The Research On Polymorphism Of MHCⅡB Genes Of Alauda Arvensis And Genotyping

The genetic diversity of the major histocompatibility complex(MHC) has been paid increasing attention on the study of conservation biology. Researchers believed that MHC genes in the mammals and fish are related to disease resistance, mate choice and kin recognition. However, more and more studies showed that MHC genes worked as rich as mammals in birds. MHC genes could be expressed in a large number of birds, which quantity and structure has variability in vivo. However, the mechanism that how MHC genes affect the birds behavior is not clear. In this paper, the polymorphism of Alauda arvensis MHC class II B gene exon 2 was studied, which provided the reference data for the researchers in-depth exploration of birds.Ten blood samples of Alauda arvensis individuals collected in Inner Mongolia Autonomous Region were investigated for MHC class II B gene exon 2 polymorphism. Firstly, Alauda arvensis blood genome was extracted. The target genes were analysised by SSCP genotyping after PCR amplification. We got 15 genotypes which were named A to O。We found that the electrophoretic bands of SSCP were complex, so we selected a large number of positive clone sequencing in order to clarify the number of specific alleles. 197 sequences were successfully sequenced. The length was 213 bp, in which we got 55 unique sequences by BioEdit v7.0.5.3, ClustalX v2.0 editing and comparing sequences, in which 23 sequences had been confirmed(the same sequence at least appeared in two individuals). It proved that the Alauda arvensis MHC class II B gene exon 2 contains 6 loci at least according to the distribution of alleles.By the MEGA and the Dna SP software, we found that the average genetic distance between 55 unique sequences is 0.2252±0.0161, nucleotide diversity level was 0.22517 and that 130 nucleotide polymorphism sites were found in these sequences, moreover, neutral test(Tajima’s test) D=-0.72507, P > 0.10, it showed that there existed a non-neutral evolution. While the average genetic distance between the 23 confirmed sequences was 0.2219±0.0169, nucleotide diversity level is 0.22192 and 118 nucleotide polymorphic loci were found, neutral test(Tajima’s test) D=-1.06244, P > 0.10, it showed that there existed a non-neutral evolution. In addition, positive selection model M2 a and M8 were both detected 4 positive selected sites by CodeML in PAML14.4 program package(Bayesian posterior probability is higher than 99%).Sequencing results were consistent with the conclusions drawn from the SSCP analysis genotyping methods developed target gene polymorphism results, both of them proved that the MHC class II B genes exon 2 of Alauda arvensis has high polymorphism.