Larvae Mediated Sacbrood Virus Resistance Using SNP Markers By Re-sequencing In Apis Cerana Cerana

The prevalence of honeybee viral diseases has recently been causing major problems in the beekeeping industry,causing economic losses worldwide.Honeybees are susceptible to a variety of diseases and various pathogens.Among these pathogens,the prevalence of Sacbrood virus(SBV)along with other factors,are seriously threatening to the health of bee species.SBV infects mostly larvae,including adult bees.However,pupae are not infected by this virus.SBV is a widespread virus among honeybee species,especially in Apis cerana cerana(A.c.cerana).At the present,there is no specific drug available for the treatment of SBV diseases,which does not affect the quality of the bee products.Therefore,the research program was conducted on the whole genome sequencing to screen single nucleotide polymorphisms(SNPs)molecular markers to SBV resistance in A.c.cerana.In the present study,88 A.c.cerana colonies from Fuan,Cangshan,Yongtai,and Minhou,in the Fujian Province,were screened using RT-PCR method across seven honeybee viruses,namely,Chinese Sacbrood virus(CSBV),deformed wing virus(DWV),Israeli acute paralysis virus(IAPV),blackqueen cell virus(BQCV),chronic bee paralysis virus(CBPV),acute bee paralysis virus(ABPV),and Kashmir bee virus(KBV).The results were showed that CSBV,was the most prevalent as it was detected in 85.22%,followed DWV in 78.40%and IAPV in 25%.In contrast,insignificant prevalence results were obtained from all apiaries no detection in 4.54%,BQCV,CBPV,APBV,and KBV,which were not detected in any sample.To isolate and purify of SBV,diseased larvae were sampled from infected colonies,SBV was purified using high-speed centrifugation with Cesium Chloride(CsCl)density gradients.The results were confirmed by TEM,RT-PCR,sequencing,and phylogenetic analysis.Three healthy bee colonies without detectable virus and other diseases were selected to conduct the experiments.Larval rearing was performed in vitro.Three days old larvae were inoculated with SBV at 1.32 x10~4copies/larva.Susceptible larvae and resistance larvae were separately collected and stored at-80℃.90 samples were randomly selected from three A.c.cerana colonies(each colony 15 susceptible larvae and 15 resistance larvae).Genomic DNA was extracted using the CTAB method.The qualified DNA sample was used in the whole-genome sequencing by Biomarker Technologies Corporation performed sequencing of samples,Beijing using commercial Illumina NOVA HisSeq X-Ten service then,the bioinformatics analysis was performed.The data analysis resulted in 337.47 G bp clean data;average Q30reached to 92.73%.The average ratio of samples after compared to the A.c.cerana genome as a reference genome was in 87.92%,the average coverage depth was 14X,and the genome coverage was 99.04%.Based on the SNP data of the mutation detection,the filtering and selective sweeps,minor allele frequency,(MAF≤0.05)and site completeness(INT≤0.5)analysis,84 SNP markers related to Sacbrood virus resistance have been screened out,which showed significant difference with their alleles compared to the reference genome of A.c.cerana.Among them,10 SNP appeared to be significantly different between resistance and susceptible samples.Also,7 candidate genes were detected in the genome(scaffold)of A.c.cerana,which may have potentially related to Sacbrood virus resistance.The results revealed a large number of SNPs and genes related to Sacbrood virus resistance exist in the specific position of the genome,providing new scientific evidence for further studies on assistant selection breeding on SBV resistance,bee species via molecular markers.