Comparative and phylogenetic analysis of the chloroplast genomes of four commonly used medicinal cultivars of Chrysanthemums morifolium.

'Boju' and 'Huaiju' are cultivars of the Chrysanthemum (Chrysanthemum morifolium Ramat.) in the family Asteraceae, valued for their medicinal, tea, and ornamental properties, and valued by individuals. However, the yield and quality of medicinal chrysanthemums are limited by the characteristics of the germplasm resources, including the identification at the varieties and cultivation levels. Currently, research characterizing the chloroplast genomes of medicinal Chrysanthemum flowers is relatively limited. This study conducted chloroplast whole-genome sequencing on two cultivars of Chrysanthemum, 'Boju' and 'Huaiju', and compared them with the previously published chloroplast genomes of 'Hangbaiju' and 'Gongju'. The study analyzed the chloroplast genome structures of these four medicinal Chrysanthemums, identifying mutation hotspots and clarifying their phylogenetic relationships. The chloroplast genome sizes of four medicinal Chrysanthemum cultivation products ranged from 151,057 to 151,109 bp, with GC content ranging from 37.45% to 37.76%. A total of 134 genes were identified, including 89 protein-coding genes, 37 ribosomal RNA genes, and 8 transfer RNA genes. Comparative genomic analysis revealed 159 large repeat sequences, 276 simple sequence repeats, 1 gene, and 8 intergenic regions identified as highly variable regions. Nucleotide diversity (Pi) values were high (≥ 0.004) for the petN-psbM, trnR-UCU-trnT-GGU, trnT-GGU-psbD, ndhC-trnV-UCA, ycf1, ndhI-ndhG, trnL-UGA-rpl32, rpl32-ndhF, and ndhF-ycf1 fragments, aiding in variety identification. Phylogenetic analysis revealed consistent results between maximum likelihood and Bayesian inference trees, showing that the four medicinal Chrysanthemum cultivars, along with their wild counterparts and related species, evolved as a monophyletic group, forming a sister clade to Artemisia and Ajania. Among the six Chrysanthemum species, the wild Chrysanthemum diverged first (Posterior probability = 1, bootstrap = 1,000), followed by Ajania, while C. indicum and C. morifolium clustered together (Bootstrap = 100), indicating their closest genetic relationship. The chloroplast whole-genome data and characteristic information provided in this study can be used for variety identification, genetic conservation, and phylogenetic analysis within the family Asteraceae.