Confirmation And Application Of Chalkbrood Resistance-Associated SNP Molecular Markers In Honey Bee,Apis Mellifera Ligustica

Chalkbrood is a contagious fungal disease caused by Ascosphaera apis in honey bees larvae(Apis mellifera L.).It’s caused severe losses for bee keeping in certain places.Recent molecular technology development accelerate genetic markers selection,and improvement of the honey bee genetic phenotypes to minimize the damages of chalkbrood.Though the potential genetic interval and large number of candidate single nucleotide polymorphnisms(SNPs)related to chalkbrood-resistance were previously shown and selected,there were no molecular markers that can be applied in beekeeping and chalkbrood-resistance breeding.This study thus aimed to screen reliable SNP markers that closely associated with chalkbrood-resistance and then confirmed their credibility.Two other genotyping methods based on Probe and allelic specific PCR were tested to identify accurate,convenient,and cost-effective method to distinguish the SNP alleles.The chalkbrood-resistant(CR)and-susceptible(CS)colonies were first selected to provide resistant(Res)and susceptible(Sus)larval samples screened by using in vitro larval rearing.The selected samples were used to discover reliable SNPs commonly shared by Res samples,and exclude false-positive SNPs appeared both in Res and Sus samples using PCR and Sanger sequencing.Symptomless larva samples were randomly collected from three other CR and CS colonies to further confirm the screened SNPs.Larva samples from different geographical locations were then analyzed to investigate whether the high yield and quality of royal jelly production and chalkbrood-resistance honey bee stocks(Fengqiang I,A.mellifera.L)exhibited traits of chalkbrood resistance or not.The confirmed SNPs were analyzed for whether could be used as marker for molecular marker assisted breeding.Last,appropriate methods,including TaqMan-MGB probes and modified allelic specific PCR(AS-PCR),were tested to genotype the SNP markers.One SNP located in the second intron of MRJP5,termed as SNP-3,was closely related to chalkbrood-resistance and verified though rigorous experiments.The frequency of C allele(PC)of SNP-3 was significantly higher in CR colonies than that in CS(P<0.05).CR colonies that had lower chalkbrood incidence can be characterized by higher PC.The capacities of chalkbrood-resistance in different colonies,which were from Fengqiang I,were in accordance with the value of PC.SNP-3 was further supported to identify the resistant level of the colonies using the PC.The colonies headed by C/C queens might have more resistance to chalkbrood infection.These results suggested that SNP-3 could serve as the genetic marker to breed chalkbrood resistant colonies and queens conferred by resistant larvae themselves.According to the exploration of SNP genotyping on the SNP-3 site,we found the appropriate genotyping method was Sanger sequencing.These results provided one convenient molecular marker for selecting honey bee chalkbrood resistant line,which was promising to reduce impacts of chalkbrood on apiculture and present cues related to mechanism of chalkbrood resistance.