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Article

De Novo Hybrid Assembly of the Tripterygium wilfordii Mitochondrial Genome Provides the Chromosomal Mitochondrial DNA Structure and RNA Editing Events

by
Yisha Cai
1,2,†,
Suxin Yang
1,†,
Haimei Chen
2,
Yang Ni
2,
Jingling Li
2,
Jinghong Zhang
1,* and
Chang Liu
2,*
1
School of Medicine, Huaqiao University, Quanzhou 362021, China
2
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(15), 7093; https://doi.org/10.3390/ijms26157093
Submission received: 25 May 2025 / Revised: 6 July 2025 / Accepted: 15 July 2025 / Published: 23 July 2025
(This article belongs to the Collection Feature Papers in Molecular Informatics)
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Abstract

Tripterygium wilfordii has extremely important pharmaceutical value in both traditional and modern medicine. The mitogenome of T. wilfordii was subjected to assembly and annotation with Nanopore long reads and Illumina short reads in this study. The mitogenome is 720,306 bp in length and is responsible for encoding 55 specific genes, including 35 protein-coding genes (PCGs), 17 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes. Upon repetitive sequence analysis, 223 simple sequence repeats (SSRs), 24 long tandem repeats (LTRs), and 47 dispersed repetitive sequences (DRSs) were identified. The 24 common PCGs were used for phylogenetic analysis, which revealed that T. wilfordii is more closely related to Euonymus alatus. Moreover, mitochondrial plastid DNA (MTPT) analysis revealed eight MTPTs in the mitochondrial genome. Furthermore, 600 RNA-editing sites were detected in the protein-coding genes according to RNA-seq results. Among these genes, the ccmB gene contained the greatest number of sites, followed by the nad4 gene. This is the first study to report the T. wilfordii mitogenome and illustrate its linear structure. The findings of this study will help elucidate the evolution of the T. wilfordii mitogenome and facilitate its potential application in genetic breeding.

1. Introduction

Tripterygium wilfordii Hook. f. is a perennial twining shrub belonging to the Celastraceae family and is distributed widely across East Asia [1]. The species is recognized as a Chinese medicinal herb that is used for managing various autoimmune conditions, such as rheumatoid arthritis and systemic lupus erythematosus [2,3]. T. wilfordii has numerous secondary metabolites, including triptolide and celastrol, which possess anti-inflammatory, immuno-suppressive, and antitumor activities and, therefore, have great development potential in the field of modern medicine [4,5]. Moreover, triptolide and celastrol are important in managing central nervous system disorders, such as Parkinson’s disease and Alzheimer’s disease [6,7]. Celastrol can also be used for the treatment of metabolic disturbances, such as high-fat-diet-induced obesity and type 2 diabetes [8,9]. High-throughput sequencing substantially contributes to the study of plant transcriptomes and genome architecture and is also useful when exploring plant genetic variations and secondary metabolites at the molecular level. Many transcriptome and genome studies conducted to date have revealed the key enzyme-encoding genes in the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, which regulate the biosynthesis of triptolide and celastrol through transcriptome data, as well as various transcription factors or miRNAs that regulate the biosynthesis of secondary metabolites in T. wilfordii, such as the negative regulation of triptolide biosynthesis by MYC2 [10]. In addition, the high-quality reference T. wilfordii genome has been illustrated, highlighting the regulatory role of CYP450 in the biosynthesis of triptolide [11]. The elucidation of the complete T. wilfordii chloroplast genome will be useful for molecular-assisted breeding in future studies [1]. However, the T. wilfordii mitochondrial genome sequence remains unreported to date, greatly limiting subsequent studies on this species.
In plants, the mitochondrial genome, which has many kinds of rearrangements, can be quite complex, although it may be used for various beneficial applications. First, as the mitochondrial genome has an extremely high rate of evolution, it has been extensively used as a molecular marker in modern molecular biology research, such as for reconstructing the phylogenetic relationships of species. Second, as cellular organelles, mitochondria are responsible for generating ATP and various metabolites, and the mitochondrial release of the metabolites of the tricarboxylic acid (TCA) cycle controls cell activity and fate [12]. Third, the plant mitogenome is huge and has a complicated structure and genetic diversity, although the stability of a plant mitogenome is protected through certain DNA repair mechanisms, particularly homologous recombination (HR) and base excision repair (BER) [13]. Finally, RNA editing, such as the conversion of cytidines (C) to uridines (U), which is important for the normal functioning of protein-encoding genes, is frequently detected in plant mitochondria [14]. In addition, T. wilfordii has long been used for asexual reproduction, resulting in many problems, such as confusion in terms of variety, serious degradation, and a low content of active ingredients. Consequently, exploring the T. wilfordii mitochondrial genome is necessary. This work, therefore, involved the sequencing and assembly of the complete T. wilfordii mitochondrial genome; analysis of the potential T. wilfordii mtDNA structure; and comparative analyses of repetitive sequences, homologous recombination, phylogenetic relationships, and RNA editing for this species. The findings of this study are expected to contribute to the further exploration of scientific issues encountered in relevant research at the molecular level and lay a certain theoretical basis for the genetic engineering and breeding of medicinal plants.

