Limited polymorphism of the functional MHC class II B gene in the black-spotted frog (Pelophylax nigromaculatus) identified by locus-specific genotyping.

Amphibians can be more vulnerable to environmental changes and diseases than other species because of their complex life cycle and physiological requirements. Therefore, understanding the adaptation of amphibians to environmental changes is crucial for their conservation. Major histocompatibility complex (MHC) presents an excellent tool for the investigation of adaptive variations and the assessment of adaptive potential because it can be under strong diversifying selection. Here, we isolated the MHC class II B (MHCIIB) gene from cDNA sequences of the black-spotted frog (Pelophylax nigromaculatus), a widespread amphibian species in China, and designed locus-specific primers to characterize adaptive variability of this amphibian. Ten alleles were identified from 67 individual frogs of three populations and no more than two alleles were present in each individual animal. Furthermore, none of the sequences had indels or/and stop codons, which is in good agreement with locus-specific amplification of a functional gene. However, we found low polymorphism at both nucleotide and amino acid levels, even in the antigen-binding region. Purifying selection acting at this locus was supported by the findings that the dN/dS ratio across all alleles was below 1 and that negatively selected sites were detected by different methods. Allele frequency distributions were significantly different among geographic populations, indicating that physiographic factors may have strong effect on the genetic structure of the black-spotted frog. This study revealed limited polymorphism of three adjacent black-spotted frog populations at the functional MHCIIB locus, which may be attributed to region-specific differences. The locus-specific genotyping technique developed in this study would provide a foundation for future studies on adaptive divergence among different frog populations.