Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.)

• ITS1, ITS2 and matK as candidates barcode loci for Crocus spp. differentiation.
• Real-time PCR coupled to high resolution melting (HRM) analysis.
• HMR analysis targeting ITS1 and matK loci were Crocus genus specific.
• ITS2 locus was specific of Crocus sativus L. and Crocus cartwrightianus.
• DNA-barcoding with HRM analysis as a reliable tool for saffron authentication.

Saffron (Crocus sativus) is the most expensive spice in the world. As a consequence, saffron has frequently been associated with an unrivalled degree of adulteration. The aim of this work was searching for DNA-barcode candidates as molecular markers to differentiate C. sativus from allied species and assessing the authenticity of saffron. New primers were specifically designed targeting three DNA barcode loci, namely ITS1, ITS2 and matK. A real-time PCR assay combined with high resolution melting (HRM) analysis was carried out in order to discriminate different Crocus spp., being further confirmed by sequencing. Data from HRM analysis showed that ITS1 and matK loci were Crocus genus specific, while ITS2 locus was species-specific for C. sativus and Crocus cartwrightianus. This study revealed that genomic tools, and specifically DNA mini-barcodes combined with HRM analysis, can provide simple, fast and highly reliable methods for saffron authentication.