Mitochondrial genome of Isatis indigotica reveals repeat-mediated recombination and phylogenetic insights in Cruciferae

<i>Isatis indigotica</i> is an important medicinal plant extensively used in traditional Chinese medicine for its antiviral and anti-inflammatory properties. While previous studies have elucidated its nuclear and plastid genomes, the mitochondrial genome (mitogenome) -critical for understanding organellar evolution, intracellular DNA transfer, and stress response mechanisms-has remained uncharacterized. Here, we present a complete <i>de novo</i> assembly and comprehensive analysis of the <i>I. indigotica</i> mitogenome, generated using high-fidelity long-read sequencing technologies. The circular mitogenome spans 260,864 bp and encodes 31 protein-coding genes, 21 transfer RNAs, and 3 ribosomal RNAs. Repetitive sequences constitute 12.3% of the genome, with large repeats mediating homologous recombination and generating alternative conformations. A total of 488 RNA editing sites were identified, predominantly of the cytidine-to-uridine (C-to-U) type, indicating extensive post-transcriptional modification. We also detected 36 regions homologous to the plastid genome, reflecting active inter-organellar DNA transfer. Codon usage analysis revealed a preference for A/U-ending codons, and Ka/Ks analysis suggested strong purifying selection in most mitochondrial genes. Phylogenomic analysis based on 24 conserved mitochondrial genes placed <i>I. indigotica</i> in close proximity to <i>Brassica</i> species, supporting its taxonomic placement within the Brassicaceae family and aligning with plastid-based phylogenies. This study provides the first complete mitogenome of <i>I. indigotica</i>, offering valuable insights into mitogenome architecture, RNA editing dynamics, and plastid-mitochondrial interactions, while contributing to broader evolutionary and genomic understanding of cruciferous medicinal plants.