Authentication of the Botanical Origin of Honey: In Silico Assessment of Primers for DNA Metabarcoding

Abstract

Honey is a valuable food product that is often mislabeled as to its botanical composition, affecting both trade transparency and consumer safety. DNA metabarcoding is a promising method for authenticity verification, although the results are affected by the choice of molecular markers and primer pairs. This study selected 43 primer pairs from the literature covering several genes (chloroplast rbcL, matK, and trnL and nuclear ITS) and length of <600 bp. Performance of these primers was in silico assessed for all Streptophyta and a subset of them representing the honeybee foraging plants included in the European list. The results showed that rbcL primers provided the highest taxonomic coverage, which increased significantly when combined with ITS2, especially for key families, such as Asteraceae, Fabaceae, Lamiaceae, and Rosaceae. These results provide valuable insights for the design and chemical setup of molecular steps in metabarcoding experiments applied to honey authenticity also supporting the standardization of official control protocols.

Keywords: honey authenticity; in silico PCR; land plants; next-generation sequencing; primer collection.

1

Location of the 43 primer pairs on the target gene: (a) chloroplast trnL with three primer pairs, (b) chloroplast rbcL with 14 primer pairs, and (c) nuclear ITS with 26 primer pairs (9 belonging to the ITS1 region and 17 belonging to the ITS2 region).

2

Venn diagrams showing numbers and percentages of exclusive and shared classes, orders, families, genera, and species amplified in silico by the four molecular regions.

3

Barplot representing the number of class, order, family, genera, and species amplified in silico by each primer pair for each molecular region: (A) rbcL and trnL barcode region and (B) ITS1 and ITS2 barcode region.

4

Heatmap of the top 40 families amplified in silico by the primer pairs.

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Venn diagrams for the honeybee plants data set showing the number and percentage of exclusive and shared classes, orders, families, genera, and species among the four molecular regions.