Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species

Background Medicago is an economically important forage genus widely distributed across China, yet its mitochondrial genomes remain poorly characterized. Comprehensive analysis of mitochondrial genome structure, function, and evolution is essential for uncovering plant biological mechanisms, enhancing germplasm utilization, and supporting molecular breeding efforts. Results Here, we assembled and compared the mitochondrial genome of eight Medicago species, including six newly sequenced genomes. Our results revealed that five species possess typical single circular mitochondrial genome structures, while M. falcata , M. platycarpos , and M. sativa exhibit complex multipartite circular conFigureurations. These mitochondrial genome sizes ranged from 281,240 to 356,577 bp, containing 55–74 functional genes. Repetitive sequence analysis identified 141 simple sequence repeats (SSRs) and 76 tandem repeats (TSRs), dominated by A/T-rich mononucleotide motifs, while dispersed repeats were mainly 30–49 bp in length. Codon usage analysis showed strong A/T bias and a preference for leucine, serine, and isoleucine. RNA editing sites were predominantly C-to-U substitutions, primarily located at the first and second codon positions. Phylogenetic reconstruction based on 31 conserved mitochondrial protein-coding genes (PCGs) strongly supported the monophyly of Medicago and resolved interspecific relationships consistent with previous chloroplast and nuclear genome studies. Most PCGs were under purifying selection, whereas a few genes, such as matR , exhibited signals of positive selection, suggesting lineage-specific adaptive evolution. Conclusions Altogether, this study enriches the mitochondrial genomic resources of Medicago and deepens the understanding of its structural evolution and phylogenetic relationships, providing valuable insights for evolutionary and functional studies in Fabaceae plants.