基于mtDNA D-Loop区和Cytb基因的清水江斑鳜群体遗传多样性分析

Analysis of genetic diversity of Siniperca scherzeri population in Qingshui River based on mitochondrial Cytb and D-Loop sequences

为了解贵州省境内清水江流域野生斑鳜(Siniperca scherzeri)的遗传多样性现状,利用mtDNA Cytb基因和D-Loop区测序技术,分析了清水江287尾野生斑鳜的遗传结构及其遗传多样性。结果表明:分别获得287条斑鳜的完整Cytb基因序列(1141 bp)和部分D-Loop区序列(840~841 bp);两个基因序列的碱基组成中A+T含量分别为51.6%和64.1%,均高于C+G含量的48.4%和35.9%,多态位点分别有23和44个,单倍型数分别为25和43种;单倍型多样性指数(核苷酸多样性指数)分别为0.821±0.018(0.0020±0.0001)和0.919±0.009(0.0083±0.0003);群体内平均遗传距离为0.0021和0.0087;AMOVA分析显示,群体内个体间变异是变异的主要来源(Cytb基因为97.54%,D-Loop区为97.32%);NJ系统发生树显示,Cytb和D-Loop区基因都聚为两支,两支的单倍型在河流的上、中、下游均有分布;清水江野生斑鳜群体的mtDNA Cytb的Fu’s Fs值呈显著负值(-0.9553,P=0.007),检测出了种群扩张,据公式推算,清水江斑鳜种群于0.176Ma年前发生了种群扩张,而D-Loop区符合中性进化的假设,未检测到种群扩张。综上所述,清水江野生斑鳜没有发生明显的遗传分化现象,遗传多样性高,呈现出较高的单倍型和核苷酸多态性。研究可为清水江野生斑鳜资源保护和持续发展与利用提供基础参考材料。

Qingshui River is located in the northern part of the eastern section of Miaoling Mountain range,which is the slope of the transition from the Yunnan-Guizhou Plateau to the hills of Hunan and Guangxi,and it is the upper reaches of Yuanjiang River in Dongting Lake Water System of the Yangtze River basin,and it is also the second largest river in Guizhou Province.In order to analyze the genetic diversity and population genetic structure of wild Siniperca scherzeri population in Qingshui River basin,and to provide a theoretical basis for conservation management of the wild resources,we have set up a total of ten sampling sites in the upper,middle and lower reaches of Qingshui River and collected 287 individuals of wild Siniperca scherzeri.Genetic diversity and phylogenetic relationshihs of 287 wild Siniperca scherzeri were investigated based on mitochondrial cytochrome b(Cytb)and control region(D-Loop)sequences.The completed mt DNA Cytb gene sequence(1141 bp)and partial D-Loop control region sequence(840-841 bp)of wild Siniperca scherzeri were PCR amplified,sequenced,and analyzed.The preliminary findings were as follows:1)The average contents of A,T,G and C in Cytb sequences were 24.3%,27.3%,15.5%and 32.4%,respectively,and the corresponding contents off our bases in D-Loop sequences were 34.0%,30.1%,15.8%and 20.1%,respectively.Meanwhile,the average content of A+T was 51.6%(Cytb)and 64.1%(D-Loop)respectively,both higher than the average C+G content of 48.4%(Cytb)and 35.9%(D-Loop),and the proportion of base G was the lowest,respectively,the data showed a bias against G,and the proportion of A to T in Cytb and D-Loop gene sequence was a little different.In addition,23 variation sites were detected in Cytb gene sequences,of which 16 were parsimony informative sites and 7 were singleton variable sites,no base insertions or deletions,and a base conversion/reversal ratio of 4.60.While 44 variation sites were detected in D-Loop gene sequences,of which 39 were parsimony informative sites and 5 were singleton variable sites,no base insertions or deletions,3 base deletions,no base insertions and a base conversion/reversal ratio of 10.19.2)25 haplotypes were defined in Cytb gene sequences,the haplotype diversity of 3 populations(UQ,MQ and LQ)ranged from 0.793±0.037 to 0.829±0.024,and the nucleotide diversity ranged from 0.0019±0.0001 to 0.0021±0.0001,the average haplotype diversity and nucleotide diversity were 0.821±0.018 and 0.0020±0.0001,respectively,the average number of nucleotide differences was 2.311.At the same time,43 haplotypes were identified in D-Loop gene sequences,the haplotype diversity of 3 populations(UQ,MQ and LQ)ranged from 0.884±0.031 to 0.921±0.017,and the nucleotide diversity ranged from 0.0074±0.0005 to 0.0091±0.0004,the average haplotype diversity and nucleotide diversity were 0.919±0.009 and 0.00831±0.00025,respectively,the average number of nucleotide differences was 6.968.The genetic diversity of 3 populations showed a decreasing trend of UQ>MQ>LQ in D-Loop gene,while Cytb gene had the highest genetic diversity in MQ,with the order of size being MQ>LQ>UQ.3)Within populations,the genetic distance decreased from 0.0092±0.0017 to 0.0074±0.0015 within UQ,MQ and LQ populations in D-Loop gene,and the degree of genetic variation became progressively lower.However,Cytb gene had the highest variation in MQ,with the same degree of variation in UQ and LQ.Among populations,the greatest genetic differences were found between UQ and MQ in D-Loop gene,and the smallest between MQ and LQ,while Cytb gene differed to a similar extent between the two populations of three populations.And the mean genetic distances within the population were 0.0021 and 0.0087 in D-Loop and Cytb genes,respectively.Fst values among 3 populations were not significant both in Cytb and D-Loop genes.The level of gene flow between UQ and LQ of Qingshui River was higher,and the individuals’exchanges were frequent.Analysis of molecular variance(AMOVA)demonstrated that variation in individuals within the population accounted for 97.54%(Cytb)and 97.32%(D-Loop)of total variation,genetic differentiation among populations was not significant.4)The results of phylogenetic analysis and haplotype evolution network showed that the haplotypes of both Cytb and D-Loop genes clustered into two branches,and haplotypes of both branches distributed in the upper,middle and lower reaches of the river,there was no clear geographical pattern of distribution.And it had the closest evolutionary relationship with Siniperca scherzeri from Yangtze River(Dongting Lake water system)according to Cytb phylogenetic study.5)Tajima’s D and Fu’s Fs values of wild Siniperca scherzeri were-0.9553 and showed extremely significant difference(P<0.01)in Cytb gene,and mismatch distribution analysis showed a poisson distribution,the results of neutral test and nucleotide mismatch analysis in Cytb gene showed that there had been a significant population expansion event recently,the equation estimated that the population expansion of wild Siniperca scherzeri population in Qingshui River occurred 0.176 million years ago.In contrast,the D-Loop gene was consistent with the hypothesis of neutral evolution and no population expansion was detected.In summary,these findings indicated that wild Siniperca scherzeri population in Qingshui River showed no obvious genetic differentiation among populations,and had higher genetic diversity,as high haplotype diversity and low nucleotide diversity.The results can complement basic genetic diversity background of wild Siniperca scherzeri in Yangtze River basin,and will be usefully applied to scientific strategies for the protection,sustainable development and commercial utilization of germplasm resources of wild Siniperca scherzeri in Qingshui River.