2. Results

2.1. Graph-Based Mitogenome and General Genomic Features

The hybrid assembly method was used to construct the T. wilfordii mitogenome by adopting long/short reads. First, 10 Gb of Illumina short reads and 7 Gb of Nanopore long reads of the total DNA were sequenced (Table A1). GetOrganelle software (v1.7.0) was then used for extracting and elongating the mitochondrial genome short reads, and SPAdes software (v3.8.2) in Unicycle was usyed for assembly into a unitig graph using the whole-genome sequencing results (Figure 1a). Moreover, Illumina short reads together with Nanopore long reads were used for calculating the sequencing depth at each genomic locus, thereby validating the accuracy of the assembly. The sequencing depth remained consistent among the genomic locations, without detectable gaps (Figure A1a,b). The T. wilfordii mitochondrial genome was revealed to have 15 contigs, including 4 double-bifurcation structures (Table 1 and Figure 1a). The longest contig was contig 3, with a length of 186,151,728 bp, and the shortest contig was contig 8, a double-bifurcation structure with a length of 1728 bp. Second, the Nanopore long reads resolved into double-bifurcation structures, with each contig merged using Bandage software (v0.8.1) (Figure 1b). Finally, one chromosome was obtained independently. The Nanopore long reads were then used to validate the T. wilfordii mitochondrial genome structure (Figure A2a–e).
The T. wilfordii mitochondrial genome was revealed to contain one chromosome 720,306 bp in length. The GC content was 45.54% (Figure 2), and it was revealed to be closely related to the Euonymus alatus mitochondrial genome in terms of size (genome size: 1,045,106 bp; NCBI accession number: NC_053921.1). In addition, the T. wilfordii mitochondrial genome was annotated as having a total of 61 genes (55 unique genes), including 35 PCGs, 22 tRNA genes (17 unique genes), and 4 rRNA genes (3 unique genes). The PCGs were classified into 10 functional groups (Table 2). The annotation of the core genes in the T. wilfordii mitochondrial genome was completed, revealing three variable gene types, including seven small subunit ribosome genes (rps1, rps10, rps12, rps13, rps19, rps3, and rps4), four large subunit ribosome genes (rpl10, rpl16, rpl2, and rpl5), and one subunit succinate dehydrogenase gene (sdh4).

2.2. Tandem Repeat Analysis

SSRs, also referred to as short tandem repeats or microsatellites, generally represent repetitive DNA sequences containing 1–6 nucleotides [15,16]. SSRs have been extensively used as genetic markers in forensics, disease diagnosis, and population genetics [17]. This work used the MISA online platform [18] for predicting the SSRs present in the T. wilfordii mitogenome. A total of 223 SSRs were identified on the chromosome of the mitogenome, including 66 monomers, 49 dimers, 29 trimers, 70 tetramers, 7 pentamers, and 2 hexamers (Table A2). These identified SSRs might be crucial molecular markers for subsequent analyses. LTRs, in which the repeat units contain ≥7 nucleotides, are crucial for numerous biological events, such as translation, transcription, promoter activity regulation, genome evolution, and chromosome stability [19,20,21]. The LTRs in the T. wilfordii mitochondrial genome were predicted using the Tandem Repeats Finder program [22] in this study. A total of 24 LTRs were predicted from the results (Table S1). LTR1 presented the greatest number of repeat units at 48 bp, whereas LTR16 was only 13 bp long.

2.3. Recombinations Mediated by Repetitive Sequences

Dispersed repetitive sequences (DRSs), or scattered repetitive sequences, are repeated at multiple positions across a genome and are used for determining biological evolution, individual identification, and disease diagnosis [23]. Furthermore, DRSs are crucial for determining mitogenome structural variations [24]. In this study, ROUS Finder 2.0.py [25] was used for predicting DRSs. A total of 47 DRSs were detected in the T. wilfordii mitochondrial genome, including 18 palindromic repetitive sequences and 29 direct repetitive sequences (Table 3). The DBSs were then compared to these DRSs, revealing four DBS sequences similar to DRS02–DRS05 (refer to Table 3, Figure S1, and Table S2). First, every repetitive sequence pair with 500 bp flanking sequences was isolated and used to create reference sequences representing the four possible conformations to be recombined. Second, the 47 DRSs were assessed for their recombination frequency with Nanopore long reads, revealing that no recombined conformation was supported by the Nanopore long reads (Table S2). Using the conformation as the baseline, it was determined that T. wilfordii has no four repetitive sequence-mediated recombinations.

2.4. Analysis of the Homologous Sequences Between Plastids and Mitochondria

Mitochondrial plastid DNAs (MTPTs) refer to plastid-derived DNA fragments in mitochondrial genomes [26]. These sequences originate from rare inter-organellar DNA transfer events, where fragments of the chloroplast genome are inadvertently integrated into the mitochondrial genome. Such transfers can cause misattribution of organelle DNA in taxonomic studies and complicate genome assembly/annotation, owing to chimeric origins. Therefore, the rigorous identification of MTPTs is essential for resolving these complexities. MTPTs have been reported in numerous species, such as Coffea arabica [27], Salvia miltiorrhiza [28], and Caragana spinosa [29]. In this study, the dataset adopted in the mitochondrial genome (OR538545.1/NC_082972.1) assembly was used for assembling the T. wilfordii chloroplast genome (OR538544.1), which is 156,692 bp long, with a GC content of 37.47%. The chloroplast genome included 131 genes (111 unique genes), comprising 87 PCGs (78 unique genes), 36 tRNA genes (29 unique genes), and 8 rRNA genes (4 unique genes) (Table S3 and Figure 3).
A total of eight MTPTs were identified in T. wilfordii by comparing the complete mitochondrial DNA with the chloroplast DNA from T. wilfordii using BLAST with default parameters [28]. These eight MTPTs were 3297 bp long, accounting for 0.46% of the entire mitochondrial genome. Among these, MTPT1 had the greatest length (1048 bp), whereas MTPT8 had the smallest length (30 bp). MTPT2 and MTPT7 were located in the inverted repeat (IR) region of the chloroplast genome (Figure S2). In addition, the locations of the eight MTPTs within the chloroplast and mitochondrial genomes are provided in Table S3 and Figure 3. In order to investigate the functions of these eight MTPTs, the DNA fragments were subjected to annotation, revealing that the MTPTs contained complete and functional ptDNA-encoded genes (trnW–CCA, trnP–UGG, trnS–GGA, and trnM–CAU) and partial-protein-encoding genes (psbD, psbC, and rpl2) (Figure 4 and Table S3). In order to further confirm that total MTPTs were present, Nanopore long reads were aligned to the reference sequences containing MTPTs as well as to the MTPT sequence and the corresponding 1000 bp upstream/downstream sequences. The mapping results revealed that the Nanopore long reads supported all eight MTPTs (Figure 4).

