贵州省不同地理株中华按蚊mtDNA-COⅠ基因遗传多态性分析

Genetic polymorphism of mt DNA-COⅠgene of Anopheles sinensis from different geographical strains in Guizhou province,China

目的了解贵州省不同地理株中华按蚊种群线粒体DNA细胞色素C氧化酶亚基Ⅰ(mtDNA-COⅠ)基因序列的多态性,探讨其遗传多样性、遗传分化和种群扩张情况以及遗传距离与地理距离及其相关关系等种群遗传特征。方法于2017年7月-2018年8月采用灯诱法在贵州省东、西、南、北、中5个方位的12个采样点采集稻田周边的成蚊,经过形态学确认为中华按蚊后,用试剂盒法提取单只蚊虫DNA样本并对mtDNA-COⅠ基因扩增后测序,测序结果用DNAStar 5.0软件进行拼接,在美国国立生物技术信息中心经BLAST比对进行蚊种的分子鉴定,对确认为中华按蚊mtDNA-COⅠ的基因序列使用DnaSP 5.1软件分析其单倍型多样性、核苷酸多样性,并计算各种群间分化系数(Fst)和基因流(Nm)大小,进行中性检验,以明确各种群之间的分化程度和种群扩张情况。使用MEGA 7.1软件计算各种群间和种群内的遗传距离,判断种群之间的遗传变异程度;根据采样点经纬度计算种群间的地理距离,与对应的遗传距离进行相关性检验,以分析两者的相关关系。结果经分子生物学鉴定,共获得贵州省12个地理株中华按蚊mtDNA-COⅠ基因序列551条,长度为709 bp,其中,碱基(A+T)和(G+C)平均含量分别为68.57%和31.43%,709个位点中保守位点有586个,变异位点有123个,单态位点有189个,简约性信息位点有278个,无插入和缺失位点。551条序列共检出329种单倍型,单倍型多样性指数为0.992,核苷酸多样性为0.01522以上,12个种群的Fst值在0.0008~0.4207,Nm值在0.0861~75.2392,中性检验统计值均为负值,种群内遗传距离范围在0.0071~0.0529,种群间的遗传距离范围在0.0079~0.0522,织金种群和都匀种群内遗传距离较其他种群大;遗传距离和地理距离之间无相关关系,不同种群之间可能存在一定的地理隔离,但种群之间遗传距离的大小与地理距离不相关。结论贵州省不同地理株中华按蚊种群遗传多样性丰富,在历史上经历了区域性的种群扩张,可能存在一定程度的遗传分化和地理隔离,但在一定范围内种群间也有基因交流,呈现出交流与隔离并存的分布格局。

Objective To determine the polymorphism of mitochondrial cytochrome c oxidase subunitⅠ(mtDNA-COⅠ)gene sequence in different geographical strains of Anopheles sinensis population in Guizhou province,China,and to explore the genetic diversity,genetic differentiation,population expansion,and the correlation between genetic distance and geographical distance.Methods From July 2017 to August 2018,adult mosquitoes around the rice fields at 12 sampling sites in the East,West,South,North,and central area of Guizhou province were captured by light trapping method.The DNA sample of a single mosquito was extracted by kit method after the mosquito was confirmed as An.sinensis by morphology,and the mtDNA-COⅠgene was amplified and sequenced.The sequencing results were spliced by DNAStar 5.0 software,and the molecular identification of mosquito species was carried out by BLAST comparison in National Center for Biotechnology Information.The haplotype diversity and nucleotide diversity of the mtDNA-COⅠgene sequence of An.sinensis were analyzed by DnaSP 5.1 software,and the differentiation coefficient(Fst)and gene flow size(Nm)among various populations were calculated.The neutrality test was carried out to clarify the differentiation degree and population expansion among various populations.The genetic distance among various populations and within populations was calculated by MEGA 7.1 software to determine the degree of genetic variation among populations.The geographical distance between populations was calculated according to the latitude and longitude of sampling sites,and the correlation with the corresponding genetic distance was analyzed by the correlation test.Results In this study,551 sequences of mtDNA-COⅠgene were obtained from 12 geographical strains of An.sinensis in Guizhou province,through molecular biological identification,with a length of 709 bp.Among them,the average content of base A+T was 68.57%,and the average content of G+C was 31.43%.Among the 709 base sites,there were 586 conserved sites,123 variable sites,189 monomorphic sites,278 parsimony-informative sites,and no insertion or deletion sites.A total of 329 haplotypes were detected from 551 sequences,with a haplotype diversity index of 0.992and a nucleotide diversity of more than 0.01522.The Fst value of 12 populations was between 0.0008 and 0.4207,and the Nm values ranged from 0.0861 to 75.2392.The neutrality test statistic values were all negative values.The genetic distance within populations ranged from 0.0071 to 0.0529,and the genetic distance between populations ranged from 0.0079 to 0.0522.The genetic distances within Zhijin and Duyun populations were larger than those within other populations.There was no correlation between genetic distance and geographical distance.There might be a certain geographical isolation between different populations,but the genetic distance between populations was not related to the geographical distance.Conclusion There is abundant genetic diversity among different geographical strains of An.sinensis populations in Guizhou province,in which regional population expansion has been experienced in history.There may be some degree of genetic differentiation and geographical isolation,but there is also gene exchange among populations in a certain range,showing a distribution pattern of coexistence of exchange and isolation.