Candidate Gene Analysis Of Resistance To White Spot Syndrome Virus (WSSV) In Chinese Shrimp Fenneropenaeus Chinensis

The industry of Fenneropenaeus chinensis, which is a kind of major economicaquaculture species in China, was seriously affected by white spot syndrome virus(WSSV) since the first outbreak in the1990s. In recent years, the shrimp industry inChina is developing with improvement of breeding and cultivation. However, WSSVis still one of the main pathogens affecting shrimp industry. The key point to solve theproblem of WSSV in shrimp industry is to breed new varieties of F. chinensis bothwith disease resistance and fast growth traits. For threshold and low heritability traits,combining the traditional breeding methods and new advanced molecular biologytechnology can improve the selection accuracy and speed up the process of breeding.This study of candidate genes and SNP analysis of resistance to WSSV in F. chinensiswas based on the “Huanghai No.2” F. chinensis454transcriptome sequencing anddone with techniques of RACE, gene cloning, conventional sequencing, HRM,ELISA, real-time PCR, etc. to provide theoretical and practical materials forWSSV-resistant shrimps breeding. This research mainly included for the first timeobtaining MEK, ERK, Ras and cathepsin B gene sequences in F. chinensis; SNPmining in each gene; gene expression level in different tissues of normal and WSSVinfected F. chinensis; characterization of WSSV replication in F. chinensis and effectof inhibition of MEK/ERK signaling transduction pathway on WSSV replication;selecting SNP candidates identified by454transcriptome sequencing; SNP genotypesin the WSSV-resistant group and WSSV-susceptible group by HRM; genetic polymorphism information; correlation analysis of disease resistance; etc.. The detailsare as follows:1. MEK gene mRNA sequence from F. chinensis (FcMEK) was reported by thisstudy (GenBank accession number: KJ135020) and the results suggested that FcMEKtranscription level was affected by WSSV infection. In WSSV infected F. chinensis,FcMEK mRNA transcription levels in the hepatopancreas increased at6h and48h,decreased at12h and24h. FcMEK mRNA transcription levels in the gill increased at12h,24h and48h, decreased at6h. FcMEK mRNA transcription levels in themuscle increased at6h,12h,24h and48h. The results suggested that FcMEKtranscription level might be affected by WSSV infection. Three SNPs were identifiedin FcMEK ORF by direct sequencing, which located at762bp,765bp and972bpafter the initiation codon respectively. All of the three SNPs were C/T transition andwere named as C-762T, C-765and C-972T respectively. The results showed thatC-762T and C-765T constituted a haplotype. The three SNPs in the resistant groupand susceptible group were further genotyped by the HRM technology. At C-762Tand C-765T loci there were readable melting curves, suggesting no intron existed nearthe two SNPs. For the C-762T and C-765T loci in the resistant group, Ho was0.604,He was0.424and MAF was0.302. For the C-762T and C-765T loci in the susceptiblegroup, Ho was0.583, He was0.424and MAF was0.302. Both of C-762T andC-765T loci in the two groups deviated from the HWE. However, chi square testresults showed no significant difference of genotype distribution between the twogroups. The deviating from the HWE suggested that these SNPs might be selected inthe “Huanghai No.2” breeding population.2. ERK gene mRNA sequence from F. chinensis (FcERK) was reported by thisstudy (GenBank accession number: KC537791) and the results suggested that FcERKtranscription level was affected by WSSV infection. In WSSV infected F. chinensis,FcERK mRNA transcription levels in the hepatopancreas decreased at6h,12h,24hand48h. FcERK mRNA transcription levels in the gill increased at6h,12h,24hand decreased at48h. FcERK mRNA transcription levels in the muscle increased at48h and fluctuated during6h to24h. FcERK protein level in the hepatopancreas increased slowly from3h to24h and decreased at48h. FcERK protein level in thegill increased at3h,12h and48h and decreased at6h and24h. FcERK protein levelin the muscle increased at12h,48h and decreased at3h,6h and24h. Changes ofFcERK expression levels after WSSV infection suggested that FcERK might beaffected by WSSV infection. One SNP was identified in the ORF and another SNPwas identified in the3’ UTR, both of which were C/T transition and named as C-544T、C-1133. The two SNPs in the resistant group and susceptible group were furthergenotyped by the HRM technology. For the C-544T locus in the resistant group, Howas0.146, He was0.154and MAF was0.083. For the C-544T locus in thesusceptible group, Ho was0.208, He was0.204and MAF was0.115. For the C-1133locus in the resistant group, Ho was0.177, He was0.162and MAF was0.089. For theC-1133locus in the susceptible group, Ho was0.156, He was0.145, MAF was0.078.No locus in the two groups deviated from the HWE and no significant difference ofgenotype distributed between the two groups, which suggested that although FcERKmight be affected by WSSV infection, the two mutation loci did not affect thefunction of FcERK in the anti-WSSV process. MAFs of both the two SNPs weregreater than0.05, which suggested that the two SNPs were qualified for associationanalysis. As an important kinase in the signaling transduction pathway, SNPs inFcERK should be further investigated.3. In the WSSV challenge test, the