2.5. Phylogenetic Analysis

The NCBI nucleotide database has 136 mitochondrial genomes for Malpighiales, 2 for Celastrales, and 251 for Fabales. In order to analyze the phylogenetic relationships among the nine Celastrales and Malpighiales species, phylogenetic trees were created using the CDSs among the 24 common PCGs in the mitochondrial genomes (atp1, atp4, atp6, atp8, atp9, ccmB, ccmC, cox1, cox2, cox3, cob, matR, mttB, nad1, nad2, nad3, nad4, nad5, nad6, nad7,nad9, rps3, rps4, and rps12). It was found that T. wilfordii and Euonymus alatus were clustered under 100 bootstrap supports (Figure 5). These results showed that T. wilfordii and Euonymus alatus are more closely related. Additionally, each node showed a bootstrap support of >90, suggesting that these nine mitochondrial genomes of Celastrales and Malpighiales species have strongly reliable phylogenetic relationships.

2.6. RNA-Editing Event Analysis

RNA editing is an important supplement to the central dogma, and it differs from the corresponding DNA templates and exerts critical effects on various processes, including chloroplast and mitochondrial biogenesis, hormone and stress responses, and seed growth [29,30]. C-to-U conversion within plant organelles is the major RNA-editing pattern [30]. In this study, a total of RNA editing sites were identified in the T. wilfordii mitogenome following RNA-seq analysis (Table 4 and Figure S3). The false positives in the analysis of mitochondrial genomic polymorphic sites were eliminated by identifying the SNPs using the DNA-seq analysis of the identical samples of RNA-seq data. Three overlaps, cox2–698, cox2–721, and nad6–26, were obtained through the comparison of the predicted RNA-editing sites and SNP sites (Table S4 and Figure S3). Consequently, 600 RNA-editing sites were obtained from the T. wilfordii mitogenome (Table S4), among which 99 (16.50%) and 501 (83.50%) sites were altered, resulting in 99 synonymous and 501 non-synonymous codons, respectively. Variations in the number of non-synonymous codons for the Ser, Pro, and Arg amino acids were observed, among which 116 (19.33%) RNA-editing sites had Ser altered to Leu, 112 (8.67%) had Pro altered to Leu, 72 (12.00%) had Ser altered to Phe, 38 (6.33%) had Pro altered to Ser, 35 (5.83%) had Arg altered to Cys, and 34 (5.67%) had Arg altered to Trp (Table S4 and Figure 6a). The RNA-editing events within T. wilfordii mitochondria occurred mostly at the first and second codon base positions, accounting for 30.83% (185) and 52.67% (316) of the overall RNA-editing sites, respectively. Additionally, the RNA-editing events occurred in the most unique PCGs within the T. wilfordii mitochondrial genome. Among these genes, ccmB represented the highest number of RNA-editing sites (n = 45), followed by nad4 with 42 sites (Figure 6b). Notably, RNA-editing events resulted in the generation of both start and stop codons. Specifically, codon changes at positions atp6-718, atp9-223, and rps19-7 led to the conversion of CAA to TAA and CGA to TGA, effectively producing stop codons.

3. Discussion

3.1. Overview of the T. wilfordii Mitochondrial Genome

T. wilfordii is a traditional Chinese medicine that exhibits excellent medicinal effects in terms of curing nephrotic syndrome, rheumatoid arthritis, and systemic lupus erythematosus; in addition, this species has been extensively adopted in China as a folk medicine [31]. Acquiring the genomic data of this species constitutes a crucial step toward comprehending its active constituents’ physiological traits and biosynthetic processes. In this study, using hybrid assembly and annotation methods involving Illumina short reads and Oxford Nanopore long reads, the complete T. wilfordii mitogenome was identified. Subsequently, the gene contents and numbers, SSRs, TRSs, DRSs, homologous sequences, and RNA-editing events of the genome were determined. Finally, the phylogenetic relationships of the genome with eight Celastrales and Malpighiales species were explored according to the conserved PCGs in the mitochondrial genomes. The mitogenome of T. wilfordii will serve as a reference for investigating the genomes of other plants in the Celastraceae family and for Tripterygium mitogenome evolution and diversity research.

3.2. Architecture of One Molecule of the T. wilfordii Mitogenome

Plant mitogenome assembly is generally achieved and presented in circular maps according to the extensively accepted concept among living scientists that plant mitochondrial DNA exists mainly as molecules of circular genomes [32]. However, the physical structure of the real mitochondrial genome may involve different circles, complex branching structures, and linear molecules [33]. The results of this study revealed that the T. wilfordii mitochondrial genome is composed of one molecule that is 720,306 bp in length (Figure 1a,b). The molecular configuration, as depicted in Figure 1a, is characterized by the presence of two distinct bubble structures, one large and one small. In contrast, Figure 1b illustrates a linear structure. However, the linear structure is supported by the nanopore long reads (Figure A1 and Figure A2). The mitochondrial genomes of plants usually exhibit diverse alternative conformations because of the presence of many repetitive sequences [34,35]. According to this study, the T. wilfordii mitochondrial genome does not undergo recombination mediated by repetitive sequences. By extracting repetitive elements along with their 500 bp flanking regions as reference sequences and mapping them onto Nanopore long reads, it was confirmed that no recombination conformations associated with repetitive sequences are present in the T. wilfordii mitochondrial genome.

