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

Marcelino, J. A.; Boardman, L.; Moore, M. R.; Fulton, J. C.; Urbina, H.; Patterson Rosa, L.; Grunewald, B.; Allsopp, M.; Pirk, C.; Cambray, G.; Ellis, J. D.

doi:10.64898/2026.03.05.709340

bioRxiv · 10.64898/2026.03.05.709340

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

Marcelino, J. A.·Boardman, L.·Moore, M. R.·Fulton, J. C.·Urbina, H.·Patterson Rosa, L.·Grunewald, B.·Allsopp, M.·Pirk, C.·Cambray, G.·Ellis, J. D.

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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 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.

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Marcelino, J. A., Boardman, L., Moore, M. R., Fulton, J. C., Urbina, H., Patterson Rosa, L., Grunewald, B., Allsopp, M., Pirk, C., Cambray, G., Ellis, J. D.. 2026-03-07. Development of molecular markers for western honey bee (Apis mellifera L.) subspecies of regulatory concern in the United States. https://doi.org/10.64898/2026.03.05.709340

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