F. chinensis which survived longer containedmore WSSV than the ones which survived shorter. This result suggested that theresistant ability to WSSV can be reflected in tolerance to the number of virus,therefore for the breeding object, tolerance to WSSV, inhibiting viral replication, eventhe ability to kill virus, is the key to the survival of shrimps. In addition, developingnew drugs to inhibit the virus replication can improve the survival rate of infectedshrimps. After inhibiting MEK/ERK pathway in F. chinensis by using U0126, WSSVreplication level was detected. Compared with the control group, WSSV replicationlevel in the experimental group, which was added U0126, was inhibited. At24h, theWSSV replication level in the experimental group was about two-fifth of that in thecontrol group. At48h, the WSSV replication level in the experimental group was about three-fifth of that in the control group. Synthesizing the results of FcMEK andFcERK gene expression level in WSSV infected F. chinensis mentioned above, it issuggested that WSSV can hijack the host MEK/ERK signaling transduction pathwayfor viral replication.4. Ras gene mRNA sequence from F. chinensis (FcRas) was reported by this study(GenBank accession number: KC522602) and the results suggested that FcRastranscription level was affected by WSSV infection. In WSSV infected F. chinensis,FcRas mRNA transcription levels in the hepatopancreas increased at6h and48h,decreased at12h and24h. FcRas mRNA transcription levels in the gill and muscleall increased at6h,12h,24h and48h. Changes of FcRas transcription levels afterWSSV infection suggested that FcRas might be affected by WSSV infection. No SNPlocus was detected by sequencing26F. chinensis’s FcRas mRNA. In the five genesresearched by this study, FcRas was the only one which contained no SNP locus. Thisresult further indicated the high conservation of Ras.5. Cathepsin B gene mRNA sequence from F. chinensis (FcCathepsin B) wasreported by this study (GenBank accession number: KC537790) and the resultssuggested that FcCathepsin B transcription level was affected by WSSV infection. InWSSV infected F. chinensis, FcCathepsin B mRNA transcription levels in thehepatopancreas increased at6h,48h and decreased at12h and24h. FcCathepsin BmRNA transcription levels in the gill increased at6h,12h,24h and decreased at48h. FcCathepsin B mRNA transcription levels in the muscle increased at6h,12h,48hand decreased slightly at24h. Changes of FcCathepsin B transcription levels afterWSSV infection suggested that FcMEK might be affected by WSSV infection. ThreeSNPs were identified in the ORF, which located at42bp,756bp and984bp after theinitiation codon respectively. All of the three SNPs were C/T transition and named asC-42T, C-756T and C-984T respectively. The results showed that the three SNPsconstituted a haplotype. The three SNPs in the resistant group and susceptible groupwere further genotyped by the HRM technology. At the C-984T locus there werereadable melting curves, suggesting no intron existed near the SNP. For the C-984Tlocus in the resistant group, Ho was0.375, He was0.344and MAF was0.219. For the C-984T locus in the susceptible group, Ho was0.417, He was0.355and MAF was0.229. No locus in the two groups deviated from the HWE and no significantdifference of genotype distributed between the two groups, which suggested theseSNPs did not affect the function of FcCathepsin B in the anti-WSSV process. Theresult that MAFs of the two SNPs were all greater than0.05suggested that the twoSNPs were qualified for association analysis.6. The candidate SNP loci in the F. chinensis “Huanghai No.2”454transcriptomesequencing results were validated and analyzed by HRM technology.81SNP lociwere identified, in which one locus was found to be associated with WSSV resistance.480candidate SNP loci were selected.480pairs of primers for HRM were designed,in which243pairs were qualified after screening. After designing unlabeled probes,the243SNPs in the resistant group and susceptible group were genotyped by theHRM technology.81locus were confirmed to be SNP loci and Ho、He、Ne、MAF、I、HWE information was calculated. For the81SNPs, MAFs of55SNPs in theresistant group were greater than0.05and MAFs of56SNPs in the susceptible groupwere greater than0.05. All of these SNPs had high polymorphism and were qualifiedfor association analysis. For the81SNPs,65SNPs in the resistant group accordedwith HWE and60SNPs in the susceptible group accorded with HWE. For the81SNPs, there were4A/C mutations,27A/G mutations,11A/T mutations,9C/Gmutations,28C/T mutations and2T/G mutations. The transition/transversion=2.115, which accorded with the “transition bias” principle. The association analysisresult showed that the C/C genotype of312C1298-154C/T mutation locus distributeddifferently between the resistant group and susceptible group both in2011and2012,which suggested that the SNP associated with WSSV resistance. The gene sequence,which contained the SNP locus accociated with WSSV resistance, was obtained bygene cloning and named as FcPH. FcPH mRNA contained a1305bp ORF encoding434amino acids. By real-time PCR, the results showed that the FcPH mRNAtranscription levels increased in the gill and muscle post WSSV challenge. In thisstudy, the results showed that FcPH was affected by WSSV infection; the312C1298-154C/T mutation locus in this gene was associated with the WSSV resistance.