3.3. Research Trends for the T. wilfordii Mitogenome

The mitogenomes of plants differ significantly in length compared to chloroplast genomes due to frequent exchange with nuclear and chloroplast DNA [36,37]. In this study, the MTPTs of T. wilfordii were identified. These MTPTs were then subjected to functional annotation. MTPTs in the T. wilfordii mitochondrial genome included four complete and functional ptDNA-encoded genes (trnW–CCA, trnP–UGG, trnS–GGA, and trnM–CAU) and three partial-protein-coding genes (psbD, psbC, and rpl2). Furthermore, MTPT2 and MTPT7 were localized to the inverted repeat (IR) region of the chloroplast genome. Nonetheless, MTPT2, the partial rpl2, could be detected within the chloroplast and mitochondrial genomes. Thus, it was impossible to precisely determine whether MTPT3 originated from the T. wilfordii chloroplast or mitochondrial genome.
RNA editing, an extensive phenomenon observed in the organelles of higher plants, is used in several processes with different mechanisms that change the nucleotide sequences of RNA molecules so that the RNAs are different from the corresponding gene sequences [38,39]. In addition, RNA-editing events frequently occur at the first and second codon positions within the plant mitochondrial genome. In the T. wilfordii mitochondrial genome, this study identified 600 RNA-editing sites with alterations in one base, producing synonymous and non-synonymous amino acid codons, which in turn affected the translation of proteins. Ser, Pro, and Arg were the most frequently altered amino acids resulting from non-synonymous codon changes. Among the identified RNA-editing sites, only 99 (16.50%) occurred at the third codon position, while 501 (83.50%) were located at the first or second positions, indicating a predominant localization of editing events at the first and second codon positions. Furthermore, most PCGs in T. wilfordii contained RNA-editing sites, with the exception of rps13. These RNA-editing events can modify and refine genetic information, enhance the diversity of gene products, and thereby contribute to plant evolutionary adaptation.

4. Materials and Methods

4.1. Plant Materials, DNA and RNA Isolation, and Sequencing

Fresh T. wilfordii leaf samples were obtained from the nursery of the medical school of Huaqiao University (Quanzhou, China). Thereafter, a DNA extraction kit and an RNAprep Pure Plant Plus Kit (Tiangen Biotech, Beijing, China) were used to extract the total DNA and RNA, respectively, which were preserved at −80 °C until use. Specifically, the extracted total DNA was used for Illumina sequencing and Oxford Nanopore sequencing (Table A1). For Illumina sequencing, 1 µg of the genomic DNA was used to construct a DNA library with the NEBNext library building kit, followed by Illumina 2500 platform sequencing (Illumina, San Diego, CA, USA). For Oxford Nanopore sequencing, a 10 kb DNA library was established prior to sequencing using a PromethION sequencer (Oxford Nanopore Technologies plc, Oxford, UK). The TruSeq Stranded mRNA Library Prep kit (Illumina) was used to construct a strand-specific RNA-seq library. Illumina sequencing was performed using PE150 sequencing for the quantified library.

4.2. Genome Assembly, Annotation, and Validation

GetOrganelle software (3.8.2)was used to complete the organelle genome assembly [40] In this study, the parameters “–R 15–k 21,45,65,85,105–F embplant_pt” were adopted for assembling the chloroplast genome. However, mitochondria-derived nuclear and plastid DNA can result in false positives during mitogenome polishing [41]. Thus, mitochondrial genome assembly was completed using the single hybrid assembly strategy. First, the parameters “–R 20 –k 21,45,65,85,105 –P 1000,000 –F embplant_mt” were used to obtain mitochondrial short reads using GetOrganelle (3.8.2) [40]. Second, long and short reads were assembled through de novo assembly into a unitig graph using SPAdes [42], miniasm, and Racon packages [43] included in Unicycler software (v0.5.0). Finally, using Unicycler software, the Nanopore long reads were adopted to address the double-bifurcating structures (DBSs) in the unitig graph [44]. The bandage software was applied to visualize the connections of contigs to allow for the manual removal of nodes derived from the chloroplasts and nuclei [45]. CPGAVAS2(v2.0) [46] was used for chloroplast genome annotation, whereas the CPGView(v1.0) web server [47] was used for potential annotation errors in the chloroplast genome”. However, the annotation of the mitogenome was completed using the GeSeq [48] and PMGA (http://www.1kmpg.cn/mgavas/, accessed on 14 July 2025) web servers. Thereafter, Apollo software (v1.11.8) [49] was used for the manual correction of the annotation results. The chloroplast and mitochondrial genome structures were subsequently drawn with PMGmap (http://www.1kmpg.cn/pmgmap, accessed on 14 July 2025). Finally, the organelle genome sequences and the annotations were deposited into GenBank (accession nos. OR538544.1 and OR538545.1 (NC_082972.1) for the chloroplast and mitochondrial genomes, respectively) [50].

4.3. Identification of Tandem Sequences

MISA [18] and Tandem Repeats Finder [22] are two web servers that are used to analyze simple sequence repeats (SSRs) and long tandem repeats (LTRs), respectively. SSRs, also referred to as short tandem repeats or microsatellites, were analyzed with MISA, using the thresholds of below 10, 5, 4, and 3 units for mononucleotides, dinucleotides, trinucleotides, and tetra-/penta-/hexa-nucleotides, respectively. Moreover, LTRs were predicted with Tandem Repeats Finder using the default parameters of 7 and 2 for mismatches/indels, which were matched, respectively, with a maximal period size of 500 and a minimal alignment score of 50.

