Mitochondrial Genome Analysis of <i>Isatis tinctoria</i> L. (Brassicaceae) Reveals Strengthened Purifying Selection Resulting From Recombination‐Driven Gene Duplication

Plant mitochondrial genomes exhibit remarkable structural diversity and active recombination, yet their influence on the molecular evolutionary patterns of functional genes requires further empirical evidence across diverse lineages. Here, we assembled and annotated the complete mitochondrial genome of <i>Isatis tinctoria</i> L. (Brassicaceae), a phylogenetically informative species. The circular genome is 260,912 bp in length and contains 65 genes, including a pair of gene duplication events, <i>ccmB</i> and <i>trnK-UUU</i>, situated between large direct repeats. Comparative analysis across 73 mitochondrial genomes-including 71 Brassicaceae accessions and 2 outgroups-revealed that genome size is significantly correlated with SSR abundance. This result supports the "repeat-driven genome expansion" hypothesis. Using the <i>ccmB</i> gene as a representative, pairwise Ka/Ks analysis across 2850 species pairs revealed that Ka is significantly correlated with both Ks and GC content, suggesting that regional mutation rates and nucleotide composition jointly shape nonsynonymous substitution rates. Notably, species with <i>ccmB</i> gene copies displayed significantly lower Ka values than those without, whereas Ks values remained unchanged. This supports a "functional redundancy-purifying selection" model, in which gene duplication enhances system robustness and strengthens purifying selection on essential genes. Together, this study highlights the interplay among repeat architecture, recombination, and evolutionary constraint in the mitochondrial genome of <i>I. tinctoria</i>, and provides new insights into the structural evolution and selective dynamics of plant mitochondrial genomes.