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Research of genetic diversity based on the mitochondrial COI gene sequences in four populations of Paramisgurnus dabryanus

  • Corresponding author: Lei WANG, hnnuwl@163.com
  • Received Date: 2018-09-16
    Accepted Date: 2019-04-26
  • In order to research the genetic diversity of different populations of Paramisgurnus dabryanus, four populations from Liaoning, Henan, Hubei and Taiwan were analyzed by sequencing the mitochondrial cytochrome oxidase subunit Ⅰ(COI) genes. The results showed that 642 base pair(bp)fragment was consisted of A, T, C and G base with 23.5%, 30.6%, 26.7% and 19.2%, respectively, indicating a preference for A and T base. A total of 44 mutations of nucleotide and 23 haplotypes were identified in 4 populations. The haplotype diversity index ranged from 0.424~0.855, and the nucleotide diversity ranged from 0.000 84~0.016 59, the results indicated that Paramisgurnus dabryanus from Taiwan population had a higher genetic diversity than Liaoning, Henan and Hubei population. The genetic differentiation index (Fst) and genetic distance showed that genetic differentiation among Taiwan and other populations was represents extremely significant difference (P<0.01), also had a further genetic distance with other populations. In general, the results showed that there was a certain genetic difference between the different geographical groups, AMOVA analysis revealed that 52.11% of genetic variations derived among populations and 47.89% of genetic variations occured within populations. In terms of the negatively selective neutrality test, the results indicated that a population expansion occurred in the populations of Liaoning, Henan and Hubei population. Which provides a reference for protection of the genetic diversity and breeding work of Paramisgurnus dabryanus.
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Research of genetic diversity based on the mitochondrial COI gene sequences in four populations of Paramisgurnus dabryanus

    Corresponding author: Lei WANG, hnnuwl@163.com
  • College of Fisheries, Henan Normal University, Xinxiang 453007, China

Abstract: In order to research the genetic diversity of different populations of Paramisgurnus dabryanus, four populations from Liaoning, Henan, Hubei and Taiwan were analyzed by sequencing the mitochondrial cytochrome oxidase subunit Ⅰ(COI) genes. The results showed that 642 base pair(bp)fragment was consisted of A, T, C and G base with 23.5%, 30.6%, 26.7% and 19.2%, respectively, indicating a preference for A and T base. A total of 44 mutations of nucleotide and 23 haplotypes were identified in 4 populations. The haplotype diversity index ranged from 0.424~0.855, and the nucleotide diversity ranged from 0.000 84~0.016 59, the results indicated that Paramisgurnus dabryanus from Taiwan population had a higher genetic diversity than Liaoning, Henan and Hubei population. The genetic differentiation index (Fst) and genetic distance showed that genetic differentiation among Taiwan and other populations was represents extremely significant difference (P<0.01), also had a further genetic distance with other populations. In general, the results showed that there was a certain genetic difference between the different geographical groups, AMOVA analysis revealed that 52.11% of genetic variations derived among populations and 47.89% of genetic variations occured within populations. In terms of the negatively selective neutrality test, the results indicated that a population expansion occurred in the populations of Liaoning, Henan and Hubei population. Which provides a reference for protection of the genetic diversity and breeding work of Paramisgurnus dabryanus.

  • 大鳞副泥鳅(Paramisgurnus dabryanus)隶属鲤形目(Cypriniformes)、鳅科(Cobitidae)、花鳅亚科(Cobitinae)、副泥鳅属(Paramisgurnus),其分布广泛,是我国常见的一种小型淡水养殖鱼类,具有较好的经济、食用及药用价值。因其肉质鲜美,营养丰富,素有“水中人参”的美誉,深受消费者的喜爱与推崇。然而,近年来随着人工捕捞及栖息地环境的破坏,大鳞副泥鳅面临着野生资源减少,种质资源衰退,群体遗传多样性降低等问题[1-2]。白晓慧等[3]对天津5个大鳞副泥鳅自然群体的线粒体DNA D-loop序列进行遗传变异分析发现,大鳞副泥鳅渔业资源不容乐观,要适时开展良种选育工作,减少对其野生资源的破坏。因此对大鳞副泥鳅开展进一步遗传研究保护工作迫在眉睫。