4.4. Identification of Repeat-Mediated Recombination

In order to identify repeat-mediated recombination, the dispersed repeat sequences of the mitochondrial genome were detected with ROUS Finder 2.0.py [25]. Thereafter, the repetitive sequences and their bilateral flanking sequences (500 bp) were extracted; these two sequences corresponded to one conformation. Later, the two sequences were integrated in silico to generate the sequences associated with the other conformations that were recombined. Next, the Nanopore long reads were mapped to four DNA sequences in two conformations, followed by counting the number of mapped reads with BWA (v0.7.17) [51] and SAMtools (v1.17) [52]. Finally, IGV software (v.2.8) was adopted to visualize the read mapping results [53]. In order to further confirm the T. wilfordii mitochondrial genome structure, the initial and terminal 1000 bp sequences of the genome were extracted and concatenated. Next, 1000 bp sequences were extracted and concatenated from the junction of contig10 and contig11, the junction of contig6 and contig12, and the junction of contig14 in the Tripterygium wilfordii mitochondrial genome. Thereafter, the Nanopore long reads were mapped to these DNA sequences using BWA (v0.7.17) [51] and SAMtools (v1.17) [52]. Finally, IGV software (v.2.8) [53] was adopted to visualize the read mapping results and verify the T. wilfordii mitochondrial genome structure.

4.5. Identification of Mitochondrial Plastid Sequences (MTPTs)

Plastid genomes are transferred to plant mitogenomes to generate MTPTs, which can influence the complexity of mitochondrial genomes and can induce the false-positive DNA barcoding paradox [54,55]. Therefore, MTPTs were identified in this study by comparing the cpgenome (OR538544.1) with the mitogenome (OR53 8545.1/NC_08 2972.1) of T. wilfordii using BLASTN software (v2.2.30+) [56] with default parameters. In order to confirm the identified MTPTs, the MTPT sequence and its upstream/downstream sequences (1000 bp) were obtained to create the reference genome. Thereafter, using default parameters, the Nanopore long reads were aligned to these reference sequences with BWA software (v0.7.17) [52]. Finally, IGV software (v.2.8 ) was used to visualize the Nanopore long-read mapping results to the MTPT regions [53]. The Circos package of TB tools (V1.098) was then applied to visualize the MTPT results [57,58].

4.6. Phylogenetic Tree Construction

A total of 9 mitochondrial genomes of Celastrales and Malpighiales species were used in the phylogenetic analysis, and Caragana spinosa (OQ785640.1) was selected as the out group. The following mitochondrial genomes were downloaded from the NCBI GenBank database: Tripterygium wilfordii (OR538545.1/NC_082972.1), Euonymus alatus (N C_053921.1), Populusdavidiana (NC_035157.1), Populusrotundifolia (MW566588.1), Salixsu chowensis (NC_029317.1), Passiflora edulis (NC_050950.1), Hevea brasiliensis (AP014526.1), Manihot esculenta (NC_0451 36.1), and Jatropha curcas (OQ603497.1). For phylogenetic analysis, the common coding sequences (CDSs) among the afore-stated species were obtained with PhyloSuite software3 (v.1.2.1) [59] and then aligned using MAFFT software4 (v7.505) [60]. Using the maximum likelihood approach, the aligned sequences were subsequently used to construct a phylogenetic tree with IQ-Tree(v2.1.4-beta) [61]. UFBoot was then used for performing bootstrap analysis involving 1000 replicates [62]. Finally, iTOL (https://itol.embl.de/, accessed on 14 July 2025) was used for visualizing the resulting phylogenetic tree [63].

4.7. RNA-Editing Site Identification in the PCGs of the T. wilfordii Mitogenome

In this study, to identify the RNA-editing sites within the T. wilfordii mitogenome, strand-specific RNA-seq data were mapped to 100 bp of the sequences in 35 PCGs and the corresponding 3′and 5′ flanking regions using BWA (Burrows–Wheeler Alignment tool) [51]; moreover, reference sequence-matching alignment reads were obtained with SAMtools (v1.17) [52]. Thereafter, REDItools [64] was used to predict the RNA-editing sites using specific parameters of frequency ≥ 0.1 and coverage ≥ 5 [15]. In order to increase the prediction accuracy, the RNA-editing sites were visualized using IGV software (v.2.8 ) [53], with a special focus on sites with frequency > 0.2. In order to exclude the impact of single-nucleotide polymorphism (SNP) sites on the findings, SNPs were also predicted, and RNA-editing sites overlapping with the results of the SNP analysis were eliminated. Similarly, when the SNPs of the T. wilfordii mitogenome were identified, the Illumina short reads in the whole genome were mapped to 100 base pairs of CDSs in the PCGs, the corresponding 3′and 5′ flanking regions were mapped with BWA software (v0.7.17) [51], and isolated alignment reads matching the reference sequence were mapped with SAM tools (v1.17) [52]. Subsequently, the SNP sites were analyzed using REDItools (http://code.google.com/p/reditools/m accessed on 14 July 2025) [64], with parameters requiring frequency ≥ 0.1 and coverage ≥ 5.

5. Conclusions

This study is the first to report the T. wilfordii mitochondrial genome according to the sequences obtained from Illumina short reads and Nanopore long reads. The T. wilfordii mitogenome is a unique chromosome. This study revealed the general features of the T. wilfordii mitochondrial genome through various comparative analyses, including repetitive sequence analysis, recombination analysis, homologous sequence analysis, phylogenetic analysis, and RNA-editing event analysis. Furthermore, it was found that the T. wilfordii mitochondrial genome exhibits no recombination conformation mediated by repetitive sequences.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms26157093/s1.

