Development Of A Microsatellite-based Technique For Individualization Of The Asiatic Black Bear

Widespread poaching and illegal trading are constant threats to Asiatic black bear (Selenarcios thibetanus or Ursus thibetanus) populations. In attempts to control the illegal trade of bear parts and products, individualization of bears involved in poaching and smuggling are important issues for determining a penalty, an individualization typing method is therefore needed. In this study, ten tetranucleotide microsatellites, designated UamA107, UamBl, UamD2, UamD11, UamD103, UamD112, UamD113, UamD116, UT4 and UT38, were selected. A wild population containing 36 individuals from the Russian Far East and Northeastern China and a farm population containing 60 bears from farms in southern China were used to characterize and validate these loci. They were labeled with different fluorescent dyes and divided into four sets for capillary electrophoresis. The PCR system and reaction conditions were optimized. Extensive sequence analysis of cloned homozygote and heterozygote alleles was performed for each locus to authenticate the allelic designation and determine the structural conformation, and 128 alleles were successfully cloned and sequenced. The results revealed that different repeat blocks, incomplete repeat units or insertions were widely present among these ten loci. Four loci (UamA107; UamD2, UamD103, UamD113) were relative simple STR having a single type of alleles each. Other five loci (UamB1, UT4, UT38, UamD11, UamD112) were much more complex STR with two to three main types of alleles. UamD116 was too complex to genotype correctly, and was discarded. The inaccurate genotyping including stutter, null alleles and allele dropout were not significant in remaining 9 microsatellites. All alleles of these 9 loci were named according to the nomenclature principles recommended by International Society of Forensic Genetics. Nine standard allelic ladders for Asiatic Black Bear were successfully constructed. Using allelic ladders as standard size markers is a reliable and precise method of allele designation in test samples, and would prevent nomenclature problems when different population studies are carried out by different labs. Thus, with these nine allelic ladders, our data could be shared more effectively and would be better used in forensic cases or population studies. Population genetic parameters including allelic frequencies, genotype frequencies, observed heterozygosities (Ho), expected heterozygosities (He), observed number of alleles (Na), effective number of alleles (Ne), polymorphic information content (PIC), discrimination power (DP) and cumulative discrimination power (CDP) were calculated for the two populations. Results indicated all loci were highly polymorphic. The 9 microsatellites had a CDP as high as 0.999999999326 and 0.999999999998405 in the wild and farm populations respectively. The probability of identity between sibs (P(ID)sib) was 0.00003 for wild populations and 0.00001 for farm population and met the criteria of PID (0.001-0.0001) for most law enforcement forensic applications in populations with related individuals or substrucuture. The average probability of identity (Plave) estimated for sib proportions of 0,0.5 and 1.0 were respectively 3.029×10-10, 3.481×10-6 and 2.552×10-4 in the wild population, and 1.287×10-12,5.336×10-7 and 7.856×10-5 in farm population. A blind test of wild bears showed the probability of identity (P(ID)) of each sample ranged between 1.164×10-11 and 3.302×10-9. The allele nomenclature of the microsatellite panel and population data presented here facilitates the assembly of an open standardized database for forensic identification of the Asiatic black bear. Sensitivity of all loci was tested using stepwise dilutions of DNA template isolated from a muscle sample to concentrations of 10ng/μl,5ng/ul,2ng/μl, 1ng/μl,0.5ng/μl,0.1ng/μl and 0.01ng/μl. The minimum template quantity to produce correct genotype results was 0.1ng in 10μl reaction system for all loci. Specificity was tested using DNA template of 13 different species including carnivore species, ungulate species and humans (Homo sapiens). All loci were able to generate strong and definite PCR products in the brown bear (Ursus arctos) and Malayan bear (Helarctos malayanus), indicating they are potentially applicable to these species after validation. For other species, they presented good specificity in general. The sensitivity and specificity of all nine markers meet the requirements of forensic application.