利用RNA-seq技术分析棘腹蛙编码区微卫星特征
Analysis of Microsatellite Composition in Paa boulengeri using RNA-seq

中文摘要：棘腹蛙 Paa boulengeri的遗传研究和基因组信息比较匮乏,致使可有效利用的分子标记非常有限。以棘腹蛙RNA-seq高通量测序数据为基础进行微卫星分子标记的大规模发掘和特征分析,结果显示:在121.6 Mb的棘腹蛙转录组序列中发现微卫星位点3165个,包含于3034条Contig序列中。在筛选到的1~6碱基重复核心的微卫星中,单碱基重复核心的比例最高,之后为三碱基、二碱基、四碱基、六碱基和五碱基重复核心,分别占29.0%、25.2%、21.7%、10.0%、10.0%和3.0%。其中A/T、AC/GT、AGG/CCT、ACAT/ATCT、AAAAT/ATTTT和AAAAAG/CTTTTT分别是单碱基、二碱基、三碱基、四碱基、五碱基、六碱基重复类型中对应的优势重复单元。棘腹蛙编码区微卫星多为重复长度小于24 bp的短序列,长度大于24 bp的微卫星仅占总数的0.92%。对编码区微卫星的侧翼序列分析发现,微卫星侧翼序列的GC含量显著低于转录组整体GC含量,且在含有微卫星上下游侧翼序列的Contig中,71.9%的序列可以设计特异引物扩增出含有微卫星序列的位点。研究结果为棘腹蛙的遗传研究和分子系统地理学研究提供了丰富的序列信息和标记资源。
英文摘要：The genetic and genomic information of Paa boulengeri was relatively lacking, which have caused a limited number of effective DNA markers. Based on the RNA-seq database, microsatellite markers in P. boulengeri were analyzed by the Misa program. A total of 3165 microsatellite loci that occurred in 3034 Contig sequences were identified in a total of 121.6 Mb nucleotides. Mononucletide repeats was the major types, followed by the trinucleotide, dinucleotide, tetranucleotide, hexanucleoide and pentanucleotide, accounting for 29.0%, 25.2%, 21.7%, 10.0%, 10.0% and 3.0%, respectively. A/T, AC/GT, AGG/CCT, ACAT/ATCT, AAAAT/ATTTT and AAAAAG/CTTTTT were the most frequent motifs in mon-, din-, tri-, tetra-, penta- and hexa-nucleotide repeats, and all of them were rich in A or T. The average length of microsatellites in the coding region of P. boulengeri was 18-24 bp, and the length more than 24 bp only accounted for 0.92%. In addition, we found that the GC content of the flanking sequences was significantly lower than that of transcriptomic sequences. For the Contig sequences with paired flanking sequences longer than 50 nt, 71.9% of them contained the sequences corresponding to the predicted microsatellite loci as determined by PCR. These results provided abundant sequences and microsatellite markers for molecular phylogeography and genetic research on P. boulengeri. 2016,(1): 24-30 收稿日期：2015-04-24 DOI：10.11984/j.issn.1000-7083.20150147 分类号：Q78;Q959.5 基金项目：重庆文理学院人才引进项目(R2014LX07,R2013LS13); 重庆市前沿与应用基础研究(一般)项目(cstc2014jcyjA80042); 重庆市科技攻关计划(cstc2012gg-yyjs80004) 作者简介：姜玉松(1984-), 博士, 讲师, 主要从事基因的分子生物学研究, E-mail:jysong@126.com *通讯作者：徐敬明, 博士, 教授, 主要从事两栖动物的分子系统地理学研究, E-mail:proteomics@163.com 参考文献： 费梁, 叶昌媛, 江建平. 2005. 中国两栖动物检索及图解[M]. 成都: 四川科学技术出版社: 136-137. 廖小林, 俞小牧, 谭德清, 等. 2005. 长江水系草鱼遗传多样性的微卫星DNA分析[J]. 水生生物学报, 29: 113-119. 鲁翠云, 孙效文, 梁利群, 等. 2005. 鳙鱼微卫星分子标记的筛选[J]. 中国水产科学, 12(2): 192-196. 孙国华, 杨建敏, 宋志乐, 等. 2010. 刺参( Apostichopus japonicus) EST序列中微卫星分布分析及其标记的筛选[J]. 海洋与湖沼, 41(1): 133-138. 袁慧, 张修月, 宋昭彬. 2008.