Abstract:

【Aim】 Based on the single nucleotide polymorphism (SNP) locus C2587245T related to the  chalkbrood resistance in drone larvae of Apis mellifera ligustica, the genetic stability and disease resistance of C2587245T were verified, to provide technical support for the direct application of molecular marker-assisted breeding in breeding production. 【Methods】 Spore suspension of the chalkbrood fungus was prepared from infected brood under laboratory conditions, and Ascosphaera apis was identified through morphological and molecular biological methods. DNA of queen bees of A. mellifera ligustica was extracted by noninvasive method, and queen bees with C/C and T/T genotypes at the SNP locus C2587245T were screened. Virgin queens of A. mellifera ligustica with C/C and T/T genotypes were reared, and their eggs were stimulated to develop into drone larvae using carbon dioxide. The 2-day-old drone larvae of A. mellifera ligustica with C and T genotypes were selected for laboratory cultivation and the 3-day-old drone larvae were inoculated with 5×10⁴ spore/μL spore suspension of A. apis for 10 d. The growth and survival rate of the drone larvae of A. mellifera ligustica with C and T genotypes after inoculation with A. apis were observed and recorded. 【Results】 High-quality DNA could be extracted using the noninvasive method without affecting the normal life of drone larvae of A. mellifera ligustica. The rearing method used in this study ensured the normal growth and development of drone larvae of A. mellifera ligustica under laboratory conditions. There were extremely significant differences in the onset time and symptoms of the disease between C-genotyped and T-genotyped drone larvae of A. mellifera ligustica after inoculating the 3-day-old drone larvae with A. apis. The C-genotyped drone larvae of A. mellifera ligustica showed the typical disease symptoms approximately 2 d later in onset time than the T-genotyped drone larvae. At 6 d after inoculation with A. apis, the symptom differences between the two genotypes of drone larvae were the most pronounced. 【Conclusion】 The SNP locus C2587245T in drones, which is homozygous haploidy due to its biological characteristics, provides a convenient means to verify the homozygous disease resistance. Drone larvae with the C genotype at the SNP locus C2587245T exhibit strong resistance to chalkbrood, and the SNP locus C2587245T demonstrates stable heritability. These research findings can be served as a reliable molecular marker in the field of breeding disease-resistant honeybees, and further provide a basis for subsequent studies such as transcriptome sequencing of male bees, investigating the differential expression of immune-related genes between drones and drone bees, and exploring the specific mechanisms underlying the resistance to chalkbrood conferred by the SNP locus C2587245T.

Key words: Apis mellifera ligustica; drone, chalkbrood; Ascosphaera apis, single nucleotide polymorphism; chalkbrood resistance