Investigation Of Genetic Variability Of MHC B-LB Ⅱ,B-G Genes And Their Genetic Structure In Some Chinese Indigenous Chicken Populations

The chicken major histocompatibility complex (B complex) is comprised of three classes of tightly linked, highly polymorphic loci, B-F(class Ⅰ), B-L(class Ⅱ), B-G(class Ⅳ), which is located at chromosome 16. The histocompatibility antigens encoded by the genes have been shown to determine the histocompatibility in chicken. Not only has MHC been reported to closely associate with initiation of immune response and adjustment of immunological function, but it also play a very important role in control the main economic traits of chicken. It will become the focus to investigate MHC by the method of molecular biotechnology combining with immune genetics and population genetics, particularly in the field of disease resistance breeding of poultry. The objective of this thesis is to study the genetic variation of MHC B-G and B-LB Ⅱ genes and their genetic structure of Chinese indigenous chicken populations with the methods of bioinformatics, population genetics and molecular biotechnology. And the association of the genes with some economic traits is also studied. The main results are listed as follows:1. The full-length cDNA of MHC B-G gene was obtained using the techniques of RT-PCR and RACE. The antigenicity value of the deduced amino acid sequences of B-G antigen molecule encoded by B-G gene was calculated preliminarily.2. Alignment of the nucleotide sequences of exon 2 of B-G gene revealed a total of 37 variable sites, of which 29 sites was parsimony informative and 8 sites singleton variable in the eight Chinese chickens sampled. The variation index for per variable site was calculated as the entropy of 0.206-1.462. To investigate the pattern of nucleotide substitutions, the relative frequencies of non-synonymous substitutions(dN) and synonymous substitutions(dS) per variable site in the region were estimated based on the method of Nei and Gojobori as 9.26+ 1.92% and 2.34±0.90%, respectively.3. 31 novel B-G alleles that belong to 10 allelic major types were found in the Chinese breeds sampled, the new alleles were all nominated on the basis of the rules reported in Homo sapiens and mammals. The allelic sequences were submitted to the NCBI GenBank and assigned the accession numbers of AY744339-AY744348, AY792330-AY792338, AY792340 and AY792342-AY792352. The distribution of alleles in the breeds sampled appeared to be notably nonsymmetrical and the phylogenetic analysis between the alleles was carried out.4. In this study, extensive variation of chicken B-LB Ⅱ gene was detected in eight Chinese indigenous breeds sampled. Alignment of the nucleotide sequences of exon 2 of B-LB Ⅱ gene revealed a total of 68 polymorphic sites, of which 51 sites was parsimonyinformative and 17 sites singleton variable. The homology between the sequences was estimated as 90.6%-99.5%. The relative frequencies of non-synonymous substitutions of nucleotide (dJV) and the frequencies of synonymous substitutions per site (d5) in the region were estimated based on the method of Nei and Gojobori as 14.64% ±2.67% and 2.92%+ 0.94%, respectively. It provided reference data for investigation of evolutionary mechanism of the polymorphisms within B-LBII genes.5. Totally, 34 novel B-LB II alleles were identified in the Chinese breeds, the new alleles were all nominated on the basis of the rules utilized in Homo sapiens and mammals. The allelic sequences were submitted to the GenBank and assigned the accession numbers of AY744338, AY744349-AY744363, AY770594-AY770606 and AY7786309-AY7786313, and the phylogenetic relationship between the alleles was analyzed.6. Genetic variation of the amino acid sequences of the P 1 domain encoded by exon 2 of B-LB II gene, and sequences of B-G immunoglobulin Variable-region-like (Ig-V-like) domains encoded by exon 2 of B-G gene were studied. The profiles of antigenicity, hydrophilicity and hydrophobicity of the both domains were displayed primarily.7. It was firstly demonstrated that alignment of exon 2 sequences of B-LB II genes revealed 91.1-97.8% similarity to each other in the chickens sampled; and chickens shared 