| Tilapia is an important fish species in freshwater aquaculture.But in recent years,the high mortality rate caused by the outbreak of streptococcal disease has caused great losses to the tilapia culture industry.In order to control the occurrence of disease,it is very important to screen molecular markers related to disease resistance and to cultivate disease resistant strains.In this paper,MHC genes were selected as the research objects.The relationship between MHC gene polymorphism and disease resistance and the polymorphism and genetic characteristics of MHC IIA in different populations were analyzed.The purpose is to lay a foundation for the screening of Nile tilapia resistance genes and disease-resistant breeding.The major results were as follws:1.MHC Class IIA polymorphism and disease-resistance to Streptococcus agalactiae of Nile Tilapia(Oreochromis niloticus)The association between major histocompatibility complex(MHC)class IIA polymorphism and the severity of infection by S.agalactiae was investigated using 40 susceptible and 40 resistant individuals of Nile tilapia.The results showed that the size of the amplified fragment was 647-775 bp.In total,25 alleles were identified from 80 individuals,which belong to 23 major allele types.High polymorphism of the second exon region of MHC IIA gene and at least two loci were discovered in Nile tilapia.In in PBR and non-PBR,the ratio of nonsynonymous(dn)substitutions to synonymous(ds)substitutions was 1.294(>1)and 1.240(>1),indicating that the second exon region of the MHC IIA gene are evolving under positive balancing selection.Association analysis showed that the allele Orni-DAA*0501 was significantly associated with resistance to S.agalactiae,while the alleles Orni-DAA*1101,Orni-DAA*1301,Orni-DAA*1401 and Orni-DAA*1201 were associated with susceptibility to S.agalactiae.To confirm these correlations,an another independent challenge experiment was performed in the Huizhou population of the Nile tilapia.The frequency distribution showed that the Orni-DAA*1101 allele was significantly more frequent in the Huizhou-Susceptible group(HZ-SG)than in the Huizhou-Resistant group(HZ-RG)(P<0.05),which was consistent with the first challenge.However,the Orni-DAA*0501 did not present in HZ-SG and HZ-RG,and the distribution frequencies of the Orni-DAA*1201,Orni-DAA*1301 and Orni-DAA*1401 alleles was not significantly more frequent in the HZ-SG than in the HZ-RG.These results indicate that the Orni-DAA*1101 allele confers susceptibility to S.agalactia infection.Therefore,the Orni-DAA*1101 alleles could be used as a disease susceptible-related MHC marker in the molecular marker-assisted selective breeding program of Nile tilapia.2.MHC Class ⅡB polymorphism and disease-resistance to Streptococcus agalactiae of Nile Tilapia(Oreochromis niloticus)One thousand and eight hundred fry offspring(F1)from twenty full-sib families were challenged with S.agalactiae,which causes significantly different mortality in Nile tilapia families.According to the disease resistance ability,12 families were selected for research.Twenty fry(P0 of parents)from each of the twelve families were selected to study the polymorphisms of MHC class ⅡB gene.The results showed that the size of the amplified fragment was 770-797 bp.1423 sequences from 240 individuals revealed 22 different alleles,which belong to 9 major allele types.Nucleotide variation loci of up to 63.58%,while the proportion of the amino acid mutation sites up to 68.73%.The rate of non-synonymous substitution(dn)was 1.4595 times higher than synonymous substitution(ds)in the partial β1 and β2 domain of whole families,respectively,which suggested balancing selection on the partial MHC Class ⅡB gene in Nile tilapia.According to the survival rate of offspring(F1)from twelve full-sib families,we deduced that the alleles Orni-DAB*0107,Orni-DAB*0201 and Orni-DAB*0302 were highly associated with resistance to S.agalactiae,while the allele Orni-DAB*0701 was associated with susceptibility to S.agalactiae.However,our previous study found that the allele Orni-DAB*0201 was associated with resistance to S.agalactia.Therefore,the allele Orni-DAB*0201 could be used as a disease resistance-related MHC marker in the molecular marker-assisted selective breeding program of Nile tilapia.3.Single nucleotide polymorphism of β2m gene and disease-resistance to Streptococcus agalactiae of Nile Tilapia(Oreochromis niloticus)β2-microglobulin plays important role in the immune system of fish as an essential subunit of MHC class I molecules.We identified 30 SNPs in the β2m gene by direct sequencing method from the parents(P0).The results showed that 22 SNPs located in the exon region(1 SNP located in 5’UTR,4 SNPs located in the 3’UTR,16 synonymous mutations,a synonymous mutation site)and 9 SNPs located in the intron region.Snapshot method was used to genotype 102 resistant and 102 susceptible individuals in the F1 generation.The genetic parameters such as He,Ho,Ne and PIC of SNPs of β2m gene in Nile tilapia were statistically analyzed by Popgen 32 and PIC-CALC software,which indicated that 7 SNPs in the susceptible group belonged to moderate polymorphism level(0.25<PIC<0.5),and 25 SNPs in the resistant group belonged to moderate polymorphism(0.25<PIC<0.5).The genotype and allele frequencies of 30 SNPs in two F1 populations were analyzed by SPSS 23.0 software,which revealed its correlation with streptococcal resistant or susceptible traits.These results illustrated that 24 SNPs were significantly associated with the resistant/susceptible against S.agalactiae(P<0.05).Linkage disequilibrium analysis showed that 30 SNPs formed 4 haplotype blocks and 14 haplotypes.And 4 of the haplotypes were significantly associated with susceptible to S.agalactiae,4 haplotypes were significantly associated with resistance to S.agalactiae(P<0.05).And the results also showed that 4 SNPs of the haplotype block 2 and 13 SNPs in the haplotype block 3 highly linkaged with each other(r2>0.9).So it meant that we identified two htSNPs inβ2m gene.In this study,the SNPs and haplotypes associated with streptococcal resistant or susceptible traits have the potential to support the breeding of Nile tilapia against streptococcal disease.4.MHC ⅡA gene polymorphism and genetic differentiation in three different populations of Nile tilapia(Oreochromis niloticus)In this study,501 valid clones of the exon 2 region of MHC ⅡA were obtained from the 91 individuals of Nile tilapia(Oreochromis niloticus)from three different farming areas in Guangdong province.The sequence of exon 2 was 647～742 bp in length.Sequence analysis showed that there are 12 alleles.The percentage of nucleotide variation among the three populations ranged from 34.73%to 60.78%,and the percentage of amino acid variation was 68.24%to 78.82%.Among them,Huizhou population had the highest percentage of variation(60.78%and 78.82%).The analysis of haplotype diversity and nucleotidediversity showed that the genetic diversity of Huizhou population was relatively abundant.The FST value,Tajima test,Nm value,AMOVA analysis and average Kimura 2-parameter distances revealed the second exon region of MHC IIA gene was not significantly differentiated among three different populations and there was some gene exchange.The high polymorphism and genetic analysis of MHC IIA gene found in this study provide important basic information for the disease resiatance breeding of Nile tilapia and the protection of germplasm resources. |
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