    鱼类线粒体DNA(mt DNA)作为一种重要的分子标记被广泛地应用于鱼类的系统学及遗传多样性研究中[4-6]。其中,细胞色素氧化还原酶 Ⅰ(COI)是线粒体13个蛋白编码基因中研究最为清楚的基因之一,其进化速率适中,在种属进化的研究中具有很高价值。目前对于大鳞副泥鳅遗传多样性的研究已有报道,但基于线粒体COI基因序列进行的研究较少[7-10]。本实验拟通过对4个不同群体的大鳞副泥鳅COI基因序列进行分析,以期揭示不同群体之间的遗传多样性及遗传结构差异,为大鳞副泥鳅遗传多样性保护及优良品种选育等工作提供依据。

1.   材料与方法
  • 大鳞副泥鳅群体于2016年分别取自辽宁大连、河南范县和湖北武汉三地,台湾群体则采购于豫北农产品水产大世界。样本活体运输至河南师范大学水产养殖基地,取鳍条组织于75%的乙醇中保存24 h,更换为95%乙醇保存24 h,最后于无水乙醇中常温保存。

  • 采用酚/氯仿抽提法[11]提取4个大鳞副泥鳅群体的基因组DNA,其中辽宁群体22个,河南群体29个,湖北群体29个,台湾群体24个。所得DNA经1%的琼脂糖凝胶电泳检测后,保存于−20 °C冰箱中备用。

  • 大鳞副泥鳅线粒体COI基因扩增所用引物序列如下[12]

    COIF:5′-CACGACGTTGTAAAACGACCAACYAATCAYAAAGATATYGGCAC-3′

    COIR:5′-GGATAACAATTTCACACAGGACTTCYGGGTGRCCRAARAATCA-3′

    引物由上海生工生物工程股份有限公司合成。PCR反应体系为50 μL,包括Mix 25 μL,上下游引物各1 μL,模板DNA 3 μL,用双蒸水补齐至50 μL。PCR反应程序为:94 °C预变性3 min;94 °C变性40 s,52 °C退火1 min,72 °C延伸1 min,共运行35个循环;72 °C终延伸10 min,PCR产物在4 °C条件下保存。PCR产物经1%琼脂糖凝胶电泳检测后,送至上海生工生物工程有限公司进行测序。

  • 测序完成后,通过Clustal X[13]软件对测序结果进行比对分析,同时进行人工核查与校正。采用软件DNASPv.5[14]计算序列变异位点数、单倍型数目、单倍型多样性和核苷酸多样性等遗传参数;利用MEGA5.1[15]软件分析序列的碱基组成,计算群体内及群体间的遗传距离,采用邻接法[16] (Neighbor-Joining,NJ)并基于Kimura双参法构建大鳞副泥鳅单倍型邻接关系进化树,节点支持率采用Bootstrap 1 000次重复检验置信度;利用Arlequin3.11[17]软件计算两两群体间的遗传分化指数(FST)并进行分子方差分析(Analysis of molecular variance,AMOVA),用于遗传变异和地理结构特点研究;利用Network 5.0[18]软件构建单倍型网络图,分析单倍型之间的进化关系。

2.   结果与分析
  • 利用Clustal X软件对测序所获目的片段进行手工校对剪切,获得长度为642 bp的同源序列。在642个位点中,共检测到44个多态性位点,占总位点数的6.85%,其中简约信息位点35个,单碱基变异位点9个,变异均为转换或颠换,转换明显多于颠换,没有检测到插入或缺失现象(图1)。COI序列中碱基A、T、C、G的平均含量分别为23.5%、30.6%、26.7%、19.2%,其中T碱基含量最高,G碱基含量最低,且A+T的平均含量高于C+G的平均含量(54%>46%),表现出了碱基组成的偏向性。

    Figure 1.  Variable sites of mitochondrial COI gene of Paramisgurnus dabryanus.

  • 在104个大鳞副泥鳅样本中共检测到23个单倍型,其中2个为共享单倍型,其余21个为独有单倍型。单倍型Hap1为4个群体的共享单倍型,出现频次最高,分布于63个样本中,占总样本数的60.58%,可能为大鳞副泥鳅的原始单倍型。单倍型Hap3为两个群体所共享(辽宁、湖北),分布于3个样本中。在独有单倍型中,辽宁群体有4个(Hap2、Hap4、Hap5、Hap6),河南群体有6个(Hap7、Hap8、Hap9、Hap10、Hap11、Hap12),湖北群体有4个(Hap13、Hap14、Hap15、Hap16),台湾群体有7个(Hap17、Hap18、Hap19、Hap20、Hap21、Hap22、Hap23)。运用K-2-P模型构建23个单倍型的NJ系统发育树(图2),由NJ系统树发现:台湾群体聚为一支,其余三个群体聚为一支,且节点支持率较高,具有较为明显的遗传差异。利用Network 5.0软件分析大鳞副泥鳅单倍型之间的进化关系:4个群体的单倍型网络分布图呈现一种以4个群体的共享单倍型(Hap1)为中心的放射状结构;台湾群体的单倍型与其他3个群体的单倍型基本处于完全分割状态,显示台湾群体和其余3个群体具有较大的遗传距离,这与NJ系统发育树的结果相一致(图3)。

