Correlation Studies Between Single Nucleotide Polymorphisms Of TLR7 And TLR8 Gene And The Hemorrhage Disease Of Grass Carp (Ctenopharyngodon Idella)

Grass carp(Ctenopharyngodon idella)is one important member of the so-called “four major fish species” in freshwater aquaculture,the development of its breeding industry is faced with great threat owing to various microbial pathogens,of which hemorrhagic disease of grass carp triggered by grass carp reovirus(GCRV)is the most harmful.The special aquatic environment and the high incidence rate and low-controllability of viral disease greatly increase the difficulties in drug therapy and prevention.Genetic polymorphisms study of immunity genes has been dedicated to detect the disease-associated polymorphic sites.And,the identification of variety of crucial molecular markers,such as single nucleotide polymorphisms(SNPs),increases the possibility for constructing highly dense genetic linkage map and large-scalely breeding improved varieties.Toll-like receptor 7(TLR7)and Toll-like receptor 8(TLR8),members of endosomal TLRs,have been proved to involve in the anti-GCRV innate immune responses of grass carp,while their regulatory mechanism still remains unclear.SNPs of C.idella TLR7(CiTLR7)and C.idella TLR8(CiTLR8)were detected in this reseatch,and then,all the samples were genotyped based on each sites.Association analysis between SNPs and susceptibility/resistance to GCRV,linkage disequilibrium(LD)analysis,haplotype analysis and SNPs(CiTLR7)-SNPs(Ci TLR8)analysis were all performed based the genotype data.Besides,mRNA expression of CiTLR7/Ci TLR8 was explored and carried on the correlation study with the significant correlative sites,respectively.Additionally,bioinformatics analyses based on SNPs in different genetic regions were introduced to explore the potential functional mechanism of both correlated and uncorrelated SNPs.The main results gained were showed as follow:1.11 and 8 identified SNPs sites were detected in CiTLR7 and CiTLR8,respectively.2.Case(susceptibility)-control(resistance)analysis showed that both 820 A/G in the single intron and 1726 A/G in coding sequence(CDS)of CiTLR7 were significantly associated with the susceptibility/resistance to GCRV in genotype(P < 0.05).3.SNPs-based susceptibility/resistance-related analysis in CiTLR8 revealed that 4062A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele(P < 0.05),while 4168 C/T was extremely significantly associated with that(P < 0.01).Both the two sites were located in the 3’-untranslated regions(UTR).4.Haplotype analysis uncovered no significantly correlative haplotype blocks in the11 identified SNPs of CiTLR7 in anti-GCRV immune response;while “SNP pair” blocks(4062 A-4178 T,4062 T-4178 T,4062 A-4231 G and 4062 T-4231 G)of the 8 identified SNPs in CiTLR8 showed significantly/extremely significantly association with the susceptibility/resistance to GCRV,suggesting SNPs linkage might play a crucial role in the anti-GCRV immune response.5.Post-GCRV infection,in spleen tissues,the average mRNA expression of CiTLR7 and CiTLR8 in resistant group were both extremely significantly higher than that of susceptible group(P < 0.01),revealing that these two genes were positive-correlated with the resistance to GCRV in mRNA level.6.SNPs(-758 A/C and 820 A/G)of Ci TLR7 and SNPs(1559 C/T and 4168 C/T)of CiTLR8 had extremely significant interaction(P < 0.01),indicating these two genes-regulated anti-GCRV immune responses might involve the SNPs-based gene-gene interaction.The SNPs gained in this research provided molecular markers for the future breeding for disease resistance.The results of SNPs-based analyses could also provide new perspectives for the anti-GCRV mechanism study of CiTLR7 and CiTLR8.