Author Contributions

H.C. conceived the study; Y.N. and J.L. assembled the mitogenome; Y.C. and S.Y. annotated the genome, performed the comparative analysis and wrote the manuscript; C.L. and J.Z. reviewed the manuscript critically. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Science & Technology Fundamental Resources Investigation Program of China (2018FY100705), guiding projects in Fujian Province (2018Y0062) and Natural Science Foundation of Xiamen, China(3502Z202373044). The funders were not involved in the study design, data collection, data analysis, decision to publish, or manuscript preparation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The organelle sequences supporting the conclusions of this article are available in GenBank (https://www.ncbi.nlm.nih.gov/, accessed on 14 July 2025), with accession numbers: OR538545.1/NC_082972.1 (mitogenome) and OR538544.1 (plastome). The sample was deposited in the Institute of Medicinal Plant Development (Beijing, China), with accession number 2023050143. The raw data have been released through GenBank, with the following accession numbers: BioProject PRJNA1067867, BioSample SAMN 39554832, and SRA database SRR27708480 (Nanopore DNA reads) and SRR27708481 (Illumina DNA reads).

Conflicts of Interest

The authors declare that they have no competing and conflicting interests.

Abbreviations

The following abbreviations are used in this manuscript:
PCGprotein-coding gene
tRNAtransfer RNA
rRNAribosomal RNA
SSRsimple sequence repeat
LTRlong tandem repeat
DRSdispersed repetitive sequence
MTPTmitochondrial plastid DNA
CDScoding sequence
MVAmevalonate
MEPmethylerythritol phosphate
TCAtricarboxylic acid
HRhomologous recombination
BERbase excision repair
Ccytidines
Uuridines
DBSdouble-bifurcating structure
SNPsingle-nucleotide polymorphism
MCmitochondrial chromosome
IRinverted repeat