    Figure 2.  Distribution of 23 haplotypes among 4 Paramisgurnus dabryanus populations and its NJ molecular phylogenetic tree

    Figure 3.  Median-joining network of COI gene haplotypes of Paramisgurnus dabryanus

  • 大鳞副泥鳅4个群体的遗传多样性参数如表1所示。单倍型多样性指数(h)在0.424~0.855之间,核苷酸多样性指数(π)在0.000 84~0.016 59之间。由单倍型参数可知,台湾群体的单倍型数及单倍型多样性均较高,湖北群体的单倍型数及单倍型多样性均较低;由核苷酸参数可知,台湾群体的核苷酸多样性较高,辽宁和湖北群体核苷酸多样性较低。以上结果表明,台湾群体具有较高的遗传多样性,河南群体其次,湖北群体和辽宁群体的遗传多样性则较低。

    群体
    population
    样本数
    sample size
    单倍型数
    number of
    haplotypes
    多态位点
    number of
    polymorphic sites
    单倍型多样性
    haplotype diversity (h)
    核苷酸多态性
    nucleotide diversity (π)
    Tajima’s DFu’s Fs
    辽宁群体 22 6 5 0.476±0.128 0.000 84±0.000 26 −1.809 01* −4.180
    河南群体 29 7 10 0.475±0.111 0.001 67±0.000 60 −1.851 50* −2.561
    湖北群体 29 6 6 0.424±0.111 0.000 84±0.000 27 −1.866 44* −3.730
    台湾群体 24 8 35 0.855±0.040 0.016 59±0.003 38 0.518 45 4.939
    合计 104 23 44 0.627±0.055 0.011 33±0.001 67 −0.491 31 −1.005
    注:“*”表示差异显著(P<0.05)
    Note: “*” means significant difference (P<0.05)

    Table 1.  Parameters of genetic diversity of different populations of Paramisgurnus dabryanus

    群体间FST分析结果显示:两两群体间的FST值在0.004 29~0.699 56之间(表2),台湾群体与其他群体间的遗传分化系数FST较大,在0.678 25~0.699 56之间,且均存在极显著差异(P<0.01)。4个群体的遗传距离分析显示:群体内遗传距离在0.000 8~0.017 1之间,群体内遗传距离最大的为台湾群体(0.017 1),遗传距离最小的为辽宁和湖北群体,均为0.000 8;两两群体间的遗传距离在0.001~0.028之间,台湾群体与其他群体间的遗传距离均较远,与FST分析结果相一致。AMOVA分析结果表明来自于群体间的遗传变异(52.11%)高于来自群体内的遗传变异(47.89%)(表3)。

    群体
    population
    群体间遗传距离及FST值 genetic distance between populations and FST群体内遗传距离
    genetic distance within populations
    辽宁河南湖北台湾
    辽宁群体 0.001 0.001 0.028 0.000 8
    河南群体 0.021 63 0.001 0.028 0.001 7
    湖北群体 0.004 29 0.009 84 0.028 0.000 8
    台湾群体 0.678 25** 0.687 18** 0.699 56** 0.017 1
    注:对角线以上为群体间的遗传距离,对角线以下为群体间FST。“**”表示差异极显著(P<0.01)
    Note: Figures above the diagonal are genetic distance,and those below the diagonal are FST. “**”represents extremely significant difference(P<0.01)

    Table 2.  Pairwise genetic distances within population, and genetic distance, fixation index (FST) between every two population of Paramisgurnus dabryanus

    变异来源
    source of variation
    自由度
    df
    平方和
    sum of squares
    方差组分
    variance components
    变异百分比%
    percentage of variation
    群体间 among populations 3 69.982 2.575 81 Va 52.11
    群体内 within populations 30 71.018 2.367 27 Vb 47.89
    总数 total 33 141.00 4.943 08

