Evaluation of a rapid single multiplex microsatellite-based assay for use in forensic genetic investigations in dogs

OBJECTIVE: To develop a set of microsatellite markers, composed of a minimal number of these markers, suitable for use in forensic genetic investigations in dogs. SAMPLE POPULATION: Blood, tissue, or buccal epithelial cells from 364 dogs of 85 breeds and mixed breeds and 19 animals from related species in the family Canidae. PROCEDURE: 61 tetranucleotide microsatellite markers were characterized on the basis of number and size of alleles, ease of genotyping, chromosomal location, and ability to be coamplified. The range in allele size, number of alleles, total heterozygosity, and fixation index for each marker were determined by use of genotype data from 383 dogs and related species. Polymorphism information content was calculated for several breeds of dogs. RESULTS: 7 microsatellite markers could be coamplified. These markers were labeled with fluorescent dyes, multiplexed into a single reaction, and optimized for resolution in a commercial genetic analyzer. The multiplex set was used to identify sires for 2 mixed litters. The test was not species specific; genotype information collected for wolves, coyotes, jackals, New Guinea singing dogs, and an African wild dog could not distinguish between these species. CONCLUSIONS AND CLINICAL RELEVANCE: This set of 7 microsatellite markers is useful in forensic applications (ie, identification of dogs and determination of parentage) in closely related animals and is applicable to a wide range of species belonging to the family Canidae.