目的 对云南省家鼠鼠疫疫源地的黄胸鼠细胞色素C氧化酶亚基Ⅰ（COⅠ）基因的序列进行分析,探讨云南省不同地理种群黄胸鼠的遗传特性。方法 2016-2019年对在大理白族自治州（大理州）、保山市、德宏傣族景颇族自治州（德宏州）、临沧市、玉溪市、文山壮族苗族自治州（文山州）和西双版纳傣族自治州（西双版纳州）7个地区捕获的黄胸鼠408份肝、脾标本进行基因组DNA提取。应用PCR技术扩增COⅠ基因,通过生物信息学分析软件开展COⅠ基因序列的核苷酸、单倍型分析,采用邻接法构建分子系统发育树,并作群体分子方差分析（AMOVA）、基因流、遗传分化指数（Fst）的计算。结果 共成功扩增云南省7个地区黄胸鼠种群408份肝、脾标本的COⅠ基因序列,分析结果显示,在639 bp的COⅠ基因序列中,碱基T、C、A、G的平均含量分别为29.67%、25.67%、27.67%和16.99%,黄胸鼠种群COⅠ基因的碱基含量A+T高于C+G。7个种群共定义了23个单倍型,单倍型多样性为0.433～0.846,核苷酸多样性为0.002 82～0.019 73,各种群整体的单倍型多样性排序由高到低分别为德宏州>玉溪市>保山市>文山州>西双版纳州>临沧市>大理州,核苷酸多样性较高的为文山州、德宏州和玉溪市种群。分子系统发育分析显示,7个种群单倍型共分成3个大支；单倍型网络关系图显示,不同地理种群的单倍型交叉混杂分布；群体Fst分析表明,除保山市与西双版纳州、临沧市、文山州,西双版纳州与临沧市、文山州,临沧市与文山州种群间外,其余各地理种群间遗传差异均有统计学意义（均P<0.05）；基因流数据显示,玉溪市与大理州、保山市、西双版纳州种群出现了一定程度的遗传分化,其他种群间基因交流缺乏。AMOVA分析结果表明,7个黄胸鼠种群的遗传变异主要来自种群内,种群间的变异程度较低。结论 7个地区的黄胸鼠种群表现出丰富的遗传多样性,部分地理种群间出现遗传分化,研究为黄胸鼠的分子生态学研究提供了参考依据。
Objective To sequence and analyze the cytochrome c oxidase subunitⅠ(COⅠ) gene of Rattus tanezumi in the domestic rodent plague foci of Yunnan Province, China, and to explore the genetic characteristics of R. tanezumi populations in different geographical areas of Yunnan Province.Methods From 2016 to 2019, R. tanezumi was captured in Dali Bai Autonomous Prefecture (Dali), Baoshan, Dehong Dai and Jingpo Autonomous Prefecture (Dehong), Lincang, Yuxi, Wenshan Zhuang and Miao Autonomous Prefecture (Wenshan), and Xishuangbanna Dai Autonomous Prefecture (Xishuangbanna). A total of 408 liver and spleen specimens of R. tanezumi were collected to extract genomic DNA and then amplify the COⅠgene by PCR. Bioinformatics methods were used for nucleotide and haplotype analysis of the COⅠ gene sequence. A molecular phylogenetic tree was constructed by using the neighbor-joining method. An analysis of molecular variance (AMOVA) among populations were performed, and gene flow and the genetic differentiation index (Fst) were caculated.Results The COⅠgene sequences of 408 R. tanezumi specimens from the seven areas of Yunnan Province were successfully amplified. In the 639 bp COⅠgene sequence, the average percentages of T, C, A, and G bases were 29.67%, 25.67%, 27.67%, and 16.99%, respectively, and the proportion of A+T was higher than that of C+G. A total of 23 haplotypes were identified in the seven geographical populations. The haplotype diversity was 0.433-0.846, and the nucleotide diversity was 0.002 82-0.019 73. The overall haplotype diversity of 7 populations from high to low was: Dehong > Yuxi > Baoshan > Wenshan > Xishuangbanna > Lincang > Dali. The Wenshan, Dehong, and Yuxi populations were more diverse by nucleotide. The molecular phylogenetic analysis showed that the haplotypes of the seven populations were divided into three clades. The haplotype network showed that the haplotypes were distributed in a mixed pattern across different geographical populations. The Fst showed significant differences between different geographical populations (all P<0.05), except between Baoshan and Xishuangbanna, Lincang, and Wenshan, between Xishuangbanna and Lincang and Wenshan, and between Lincang and Wenshan. Gene flow data revealed a certain degree of genetic differentiation between Yushan populations and Dali, Baoshan, and Xishuangbanna populations, while there was a lack of gene exchange among other populations. The AMOVA analysis showed that the genetic variation among the seven R. tanezumi populations mainly was intra-population variation, with a low level of inter-population variation.Conclusions The R. tanezumi populations of the seven cities or prefectures have high genetic diversity, and genetic differentiation is present between some geographical populations. This study provides a reference for the study of molecular ecology of R. tanezumi.
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