A genomic approach for accurate identification and authentication of Alpinia galanga (L.) Willd. using SSH and NGS technologies

The authenticity of herbal products is a growing global concern due to frequent mislabeling, substitution, and adulteration, especially among high-value medicinal plants. Alpinia galanga (A.galanga) is widely used in traditional medicine to treat various ailments. However, it is frequently substituted with Alpinia calcarata (A.calcarata) , which can compromise the safety and therapeutic efficacy of herbal formulations. Currently, no standardised method exists that is both rapid, sensitive, and cost-effective for distinguishing A. galanga from A. calcarata. Therefore, our study aimed to identify differential metabolites and employ suppression subtractive hybridisation (SSH) coupled with next-generation sequencing (NGS) in A. galanga and A. calcarata to develop species-specific DNA markers for the accurate authentication of A. galanga . The plant samples were collected and authenticated through taxonomic identification and DNA barcoding. Untargeted metabolite profiling was performed using UPLC–MS/MS. SSH, coupled with NGS, was used to develop species-specific markers, which were validated using PCR and Sanger sequencing. Taxonomic identification and DNA barcoding were successful, and the sequences were submitted to NCBI GenBank. A total of 152 metabolites were identified, and 50 significant differential metabolites were identified between A. galanga and A. calcarata , indicating chemical distinctions. The developed species-specific DNA markers demonstrated high sensitivity and specificity in distinguishing between the two species, and validation in commercial Rasna raw drug samples revealed adulteration. This study demonstrates the use of the SSH-NGS platform to develop species-specific DNA markers and provides a reliable tool for the authenticati on of A. galanga in both fresh plant materials and commercial products.