东北虎微卫星DNA遗传标记的筛选及在亲子鉴定中的应用

Identification of polymorphic microsatellite DNA loci and paternity testing of Amur tigers

利用 18个家猫微卫星基因座 ,在东北虎 (Pantheratigrissibilia)DNA中扩增结果有 4个基因座没有产物 ,8个基因座为单态 ,6个基因座为多态性。同时利用苏门答腊虎的微卫星序列设计了 8对引物 ,在东北虎DNA中有 4对具有多态性。微卫星基因座的多态性百分率为 38 5 %。在供试的 2 7只东北虎中 ,发现等位基因间的变异均为偶数碱基长度变化 ,对有准确谱系记录的个体研究表明 ,这 10个微卫星DNA遗传标记符合孟德尔遗传规律 ,所以这些微卫星DNA可以有效的应用于东北虎的亲子鉴定。利用这 10对多态性引物 ,我们成功地鉴定了 7个父子关系不清的后代。收集的样品包括 2 3只毛发样品和 4只血液样品 ,实验结果表明 ,毛发和血液样品均可以得到清晰的微卫星条带 [动物学报 49(1) :118～ 12 3,2 0 0 3]。

We identified the paternity of some Amur tigers (Panthera tigris altaica) in Ha'erbin Tiger Park and constructed clear pedigrees using microsatellite loci.Four blood (No.209, No.232, No.233, No.244) and twenty-three hair specimens from Amur tigers were examined. Eighteen polymorphic microsatellite DNA loci identified in the domestic cat (Felis catus) were amplified in the Amur tiger genome. It was found that four of the loci failed to be amplified, eight were monomorphic and six (Fca005, Fca075, Fca094, Fca152, Fca161, Fca294) were polymorphic. At the same time, we designed eight pairs of microsatellite primers based on Sumatran tiger (Panthera tigris sumatrae) gene sequences for amplification in the Amur tiger. The results show that four pairs of primers produced a monomorphic pattern and the other four (Pti002, Pti003, Pti007, Pti010) gave a polymorphic pattern. Altogether, 38.5% of the twenty six microsatellite markers were polymorphic, and inheritance patterns of alleles at these loci show close agreement with the well documented pedigrees.Seven tigers (No.067, No.130, No.132, No.209, No,232, No.233, No.244) had ambiguous paternity. We identified their biological fathers (No.048, No.048, No.057, No.048, No.019, No.081, No.075, respectively) according to the inheritance patterns of ten polymorphic microsatellite loci. This analysis revealed that male tiger No.048 had a relatively high propagation competence while some other males had fathered relatively few progeny. In this study, we obtained the genome DNA from hairs using the non-invasive DNA technology. We demonstrated that DNA derived from hair samples is as good as that obtained from blood for the analysis of microsatellite polymorphism. These results imply that microsatellite DNA markers and non-invasive DNA technology can help establish the detailed pedigrees required for the captive husbandry of the Amur tiger. We suggest that this method could be useful in the captive management of other endangered species[Acta Zoologica Sinica 49(1):118-123,2003].