Identification And Adaptive Evolution Analysis Of Functional MHC Class Ⅱ Genes For The Giant Panda

The major histocompatibility complex(MHC) plays a central role in the adaptive immune system of vertebrates for its function of presenting pathogen peptides to T cells. The exon2 of MHC classⅡclasscical DR and DQ genes encodes antigen binding sites and displays extreme polymorphism,which is believed to be essential for vertebrates to resist pathogen infection.Thus this study aims to:1) isolate all functional classical MHC classⅡloci for the giant panda;2) investigate variations of antigen-binding exon2 of all functional classical MHC classⅡgenes in the giant panda;2) exploring the evolutionary history of immunity-related genes in two subspecies of the giant panda;and 3) reveal the maintaining mechanism of MHC diversity in the giant panda.The results are as follows:1) This study developed a novel magnetic bead hybridization method to isolate cDNA sequences of MHC genes.In the giant panda,a total of 10 full-length MHC cDNA sequences were isolated,nine of which were assigned to 6 known classical MHC classⅡgenes identified from the giant panda MHC genome project.Thus,the othe one DRB cDNA sequence represents a novel locus,which was designated DRB3. The results demonstrated the novel magnetic bead hybridization method is efficient and reliable and thus could be applied to other mammals.2) The complete coding regions in 9 out of the 10 full-length Aime-MHC cDNA sequences revealed no frame-shift mutations,stop codons or indel,which were in good agreement with functional genes.However,the DQA2 was truncated in both the leader peptide and the cytoplasmic tail as compared to Aime-DQA1 and homologs in other mammals,which implies loss of function or an evolving pseudogene.3) Genetic variations and evolutionary patterns of all 7 functional MHC classⅡgenes (1 DRA,2 DRB,2 DQA and 2 DQB) were investigated for 121 giant pandas,using locus-specific single-strand conformation polymorphism genotyping and sequencing techniques.The results revealed the presence of 2 monomorphic loci(DRA and DQB2) and 5 polymorphic loci with different numbers of alleles(7 at DRB1,6 at DRB3,7 at DQA1,4 at DQA2,6 at DQB1).Compared to other endangered animals and species experiencing severe bottlenecks,the giant panda exhibited ample variations in the adaptive immune systems.4) Despite sharing the identical alleles across all MHC loci,the Wolong and the Chengdu captive populations of the Sichuan(i.e.nominal) subspecies exhibited large differences in the distribution of allelic frequencies,reflecting respective founder effects.The main founder's alleles were distributed widely in the captive population, while rarer alleles not harbored in the founder may have drifted out.For sustainable development of the giant panda,it will be necessary to increase the mating success of giant pandas carrying rare alleles,in order to enhance pathogen resistance in captive populations.5) The two giant panda subspecies,the Sichuan(i.e.nominal) and the Qinling subspecies,exhibited substantial differences in allele distribution and allele richness. The former has more alleles in DQ genes than the latter(16:10),and by contrast,the latter has more alleles in DR genes than the former(11:9).In total,the Sichuan shaped 9 specific alleles,and the Qinling subspecies 5.These differences in allelic distribution and the presence of subspecies-specific alleles implies that the two giant panda subspecies have experienced different evolutionary histories since their division,and have evolved their own functional MHC molecules to resist different habitat-specific pathogens.6) Balancing selection in the giant panda was supported by the following pieces of evidence:(a) the observed heterozygosity was higher than expected.(b) Amino acid heterozygosity was significantly higher at antigen binding sites(ABS) compared to non-ABS sequences(P＜0.01).(c) The selection parameterω(d_N/d_S) was significantly higher at ABS compared to non-ABS sequences(P＜0.01).(d) Approximately 95.45%of the positively selected codons(P＞0.95) were located at or adjacent to an ABS.7) The Sichuan subspecies displayed lowωvalues at DRB1(ω＜0.72) and DQA2(ω＜0.48),suggesting that these sites underwent strong purifying selection.On the contrary,the Qinling subspecies exhibited highωvalues across each locus(all＞1), supporting its extensive positive selection.This contrasting pattern revealed that the Sichuan and the Qinling subspecies developed different evolution-driven mechanisms in the diversity patterns of MHC.8) In both Sichan and Qinling subspecies,the entire exon2 of the DRB3 gene underwent intense positive selection at both ABS(ω＞9.27) and non-ABS(ω＞3.49).For the DRA and DQB2,both subspecies exhibited monomorphism,showing strong purifying selection.On the other hand,intragenic recombination was detected in DRB1,DQA1 and DQB1,providing evidence of recombination serving as one important diversification mechanism.In sum,our present results suggest that balancing,positive and purifying selection accompanied by recombination drive the contrasting diversity patterns of the MHC classⅡgenes between 2 giant panda subspecies.