Studies On The Major Histocompatibility Complex Class Ⅱ Of Giant Panda (Ailuropoda Melanoleuca)

Giant Panda (Ailuropoda melanoleuca) is an endangered species that is endemic to China. Currently, the giant pandas are restricted to the isolated Qinling Mountain region of Shaanxi Province and the Minshan, Qionglai, Xiangling and Liangshan Mountains of Sichuan Province on the edge of the Tibetan plateau (Schaller et al. 1985;MacKinnon et al. 1989;O'Brien et al. 1994) . The historical separation between the Qinling and other populations has yielded a new Qinling subspecies (Ailuropoda m. qinlingensis) from the nominate Sichuan subspecies (Ailuropoda m. melanoleuca) (Wan et al. 2003;Wan et al. 2005).A captive breeding program was initiated in the 1980s, and now there 183 giant pandas in the captive program. The giant panda breeding bases in Ya'an-Wolong and Chengdu now contain large captive populations of the Sichuan subspecies, comprising 143 individuals, respectively (Xie & Gipps, 2004).In this study, we collected samples from 63 individual. The samples covered the both subspecies of Giant Panda and both the wild and the captive populations. We selected major histocompatibility complex (MHC) as genetic makers, and did comprehensively research on genetic diversity of this species with functional loci. We also developed a reliable method for isolating DNA from giant panda feces, as this would allow us to analyze a larger, fecal-based sample size. Our major results were:1. We developed a reliable and efficient method for isolating and analysis DNA from giant panda feces. This series of method include the collecting, conserving, extracting and analysis of feces of giant panda. Construction of this method would be benefit to future research without intrusion on giant panda and other rare and cryptic species.2. We found 7 alleles on locus DRB and named them as Aime-DRB*\ ^ Aime-DR&a , Aime-DR&t-Z , Aime-DRB*A ^ Aime-DRB*b > Aime-DRB*6 and Aime-DRB*1. The Qinling subspecies presented five alleles (Aime-DRB*\, Aime-DR&e-A^ Aime-DRB*5, Aime-DRB*G and Aime-DRB*!), in which alleles Aime-DRB*5 ^ Aime-DRB*Q and Aime-DRBrt are private. The Sichuan subspecies presented four alleles {Aime-DRB*\ â– , Aime-DRB*2^ Aime-DRBQ and Aime-DRB*4), in which alleles Aime-DR&e-i and Aime-DRB*Z are private. This result gives further proof to the subspeciation.3. We found 6 alleles on locus DQA and named them as Aime-DQA*\, Aime-DQA*2, Aime-DQA*3, Aime-DQA*4, Aime-DQA*5 and Aime-DQA*6. The Qinling subspecies presented all the six alleles while Sichuan subspecies presented five of them (Aime-DQA*\ , Aime-DQA*2 ^ ^/me-Z>^4*3 x Aime-DQAH and ,4/me-Z)ï¿¡M*5). In Sichuan subspecies, Chengdu population present five alleles (Aime-DQA*\, Aime-DQA*2, Aime-DQA*3 , Aime-DQA*4 and Aime-DQA*5), while Ya'an-wolong population presented four of them (Aime-DQA*\ ,Aime-DQA*2,Aime-DQA*4 and Aime-DQA *5) only.4. Diversity of giant panda's MHC gene was evaluated with respect to the number of alleles as well as the number of substituteions between alleles. The giant panda DRB and DQA diversity is minute compared with that of non-bottlenecked vertebrates and at the same level with other bottled vertebrates.5. In contrast to the theoretical expectations in large and outbreed populations, we observed lower than neutral levels of heterozygosity in all three giant panda populations (Chengdu, Ya'an-wolong and foping-louguangtai). All the findings suggest that inbreeding might account for lower observed heterzyogosity. Ewens-Watterson test revealed that the captive Ya'an-Wolong population exhibited a more even distribution of alleles than predicted by neutral theory, as reflected in the wild Foping-Louguantai population. Despite more captive-born pandassampled, the captive Chengdu population indicated that the distribution of its four alleles was consistent with the neutral expectation.6. We found three types of evidence for balancing selection for the giant panda alleles. First, we found an obvious excess of nonsynonymous substitutions over synonymous at the ABS positions, whereas a lower estimate of nonsynonymous ones than synonymous was present in the non-ABS positions. Second, transspecies evolution of the alleles Aime-6 and Aime-7 was revealed between the giant panda and other feline species. Third, we found a more even distribution of alleles than expected from neutrality.7. A neighbor-joining (NJ) tree was constructed together with sequence data from other carnivores. Results revealed that the Aime-DR&*6 and Aime-DR&n alleles showed higher ds values against other panda alleles that against cat MHC alleles, strongly supporting a trans-species evolution of the DRB loci under study, i.e. the origin of the alleles Aime-6 and Aime-7 predated the phylogenetic split between the giant panda and other felids.8. RT-PCR from blood cDNA using the exon-located primerpair showed that the cDNA product did represent the MHC II DRB and DQA target locus, demonstrating that the MHC gene examined here is expressed loci.9. According to the results above, we provided some advices for the management of captive giant panda population including efforts undertaken to increase the abundance of less frequent alleles across all panda populations.