三角帆蚌四核苷酸重复微卫星的开发及特征分析

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

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.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. 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/Abstract

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

韩学凯, 李家乐, 王照旗, 白志毅. 三角帆蚌四核苷酸重复微卫星的开发及特征分析[J]. 生物技术通报, 2014, 0(6): 139-144.

Han Xuekai,Li Jiale,Wang Zhaoqi,Bai Zhiyi. Development and Characteristics of Tetranucleotide Repeat Microsatellite Loci in Hyriopsis cumingii[J]. Biotechnology Bulletin, 2014, 0(6): 139-144.

参考文献

[1] Hua D, Gu R. Freshwater pearl culture and production in China[J] . Aquaculture Asia, 2002, 7（1）：6-8.
[2] Li JL, Li YS. Aquaculture in China—Freshwater pearl culture[J] . World Aquaculture, 2009, 40（1）：60-62.
[3] 刘月英, 张文珍, 王跃先, 等.中国经济动物志———淡水软体动物[M] . 北京：科学出版社, 1979：114-115.
[4] 张元培.淡水珍珠养殖技术[M] .长沙：湖南科学技术出版社, 1981.
[5] Lagercrantz U, Ellegren H, Andersson L. The abundance of various polymorphic microsatellite motifs differs between plants and vertebrates[J] . Nucleic Acids Research, 1993, 21（5）：1111-1115.
[6] 盛岩, 郑蔚红, 裴克全, 等.微卫星标记在种群生物学研究中的应用[J] . 植物生态学报, 2002, 26（s）：119-126.
[7] Sato M, Kawamata K, Zaslavskaya N, et al. Development of microsatellite markers for Japanese scallop（Mizuhopecten yessoensis）and their application to a population genetic study[J] . Mar Biotechn, 2005, 7（6）：713-728.
[8] Geist J, Geismar J, Kuehn R. Isolation and characterization of the first microsatellite markers for the endangered swan mussel Anodonta cygnea L.（Bivalvia：Unionoidea）[J] . Conserv Genet, 2009, 11（3）：1103-1106.
[9] Jones JW, Culver M, David V, et al. Development and characterization of microsatellite loci in the endangered oyster mussel Epioblasma capsaeformis（Bivalvia：Unionidae）[J] . Mol Ecol Not, 2004, 4（4）：649-652.
[10] Shaw KM, King TL, Lellis WA, et al. Isolation and characterization of microsatellite loci in Alasmidonta heterodon（Bivalvia：Unionidae）[J] . Molec Ecol Not, 2006, 6（2）：365-367.
[11] Bai ZY, Yin YX, Hu SN. Identification of genes involved in immune response, microsatellite, and SNP markers from expressed sequence tags generated from hemocytes of freshwater pearl mussel （Hyriopsis cumingii）[J] . Mar Biotechnol, 2009, 11（4）：520-530.
[12] Li JL, Wang GL, Bai ZY. Genetic diversity of freshwater pearlmussel（Hyriopsis cumingii）in populations from the five largest lakes in China revealed by inter-simple sequence repeat（ISSR）[J] . Aquacult Int, 2009, 17（4）：323-330.
[13] 许巧情, 谢骏, 张书环, 等.三角帆蚌微卫星富集文库的构建、鉴定及多态性分析[J] .中国水产科学, 2010, 17（6）：1200-1207.
[14] 罗明, 白志毅, 李应森, 等.三角帆蚌微卫星位点筛选及多态性分析[J] .淡水渔业, 2012, 42（1）：81-83.
[15] Xu B, Li JL, Wang GL. Development and characteriza tion of microsatellite loci in Lamprotula leai, with cross-amplication in Hyriopsis cumingii[J] . Conserv Genet Resour, 2011, 3（3）：545-547.
[16] Jia MJ, Li JL, Wang SL. Isolation and characterization of 18 polymorphic microsatellite loci from freshwater pearl mussel（Cristaria plicata）[J] . Conserv Genet, 2010, 11（3）：1131-1133.
[17] 汪桂玲, 苏翔, 李家乐, 等.背角无齿蚌基因组（GT）n微卫星DNA特征[J] .生态学杂志, 2011, 30（1）：1-6.
[18] Edwards A, Civitello A, Hammond HA, et al. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats[J] . American Journal of Human Genetics, 1991, 49（4）：746-756.
[19] 鲁翠云, 毛瑞鑫, 李欧, 等.鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析[J] .农业生物技术学报, 2009, 17（6）：979-987.
[20] 谭照君, 张天奇, 鲁翠云, 等.鲢三、四核苷酸重复微卫星标记的筛选及其特征分析[J] .上海海洋大学学报, 2011, 20（3）：329-335.
[21] Li JL, Wang GL, Bai ZY, et al. Ten polymorphic microsatellites from freshwater pearl mussel Hyriopsis cumingii[J] . Mol Ecol Notes, 2007, 7（6）：1357-1359.
[22] Hauge XY, Litt M. A study of the origin of‘shadow bands’seen when typing dinucleotide repeat polymorphisms by the PCR[J] . Human Molecular Genetics, 1993, 2（4）：411-415.
[23] Murray V, Monchawin C, England PR. The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR[J] . Nucleic Acids Research, 1993, 21（10）：2395-2398.
[24] Gastier JM, Pulido JC, Sunden S, et al. Survey of trinucleotide repeats in the human genome：assessment of their utility as genetic markers[J] . Human Molecular Genetics, 1995, 4（10）：1829-1836.
[25] 曹柱, 鲁翠云, 邓先虎, 等.磁珠富集法筛选方正银鲫的三、四核苷酸重复微卫星 DNA[J] .淡水渔业, 2011, 41（1）：23-28.
[26] 李文升, 刘翠, 鲁翠云, 等.草鱼三、四核苷酸重复微卫星标记的分离与特征分析[J] .中国水产科学, 2011, 18（4）：742-750.
[27] 田艳苓, 毕伟伟, 杨海玲, 等.济宁百日鸡群体遗传多样性的微卫星标记分析[J] .经济动物学报, 2010, 14（1）：41-45.
[28] Botstein D, White RL, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J] . American Journal of Human Genetics, 1980, 32（3）：314-331.
[29] 高焕, 孔杰.串联重复序列的物种差异及其生物功能[J] .动物学研究, 2005, 26（5）：555-564.