Artificial breeding is an important project to protect, recover and reintroduce endangered species. Knowledge of the population’s genetic diversity at functional loci is important for the establishment of effective captive breeding programs. The major histocompatibility complex (MHC) genes are ideal candidate genetic markers to inform planned breeding, due to their high levels of polymorphism and importance in the main immune coding region of the vertebrate genome. In this study, we constructed BAC-based contigs and isolated six functional MHC class I genes from the giant panda (Ailuropoda melanoleuca), which we designated Aime-C, Aime-F, Aime-I, Aime-K, Aime-L and Aime-1906. Analyses of the tissue expression patterns and full-length cDNA sequences of these class I genes revealed that Aime-C, -F, -I and -L could be considered classical class I loci, due to their extensive expression patterns and normal exonic structures. In contrast, Aime-K and -1906 appeared to be nonclassical genes based on their tissue-specific expression patterns and the presence of an abnormal exon 7 in both genes. We established techniques for genotyping exons 2 and 3 of the classical loci using locus-specific single strand conformation polymorphism (SSCP) and sequence analysis. In the Chengdu captive population, we identified one monomorphic locus (Aime-F) and three polymorphic loci with different numbers of alleles (4/4/4 exon 2 alleles at Aime-C/I/L and 6/5/5 exon 3 alleles at Aime-C/I/L). The distributions of the Aime-C, -I and -L alleles among members of different families were in good agreement with the known pedigree relationships, suggesting that the genotyping results are reliable. Therefore, the MHC-I genotyping techniques established in this study may provide a powerful tool for the future design of scientific breeding or release/reintroduction programs.
giant panda MHC class I isolation locus-specific genotyping
1.The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, State Conservation Center for Gene Resources of Endangered Wildlife, College of Life SciencesZhejiang UniversityHangzhouChina