Biodiversity Science ›› 2012, Vol. 20 ›› Issue (2): 184-192.

• Original Papers •

Polymorphism of exon 2 of MHC Class II B gene in the Chinese concaveeared torrent frog (Odorrana tormota)

Fang Li, Yilin Shu, Hailong Wu*

1. Key Laboratory for the Conservation and Utilization of Important Biological Resources, Anhui Province; College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000
• Received:2011-11-22 Revised:2012-01-31 Online:2012-04-09
• Hailong Wu E-mail:whlong@mail.ahnu.edu.cn

Currently, amphibians are experiencing global population decline, and it is believed that several amphibian mass extinction events were caused by environmental pathogens (such as chytrid fungus). Major histocompatibility complex (MHC) genes play a critical role in the course of immune response in all jawed vertebrates. MHC gene is considered to be one of the best candidates to analyze animal’s adaptive evolution because its polymorphisms are usually associated with resistance or susceptibility to animal diseases. Here, we report our preliminary research on the allelic diversity of MHC class II B gene from the Chinese concave-eared frog (Odorrana tormota), a species endemic to eastern China. We initially amplified a 180-bp fragment of MHC II exon2 gene in O. tormota using published polymerase chain reaction primers. Based on these results, we successfully obtained sequences of the gene’s flanking regions using a ligation-mediated PCR method. After splicing, we obtained a sequence with length of 2,030 bp including whole exon2 and partial sequences of intron1 and intron2. Then two exon2-specific primers (IIQ1BU/IIQ1BD) were designed for the species and were used to investigate the B gene diversity of a wild population (Huangshan Mt., n=32) using PCR, cloning and sequencing. In total, 34 distinct alleles were obtained and 2 to 5 alleles were found per individual. The proportion of variable sites for nucleotide and amino acid sequences across the 34 alleles was 16.17% (33/204) and 26.87% (18/67), respectively, and the majority of variable amino acids were located in antigen binding sites (ABS). Based on cDNA data and individual allelic diversity, we conclude that O. tormota possesses at least three class II B loci. These results showed that though the species exhibits a restricted distribution, the Chinese concave-eared frog displays high diversity at the B loci compared to that of other species in Ranidae. Patterns of nucleotide substitution exhibited the signature of positive selection. For example, in the ABS, dN was significantly greater than dS (P <0.05). This pattern was also echoed by results of a likelihood ratio test, and five amino acid sites under significant positive selection were detected by Bayesian analyses. Finally, topology of a Bayesian phylogenetic tree showed that alleles from different anuran families clustered into unique monophyletic groups, but that alleles from different genera within Ranidae did not form monophyletic groups. For example, an allele of Rana clamitans shared a common lineage with alleles of O. tormota. These results suggested that trans-species evolution may be a mechanism that maintains allelic polymorphism of the MHC II B gene across genera in the family Ranidae.