Plant mitochondrial genomes are difficult to analyze because of their structural dynamism and frequent annotation errors. To address these challenges, we first constructed a high-confidence mitochondrial reference library for Rhodiola by integrating transcriptomic evidence, public sequence resources, and experimental validation. This curated resource defined 30 mitochondrial protein-coding genes (PCGs), including corrected exon–intron boundaries and validated 5′-terminal variants in ccmC, ccmFn, and nad9. Leveraging this curated dataset, we developed the RhoMitoAnnotator, which integrates three novel algorithms, EBAnno, REAnno, and NCAnno, to accurately annotate trans-splicing, RNA editing, and non-canonical start/stop codons. Using long-read sequencing guided by the RhoMitoAnnotator, we completed the mitogenomes of R. rosea, R. crenulata, and R. sacra, systematically re-annotated seven publicly available mitogenomes, revealing cross-chromosomal gene arrangement, and widespread structural misannotations. To enable scalable analysis with short-read data, we built Polypods, an integrated pipeline that successfully assembled mitochondrial PCGs from 108 samples across 39 Rhodiola species, and identified variant genes, stop codon-lacking regions in nad6, and internal stop codons in rpl16. Phylogenetic analyses based on mitochondrial and chloroplast PCGs showed a lineage pattern consistent with the hypothesis of an evolutionary transition from hermaphroditism to dioecy in Rhodiola, and consistently supported six species as monophyletic lineages. Overall, this study provides a curated mitochondrial gene atlas for Rhodiola and a reference-guided analytical framework for mitochondrial PCG annotation and recovery in this genus, with potential adaptability to other plant lineages after lineage-specific database construction and parameter optimization.
Zang et al. (Fri,) studied this question.