Cytb gene sequences of Fejervarya species from Lesser Sunda, Indonesia and other Asian countries

A total of 27 frogs belonging to the genus Fejervarya were used for genetic research. Genomic DNA for PCR was extracted from clipped toes using the DNAeasy Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The extracted DNA solutions were used to amplify a partial region of the Cytb gene corresponding to nucleotide positions 14396–15063 in the Hoplobatrachus tigerinus complete mtDNA sequence (accession no. AP011543, Alam et al. 2010). PCR amplification and sequencing were performed using the primers Fow-1-1 and Rev-1 (Hasan et al. 2012) to obtain a fragment of ca. 667 bp. The resultant nucleotide sequences were aligned using the ClustalW program (Thompson et al. 1994). A phylogeny was constructed using the maximum likelihood (ML) method implemented in Treefinder (Jobb 2008) and branch support was evaluated by a bootstrap analysis with 100 replicates. The resultant sequence data were deposited in the DDBJ database under accession nos. LC706527–LC706542. Additional Cytb data were obtained from GenBank for tree construction.

本研究共选用27份隶属于扁手蛙属（Fejervarya）的蛙类样本用于遗传学研究。按照试剂盒说明书的操作步骤，使用DNAeasy组织提取试剂盒（Qiagen，美国加利福尼亚州巴伦西亚）从剪取的蛙趾组织中提取用于聚合酶链式反应（PCR）的基因组DNA。以提取得到的DNA溶液为模板，扩增虎纹蛙（Hoplobatrachus tigerinus）完整线粒体DNA（mtDNA）序列中对应核苷酸位置14396至15063的细胞色素b（Cytb）基因部分区域，该参考序列的登录号为AP011543，相关研究见于Alam等人2010年的报道。使用引物Fow-1-1与Rev-1（相关方法见于Hasan等人2012年的研究）完成PCR扩增与测序，最终获得长度约为667 bp的DNA片段。使用ClustalW程序（相关方法见于Thompson等人1994年的研究）对得到的核苷酸序列进行多序列比对。基于Treefinder软件（Jobb 2008）所实现的最大似然法（ML）构建系统发育树，并通过100次重复的自举检验评估分支支持率。本研究得到的序列数据已提交至日本DNA数据库（DDBJ），登录号为LC706527至LC706542。为构建系统发育树，另外从GenBank数据库获取了部分细胞色素b基因序列数据。