Characterization and Comparative Analysis of the Actinidia rufa Mitochondrial Genome

Background/Objectives: Actinidia rufa provides a wild kiwifruit resource for examining mitochondrial sequence conservation and structural variation. We examined the hypothesis that coding-sequence (CDS) coordinates retain higher interspecific alignment coverage than non-CDS coordinates despite differences in block organization. We also assessed reference support, repeat-associated junctions, and mitochondrial gene-tree concordance. Methods: NC_086781.1 was evaluated using PacBio HiFi reads (CRR3429456), uniform repeat-junction screening, six interspecific one-to-one comparisons, repeat and mitochondrial plastid DNA (MTPT) masking, and single-gene trees. Results: The 1,022,459 bp reference contains 39 protein-coding loci. Across six comparisons, CDS coverage was 99.90–100%, versus 68.94–87.79% for non-CDS sequence; adjusted circular-shift P values were 0.001674–0.008745. Homologous sequence occurred in 32–61 merged blocks per comparison, and separate repeat and MTPT masks retained 94.39–98.29% and 93.60–99.39% of baseline aligned reference sequence, respectively. Stringent competitive mapping gave a mean depth of 40.35× and breadth of 73.9% at ≥1×. Of 192 repeat pairs ≥50 bp, 41 were evaluable and none met the reciprocal-alternative-support rule; R43 and R47 each had one-sided single-read support. Eight of 16 informative gene trees contained the A. rufa–A. chinensis split, but phylogenetic information was limited. Conclusions: The comparisons support higher CDS than non-CDS coordinate coverage within a reference set with variable block organization. Repeat-junction support and single-gene resolution remain limited; the data do not establish ongoing recombination, an exclusive circular physical architecture, or a mitochondrial contribution to waterlogging tolerance.