Appendix A

Table A1. Statistic summary of the sequencing data generated by Nanopore and Illumina platforms.
Table A1. Statistic summary of the sequencing data generated by Nanopore and Illumina platforms.
Sequencing DescriptorSequencing Platform
IlluminaNanopore
Total number of nucleotides13,177,504,5007,231,843,596
Total number of reads87,850,030316,356
Mean read length (bp)15022,859.8
Table A2. Statistics of simple sequence repeats in the T. wilfordii mitochondrial genome.
Table A2. Statistics of simple sequence repeats in the T. wilfordii mitochondrial genome.
IDSSR TypeSSRSizeStartEnd
SSR1p4(TCAT)31239043915
SSR2p3(TGC)41214,55214,563
SSR3p2(GA)51018,25818,267
SSR4p3(TTC)41221,60321,614
SSR5p4(TTCA)31222,66922,680
SSR6p1(T)101024,47624,485
SSR7p2(GA)51026,94726,956
SSR8p1(A)111130,36830,378
SSR9p1(A)101036,14036,149
SSR10p4(CTTA)31242,12842,139
SSR11p2(TA)61249,29249,303
SSR12p2(AT)51055,85355,862
SSR13p1(A)121256,24556,256
SSR14p2(CT)51056,50956,518
SSR15p4(ATCA)31259,47059,481
SSR16p3(CTT)41263,29463,305
SSR17p1(T)121297,61097,621
SSR18p2(TA)612101,015101,026
SSR19p2(TA)510102,475102,484
SSR20p1(T)1010102,491102,500
SSR21p1(A)1313102,503102,515
SSR22p1(T)1010103,640103,649
SSR23p1(A)1010104,561104,570
SSR24p5(TTTCT)315108,551108,565
SSR25p4(ATAG)312108,894108,905
SSR26p4(AGAA)312114,292114,303
SSR27p4(TAAG)312114,945114,956
SSR28p1(A)1111116,756116,766
SSR29p1(T)1010118,096118,105
SSR30p2(CT)510118,358118,367
SSR31p2(CT)510122,012122,021
SSR32p3(CAA)412125,275125,286
SSR33p4(CTGT)312127,624127,635
SSR34p2(CT)510128,277128,286
SSR35p1(T)1010130,920130,929
SSR36p3(TAT)412140,781140,792
SSR37p2(TA)510149,059149,068
SSR38p1(T)1212149,539149,550
SSR39p1(T)1010149,968149,977
SSR40p4(GGCA)312151,469151,480
SSR41p1(A)1212160,470160,481
SSR42p5(ACTAG)315160,484160,498
SSR43p4(TTCT)312162,501162,512
SSR44p1(A)1010163,156163,165
SSR45p4(TCTT)312170,024170,035
SSR46p2(TC)510172,086172,095
SSR47p2(AG)510173,151173,160
SSR48p4(ATTT)312177,791177,802
SSR49p2(AT)612178,785178,796
SSR50p4(CTTT)312179,931179,942
SSR51p1(T)1010179,992180,001
SSR52p1(C)1111182,801182,811
SSR53p2(CT)510186,353186,362
SSR54p1(A)1010186,457186,466
SSR55p4(TTCT)312187,055187,066
SSR56p1(A)1010188,497188,506
SSR57p1(T)1010191,228191,237
SSR58p1(T)1111192,751192,761
SSR59p4(GGCG)312197,572197,583
SSR60p3(CTT)412198,276198,287
SSR61p1(A)1111198,567198,577
SSR62p4(AGCC)312200,534200,545
SSR63p4(AGAC)312204,756204,767
SSR64p3(CTT)412205,202205,213
SSR65p1(G)1010215,294215,303
SSR66p1(A)1010218,839218,848
SSR67p4(AAAG)312226,751226,762
SSR68p4(GCCG)312231,013231,024
SSR69p4(CAAG)312234,577234,588
SSR70p2(GA)510239,681239,690
SSR71p4(GCAA)312250,096250,107
SSR72p4(TCTT)312250,875250,886
SSR73p3(CTT)412253,352253,363
SSR74p4(TACT)312253,737253,748
SSR75p2(AT)816261,274261,289
SSR76p4(GTGA)312262,669262,680
SSR77p2(AT)714265,650265,663
SSR78p1(A)1010266,208266,217
SSR79p1(A)1111266,771266,781
SSR80p3(TTA)412275,552275,563
SSR81p1(A)1010276,586276,595
SSR82p2(TC)510291,363291,372
SSR83p4(AAAG)312291,575291,586
SSR84p3(CAA)412293,222293,233
SSR85p4(AAAC)312296,155296,166
SSR86p4(TCTT)312307,547307,558
SSR87p2(TC)510317,136317,145
SSR88p3(TCT)412319,730319,741
SSR89p4(TCTT)312320,202320,213
SSR90p4(TCAA)312325,791325,802
SSR91p2(AT)510329,975329,984
SSR92p2(CT)510335,795335,804
SSR93p2(TC)510336,109336,118
SSR94p2(TC)612340,581340,592
SSR95p3(GAT)412340,901340,912
SSR96p2(TA)612342,602342,613
SSR97p4(TCAG)312344,772344,783
SSR98p3(TCT)412347,832347,843
SSR99p4(TTCT)312348,543348,554
SSR100p4(AGAA)312354,135354,146
SSR101p2(AG)510354,778354,787
SSR102p3(GGA)412357,969357,980
SSR103p2(TA)510360,398360,407
SSR104p4(CCTT)312362,330362,341
SSR105p3(CTT)412366,171366,182
SSR106p4(GAGT)312366,513366,524
SSR107p2(TA)510376,772376,781
SSR108p4(GAAT)312383,703383,714
SSR109p4(CTTT)312384,018384,029
SSR110p2(CT)510389,799389,808
SSR111p5(TTAGA)315390,482390,496
SSR112p3(CTG)515391,010391,024
SSR113p1(T)1010395,390395,399
SSR114p3(CTT)412402,562402,573
SSR115p3(AGA)412403,830403,841
SSR116p2(CT)510404,825404,834
SSR117p4(CTAT)312408,871408,882
SSR118p4(GCTA)312409,575409,586
SSR119p1(A)1010411,626411,635
SSR120p1(A)1313412,695412,707
SSR121p4(TTCT)312413,246413,257
SSR122p1(A)1010413,768413,777
SSR123p4(ATAG)312415,985415,996
SSR124p2(CT)510416,396416,405
SSR125p2(CT)510423,720423,729
SSR126p2(AT)918429,578429,595
SSR127p2(GA)510433,555433,564
SSR128p2(AG)510433,863433,872
SSR129p1(A)1111437,201437,211
SSR130p1(T)1414440,277440,290
SSR131p5(AAAGA)315450,941450,955
SSR132p4(GGAT)312452,636452,647
SSR133p4(GGAG)312453,553453,564
SSR134p4(GAAA)312455,204455,215
SSR135p5(TCTAT)315456,202456,216
SSR136p1(A)1414456,223456,236
SSR137p2(AT)510458,251458,260
SSR138p2(AT)510458,267458,276
SSR139p3(TAA)412463,152463,163
SSR140p4(CTTT)312470,520470,531
SSR141p1(G)1010472,879472,888
SSR142p1(A)1010476,424476,433
SSR143p3(ACT)412480,790480,801
SSR144p1(A)1010481,798481,807
SSR145p3(TCC)412485,671485,682
SSR146p1(A)1111487,453487,463
SSR147p2(AT)612491,773491,784
SSR148p1(A)1111495,422495,432
SSR149p1(T)1010495,583495,592
SSR150p2(TA)510497,184497,193
SSR151p1(T)1010501,990501,999
SSR152p4(TTTC)312510,724510,735
SSR153p4(AAGA)312514,161514,172
SSR154p1(T)1212514,486514,497
SSR155p2(AT)510514,761514,770
SSR156p3(TTA)412516,658516,669
SSR157p1(A)1010519,246519,255
SSR158p1(A)1111520,785520,795
SSR159p1(A)1010521,711521,720
SSR160p2(TC)510527,030527,039
SSR161p4(TCTA)312527,038527,049
SSR162p4(TAGA)312527,050527,061
SSR163p3(CTA)412527,121527,132
SSR164p1(A)1010528,215528,224
SSR165p4(AGCC)312528,751528,762
SSR166p4(AAAG)312537,670537,681
SSR167p1(T)1010553,153553,162
SSR168p4(AAGC)312556,534556,545
SSR169p4(AACA)312563,478563,489
SSR170p1(T)1010566,427566,436
SSR171p1(A)1313570,844570,856
SSR172p6(ATCTAT)318574,227574,244
SSR173p1(A)1111578,831578,841
SSR174p1(A)1212581,826581,837
SSR175p4(AATG)312583,827583,838
SSR176p2(TA)612584,679584,690
SSR177p4(CTTT)312592,151592,162
SSR178p2(TC)510593,996594,005
SSR179p1(T)1010596,355596,364
SSR180p5(GTAAT)315599,235599,249
SSR181p1(A)1111600,150600,160
SSR182p5(GCCCA)315603,387603,401
SSR183p1(T)1010606,766606,775
SSR184p1(A)1313611,183611,195
SSR185p6(ATCTAT)318614,566614,583
SSR186p4(ATTC)312623,581623,592
SSR187p4(TTAG)312625,006625,017
SSR188p4(TTTC)312625,633625,644
SSR189p1(T)1010626,533626,542
SSR190p1(T)1010626,867626,876
SSR191p1(T)1212627,314627,325
SSR192p1(T)1111627,327627,337
SSR193p3(GCT)412627,388627,399
SSR194p4(TCGA)312630,121630,132
SSR195p4(TTTC)312632,172632,183
SSR196p4(GAAT)312636,280636,291
SSR197p4(AGAA)312647,143647,154
SSR198p2(AG)510656,323656,332
SSR199p1(A)1010656,397656,406
SSR200p3(GGT)412657,497657,508
SSR201p2(GA)612657,832657,843
SSR202p3(GTT)515658,242658,256
SSR203p1(T)1111658,267658,277
SSR204p4(CTTG)312668,106668,117
SSR205p1(T)1010670,382670,391
SSR206p4(AAGA)312670,430670,441
SSR207p3(CTT)412676,856676,867
SSR208p2(CT)510684,284684,293
SSR209p4(CTTT)312686,396686,407
SSR210p4(ATTC)312689,697689,708
SSR211p4(AAAG)312694,379694,390
SSR212p1(C)1111697,246697,256
SSR213p2(CT)510697,892697,901
SSR214p1(A)1010698,667698,676
SSR215p1(T)1010699,203699,212
SSR216p4(TTAA)312699,408699,419
SSR217p4(AAGA)312700,384700,395
SSR218p4(GATA)312700,546700,557
SSR219p3(TCT)412701,847701,858
SSR220p3(TCC)412706,852706,863
SSR221p4(AAGC)312709,507709,518
SSR222p2(CT)510709,834709,843
SSR223p2(TC)510715,387715,396

