Integrated application of CBDP, iPBS, and SCoT markers with DNA barcoding enables effective classification of Chinese prickly ash (Huajiao)

Huajiao (Chinese prickly ash, CPA), referring to the edible-pericarp species within the genus Zanthoxylum , is cultivated as a valuable medicinal and aromatic plant whose industrial standardization is impeded by ambiguous classification, inconsistent nomenclature, and genetic admixture, particularly between Zanthoxylum bungeanum and Z. simulans . To clarify taxonomic relationships, we assessed 109 germplasm accessions of Zanthoxylum —encompassing key CPA species and outgroups—using three dominant molecular marker systems (CBDP, iPBS, SCoT) and DNA barcoding (ITS2, trn H- psb A). Amplification with 36 selected primers yielded 841 polymorphic bands (with a PPB of 100% among the retained primers). iPBS markers demonstrated superior informativeness over CBDP and SCoT. Mantel tests confirmed significant pairwise correlations among all systems, validating their combined reliability. Cluster and population structure analyses delineated two primary genetic clusters: Cluster I dominated by Z. bungeanum , Z. simulans , and Z. piasezkii ; Cluster II dominated by Z. armatum , Z. schinifolium , and Z. acanthopodium . ITS2 barcoding corroborated Z. armatum as a monophyletic clade, whereas trn H- psb A revealed geographic substructure within Z. armatum corresponding to Yunnan, Shaanxi-Hubei, and Sichuan populations. Crucially, integrated marker and ITS2 data robustly supported ambiguous local varieties (“Choujiao”, “Qiujiao”, “Fuguhuajiao”, “Goujiao”, “Bayuejiao”) and Shandong germplasm as closely related to Z. simulans , resolving persistent taxonomic confusion. This study establishes a coherent molecular framework for accurate classification and identification of CPA germplasm, providing a critical foundation for resource standardization and sustainable utilization of CPA germplasm resources.