It is well known that MHC genes are vital components of both the innate and adaptive immune system. They present foreign peptides to T cells. Cloning and cDNA polymorphism of the MHC II B gene has been discussed . In the present study, partial sequences of the MHC class IIB gene in different families of half-smooth tongue sole were isolated, then molecular polymorphisms as well as the link between alleles and resistance/susceptibility to V. anguillarum were analyzed.
Among the 72 mutated regions in the complete sequence of MHC IIB exon2, 36 regions were multi-nucleotide co-mutations, which indicate inter-allelic recombination took place in these regions. Moreover, no deletion, insertion or stop codon was observed, indicating that all of these alleles were functional genes. The frequency ratio of substituted nucleotides per mutation region was not equally distributed, which suggests that different regions might have different impact.
The rate of non-synonymous substitutions to synonymous substitutions (dN/dS) in the PBR and non-PBR of MHC IIB exon2 of half-smooth tongue sole was studied (Table 3). The dN/dS ratio was higher in the PBR than non-PBR, which corresponds with the results reported in other species [43, 54–56]. The dN/dS ratio in exon2 was higher than 1. The location of the PBR sites in the MHC genes of fish was not yet defined, therefore PBR sites were identified using the model of Brown et al.  to define HLA-DRB, It was also in accordance with a previous application by Xu et al.  for half-smooth tongue sole. The 23 positions were used as PBR sites for in-depth study: 3, 5, 7, 25, 27, 29, 34, 35, 44, 53, 57, 58, 62, 65, 67, 71, 74, 77, 78, 82, 83, 85 and 86 (Figure 3).
It is possible that the PBR sites in fish do not exactly correspond to those in humans . In mammals, MHC polymorphisms are maintained over long periods of time by balanced selection or positive selection at the non-synonymous sites specifying the PBR of the MHC . The ratio between non-synonymous and synonymous substitutions in PBR sites of MHC IIB exon2 genes is greater than 1 (Table 3), as would be expected if the locus were evolving under a condition of balanced selection . The number of alleles per individual ranged from 1 to 5, which showed that at least three loci existed per individual, a result is in accordance with previous studies [22, 28, 40]. Polymorphism of the 88 alleles in the 160 individuals was higher in half-smooth tongue sole than in Atlantic salmon [57, 59] and cyprinid fish , and each family had 25-38 alleles. A few hypotheses have been put forward to interpret the abundant polymorphism of the MHC genes, including overdominant selection or heterozygous advantage , negative frequency-dependent selection [61, 62] and balanced selection . Pathogen-driven selection [26, 60] is reported to be contributing to MHC gene diversity through both frequency-dependent selection and heterozygote advantage (over-dominance) . In the present study, the high rate of dN/dS score and high levels of polymorphism which occurred in half-smooth tongue sole revealed that balanced selection is responsible for presence in the PBR domain of the MHC class IIB exon2 gene. This results in the high polymorphism levels in MHC IIB genes in half-smooth tongue sole. Due to the polymorphic nature of MHC genes, certain alleles/haplotypes may be associated with increased disease resistance. In the present study, the distinct distribution pattern of the alleles exhibited a relationship between MHC class IIB alleles and resistance/susceptibility to V. anguillarum in half-smooth tongue sole.
The Cyse-DBB*3301, Cyse-DBB*4701 and Cyse-DBB*6801 alleles which was found in three families, and the Cyse-DBB*5901 allele in two families, were markedly more frequent in HR families (13.75%, 11.25%, 11.25%, 8.75% respectively) than in LR families (1.25%, 1.25%, 1.25%, 1.25%, respectively). This suggests an association of the V. anguillarum disease resistance alleles in half-smooth tongue sole. The Cyse-DBB*6501, Cyse-DBB*4002 and Cyse-DBB*5601 alleles were found in two LR families (35%, 33.75% and 16.25% respectively) and one HR family (1.25%, 1.25% and 1.25%, respectively), while the Cyse-DBB*6102 allele was found in three LR families (27.5%) and one HR family (1.25%), Cyse-DBB*2801 was found in two LR families (15%) and two HR families (2.5%), which might be associated with susceptibility to V. anguillarum in half-smooth tongue sole. In the present study, statistical analysis was used to reveal the associations between the alleles and resistance or susceptibility to V. anguillarum in half-smooth tongue sole. The observed link between alleles Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801, Cyse-DBB*5901, Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 and resistance/susceptibility to V. anguillarum supported the hypothesis that frequency-dependent selection is crucial for the maintenance of MHC variation . This experimental result was in accord with reports in Atlantic salmon  and flounder . However, it was not possible to identify a single allele which appeared in all HR families or all LR families. This might indicate the importance of multiple polymorphisms. One MHC haplotype has been reported to be significantly associated with resistance to Marek's disease in chickens , and MHC polymorphism was significantly associated with both juvenile survival and resistance to nematode parasites was also reported in Soay sheep .
A link between MHC polymorphism and resistance/susceptibility to disease in fish has been reported. Kjøglum et al.  demonstrated that fish with the genotypes UBA*0201/UBA*030 and DAA*0201/*0201 were the most resistant to infectious anaemia in Atlantic salmon, while fish with the genotypes UBA*0601/*080, DAA*0501/*0501 and UBA*0201/*030, DAA*0301/*0501 were the most susceptible, based on an analysis of the combined MHC class I and class II A genotypes. It is reported  that the allele combinations DAA*0201-*0201 and DAA*0301-*0301 displayed a significantly lower prevalence of death in homozygous fish than in Atlantic salmon containing one copy or no copy of the allele in Aeromonas salmonicida-challenged Atlantic salmon.
The Sasa-DAA-3'UTR 239 allele  was shown to be significantly associated with a decrease in the severity of amoebic gill disease in Atlantic salmon. It was also reported  that Sasa-B-04, at the non-classical class I locus, was highly associated with resistance to infectious hematopoietic necrosis in Atlantic salmon. The alleles Paol-DAB*4301 and Paol-DAB*1601 were shown to be associated with resistance and susceptibility to V. anguillarum in flounder .
In this study in half-smooth tongue sole, the alleles Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901 were found to be associated with resistance while the Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 alleles were associated with susceptibility to V. anguillarum. Associations of MHC with resistance or susceptibility to specific pathogens can also be derived through linkage disequilibrium with a resistance or susceptibility locus or gene, and may not be due to the MHC gene itself [55, 67–69].