Adaptation Evolution And Genetic Diversity Of Wolves In Qinghai Region Based On MHC Class Ⅰ Molecular Markers

Wolves(Canis lupus) are one of the most adaptable terrestrial wild mammals to the environments. With the evolution to the current arrangement, it is the world’s most successful cluster hunting carnivores. The Wolf was once the most widely distributed of terrestrial wild mammals. Because the Wolf caused a great threat to human and animal husbandry of livestock, plus human love of canine animal fur, to make them by human hunting; And as the development of human society, economic development, expanding constantly narrowing or wolves habitat loss, making the number of wolves are declining, many area has nearly died out. In our country, the Wolf are mainly distributed in the sparsely populated Inner Mongolia, Qinghai, Xinjiang, Tibet and other regions.Major Histocompatibility Complex(MHC) is one of the most polymorphic functional genes extensively in the genome of vertebrates. It plays an important role in maintaining animals adapt to the changing environment due to its ability to encoding molecules those present pathogen petides to immune cells and trigger an immune response. MHC is applied to the study population disease-resistant ability and adaption to environmental stresses, which is also as an important part of adaptive immune system. And MHC classⅠhas its own unique evolutionary history and polymorphism maintaining mechanism, making it a evaluation wildlife population genetic structure, environment, an important index of potential ability to adapt and disease resistance. But wolf in China has never been conducted studies of MHC classⅠ, even in the world about MHC classⅠas the kind of molecular markers of Wolf is blank. This study as the first time involving Canis lupus filchneri sample has carried on the exploration of MHC class Ⅰmolecular markers. Genetic variation analysis reveals diversity and evolutionary mechanisms of Canidae of the four structural integrity MHC classⅠgenes. The following are the conclusions we obtained from this study:(1) The PCR and cloning technology has been amplify the sequence of DLA-88、DLA-12、DLA-79 and DLA-64(Dog Leukocyte Antigen) genes. We have stitched the fragments of exon2 and exon3 to get the integration of DLA-88 alleles whose length is 549 bp. By comparing the 37 unique sequence, our analysis identified 24 DLA-88 haplotypes which is different from 71 known DLA-88 haploid types of domestic dogs. In the same way, we also identified 16 DLA-12 alleles. Among 12 alleles are only found in wolves, while the other four found in wolves are shared with domestic dogs. We identified 16 DLA-64 alleles which is only found in wolves. At last we identified DLA-12 alleles which is share between wolves and domestic dogs.(2) Our study found that the base composition of canine MHC class Ⅰgenes is very special. The protein coding sequence GC content is very rich. The GC content of DLA-88 exon2 and exon3 68.6% is higher than AT content 31.4%. The first position of codons of GC content is 63.8%. The second position of codons of GC content is 50.8%. The third position of codons of GC content is 91.4%. The GC content of DLA-12 exon2 and exon3 68.7% is higher than AT content 31.4%. The first position of codons of GC content is 63.9%. The second position of codons of GC content is 51.0%. The third position of codons of GC content is 91.2%. The GC content of DLA-79 exon2 and exon3 64% is higher than AT content 36%. The first position of codons of GC content is 62.9%. The second position of codons of GC content is 47.6%. The third position of codons of GC content is 81.5%. The GC content of DLA-64 exon2 and exon3 68.6% is higher than AT content 31.5%. The first position of codons of GC content is 65.6%. The second position of codons of GC content is 47.8%. The third position of codons of GC content is 92.3%.(3) Canine MHC class Ⅰgenes DLA-88 showed very high polymorphism. By comparing the Canis lupus filchneri DLA-88 exon2 and exon3 sequences, 78 variable sites were found(14.2%), including 19 single polymorphic loci and 59 parsimony informative sites. The overall nucleotide diversity(Pi) was 0.03370, and the overall haplotype diversity(Hd) was 0.937. Correspondingly, DLA-88 was found to be significantly more polymorphic than the other three genes what is called non-classical class Ⅰgenes DLA-12、DLA-79、DLA-64. It is in line with the previous definition of classical and non-classical genes.(4) By putting 24 Canis lupus filchneri DLA-88 new alleles and another six kinds of sequence known domestic dogs for population genetics analysis, we found the genetic distance relationship for DLA-88 was Basenji> Gold Retriever> Beagle> Boxer> Canis lupus filchneri> Harrier> Greyhound. The largest average genetic distance between them was Basenji and Greyhound. The smallest average genetic distance between them was Harrier and Greyhound. Canis lupus filchneri and Basenji genetic distance was the largest, next was Gold Retriever, and Canis lupus filchneri and Harrier genetic distance was the smallest.(5) Canis lupus filchneri and six kinds of domestic dogs DLA-88 exon2 and exon3 all sites non-synonymous(d N) and synonymous(d S) ratio was greater than 1, which means that all groups were under positive selection. Canis lupus filchneri peptide binding region(PBR) and non-PBR sites d N and d S ratio was greater than 1, indicating that these genes were under positive selection. All DLA-12 and DLA-79 genes sites d N and d S ratio was greater than 1, indicating that these genes were under positive selection. Canis lupus filchneri classical and non-classical genes suffered natural selection instead of neutral selection.(6) The constructions of canids all currently known DLA-88 alleles of maximum likelihood tree and bayesian tree were consistent. 24 different genotypes of wolves dispersed in evolutionary tree, indicating cross-species polymorphism.