Takehana Yusuke, Nagai Naoko, Matsuda Masaru, Tsuchiya Kimiyuki, Sakaizumi Mitsuru
Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata, Japan.
Zoolog Sci. 2003 Oct;20(10):1279-91. doi: 10.2108/zsj.20.1279.
We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.
我们对线粒体细胞色素b基因进行了聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析，以阐明日本野生青鳉（Oryzias latipes）种群详细的遗传结构。对从303个地点采集的1225个样本进行分析，共鉴定出67种线粒体类型。随后，我们测定了完整细胞色素b基因（1141bp）的核苷酸序列，以阐明这些线粒体类型之间的系统发育关系。基于核苷酸序列构建的系统发育树显示出三个主要分支（A、B和C），它们之间的差异为11.3%-11.8%，这与之前通过对整个线粒体DNA进行RFLP分析所确定的三个聚类相对应。分支A和B中线粒体类型的地理分布与通过等位酶定义的北方和南方种群完全一致。分支A可细分为三个亚分支，分支B可细分为十一个亚分支，亚分支之间的序列差异为1.3%-5.8%。每个亚分支中线粒体类型的分布大致与之前确定的亚聚类中mtDNA单倍型的分布相对应。分支C中的线粒体类型仅在关东地区发现。系统发育关系和估计的分化时间表明，日本的三个分支起源于一个共同祖先，并在上新世期间分离，亚分支的区域分化与第四纪的地质历史密切相关。本研究还证明了自然分布受到人为干扰的可能性，特别是在关东地区，以及PCR-RFLP分析作为一种检测青鳉遗传变异和人工基因流动的简单方法的卓越功效。
