ultivar-resolved assembly and characterization of the mitochondrial genome of Medicago sativa cv. AH reveal repeat-mediated alternative junctions and extensive RNA editing

Background : Plant mitochondrial genomes are characterized by extensive structural dynamics driven by repeat-mediated recombination, widespread RNA editing, and plastid-to-mitochondrion DNA transfer (MTPT). However, cultivar-resolved mitochondrial genome assemblies that integrate structural validation, RNA editing landscapes, and phylogenetic context remain limited in Medicago sativa , an important legume forage crop. Results : We assembled the complete mitochondrial genome of Medicago sativa cv. AH using PacBio HiFi long reads and a graph-resolving workflow. The circular mitogenome was 300,797 bp in length with a GC content of 45.36%, encoding 33 unique protein-coding genes, 16 tRNA genes, and three rRNA genes. Repeat analysis identified 59 simple sequence repeats, nine tandem repeats, and 112 pairs of dispersed repeats. A 4,354 bp long direct repeat was confirmed by junction PCR and Sanger sequencing to mediate homologous recombination, generating both master-circle and alternative subgenomic configurations. Six MTPT fragments totaling 725 bp were detected, harboring five intact plastid-derived tRNA genes. Maximum-likelihood phylogenetic analysis based on 23 conserved protein-coding genes placed cv. AH within the M. sativa clade with strong support (bootstrap = 96), yet synteny analysis revealed substantial rearrangements in gene-block order and orientation among intraspecific mitogenome assemblies. A total of 468 high-confidence C-to-U RNA editing sites were predicted across all 33 protein-coding genes, with nonsynonymous edits accounting for 95.73% of total events. The gene nad4 harbored the most editing sites (45), and the most frequent amino acid conversions were Ser→Leu (107) and Pro→Leu (104). Representative editing sites were experimentally validated by RT-PCR and Sanger sequencing, including the restoration of a canonical AUG start codon in nad4L through ACG-to-AUG editing. Conclusions : This study provides a cultivar-resolved mitochondrial genomic resource for M. sativa cv. AH that integrates structural isoform validation, MTPT characterization, phylogenetic placement, synteny-level rearrangement analysis, and an experimentally supported RNA editing landscape within a single framework. These findings advance our understanding of mitochondrial genome dynamics in alfalfa and provide a foundation for future investigations into cytoplasmic diversity and breeding applications.