Single Nucleotide Polymorphism in the Dopamine Receptor Type 3 (DOP3) Candidate Gene Associated with Varroa Destructor Resistance in Honeybee

Varroa infestation is undoubtedly the greatest threat and challenge facing Apiculture today. This external parasite inevitably lives in the bee colony and causes irreparable damage to its colony and the subsequent honey production. One of the proposed strategies in this regard is the use of pesticides, which have a negative impact on the health of bees and honey consumers. To avoid these negative consequences, safer alternative methods of controlling mites are needed, including the use of resistant genetic strains and breeding selection programs to establish colonies with relative resistance to mites. In honeybee colonies, there are several physiological and behavioral mechanisms for Varroa resistance that by examining their relationship with identified resistance genes, the genetic basis of Varroa mite resistance in honeybees is identified and can used in breeding programs. The aim of the present study was to identify single nucleotide polymorphisms (SNPs), in a sample of the Iranian honeybee population, in the candidate gene (the dopamine receptor gene (DOP3)) effective on defense behaviors of honeybees against the varroa mite.

For this purpose, a total of 10 drone bees (5 susceptible and 5 resistant) were selected and their DNA was then isolated using a CTAB-based method. The PCR was carried out based on specific primers in the UTR region sequence of the DOP3 gene. The quantity and quality were then determined using the nanometer method. After a single band (900 bp) of the expected size, product purification and sequencing (Sanger method) were performed. The display of the outputs and the determination of the quality of the raw sequence (phred index) took place with the FinchTV software and the alignment with BLAST and clustering with the MAFTT software.

In this study, differences were observed in several regions of the nucleotide sequence of the UTR region of DOP3 gene, the most important difference in the nucleotide sequence between sensitive and resistant individuals in the two regions. One is in the nucleotide region of 428 to 437 forward readings as many as 9 bp nucleotides and the other is in the nucleotide region of 715 to 720 forward readings as many as 6 bp nucleotides, which in both, mutations of the deletion type have been performed.

Evaluation of the final results showed significant difference, in type of deletion/addition with the size of 6 and 9 bp between two groups (sensitive and resistant to Varroa), respectively which can be used in the molecular identification of resistant colonies and breeding programs to produce Varroa mite resistant colonies. No such deletions from this gene have been reported so far.