Abstract
BACKGROUND: Medicago is an economically important forage genus widely distributed across China, yet its mitochondrial genomes remain poorly understood. Comprehensive characterization of mitochondrial genome structure, function, and evolution is crucial for elucidating plant biological mechanisms, improving germplasm utilization, and advancing molecular breeding. RESULTS: We assembled and compared the mitochondrial genomes of eight Medicago species, including six newly sequenced genomes. Five species contained typical single circular mitochondrial genomes, whereas M. falcata, M. platycarpos, and M. sativa displayed complex multipartite circular conformations. Genome sizes ranged from 281,240 to 356,577 bp, with 55-74 functional genes. Repeat analysis identified 141 simple sequence repeats (SSRs) and 76 tandem repeats (TSRs), largely composed of A/T-rich mononucleotide motifs, while dispersed repeats were mainly 30-49 bp in length. Codon usage showed a strong A/T bias and a preference for leucine, serine, and isoleucine. RNA editing events were predominantly C-to-U substitutions, primarily occurring at the first and second codon positions. Phylogenetic reconstruction based on 31 shared mitochondrial protein-coding genes (PCGs) strongly supported the monophyly of Medicago and recovered interspecific relationships consistent with previous chloroplast and nuclear genome studies. Most PCGs were under purifying selection, while a few, such as matR, exhibited signatures of positive selection, suggesting lineage-specific adaptive evolution. CONCLUSIONS: This study expands the mitochondrial genomic resources of Medicago and provides new insights into its structural evolution and phylogenetic relationships, offering a valuable foundation for evolutionary and functional studies in Fabaceae.