Abstract
Difengpi, derived from the air-dried stem bark of Illicium difengpi and enlisted in the Chinese Pharmacopoeia for its therapeutic effect against common ailments, confronts challenges due to dwindling wild resources and intentional substitution with potentially harmful botanical relatives. The imperative need to authenticate this herbal remedy has led to the development of robust methods. Here, we integrated chloroplast mini-barcoding and high resolution melting (HRM) analysis to distinguish Difengpi from the reported adulterants. We assembled the complete chloroplast (cp) genomes of I. difengpi and substituted close relatives I. jiadifengpi and I. majus, and conducted an in-depth comparative analysis to screen divergent regions for exploiting as DNA mini-barcodes. Despite the conservativeness characterizing the whole cp genomes among these Illicium species, we identified some highly variable regions with promising potential as molecular markers for species identification. Subsequently, we designed DNA mini-barcodes and subjected them to HRM analysis to assess their efficacy in species discrimination. Melting profiles unveiled that mini-barcodes designed from four divergence regions trnL-trnF, trnF-ndhJ, ycf1-ndhF and rpl32-trnL exhibited substantial discriminatory power, distinctly differentiated I. difengpi from I. jiadifengpi, I. majus and I. verum. We tested ten commercially available Difengpi products from online stores and local traditional markets using these four mini-barcodes. All ten samples clustered closely with the reference I. difengpi with genotype confidence higher than 93 %, indicating the presence of the claimed species in these samples, sans any reported toxic adulterants. Consequently, we simulated Difengpi mimic samples utilizing I. jiadifengpi, I. majus and I. verum and subjected them to evaluate the practicability of these mini-barcodes. The outcomes confirmed the precision of the four mini-barcodes in accurately discerning the mimic samples. In conclusion, integrating taxon-specific DNA mini-barcodes with HRM analysis is an efficient strategy for the authentication of species identity within commercial herbal products.