Super-barcoding of four Agrimonia species distributed in Korea based on complete plastid genomes and nuclear ribosomal DNAs.

The genus Agrimonia is widely distributed throughout temperate regions and includes species used in traditional medicine in Asia and Europe. However, their accurate identification is often challenging because the vegetative parts used, such as leaves and roots, are morphologically highly similar across species. To investigate the genetic diversity of Agrimonia species commonly distributed and traded in Korea and to develop reliable molecular tools for species authentication, we collected 36 samples primarily representing four Agrimonia species (A. pilosa, A. coreana, A. nipponica, and A. eupatoria). We sequenced and assembled complete plastid genomes (plastomes) and 45S nuclear ribosomal DNA (nrDNA) sequences from these four species. The assembled plastomes ranged from 155,128-155,313 bp, while the nrDNA sequences spanned 5,860-5,873 bp. Phylogenetic analyses based on both plastome and nrDNA datasets revealed that each species formed a distinct clade, demonstrating clear genetic differentiation among taxa. Based on plastome sequence variations, we developed eight plastome-based super-barcoding markers and validated their reliability using 36 Agrimonia accessions, including an additional closely related congeneric accession, A. gorovoii. The markers successfully classified samples into species-specific haplotype groups. This plastome-based super-barcoding approach provides a practical molecular authentication method for major Agrimonia species used as medicinal resources in Korea, thereby facilitating quality control and accurate utilization of Agrimonia materials.