The screening of microsatellite DNA loci for the identification of individual Tibetan foxes, using copro-DNA samples

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 Echinococcus 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 (H_e), observed heterozygosity (H_o), polymorphism information content (PIC), and probability of identity (PI) were calculated by allelic frequency. N ranged from 4 to 7, H_e was 0.66-0.80, H_o was 0.17-0.68, and PIC was 0.5496-0.7623. The overall copro-DNA PI values of the 11 loci were low (PI_biased=1.283×10^-11; PI_sibs=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 (PI_biased=2.775×10^-7; PI_sibs=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.