The Polymorphisms Of Candidate Genes Associated With Susceptibility/Resistance Of Zhikong Scallop (Chlamys Farreri) To Listonella Anguillarum

Zhikong scallop (Chlamys farreri) is one of the economic scallop species cultivated widely in North China and contributes enormously to the economic development of coastal provinces. Since the summer of 1997, frequent diseases have caused catastrophic economic losses to scallop aquaculture which resulted in the production decreasing drastically. Cultivating new strains of scallops with enhanced resistance to diseases is now considered to be one of the basic and ultimate solutions to disease control. Since the traditional breeding techniques are usually costly and time-consuming, and could not fulfill the urgent requirement for thoroughbred, it is necessary to accelerate and improve selective breeding of resistance strains in virtue of molecular methods. Marker assisted selection (MAS) is one of the molecular methods successfully applied in animal breeding. However, MAS is currently far from practice in mollusks as the lack of markers associated with quantitative traits. Identification of markers associated with resistance to pathogens is necessary for the development of MAS in mollusks.In this study, Zhikong scallops were classified into susceptible and resistant stocks according to the survival time after L. anguillarum challenge. The nucleotide sequence polymorphisms in CfLysG, CfC1qDC and CfLITAF genes from Zhikong scallop were investigated to explore their associations with susceptibility/resistance to L. anguillarum by PCR, PCR-RFLP and Bi-PASA PCR methods.One hundred and four sites of single nucleotide polymorphisms (SNPs) and twenty nine sites of insert/deletion (I/D) polymorphisms were identified in CfLysG gene. Seventeen polymorphisms located in its promoter region. Three of them, -753 I/D polymorphism, -391 A/G SNP and -284 I/D polymorphism were selected to analyze their distributions in susceptible and resistant stocks. Statistical analysis revealed that the genotypic frequencies of all alleles were in Hardy-Weinberg equilibrium (HWE) (P>0.05) at loci -753, -391 and -284 in both stocks. Among them, the -753 ID genotype and -284 ID genotype were more prevalent in resistant stock than those in susceptible stock, but there were no significant differences in the frequency distributions between these two stocks (P>0.05). In contrast, the frequency of -391 AG genotype in resistant stock was significantly higher than that in susceptible stock (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L. anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 AA genotype was significantly higher than those with -391 AG genotype (P=0.001), which further validate the significant association between -391 AG genotype and the resistance of Zhikong scallop to L. anguillarum. Three SNPs were found in the exons of CfLysG gene, among which +3473 A/C in exon III was a non-synonymous SNP. Statistical analysis revealed that the genotypic frequencies of alleles at locus +3473 were in HWE (P>0.05) in susceptible stock, while not in HWE (P<0.01) in resistant stock. The frequency of +3473 AA genotype in resistant stock was significantly higher than that in susceptible stock, indicating its significant association with the resistance of Zhikong scallop to L. anguillarum. Two large fragment I/D polymorphisms, +96 I/D polymorphism and +487 I/D polymorphism, were identified in intron I of CfLysG gene. The genotypic frequencies of all alleles were in HWE (P>0.05) at loci +96 and +487 in both stocks. The +96 DD genotype and +487 ID genotyp were more prevalent in resistant stock than those in susceptible stock, but there were no significant differences in the frequency distributions between these two stocks (P>0.05). The results suggested that these two polymorphisms were not significantly associated with the resistance of Zhikong scallop to L. anguillarum. Linkage disequilibrium was found between the multiple loci of CfLysG gene, indicating the existence of haplotypes. Nineteen haplotypes with frequency above 1% were identified. Among them, the frequency of -753 I/-391 G/-284 I/+96 I/+487 D/+3473 A haplotype in resistcant stock was significantly higher than that in susceptible stock (P=0.044), indicating its significant association with the resistance of Zhikong scallop to L. anguillarum.Fourteen SNPs were identified in CfC1qDC gene, and the association between +423 T/C SNP and the resistance of Zhikong scallop to L. anguillarum was investigated. The genotypic frequency of each allele was in HWE (P>0.05) at locus +423 in both stocks. The +423 TT genotype was significantly more prevalent in resistant stock than that in susceptible stock (P=0.005), suggesting its significant association with the resistance of Zhikong scallop to L. anguillarum.Three SNPs and one I/D polymorphism were found in CfLITAF gene. The association between +145 I/D polymorphism and the resistance of Zhikong scallop to L. anguillarum was investigated. All alles in locus +145 could be found in each susceptible and resistant individual, suggesting that there was no association between +145 I/D polymorphism and the resistance of Zhikong scallop to L. anguillarum.These results above suggested that, the -391 AG genotype, +3473 AA genotype and -753 I/-391 G/-284 I/+96 I/+487 D/+3473 A haplotype of CfLysG gene and +423 TT genotype of CfC1qDC gene were significantly associated with the resistance of Zhikong scallop to L. anguillarum, and could be potential markers applied in future selection of Zhikong scallop with enhanced disease resistance. These results would hopefully provide reference for MAS in mollusks. Furthermore, identification of markers associated with disease could also improve the understanding of pathogenesis and aid in the development of preventive and therapeutic measures.