Abstract: Abstract:The Tibetan fox (Vulpes ferrilata)has been identified as the main wildlife host of Echinococcus multilocularis and E. shiquicus in the eastern Tibetan plateau in China. Echinococcosis is a lethal parasitic zoonosis caused by Echinococ- cus spp. endemic to the pastures of the eastern Tibetan plateau. Thus,Echinococcus prevalence in Tibetan fox populations is of interest for studies into this disease. Consequently,there is practical significance to evaluate the population size of Tibetan foxes. Therefore we developed noninvasive microsatellite DNA identity analysis techniques using Tibetan fox feces. A total of 48 microsatellite loci were tested for effectiveness,among which 11 were selected to analyze identities of 128 qualified Tibetan fox fecal samples collected in field during July-August,2011 and 2012 (i. e. ,68 in 2011,and 60 in 2012). The number of genotypes (N),expected heterozygosity (He ),observed heterozygosity (Ho ),polymorphism information content (PIC),and probability of identity (PI)were calculated by allelic frequency. N ranged from 4 to 7,He was 0.66-0. 80,Ho was 0.17 -0. 68,and PIC was 0. 5496 - 0.7623. The overall copro-DNA PI values of the 11 loci were low(PIbiased = 1.283 × 10 ^{- 11} ;PIsibs = 7.572 ×10 ^{- 5} ). However,the amplification success rate of each microsatellite locus was quite different ranging from 0. 926 to 0.176. We then sorted the loci according to their amplification success rates from the highest to the lowest and found PI values of the first six loci with amplification success rates above 60% (i. e. ,P03,
CXX172,CPH6,CPH8,P01i,P08)were already low enough (PIbiase d = 2.775 ×10 ^{- 7} ;PIsibs = 3. 606 × 10 ^{- 3} )for individual identification. Therefore the individual identification standards were devised as follows:(1)only copro-DNA samples with successful amplification from at least the first six microsatellite loci were used for further analysis;(2)when all alleles were identical between two samples,the samples were considered to originate from the same individual;(3)in a conservative approach,if just one allele mismatch was observed between two samples,they were also judged to originate from the same individual. We then identified 30 fox individuals from fecal samples in 2011,and 21 individuals from fecal samples in 2012.

Key words: Fecal DNA, Individual identification, Microsatellite DNA, Tibetan fox

摘要： 藏狐是我国青藏高原东部多房棘球绦虫和石渠棘球绦虫最主要的野生动物终末宿主。棘球绦虫会导致一类称为棘球绦虫病的致死性人兽共患疾病,青藏高原东部牧区是该病重要的流行区。因此作为终末宿主,评估藏狐种群的棘球绦虫感染率对于该病的流行病学研究意义明显。而要获取这方面信息,首先必须了解藏狐的种群数量。为此,我们基于非损伤取样的原则,使用藏狐新鲜粪便作为研究材料,从已发布的藏狐及近缘种的48个微卫星位点中筛选了11 个用于藏狐粪便DNA 多态性分析。对2011 -2012 年7 -8 月间收集的128 份有效藏狐粪便样品(2011 年68 份,2012 年60 份)进行特异性PCR 扩增,并用琼脂糖凝胶电泳和荧光引物标记法进行基因分型,根据各位点的等位基因频率计算出各位点的基因型数(N),期望杂合度(He )、观测杂合度(Ho)、多态信息含量(PIC)以及不同个体基因型相同概率值(PI)。结果发现,各位点N 介于4 - 7,H e为0.66 - 0. 80,H o为0.17 -0.68,PIC 为0.5496 - 0.7623。11 个位点的累积PI 值满足个体识别的需要(PIbiased = 1. 283 × 10 ^{- 11} ;PIsi bs =7.572 × 10 ^{- 5} )。但是,由于粪便DNA 质量差异较大,不同位点的扩增成功率差异较大(0.176 - 0. 926)。我们发现,按照扩增成功率由高到低排列,前6 个微卫星位点(P03,CXX172,CPH6,CPH8,P01i,P08)的扩增成功率均超过0.6,且累积PI 值小于0.004 (PIbiased =2.775 × 10 ^{- 7} ;PIsibs = 3. 606 ×10 ^{- 3} ),表明这6 个位点可以对藏狐进行个体识别。因此,针对本研究的数据,制定了如下的个体识别原则: (1)只有粪便DNA 至少成功扩增出前6 个微卫星位点的样品可以进入下一步分析; (2)所有位点的信息均相同的两个样品被认为是来自同一个体;(3)保险起见,如果仅有一对位点信息不相等,此两个样品依然被判定来自同一个体。在此基础上,我们从2011 年样品中识别出30 个藏狐个体,从2012 年样品中识别出21 个个体。

关键词: 藏狐, 微卫星DNA, 个体识别, 粪便DNA