Authentication of the Botanical Origin of Honey: <i>In Silico</i> Assessment of Primers for DNA Metabarcoding

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 <i>rbcL</i>, <i>matK</i>, and <i>trnL</i> and nuclear <i>ITS</i>) and length of <600 bp. Performance of these primers was <i>in silico</i> assessed for all Streptophyta and a subset of them representing the honeybee foraging plants included in the European list. The results showed that <i>rbcL</i> primers provided the highest taxonomic coverage, which increased significantly when combined with <i>ITS2</i>, 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.