# PCR-Based Molecular Authentication Method for Sources of Agrimoniae Herba via Comparative Analyses of Complete Chloroplast Genomes

## TL;DR

This study develops a PCR-based method to accurately identify Agrimonia species using their chloroplast genomes, improving herbal medicine quality control.

## Contribution

The novel contribution is the development of PCR-based molecular markers derived from chloroplast genome variations for species-specific authentication of Agrimoniae Herba.

## Key findings

- Nine PCR-based molecular markers were developed to distinguish four Agrimonia species based on chloroplast genome variations.
- Chloroplast genome sequences of Agrimonia species serve as effective super DNA barcodes for species identification.
- Phylogenetic analysis confirmed the monophyly of the genus Agrimonia and distinct clustering of species within the subtribe Agrimoniinae.

## Abstract

Accurate species identification is essential for the quality control and standardization of herbal medicines. Agrimonia species, the authentic sources of Agrimoniae Herba, have long been used in traditional medicine, yet limited genomic resources have hindered the establishment of reliable molecular approaches for accurate species discrimination within this genus. Here, we report the newly assembled complete chloroplast genomes (155,156–155,302 bp) of four Agrimonia species, which exhibit the typical quadripartite structure and contain 112 unique genes. Comparative analysis revealed 684 variable sites, including 497 single nucleotide polymorphisms (SNPs) and 187 insertions/deletions (InDels), predominantly located in the single-copy regions. Based on these species-specific variations, we developed nine PCR-based molecular markers that distinguished the four species. The markers were validated using herbarium specimens and commercial herbal products, demonstrating reproducibility and practical applicability. Phylogenetic analysis supported the monophyly of the genus Agrimonia and resolved each species into distinct clusters within the subtribe Agrimoniinae. These results showed that chloroplast genome sequences of the genus Agrimonia can serve as effective super DNA barcodes for species identification. Our study provides fundamental genomic resources for Agrimonia and reliable molecular tools for species authentication, providing a basis for ensuring the authenticity and safety of Agrimoniae Herba.