84.1-87.0% homology to Phasianus colchicus, 78.5-81.5% similarity to Coturnix japonica. The sequences in poultry showed 62.6-68.1% identity to HLA-DRB1, 50-61.5% similarity to DQB (HLA-, SLA- and H2-BB), 53.7-60% to HLA-DPB and 53.3-57.8% similarity to HLA-DOB. The frequency of non-synonymous substitutions of nucleotide was higher than that of synonymous substitutions; and the frequencies of non-synonymous and synonymous substitutions in poultry B-LB II genes were lower than those observed in mammalian DRB1 and DQBl genes. The deduced amino acid sequences of MHC class II 61 domain exhibited extremely different conserved region and variable region patterns among the various species, but the two conserved cysteins forming disulfide-bond were shown identical in poultry with in mammalian species; and the carbohydrate attachment site was found more conserved in chicken, Homo sapiens, Bos taurus, Ovis aries and Capra hircus than in Sus scrofa and rodent animals. Compared with exon 2 of DQBl genes of Homo sapiens, ruminant species and Sus scrofa, the differentia that the deletion of six nucleotides at positionl95 to 200 of exon 2 of DQBl genes, and insertion of three nucleotides at position247 to 249 of the exon 2 existed in rodent species were found, which led to the absence of three AA residues at position 65, 66, and 67 within 61 domain of DQBl chain, and the insertion of one AA residue at position 85. The difference of the deletion of six nucleotides at position 72 to 77 of exon 2 ofDPBl geneswas observed from Homo sapiens DQB1, which caused absence of three AA residues at position 24, 25, and 26 of Bl domain ofDPBl chain. The phylogenetic tree revealed that the B-LB II sequences from poultry are not orthologous to the class II MHC 3 -chain genes of mammalian species. The tree indicated that genetic evolutionary relationship between chickens with Phasianus colchicus was much closer than with Coturnix japonica; and the DQB and DPB clusters are more tightly related to each other than to the remaining clusters.8. Six PCR-RFLP polymorphic sites of Alu I ,Cai\ , Cfr I , Hinl I , Hint I and Rsa I within exon 2 of B-LB II gene, two PCR-RFLP sites of Mspl and Tasl within exon 2 of B-G gene and one BsuRl-RFLP site in intron 1 of B-G gene were found, and their frequencies of the genotypes and alleles at the sites were estimated and compared between the different populations, respectively.9. The study of association of the genotypes at Cfr I -RFLP and Cai I -RFLP sites of B-LB II gene with laying performance in Tibetan hens has shown that hens with genotype AB at Cfr I site had significantly more laying numbers at the five laying phases of 90, 120, 150, 180 and 210 days after laying started than those with genotype AA (p<0.05); and the layers with genotype AA at the Cai I site had significantly greater average egg weight than those with genotype BB (p<0.05). It was also found that the layers with genotype AB at the BsuR I -RFLP site in intron 2 of B-G gene had significantly greater average egg laying number within 160 days after laying started than those with genotype BB (p<0.05).10. The genetic structure of populations of eight local Chinese breeds and one exotic breed was studied with the eight PCR-RFLP markers. It revealed that the average number of effective allele in the nine populations was 1.564 to 1.955; PIC (polymorphism information content) value was 0.372-0.461; The average (observed) heterozygosity of Tibetan chicken was 0.514, the highest one in the eight Chinese breeds analyzed, and that of Langshan chicken was the lowest (0.209); the coefficient of gene differentiation (Gst) at BsuR I site was the smallest (0.1152), and that at Rsa I site was the greatest (0.5375). The study has shown the genetic variability of the allelic frequencies between the nine populations sampled was estimated as 27.01%, and the 72.99% of genetic variability belonging to the heterozygosity within population. The value of gene flow (Nm) was computed, 1.3512>1; and the coancestry coefficients corresponding to the Nm value was 0.1595. The standard genetic distance Ds and genetic distance Da using Nei method were determined, and the distances have been employed to construct the UPGMA trees.