抗白垩病相关SNP位点C2587245T在意大利蜜蜂雄蜂幼虫中的抗性鉴定

Identification of resistance of the chalkbrood resistance-associated SNP locus C2587245T in drone larvae of Apis mellifera ligustica

【目的】基于抗白垩病相关单核苷酸多态性(single nucleotide polymorphism,SNP)位点C2587245T在意大利蜜蜂Apis mellifera ligustica雄蜂幼虫中白垩病抗性检验,验证该位点遗传稳定性和抗病性,为分子标记辅助育种在育种生产中直接应用提供技术支持。【方法】通过白垩病虫尸,在实验室条件下制备白垩真菌孢子悬液,通过形态学和分子生物学方法鉴定蜜蜂球囊菌Ascosphaera apis。使用无伤害方法提取意大利蜜蜂蜂蜂王DNA筛选出SNP位点C2587245T为C/C和T/T基因型的蜂王,并培育C/C和T/T基因型处女王,用二氧化碳刺激其产出雄蜂卵,选择C和T基因型2日龄意大利蜜雄蜂幼虫进行实验室培养,3日龄龄雄蜂幼虫接种5×10^(4)个孢子/μL的蜜蜂球囊菌悬液10 d,观测接种蜜蜂球囊菌后的C基因型和T基因型雄蜂幼虫的生长情况和存活率。【结果】通过无伤害提取DNA的方法可以在不影响意大利蜜蜂雄蜂幼虫正常生活的条件下提取出高质量的DNA;用本研究的饲养方法可以保证意大利蜜蜂雄蜂幼虫在实验室条件的正常生长发育。C基因型和T基因型意大利蜜蜂雄蜂3日龄幼虫接种蜜蜂球囊菌后在发病时间和发病症状等方面都有明显差异,C基因型雄蜂幼虫在表现疾病典型症状的时间上比T基因型雄蜂幼虫晚大约2 d;在接种蜜蜂球囊菌后6 d时两种基因型雄蜂幼虫表现出的症状差异最为明显。【结论】雄蜂由于其纯合单倍体的生物学特性,更方便验证SNP位点C2587245T纯合的抗病作用。SNP位点C2587245T为C基因型雄蜂幼虫具有良好的抗白垩病能力,并且SNP位点C2587245T具有稳定的遗传性,这些研究结果可为在蜜蜂抗病育种领域提供一种可靠的分子辅助标记,为后续雄蜂转录组测序、寻找雄蜂和工蜂在免疫相关基因表达方面的差异,尝试找到SNP位点C2587245T对白垩病所特有的抗病机制提供基础。

【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 5×10^(4) 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 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 that of 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 drone bees,investigating the differential expression of immune-related genes between drones and worker bees,and exploring the specific mechanisms underlying the resistance to chalkbrood conferred by the SNP locus C2587245T.