Long-Read Evidence for Repeat-Associated Structural Heterogeneity in the Mitochondrial Genome of Rhododendron spinuliferum

Background/Objectives: Plant mitochondrial genomes are structurally dynamic, and assembled paths may not reflect all molecular configurations. We characterized the mitochondrial genome of Rhododendron spinuliferum and evaluated repeat-associated structural heterogeneity and transcript support for mitochondrial RNA processing. Methods: Public PacBio HiFi reads were assembled and curated; Illumina reads from a second individual provided cross-individual sequence support. Candidate local connections were assessed by strict single-read two-junction phasing. Gene content, repeats, plastid-derived sequences, comparative genomics, phylogeny, predicted C-to-U RNA-editing sites, and mature exon junctions were also examined. Results: Three representative assembly paths totaled 786,593 bp and contained 32 protein-coding loci, three rRNA genes, and 19 tRNA types. A total of 226 HiFi reads supported four local configurations associated with two repeat pairs, and all four persisted across 24 filtering combinations. Nine mitochondrial plastid DNA transfer blocks covered 6.77 kb. Public RNA-seq data provided read-level support for 211 of 475 predicted editing sites and all 12 examined mature exon junctions of nad1, nad2, and nad5. Across six congeners, 99.7% of resolved homologous positions corresponding to RNA-supported sites retained genomic C. Conclusions: These results support repeat-associated local structural heterogeneity in the R. spinuliferum mitogenome and provide a curated DNA- and RNA-supported resource for comparative Rhododendron mitochondrial genomics.