Himalayan Coffee arabica is a unique and highly prized coffee species in the world, grown in the picturesque region of Gulmi, Nepal. As the demand for specialty coffees continues to rise globally, ensuring the authenticity and traceability of this exclusive coffee variety has become paramount. In this study, we employed DNA barcoding using three gene loci, namely rbcL, matK, and ITS, to molecularly authenticate Himalayan Coffee arabica and establish its distinctiveness for international recognition. Leaf samples were collected from multiple Himalayan Coffee arabica plants cultivated in the Gulmi region of Nepal. DNA extraction and PCR amplifications were performed for the rbcL, matK, and ITS gene regions, following standard laboratory protocols. The resulting amplicon was sequenced and analyzed using bioinformatics tools of MEGA for phylogenetic and iBOLD reference databases to identify the species and assess genetic variability. Our findings revealed high sequence similarity among all the Himalayan Coffee arabica samples for the targeted gene loci, validating the suitability of rbcL, matK, and ITS as robust DNA barcodes for molecular authentication with 99% similarity. Phylogenetic analysis further demonstrated the clear delineation of Himalayan Coffee arabica from closely related coffee species, confirming its uniqueness and distinct genetic identity. This study highlights the importance of DNA barcoding in safeguarding the authenticity of Himalayan Coffee arabica. Ultimately, the successful molecular authentication of this unique coffee species will foster its international recognition and contribute to the preservation and appreciation of Himalayan Coffee arabica.
Keywords: Coffee arabica, rbcL, matK, ITS, MEGA, iBOLD, and Molecular authentication.