Molecular marker development and phylogenetic analysis of Aconitum species based on chloroplast genomes

The genus Aconitum is composed of more than 350 species that are generally toxic, including some valuable medicinal resources. However, the classification and identification of this genus present challenges due to abundant species diversity, similar botanical structure, and wide distribution range. In this study, the complete chloroplast (CP) genomes of eight Aconitum species were newly assembled and annotated, and combined with 70 downloaded genome sequences to clarify the taxonomic and phylogenetic relationships. The results showed that the genome length ranged from 155,614 to 157,332 bp, and a typical circular quadripartite structure was formed consisting of a small single copy (SSC) and a large single copy (LSC) separated by two inverted repeats (IRs). The whole genome sequences exhibited relative conservation across species in terms of structure, GC content, codon usage, and gene composition. Mutation sites were primarily located in LSC and SSC areas while non-coding regions showed higher variability than the coding regions. Through global sequence alignment, the fragment combination of clpP and ndhC_trnV-UAC were selected as an auxiliary molecular marker for the differentiation of specific species in Aconitum. In addition, the chloroplast genomes were recommended as super DNA barcodes for the identification of closely related species due to their higher resolution. Phylogenetic analysis divided the Aconitum genus into three taxa, which supported the separation of A. gymnandrum as an independent taxon. In summary, the increasing availability of CP genome resources will deepen the understanding of the classification and systematic evolution of Aconitum, and the development of assisted molecular markers holds great significance in ensuring the safety and accuracy of clinical applications.