Towards honey authentication: Differentiation of Apis mellifera subspecies in European honeys based on mitochondrial DNA markers.

Honey is the natural sweet substance produced by Apis mellifera honeybees in Europe. Depending on the country/region, the A. mellifera subspecies native to Europe belong to three different lineages: A (A. m. iberiensis), M (A. m. iberiensis and A. m. mellifera) and C (A. m. ligustica and A. m. carnica). In this work, two DNA-based approaches were developed with the aim of entomological authentication of European honeys. A cytb specific PCR assay was proposed to identify A-lineage honeybees, while a second method based on real-time PCR coupled to high resolution melting analysis targeting the COI gene was developed to differentiate C- and M-lineages honeybees. The proposed methodologies were validated successfully with honeys of known origin and applied to the entomological authentication of 20 commercial samples from different European countries. The results highlight the predominance of honeys from C-lineage honeybees in Europe, except in Iberian Peninsula countries (honey from A-lineage honeybees).