The Polymorphisms Of Candidate Genes And Their Association With Heat Tolerance Of Scallops

Scallop aquaculture is an important economic activity in China. However,successive large-scale mortality of cultured scallop in summer has caused seriousdamage to the healthy development of scallop aquaculture. High temperature isconsidered the main environmental factor that caused scallops mass mortalities insummer. To cultivate new scallop strains with enhanced resistance to high temperatureis now considered to be one of the basic and ultimate solutions to mass mortalitiescontrol in summer. Markes assiated selection (MAS) is the fastest method of scallopsbreeding with heat tolerance, and identifing markers associated with heat tolerance isthe first step for MAS. In this study, the Bay scallops (Argopecten irradians) fromtwo geography communities in Zhanjiang and Qinhuangdao were employed as heatresistant stock and heat susceptible stock, respectively. The nucleotide sequencepolymorphisms in AiHSP70,AiMT1,AiFer and AiTLR8genes from Bay scallopwere investigated to explore their associations with susceptibility or tolerance to hightemperature by by using the techniques including PCR, sequencing, PCR-RFLP andBi-PASA PCR. The contribution of the polymorphisms in the candidate genes to theheat-resisting phenotype of scallop was explored by RT-PCR, Western blot, promoterreporting construction and recombinant protein expression.The full length of the AiHSP70genomic DNA is5622bp, consisting of promoter,5extrons and4introns. Fifty seven single nucleotide polymorphisms (SNPs) and eightinsert/deletion (I/D) polymorphisms were identified in AiHSP70gene. Eleven of themlocated in the promoter region and their frequencies in the susceptible and resistantstocks were examined by sequencing. Statistical analysis revealed that the genotypic frequencies were in Hardy-Weinberg equilibrium (HWE) at loci-1248,-1108,-1107,-999,-894and-204, while not in HWE at loci-408and-28in both stocks. At loci-967,-480and-83, the genotypic frequencies were in HWE in susceptible stock,while not in HWE in resistant stock. Among them, the frequencies of eight genotypes(-1248AA,-1108AA,-1107TT,-999CC,-967GG,-480AC,-408TT and-83AG)in resistant stock were significantly higher than that in susceptible stock, indicating asignificant association with the heat tolerance of Bay scallop. In contrast, there wereno significant differences in the frequency of different genotypes at loci-894,-204and-28between these two stocks. Thirty two SNPs were found in the exons ofAiHSP70gene, and thirteen of them were non-synonymous mutations. Three SNPs,+406A/T,+3734A/C and+3806G/T, were selected to analyze their distributions insusceptible and resistant stocks. Statistical analysis revealed that the genotypicfrequency of alleles at locus+3734was in HWE in both stocks, while not at loci+406and+3806. The frequency of genotypes+406TT,+3734AC and+3806TT inresistant stock was significantly higher than that in susceptible stock, indicating theirsignificant association with the heat tolerance of Bay scallop. Linkage disequilibriumwas found between the multiple loci of AiHSP70gene, and forty one haplotypes withfrequency above0.01were identified. The frequency of G-C-A-A-C-T-A-A-C-A-Ahaplotype in resistant stock was significantly higher than that in susceptible stock,indicating its significant association with the heat tolerance of Bay scallop.Total sixteen sites of SNPs and one site of I/D polymorphism were identified in thepromoter region of AiMT1gene. Two of them,-375T/C SNP and-337A/C SNP wereselected to analyze their distributions in susceptible and resistant stocks. Statisticalanalysis revealed that the genotypic frequency of alleles at locus-375were in HWE inboth stocks. The genotypic frequency of alleles at locus-337was in HWE insusceptible stock, while not in resistant stock. There was no significant difference inthe frequency distributions of different genotypes at locus-375between these twostocks. In contrast, the frequency of-337AA genotypes in resistant stock wassignificantly higher than that in susceptible stock, indicating its significant associationwith the heat tolerance of Bay scallop. One site of I/D polymorphism and five sites of SNPs were identified in AiFer gene,and the association between SNP1, SNP3and SNP4and the heat tolerance of Bayscallop was investigated. The frequency of SNP3GG genotype and SNP4TGgenotype in resistant stock were significantly higher than that in susceptible stock,suggesting their significant association with the heat tolerance of Bay scallop.Six SNPs were found in AiTLR8gene, and their association with the heat toleranceof Bay scallop was investigated. The frequency of SNP1AA genotype, SNP2TTgenotype, SNP3TT genotype, SNP4GG genotype, SNP5AA genotype and SNP6TTgenotype in resistant stock were significantly higher than that in susceptible stock,suggesting their significant association with the heat tolerance of Bay scallop.The contribution of the polymorphisms in the promotor of AiHSP70gene to theheat-resisting phenotype of Bay scallop was explored. Under heat shock stress, thepromoter of AiHSP70gene in heat-resistant stock can initiate the transcription of bothentogenous AiHSP70gene and the exogenous luciferase gene more effectively thanthat in heat-sensitive stock. As for the contribution of SNP+94A/C in the exon ofCfHSP22gene, the result indicated that the molecular chaperone activity of theheat-resistant CfHSP22protein encoded by+94AA genotype is slightly stronger thanthe heat-sensitive protein, however, there is no significant difference between them.The probable reason is that the contribution of SNP+94A/C to the heat-resistingphenotype of Zhikong scallop is very small.These results above suggested that, some polymorphisms in AiHSP70, AiMT1,AiFer and AiTLR8were significantly associated with the heat tolerance of Bayscallop, and they could be potential markers applied in future selection of Bay scallopwith enhanced heat tolerance. These results provided evidences to clarify the immunedefense mechanism of scallop under heat stress, and provided clues for the MAS ofscallops.