Association Between The Polymorphism Of Immune Genes And Disease Susceptible/resistance Of Scallop

Aquaculture is becoming an important source for world food economy and supplies one-third ofworld food consumption. Mollusk takes second place in the global aqua production, and over80to90%of the production is from China and Japan. Both Zhikong scallop (Chlamys farreri) and Bay scallop(Argopecten irradians irradians) are important mollusk species cultivated in China and contribute a lotto aquaculture industry. However, a scallop aquaculture has been suffering a lot due to the frequentdisease occurrences in the recent decades. Therefore, the selection of disease resistant strain is essentialis mandatory. Marker assisted selection (MAS) is one of the molecular methods successfully applied inanimal breeding. However, MAS is currently far from practice in mollusks due to the lack of markersassociated with quantitative traits. Identification of markers associated with resistance to pathogens isnecessary for the development of MAS in mollusks.In this study, Zhikong scallops and Bay scallops were classified into susceptible and resistantstocks according to their survival time after L. anguillarum challenge. The nucleotide sequencepolymorphisms in CfPGRP, CfLGBP from Zhikong scallop and AiSPI, AiDef genes from Bay scallopwere investigated to explore their associations with susceptibility/resistance to L. anguillarum by PCR,PCR-RFLP and Tetra ARMs PCR methods.Peptidoglycan recognition protein (PGRP) and Lipopolysaccharide and β-1,3-glucan bindingprotein (LGBP) are pattern recognition receptors, and they play important roles in the innate immune response against invasive pathogens. The single nucleotide polymorphism (SNP) in PGRP, LGBP gene(CfPGRP,CfLGBP) were screened in PGRP, LGBP gene from scallop Chlamys farreri to investigatetheir association with disease resistance of scallop against the gram negative marine bacteria Listonellaanguillarum. Eight and twelve mutations were found in the potential LPS and glucanase motif ofCfLGBP and PGRP domain of CfPGRP, respectively. Among them, two mutations werenonsynonymous in LPS and glucanase motif region, and one mutation was nonsynonymous for PGRPdomain. The polymorphism of the locus+4407-4408for PGRP domain and at locus+7679for LPS andglucanase domain were screened, and the frequencies of their genotypes were significantly differentiated(P <0.05) between disease susceptible and resistant group. The genotype+4407-4408CG/TA forCfPGRP,+7679G/G for CfLGBP were more towards resistant phenotype against L. anguillarum. Thegenotype frequency of CG/CG in the resistant stock was significantly lower than that in susceptiblestock (0%VS32.4%), while that of CG/TA in the resistant stock was significantly higher than that insusceptible stock for CfPGRP (P <0.01). For CfLGBP, three genotypes; G/G, G/A and A/A, wererevealed at locus+7679, and their frequencies were89.7%,7.7%and2.6%in the resistant stock, while63.2%,34.2%and2.6%in the susceptible stock, respectively. The frequency of genotypes G/G and G/Awere significantly different (P <0.05) between the two stocks. The pathogen-associated molecularpatterns (PAMP) binding activity of recombinant proteins of CfPGRP and CfLGBP, rCfPGRP-S1(R)with CG variant in4407-4408site and rCfPGRP-S1(Y) with TA variant in4407-4408site, rCfLGBP (G)with G variant at locus+7679and rCfLGBP (S) with A variant at locus+7679, were elucidated byELISA assay. LPS, PGN were used as substrate for ELISA assay for PGRP and LPS, β-glucan wereused for LGBP because these substrates are conserved pathogen-associated molecular pattern (PAMP)molecules of bacteria and fungi. The results were further validated the significant differentiation (P <0.05) between the protein variants. Besides, PGRP is a multifunctional gene and also displays the antibacterial activity. Growth inhibition assay was conducted to find the antibacterial activities of PGRPprotein variants. Although, both the variants drastically reduced the growth rate of E. coli, no significantdifference (P>0.05) was found between the protein variants. In conclusion, the polymorphism at locus+4407-4408CG/TA of PGRP domain and at locus+7679G/G of LPS and β-glucanase motif weresignificantly associated with disease resistance in Zhikong scallop, and the effect of this polymorphismmight considerably affect the PAMP binding of CfPGRP and CfLGBP but not the antibacterial activityof CfPGRP. The findings clearly suggested that the polymorphic loci could be utilized as a potentialmarker for disease resistance against L. anguillarum.Serine protease inhibitor (SPIs) and Defensin are important genes associated with rapid defenseagainst pathogen. Serine protease inhibitors (AiSPI) play a crucial role in regulation of Serine proteaseactivity, and were classified into several protein families, where Kazal-type inhibitors are one of theproteinase inhibitor families with multi-domain proteins. Similarly, Defensin (AiDef) is one of the AntiMicrobial Protein (AMPs) which posses remarkable microbicidal activity against Gram-positive, Gram-negative bacteria and fungi. In the present study, the polymorphism of AiSPI and partial sequence ofAiDef domain from Bay scallop was revealed to find possible disease resistant association. Nine SNPswere identified in the exon region of AiSPI where5SNPs were nonsynonymous mutation, and therewere no mutation found in the obtained partial sequence of AiDef domain. Among the AiSPI mutation,two mutations were analyzed and found to be associated with disease resistant. The mutation from A-Gat locus+536was found to be significantly associated, to the genotype A/A which was much prevalentin resistant group than in susceptible group, there was a significant difference (P <0.05). The genotypefrequency of A/G in the resistant stock was significantly lower than that in susceptible stock (12.8%VS35.1%), while that of A/A in the resistant stock was significantly higher than that in susceptible stock forAiSPI (P <0.05). But the other mutation C-T at locus+1312was not associated with either group, and was found to be a rare mutation (P>0.05), and the genotypic frequencies of genotypes T/T, T/C, C/C atlocus+1312were94.6%,2.7%and2.7%in the susceptible stock, while100%,0%and0%in theresistant stock, respectively. In vitro SP-inhibition assay of the protein variants of AiSPI was conductedto elucidate the effect of SNP, and the assay demonstrated that rAiSPI (N) with A variant at locus+536significantly exhibited a higher inhibition (P <0.05) than rAiSPI (S) with G variant. In conclusion, themutation at locus+536A/A significantly associated with disease resistant in Bay scallop and its effectmight significantly affect the function of SPI. Nevertheless, the research needs further downstream andin vivo assay to establish further functional association. These evidences would hopefully give sometheoretical information for MAS in mollusks.