Molecular Screening of Varroa-Resistant Trait of Honey Bee Colonies based on NorpA2 Candidate Gene Polymorphism: A Genetic Case-Control Study

In recent years the Varroa destructor mite has been a dominant challenge for honey bee colonies, world-wide. Varroa-sensitive hygiene, a behavioral characteristic of honey bee, involves the detection and removal of Varroa larvae from the colony. It has been exploited as a recordable trait for Varroa resistance. The sense of smell is a key element in Varroa sensitive hygiene behavior. In this scenario, NorpA2 candidate gene, a putative olfactory receptor, is associated with vision and smell in honey bee and is known as a candidate gene for Varroa sensitive hygiene. With this motivation, the main goal of the current work was to determine some single nucleotide polymorphism (SNP) markers of NorpA2 candidate gene employing PCR sequencing in a case-control approach. To this end, after the identification of resistant (RES) and suscepti-ble (SUS) colonies according to the percentage of infections of the drone pupae to Varroa mite, a total of 10 drone pupae (5 from SUS and 5 from RES) were selected and were subjected for DNA isolation. The poly-merase chain reaction (PCR) was performed based on two pairs of specific primers for the amplification of 5? untranslated region (5?UTR) and promoter regions of NorpA2 gene. After expected size bands were ob-served, the purification process and Sanger sequencing of the PCR products were carried out. The sequenc-ing results were quality checked and the alignment and clustering were done using the BLAST and MAFTT software, respectively. Sequences of both UTR and promoter regions displayed multiple variations (SNP, deletion). Interestingly, the results showed the existence of three specific differences in sequence in the form of SNP (C/T) at position 308 and the form of SNP/deletion at positions 504 and 563 of the nucleotide sequence region in the promoter between the SUS and RES groups. To our knowledge, this is the first re-port on the identification of biomarkers to control host-mite interaction in the honey bee Apis mellifera. Further research is required for the reported SNPs to be validated as biomarkers of resistance to Varroa.