的开发及特征分析

韩学凯1, 李家乐1,2, 王照旗1, 白志毅1   

  1. (1.上海海洋大学 农业部淡水水产种质资源重点实验室,上海 201306;2.上海高校水产养殖学 E-研究院,上海 201306)
  • 收稿日期:2013-12-16 出版日期:2014-06-25 发布日期:2014-06-25
  • 作者简介:韩学凯,男,硕士,研究方向:种质资源与遗传育种;E-mail:15618062362@163.com
  • 基金资助:
    上海市高校知识服务平台项目(ZF1206),国家科技支撑计划项目(2012BAD26B04),国家自然科学基金项目(31272657),上海市青年科技启明星(A类)(12QA1401400),上海工程技术中心能力提升项目(13DZ2280500)

Development and Characteristics of Tetranucleotide Repeat Microsatellite Loci in Hyriopsis cumingii

Han Xuekai1,Li Jiale 1,2,Wang Zhaoqi1,Bai Zhiyi1   

  1. (1. Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai  201306; 2. Aquaculture Division, E-Institute of Shanghai Universities, Shanghai 201306)
  • Received:2013-12-16 Published:2014-06-25 Online:2014-06-25

摘要: 用磁珠富集法和生物素标记(ATAC)5为探针构建了三角帆蚌基因微卫星富集文库。利用PrimerSelect软件对得到的微卫星序列进行引物设计并合成得到43对引物,初筛后发现有20对引物可以扩增出稳定、清晰的目的产物带。荧光标记这20对引物,然后用36只洞庭湖三角帆蚌扩增,发现17对引物呈现多态性。17个微卫星位点等位基因数的范围为6-28。期望杂合度(He)、观测杂合度(Ho)的范围分别为0.677 6-0.946 4、0.638 9-1.000 0。多态信息含量(PIC)在0.630-0.929之间。统计分析结果显示,其中有12个微卫星位点满足Hardy-Weinberg平衡条件。与本实验室之前开发的二核苷酸重复微卫星相比,该17对四核甘酸重复微卫星具有更高多态性,且更易于采用聚丙烯酰胺凝胶电泳等传统方法分析基因型,有利于节约成本,从而为三角帆蚌群体遗传分析、亲子鉴定等技术提供了准确、价格低廉的微卫星标记。

关键词: 三角帆蚌, 微卫星, 四核苷酸重复, 多态性

Abstract: A microsatellite-enriched library of the Hyriopsis cumingii was constructed using repeat-enrichment method with biotin-labeled oligo(ATAC)5 and streptavidin coated magnetic beads. Forty-three pairs of primers designed by software PrimerSelect according to the sequences were synthesized for the pre-screening. Twenty pairs of primers which could amplify constant and legible expected DNA products were labeled by fluorescence. Seventeen microsatellite loci showed high levels in genetic polymorphism testing on 36 individuals sampled from Dongting Lake. The number of alleles at each locus ranged from 6 to 28. The expected and observed heterozygosities varied from 0.677 6 to 0.946 4 and from 0.638 9 to 1.000, respectively. The PIC value ranged from 0.630 to 0.929. Twelve microsatellite loci fitted to Hardy-Weinberg equilibrium. Compared with the previous dinucleotide repeat microsatellite developed in our laboratory, these 17 tetranucleotide repeat microsatellite loci showed higher polymorphism. Also, these 17 microsatellite loci can make genotyping easier by conventional methods such as polyacrylamide gel electrophoresis and save cost. Therefore, it offered accurate and inexpensive microsatellite markers for population genetic analysis and paternity test in Hyriopsis cumingii.

Key words: Hyriopsis cumingii, Microsatellite, Tetranucleotide repeat, Polymophism