High resolution melting of chloroplast mini-barcode in star anise (Illicium verum) authentication

Star anise (Illicium verum), dubbed the 'King of Spices', is one of the most popular spices widely used in cuisine, cosmetics and herbal medicine. However, recent surveys have found that commercial products occasionally replace star anise with some toxic lookalike species. To address this problem, we applied a high resolution melting of chloroplast mini-barcode method to distinguish star anise from poisonous adulterants. We sequenced the complete chloroplast genomes of four reported adulterant species (i.e., Illicium lanceolatum, Illicium simonsii, Illicium henryi and Illicium brevistylum). We carried out a comparative analysis between these species and the published chloroplast genome of star anise. Although the chloroplast genomes are conserved among Illicium species, we found some divergent hotspot regions that can potentially exploit high resolution DNA mini-barcodes for species authentication. Subsequently, we used high resolution melting (HRM) assay to generate standard melting profiles of these mini-barcodes for these species. The melting data showed that the mini-barcodes derived from two divergence regions (i.e., atpF-atpH and ndhF-rpl32) have high resolution for species discrimination and could clearly distinguish star anise from four tested adulterants. We applied these two mini-barcodes to check twelve commercial products alleged or labelled as "star anise", which were purchased from China traditional markets and internet shops. The HRM curves of eleven samples are clustered with the reference star anise with confidence higher than 95%, indicating that these products contain the correct species as declared. We also found one toxic adulterant, as its melting curve clustered with Illicium simonsii. We conclude that DNA mini-barcode derived from divergent hotspot region of chloroplast genome combined with HRM analysis is an effective and rapid strategy to discriminate star anise from its toxic lookalike adulterants.