{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T15:10:14Z","timestamp":1779117014728,"version":"3.51.4"},"reference-count":50,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key project at the central government level: The ability establishment of sustainable use for valuable Chinese medicine resources","award":["2060302"],"award-info":[{"award-number":["2060302"]}]},{"name":"Research on Artificial Breeding of Rare and Endangered Chinese Medicinal Materials and Scale-up Demonstration Project of Chinese Medicine Administration","award":["BJTH-2024-02F-0008"],"award-info":[{"award-number":["BJTH-2024-02F-0008"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2013intron boundaries and validated 5\u2032-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.<\/jats:p>","DOI":"10.3390\/ijms27104440","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T14:40:31Z","timestamp":1779115231000},"page":"4440","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["RhoMitoAnnotator and Polypods, Bioinformatics Tools for the Rhodiola Mitochondrial Gene Assembly, Annotation and Phylogenetic Analysis"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2882-3685","authenticated-orcid":false,"given":"Erhuan","family":"Zang","sequence":"first","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"},{"name":"School of Pharmacy, Minzu University of China, Beijing 100081, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanda","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tingyu","family":"Ma","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Minzu University of China, Beijing 100081, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dengxiu","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lingchao","family":"Zeng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaozhe","family":"Yi","sequence":"additional","affiliation":[{"name":"School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peigen","family":"Xiao","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lijia","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linchun","family":"Shi","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China"},{"name":"Engineering Research Center of Chinese Medicine Resource, Ministry of Education, Beijing 100193, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1725-2293","authenticated-orcid":false,"given":"Jinxin","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Minzu University of China, Beijing 100081, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1146\/annurev-arplant-043015-112232","article-title":"Plant Mitochondrial Genomes: Dynamics and Mechanisms of Mutation","volume":"68","author":"Gualberto","year":"2017","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1111\/tpj.15495","article-title":"Plant mitochondria-past, present and future","volume":"108","author":"Rasmusson","year":"2021","journal-title":"Plant J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.2741\/4531","article-title":"Plant mitochondrial DNA","volume":"22","author":"Morley","year":"2017","journal-title":"Front. Biosci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1040","DOI":"10.1111\/tpj.14578","article-title":"Plant organellar RNA editing: What 30 years of research has revealed","volume":"101","author":"Small","year":"2020","journal-title":"Plant J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"754","DOI":"10.1016\/j.tplants.2023.12.014","article-title":"Plant organellar genomes: Much done, much more to do","volume":"29","author":"Wang","year":"2024","journal-title":"Trends Plant Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"uhae023","DOI":"10.1093\/hr\/uhae023","article-title":"PMAT: An efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data","volume":"11","author":"Bi","year":"2024","journal-title":"Hortic. Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Mili\u00e1n-Garc\u00eda, Y., Hempel, C.A., Janke, L.A.A., Young, R.G., Furukawa-Stoffer, T., Ambagala, A., Steinke, D., and Hanner, R.H. (2022). Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae). BMC Genom., 23.","DOI":"10.1186\/s12864-022-08743-x"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1111\/jse.13003","article-title":"Plastomic data shed new light on the phylogeny, biogeography, and character evolution of the family Crassulaceae","volume":"61","author":"Liu","year":"2023","journal-title":"J. Syst. Evol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1002\/med.21564","article-title":"Rhodiola species: A comprehensive review of traditional use, phytochemistry, pharmacology, toxicity, and clinical study","volume":"39","author":"Tao","year":"2019","journal-title":"Med. Res. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1111\/jse.13149","article-title":"Integrated analyses reveal extensive cytonuclear discordance and two new members of Rhodiola","volume":"63","author":"Huang","year":"2025","journal-title":"J. Syst. Evol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Liu, J., Zang, E., Tian, Y., Zhang, L., Li, Y., Shi, L., Xu, L., and Xiao, P. (2024). Comparative chloroplast genomes: Insights into the identification and phylogeny of rapid radiation genus Rhodiola. Front. Plant Sci., 15.","DOI":"10.3389\/fpls.2024.1404447"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1080\/07388551.2021.1901648","article-title":"Gauging the trends of pseudogenes in plants","volume":"41","author":"Garewal","year":"2021","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3304","DOI":"10.1093\/nar\/22.16.3304","article-title":"Translation of nad9 mRNAs in mitochondria from Solanum tuberosum is restricted to completely edited transcripts","volume":"22","author":"Grohmann","year":"1994","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1007\/s004380050761","article-title":"Cis-and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria","volume":"258","author":"Gualberto","year":"1998","journal-title":"Mol. Gen. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1007\/BF00352014","article-title":"Characterization of the Brassica campestris mitochondrial gene for subunit six of NADH dehydrogenase: Nad6 is present in the mitochondrion of a wide range of flowering plants","volume":"23","author":"Nugent","year":"1993","journal-title":"Curr. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lenz, H., Hein, A., and Knoop, V. (2018). Plant organelle RNA editing and its specificity factors: Enhancements of analyses and new database features in PREPACT 3.0. BMC Bioinform., 19.","DOI":"10.1186\/s12859-018-2244-9"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"W65","DOI":"10.1093\/nar\/gkz345","article-title":"CPGAVAS2, an integrated plastome sequence annotator and analyzer","volume":"47","author":"Shi","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e191","DOI":"10.1002\/imt2.191","article-title":"SeqKit2: A Swiss army knife for sequence and alignment processing","volume":"3","author":"Shen","year":"2024","journal-title":"Imeta"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Lang, B.F., Beck, N., Prince, S., Sarrasin, M., Rioux, P., and Burger, G. (2023). Mitochondrial genome annotation with MFannot: A critical analysis of gene identification and gene model prediction. Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1222186"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1818","DOI":"10.1038\/s41477-023-01538-2","article-title":"Targeted knockout of a conserved plant mitochondrial gene by genome editing","volume":"9","author":"Forner","year":"2023","journal-title":"Nat. Plants"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"977","DOI":"10.1038\/s42003-025-08418-9","article-title":"Evolution of mitochondrial RNA editing sites and stop codon-lacking transcripts in angiosperms","volume":"8","author":"Yu","year":"2025","journal-title":"Commun. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3676","DOI":"10.1093\/nar\/gkm270","article-title":"Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: T-elements contribute to 5\u2032 and 3\u2032 end formation","volume":"35","author":"Forner","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1007\/BF00312637","article-title":"Editing of rps3\/rpl16 transcripts creates a premature truncation of the rpl16 open reading frame","volume":"23","author":"Sutton","year":"1993","journal-title":"Curr. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Varr\u00e9, J.-S., D\u2019Agostino, N., Touzet, P., Gallina, S., Tamburino, R., Cantarella, C., Ubrig, E., Cardi, T., Drouard, L., and Gualberto, J.M. (2019). Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20194788"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5907","DOI":"10.1093\/nar\/gkg795","article-title":"The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): Comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana","volume":"31","author":"Handa","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4944","DOI":"10.1111\/pbi.70249","article-title":"Advance in the assembly of the plant mitochondrial genomes using high-throughput DNA sequencing data of total cellular DNAs","volume":"23","author":"Ni","year":"2025","journal-title":"Plant Biotechnol. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1093\/aob\/mcab133","article-title":"Comparative phylogeography study reveals introgression and incomplete lineage sorting during rapid diversification of Rhodiola","volume":"129","author":"You","year":"2022","journal-title":"Ann. Bot."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.ympev.2014.04.013","article-title":"Rapid radiation and dispersal out of the Qinghai-Tibetan Plateau of an alpine plant lineage Rhodiola (Crassulaceae)","volume":"77","author":"Zhang","year":"2014","journal-title":"Mol. Phylogenet. Evol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6960","DOI":"10.1073\/pnas.97.13.6960","article-title":"Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates","volume":"97","author":"Palmer","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1104\/pp.111.190231","article-title":"A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes","volume":"158","author":"Bentolila","year":"2012","journal-title":"Plant Physiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e2317240121","DOI":"10.1073\/pnas.2317240121","article-title":"Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA","volume":"121","author":"Zwonitzer","year":"2024","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e2206076119","DOI":"10.1073\/pnas.2206076119","article-title":"Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification","volume":"119","author":"Wang","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5169","DOI":"10.1093\/jxb\/erae034","article-title":"Methods for assembling complex mitochondrial genomes in land plants","volume":"75","year":"2024","journal-title":"J. Exp. Bot."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"W6","DOI":"10.1093\/nar\/gkx391","article-title":"GeSeq-versatile and accurate annotation of organelle genomes","volume":"45","author":"Tillich","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"7112","DOI":"10.1093\/nar\/gkq591","article-title":"Intron RNA editing is essential for splicing in plant mitochondria","volume":"38","author":"Castandet","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"101191","DOI":"10.1016\/j.xplc.2024.101191","article-title":"PMGA: A plant mitochondrial genome annotator","volume":"6","author":"Li","year":"2025","journal-title":"Plant Commun."},{"key":"ref_37","first-page":"555","article-title":"Applications and challenges of DNA barcoding in rapid radiation groups: Rhodiola (Crassulaceae) as a case study","volume":"17","author":"Liu","year":"2025","journal-title":"Chin. Herb. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"107863","DOI":"10.1016\/j.ympev.2023.107863","article-title":"Genomic data provide a robust phylogeny backbone for Rhodiola L. (Crassulaceae) and reveal extensive reticulate evolution during its rapid radiation","volume":"186","author":"Ren","year":"2023","journal-title":"Mol. Phylogenet. Evol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1111\/1755-0998.12616","article-title":"Modified low-salt CTAB extraction of high-quality DNA from contaminant-rich tissues","volume":"17","author":"Arseneau","year":"2017","journal-title":"Mol. Ecol. Resour."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1038\/s41587-019-0072-8","article-title":"Assembly of long, error-prone reads using repeat graphs","volume":"37","author":"Kolmogorov","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3350","DOI":"10.1093\/bioinformatics\/btv383","article-title":"Bandage: Interactive visualization of de novo genome assemblies","volume":"31","author":"Wick","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., and Madden, T.L. (2009). BLAST+: Architecture and applications. BMC Bioinform., 10.","DOI":"10.1186\/1471-2105-10-421"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: Pairwise alignment for nucleotide sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1093\/bioinformatics\/btq033","article-title":"BEDTools: A flexible suite of utilities for comparing genomic features","volume":"26","author":"Quinlan","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Wick, R.R., Judd, L.M., Gorrie, C.L., and Holt, K.E. (2017). Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol., 13.","DOI":"10.1371\/journal.pcbi.1005595"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"W59","DOI":"10.1093\/nar\/gkz238","article-title":"OrganellarGenomeDRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar genomes","volume":"47","author":"Greiner","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1186\/s13059-020-02154-5","article-title":"GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes","volume":"21","author":"Jin","year":"2020","journal-title":"Genome Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.1093\/bioinformatics\/btu033","article-title":"RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies","volume":"30","author":"Stamatakis","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e102","DOI":"10.1002\/cpbi.102","article-title":"Using SPAdes De Novo Assembler","volume":"70","author":"Prjibelski","year":"2020","journal-title":"Curr. Protoc. Bioinform."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/10\/4440\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T14:47:20Z","timestamp":1779115640000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/10\/4440"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,15]]},"references-count":50,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ijms27104440"],"URL":"https:\/\/doi.org\/10.3390\/ijms27104440","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,15]]}}}