Exploiting the mitogenomes of Apis mellifera subspecies to develop an authentication tool to verify the entomological origin of mediterranean honeys
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Honey is highly susceptible to adulteration. Currently, the assessment of its geographical origin remains one of the most difficult tasks, which is typically performed by melyssopalynology. Recently, the attention has shifted towards indirect approaches such as the entomological origin based on geographical distribution patterns of honey bee subspecies. Although queens’ trade has impacted the natural subspecies distribution, honeys produced with autochthonous bees or bearing a Protected Designation of Origin specifying the producing honey bee subspecies, offer a unique avenue for authentication.
In the MEDIBEES project, we aim to develop a DNA-metabarcoding approach to authenticate honey's entomological origin focusing on mitochondrial lineages A, M, C, and O. To achieve this goal, the DNA from 1251 honey bees representing 16 subspecies (A.m. sahariensis, A.m. intermissa, A.m. siciliana, A.m. ruttneri, A.m. iberiensis, A.m. ligustica, A.m. macedonica, A.m. adami, A.m. cecropia, A.m. cypria, A.m. caucasia, A.m. meda, A.m. anatoliaca, A.m. syriaca, A.m. jemenitica, A.m. lamarcki) was extracted and the whole genome sequenced. From those, 740 mitogenomes were assembled using the MitoZ software. The quality of the assembled mitogenome was assessed by aligning all the sequences using MEGA and 348 samples were deleted. Finally, a phylogenetic analysis was conducted to eliminate non-local subspecies, resulting in a total of 326 mitogenomes. This dataset was used for calculating the fixation index (FST) pairwise values, and a sliding window of 400bp was used to identify single nucleotide polymorphisms that effectively differentiate (FST>0.98) the four lineages, enabling the identification of promising regions for primer design. In this study, three regions were identified that discriminate the four maternal lineages while showing an appropriate length for metabarcoding, namely in the COI, ND1 gene, and CYTB genes.
