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

Abstract 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 bee’s 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.