SNPs Screening And Identification Of MHC ?A Gene In Blunt Snout Bream(Megalobrama Amblycephala) And Their Association With Disease Resistance Trait

Blunt snout bream(Megalobrama amblycephala)is one of the major freshwater fish cultured in China.In recent years,epizootic outbreaks of fish diseases are increasingly common as a result of intensive aquaculture,fish farming and bad environment.Among them,the most serious disease is bacterial septicemia,caused by the infection of Aeromonas hydrophila.The disease can cause a large number of fish deaths.At present,there is no quick and effective way to prevent and treat it.In this study,blunt snout bream were infected with Aeromonas hydrophila to distinguish susceptible and disease resistant individuals.PCR and sequencing were used to screen single nucleotide polymorphisms(SNP)in the blunt snout bream MHC ?? gene.High-resolution melting and Restriction enzyme digestion were used for SNP genotyping to analyze the association between gene polymorphism and disease resistance.A total of 35 SNP sites(1.45% of total nucleotide)were screened out of the MHC ?? gene,including 30 transition sites and 5 transition sites.In these 35 SNPs,1 was found to be located in the 5'-untranslated region(UTR),16 in exon,7 in intron,and 11 in 3' UTR.Coding sequence region contained 13 SNPs(5.56% of total amino acid),in which,TA transversion located at 839 bp and AG transition located at 1663 bp were nonsense mutations,and the other 11 SNPs were sense mutations.There were 4 variable sites in the 21 Peptide Binding Region(PBR),with the mutation rate of 19.05%,however,the mutation rate of non-PBR was just 8.20%(5/61).The results confirmed that the blunt snout bream MHC ?? gene was rich in polymorphic sites,had high specificity and had a certain impact on the structure and function of protein.The percentage of SNPs of nucleotide and coding region in ?1 domains were 4.88% and 10.98%,respectively,which were significantly higher than that in ?2 domain(1.08% and 3.22%).This was due to the high polymorphism of the antigenic peptide binding region in the ?1 domains,which was confirmed that the PBR locus existed in ?1 domains.It indicated that the polymorphism of antigen binding site region was higher than that of non antigen binding site,and it also showed that amino acid substitution and substitution appeared more in polypeptide binding sites.In the phylogenetic tree constructed by Neighbor-joining method,the blunt snout bream MHC ?? gene and teleosts were clustered into one clade,it confirmed that the teleost fish were monophyletic.In addition,teleost fish were divided into two branches,Bream and other cyprinid species clustered into a branch,salmon fish clustered into another clade,they formed their independent evolutionary monophyletic groups.It was revealed in the Neighbor-joining tree that the blunt snout bream had the closest relationship with grass carp,far from zebra fish and cyprinus carpio var.jian,still farther from salmo salar and rainbow trout,farthest from other vertebrates and mammals.The genotype and allele frequencies of the 5 successfully genotyped SNP sites in the 100 susceptible and 100 disease resistant individuals were statistically different.Chi-square tests suggested that the differences of genotype and allele frequencies of SNP(T/A)located at 1395 bp were extremely significant(P<0.01),SNPs located in 221 bp(G/T)and 1859 bp(G/T)were significant different between susceptible and resistant groups(P<0.05).Mutation in the third exon located in 1395bp(T/A)led to the conversion of aspartate to glutamic acid,which caused the change of protein structure and function,and changed the resistance of fish.Mutations in the 5'non coding region located in 221bp(G/T)and in the non coding region of 3' located in 1859bp(G/T)did not result in amino acid alterations,but the experimental results showed association with disease resistance.The results showed that the MHC ?? gene polymorphism was significant correlated with bacterial septicemia resistance.Further analysis showed that the homozygous individuals had a higher resistance to bacterial septicemia caused by the infection of Aeromonas hydrophila.The SNP markers,corresponding advantages and disadvantages of genotypes obtained in this experiment can provide theoretical basis for the selection of parents when we work for resistance breeding of megalobrama amblycephala,it also lays the theoretical foundation for molecular genetic markers and resistance gene screening.