Genetic Diversity Of The MHC-DQB Gene In The Largha Seal (Phoca Largha) From Liaodong Bay

The largha seal Phoca largha distributing in Liaodong Bay is the only pinniped that breeds in coastal areas of China. It has been listed in Order II under the Wild Animal Protection Law in China. Liaodong Bay is the southern-most end of the eight breeding areas of the largha seals, where the population has few or no genetic exchanges with the ones from the other breeding areas. During to the pollution of coastal waters, loss of habitats, and poaching, the population in Liaodong Bay is estimated to be less than 1000 individuals. The previous studies mainly focus on its morphology, habits, distribution and artificial feeding and breeding, but the conservation is still undertaking without the information on genetic researches.The major histocompatibility complex (MHC) is one of the nuclear genes with high degree of diversity, and is significantly useful to clarify the mechanisms of mutation and adaption among populations. The level of genetic diversity can be effectively assessed by the mutation analysis of MHC gene. Therefore, the gene polymorphism of the second exon of MHC-DQB loci obtaining from twenty-five specimens, and the abilities and potential threats in resisting diseases and adapting environment change within this population, was studied.141bp nucleotide sequences were extracted from more than 270 clones of the twenty-five largha seals. From the sequences, twenty-one variable sites were found and twelve alleles were defined. The variability was 25.5% at the amino acid level. The nucleotide diversity was from 0.0071 to 0.1064 (average 0.0577), and the base difference was from lbp to 15bp (average 8bp). All these results showed a relatively high level of genetic diversity in this population. Fifty-six questionable sequences with thirteen different length were found, considered to be microsatellite loci caused by non-specific amplification.High rate of non-synonymous vs synonymous (dN/ds=2.07) substitution especially in the peptide-binding region (PBR) suggested strong positive selection or balancing selection for maintaining high MHC diversity at the DQB loci. Most physical and chemical properties of the new amino acids translated after nucleotide substitutions were different from those of the original ones, which was interpreted as being relative with identifying and presenting different antigens.Phylogenetic dendrogram and TCS network indicated trans-species evolution in the pinniped MHC gene, which was also supported by the ds-value analysis. The imbalanced distribution of DQB alleles and the disproportion of the rare alleles suggested the genetic bottleneck effect in the history.Eleven individuals holding more than two alleles suggested duplication of DQB gene. The second exon of DQB contained two GC-rich regions (GCi and GCii) and one X-like sequence, showing a pure signal of gene recombination. Based on the analyses from the phylogenetic dendrogram, median-joining network, TCS network, and genetic structure analysis, it concluded that the fifty-nine alleles of the largha seals may be from two DQB loci and one DRB locus. High level of genetic diversity in every locus was proposed from the parameters of haplotype diversity, nucleotide diversity and average number of nucleotide differences, as well as from comparisons with other pinniped's parameters, viz. pairwise distance, number of alleles in each individual, mutations of the nucleotide and amino acid. The results of Fu'Fs and Tajima'D test, and the comparison between rates of non-synonymous and synonymous substitutions, showed positive selection on DQB, which supported Darwin's theory of natural selection to some extent.Gene duplication and genetic diversity per locus work together to make the MHC of the largha seal showing polymorphism, which could adapt sufficiently to the changeable pathogen environments. However, the imbalanced distribution of alleles and the possibility of losing rare alleles in genetic drift would further lead to the loss of the diversity among the populations of the largha seal and the abilities to resist different pathogens in the future. Therefore, the conservation of largha seals should not only focus on their living environments, but also their genetic diversity.