Development of molecular markers for western honey bee (Apis mellifera L.) subspecies of regulatory concern in the United States

Rapid, accurate and cost-effective identification of Apis mellifera subspecies is needed for subspecies of regulatory concern. We designed and validated subspecies markers based on single nucleotide polymorphisms (SNP) on mitochondrial cytochrome b (Cytb) and NADH dehydrogenase 4 (ND4) genes. We used a combination of established and novel real-time qPCRs in a stepwise series, with increasing discrimination power, to (1) differentiate honey bees of African (A-lineage) ancestry from those of other lineages (Cytb SNP #1), (2) identify African-derived honey bees (AHBs) (Cytb SNP #2), and (3) detect A. m. capensis exclusively in the bees indigenous region of South Africa (ND4 SNP). We also developed a restriction fragment length polymorphism assay targeting a SNP on the NADH dehydrogenase 2 (ND2 RFLP) gene to detect the specific mitochondrial A-lineage clade. These assays allow for reliable time- and cost-effective results that provide increased accuracy on subspecies assignation.