虎(Panthera tigris)微卫星位点多态性及其在圈养种群管理中的应用
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摘要
本文应用基于荧光标记的全自动DNA序列分析技术和微卫星多态性分析技术,
    从30个家猫(Felis Catus)微卫星位点中筛选了15个多态性良好、扩增结果稳定可
    靠的位点用于分析虎的3个亚种,即东北虎(Panthera tigris altaica)、孟加拉虎(P.t.
    tigris)和华南虎(P.t.amoyensis)的圈养种群的遗传多样性水平。这15位点包括
    Fca043、Fca074、Fca077、Fca066、Fca090、Foa107、Fca304、F53、F141、F42、Fca391、
    Fca453、F146、Fca441、Fca41,其中Fca043、Fca074、Fca077、Fca304、Fca453、Fca441、
    F42等7个位点通过测序分析获得了重复单位的碱基组成和重复次数。对36只虎15
    个位点的分析发现,这些位点有4-13个等位基因,等位基因频率和基因型频率分布
    不均衡,10个位点不符和H-W平衡(α=0.01)。对20只东北虎、11只孟加拉虎和
    5只华南虎的分析表明,群体条合度在物种水平上为0.4937~0.8412之间;在东北虎
    为0.3691~0.8003之间;在孟加拉虎为0.3872~0.8917之间;在华南虎为0.5341~0.8565
    之间。杂合子频率与杂合度之间基本吻合,只有少数位点杂合子频率显著低于杂合
    度。各个亚种都有自己独特的等位基因,通过15个微卫星位点的数据计算亚种间的
    遗传距离为:东北虎-孟加拉虎之间0.2449、东北虎-华南虎之间0.0687、华南虎-孟
    加拉虎之间0.1031。两个无关个体15个微卫星位点完全偶合的概率在10~(-11)~10~(-18)之
    间。用15个位点计算的父-子-母三联体的PI值为159824.94~8831296.70,RCP值在
    0.99999374~0.99999989之间;缺少母本样品时PI为498.23~1448029.34,RCP值为
    0.99799693~0.99999931之间。不同种属的扩增显示,这些微卫星位点的引物在狍、
    梅花鹿、家犬、绵羊、人等均不能成功扩增,可以用于野外样品或混合样品的分析。
    对雄森熊虎山庄的圈养种群的分析表明,圈养种群的管理和遗传多样性保护应从以
    下4个方面入手:1)严格保持亚种的独立性;2)避免近交繁殖;3)适当延长世代
    间隔时间;4)促进种群间的基因交流。本文也可为豹等其他大型猫科动物的微卫星
    遗传标记的研究与应用提供良好的技术路线和基础。
By using fluorescent-labeling-based automated DNA sequencing and microsatellite polymorphism
     analyzing techniques, of 30 microsatellite loci derived from domestic cat (Felts catus), 15 were proved
     polymorphic, reproducible and reliable in amplifying tiger genomic DNA. They were applied to study the
     genetic diversity in tiger captive population involving 3 subspecies, Siberian tiger (Pant he ra tigris altaica),
     Bengal tiger (P t. tigris) and South China tiger (P t. amoyensis). These 15 microsatellites include namely
     Fca043, Fca074, Fca077, Fca066, FcaO9O, FcalO7, Fca304, F53, F141, F42, Fca391, Fca453, F146,
     Fca441 and Fca4l, of which 7 loci, Fca043, Fca074, Fca077, Fca304, Fca453, Fca441 and F42 were
     sequenced to discover the repeating motif and repeating times. In the population analysis of 36 tigers by
     using 15 microsatellites, 4-13 alleles were found in each locus. The allelic frequency and genotypic
     frequency of 10 loci showed significant departure from Hardy-Weinberg Balance (a=0.01) indicating the
     disequilibrium of allelic frequency and genotypic frequency distribution. Heterozygosity was found
     between 0.4937-4)8412 at species level (im36), 0.3691-41.8003 in Siberian tiger (n20), 0.3872--0.8917 in
     Bengal tiger (n1 1) and 0.5341桹.8565 in South China tiger (n~5). Observed frequencies of lieterozygotes
     in each locus basically match the heterozygositv, except for a few loci whose heterozygote frequency is
     rather lower. Each subspecies o抶ns a few special alleles. Genetic distance among 3 subspecies calculated
     based on the data of 15 loci is: Siberian tiger-Bengal tiger 0.2449, Siberian tiger-South China tiger 0.0687
     and South China tiger-Bengal tiger 0.1031. The matching probability between two unrelated individual at
     15 loci is l0l1~~10.i8. P1 value in Father-child-mother set is between ]59824.94~883l296.70,
     correspondingly, RCP value is between 0.99999374-4)99999989. In the sets without mother抯 sample, P1
     is between498.23?448029.34, correspondingly, RCP value is between 0.99799693?.99999931. The
     study also showed that no successful amplification was detected when applied primer sets of these 15
     microsatellites to other species namely roe deer, sika deer, domestic dog, sheep and human DNA. It is
     suggested that the primers can be reliable in analyzing field samples and mixed or contaminated samples.
     Taking the example of the captive population of Xiongsen Bear and Tiger Village, this study suggested 4
     countermeasures to manage the population and maintain genetic diversity: 1) Keep the independence ot
     subspecies strictly; 2) avoid inhrecding~ 3) prolong the generation leneth; 4) promote the genetic exchange
     among populations. As a potential significance, this study can also provide a practical technique route and
     basis for the microsatellite research in other large felines.
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