    Table 3.  Analysis of molecular variance (AMOVA) among populations of Paramisgurnus dabryanus

  • 4个大鳞副泥鳅群体的中性检验结果见表1。中性检验结果显示:除台湾群体外,辽宁、河南及湖北三个群体的Tajima’s D及Fu’s Fs检验结果均为负值,且Tajima’s D检验结果显著(P<0.05)。D值和Fs值为负表明群体可能经历过种群扩张,据此推测除台湾群体外的其余三个群体历史上可能经历了一定的种群爆发和扩张事件。

3.   讨论
  • 遗传多样性是生物多样性研究的重要部分,是进化和适应的基础,种内遗传多样性或变异性越丰富,则物种对环境变化的适应能力越大,其进化的潜力也就越大[19]。以往研究者们利用COI基因序列分析了北极七鳃鳗(Lethenteron Camtschaticum)[20]、银鲳(Pampus argenteus)[21]、翘嘴鲌(Culter Alburnus)[22-23]等鱼类的遗传多样性及遗传结构,说明利用COI基因序列进行淡水鱼类的遗传多样性研究是可行的。目前对大鳞副泥鳅遗传多样性的研究多采用线粒体DNA、微卫星等[289]方法,由于线粒体COI基因序列具有较高的遗传多态性,因此适用于种内分析。由Grant等[24]的研究可知,单倍型多样性与核苷酸多样性的临界值分别为0.5及0.005,值越大则表明遗传多样性越高。本研究中,四个大鳞副泥鳅群体的核苷酸多样性在0.000 84~0.016 59之间,单倍型多样性在0.476~0.855之间,总体来看,除台湾群体外,其余三个群体的遗传多样性均较低。赵旭[25]基于线粒体COI基因片段对山西临汾、河南新乡、山东济南三个地区的大鳞副泥鳅群体进行遗传多样性分析,发现其单倍型多样性在0.533~1.000之间,核苷酸多样性在0.001 53~0.005 88之间,高于辽宁、河南、湖北三个群体的遗传多样性,这可能与采样地点不同或大鳞副泥鳅的遗传多样性水平降低有关。在大鳞副泥鳅COI基因序列的碱基组成中,A+T的平均含量远高于C+G的平均含量(54%>46%),表现出一定的AT偏好性,与其他硬骨鱼纲COI基因序列碱基组成的特点相似[26-28]

  • 由4个大鳞副泥鳅群体的遗传距离分析结果可知,台湾群体与其他3个群体之间遗传距离较大,且具有较高的遗传分化,这可能与其地理分布有关,台湾群体来源于地理相对隔离的台湾岛,基因交流贫乏。顾林林等[29]对南四湖大鳞副泥鳅COI基因序列进行分析,发现其种内遗传距离为0.001,与本研究中不相符,这可能是由于其样本量较少或采样地点不同造成的。

    遗传分化系数FST是反映群体间遗传分化程度的重要指标[30],较高水平的FST表明其具有较高水平的遗传分化,当FST为0~0.05时无分化,0.05~0.15为中度分化,0.15~0.25为高度分化。台湾群体与其他三个群体之间的FST均为正值,且P<0.01,具有极显著差异,说明台湾群体与其他三个群体间有明显的遗传分化,这可能是地理上的隔离造成的。

  • 单倍型多样性是评价群体遗传分化和多态程度的重要指标,单倍型多样性越高,群体的遗传多样性越高。研究发现台湾群体的单倍型多样性指数最高(0.855±0.040)。在单倍型NJ进化树中,23个单倍型分为2个分支,辽宁、河南和湖北群体聚为一支,台湾群体单独聚为一支;遗传分化系数FST分析表明台湾群体与其他群体间的遗传结构存在显著差异;结合遗传距离和单倍型网络进化图可以发现台湾群体与其他三个群体的遗传距离较远,遗传分化程度较大,具有一定的地理差异。

4.   结论
  • 本研究对4个大鳞副泥鳅群体进行遗传多样性分析发现,辽宁、河南、湖北三个群体遗传多样性较低,大鳞副泥鳅的种质资源现状并不乐观。因此建议:加大对大鳞副泥鳅种种质资源的保护力度,提高大鳞副泥鳅群体的遗传多样性;采用遗传距离适中的群体开展大鳞副泥鳅的选育工作,为大鳞副泥鳅产业健康发展提供技术支撑。

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