Appendix B

Ijms 26 07093 g0a1
Figure A1. The coverage depth of the mitochondrial genome of T. wilfordii. The x-axis represents the genome position. The y-axis is the depth of those positions. (a) The coverage depth for Illumina short reads. (b) The coverage depth for Nanopore long read.
Figure A1. The coverage depth of the mitochondrial genome of T. wilfordii. The x-axis represents the genome position. The y-axis is the depth of those positions. (a) The coverage depth for Illumina short reads. (b) The coverage depth for Nanopore long read.
Ijms 26 07093 g0a1
Ijms 26 07093 g0a2aIjms 26 07093 g0a2b
Figure A2. The validation of structure of the mitochondrial genome of T. wilfordii. (a) The junction between initial and terminal (2000 bp), The initial is the start of contig11 and terminal is the end of contig12 (b) the junction between contig10 and start of contig11 (2000 bp), (c) the junction between contig11 and end of contig10 (2000 bp), (d) the junction between contig14 and contig6 (2000 bp), and (e) the junction between contig6 and contig12 (2000 bp).
Figure A2. The validation of structure of the mitochondrial genome of T. wilfordii. (a) The junction between initial and terminal (2000 bp), The initial is the start of contig11 and terminal is the end of contig12 (b) the junction between contig10 and start of contig11 (2000 bp), (c) the junction between contig11 and end of contig10 (2000 bp), (d) the junction between contig14 and contig6 (2000 bp), and (e) the junction between contig6 and contig12 (2000 bp).
Ijms 26 07093 g0a2aIjms 26 07093 g0a2b

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Figure 1. Unitig graph showing the T. wilfordii mitogenome. (a) Green and purple contigs are the mitochondrial chromosomes (MCs), whereas purple contigs are double-bifurcation structures (DBSs). (b) The graph after resolving the DBSs using long reads.
Figure 1. Unitig graph showing the T. wilfordii mitogenome. (a) Green and purple contigs are the mitochondrial chromosomes (MCs), whereas purple contigs are double-bifurcation structures (DBSs). (b) The graph after resolving the DBSs using long reads.
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Figure 2. A sketch map showing the T. wilfordii mitogenome. PMGmap (http://www.1kmpg.cn/pmgmap, accessed on 14 July 2025) was used for graph creation. This graph represents (1) the genes on both positive and negative strands; (2) the short tandem repeat distribution on the chromosome; (3) the long tandem repeat distribution on the chromosome; (4) the relationship of the dispersed repeat sequences; (5) the dispersed repeat sequence distribution on the chromosome, with yellow and green indicating the direct and inverted dispersed repeat sequences, respectively; (6) the scale coordinate axis; and (7) the GC content of the chromosome.
Figure 2. A sketch map showing the T. wilfordii mitogenome. PMGmap (http://www.1kmpg.cn/pmgmap, accessed on 14 July 2025) was used for graph creation. This graph represents (1) the genes on both positive and negative strands; (2) the short tandem repeat distribution on the chromosome; (3) the long tandem repeat distribution on the chromosome; (4) the relationship of the dispersed repeat sequences; (5) the dispersed repeat sequence distribution on the chromosome, with yellow and green indicating the direct and inverted dispersed repeat sequences, respectively; (6) the scale coordinate axis; and (7) the GC content of the chromosome.
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Figure 3. Mitochondrial plastid DNA MTPTs in the T. wilfordii mitogenome. The blue and green arcs represent the chloroplast and mitochondrial genomes, respectively. The green and pink arcs within the circle represent homologous regions in the chloroplast and mitochondrial genomes, respectively.
Figure 3. Mitochondrial plastid DNA MTPTs in the T. wilfordii mitogenome. The blue and green arcs represent the chloroplast and mitochondrial genomes, respectively. The green and pink arcs within the circle represent homologous regions in the chloroplast and mitochondrial genomes, respectively.
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Figure 4. A schematic representation of the T. wilfordii chloroplast genome. The graph was drawn using PMGmap (http://www.1kmpg.cn/pmgmap, accessed on 14 July 2025). For the graph, the following are represented from inside-out: (1) the distribution of the GC content on the chromosome and (2) the genes located on the negative strand and the positive strand.
Figure 4. A schematic representation of the T. wilfordii chloroplast genome. The graph was drawn using PMGmap (http://www.1kmpg.cn/pmgmap, accessed on 14 July 2025). For the graph, the following are represented from inside-out: (1) the distribution of the GC content on the chromosome and (2) the genes located on the negative strand and the positive strand.
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Figure 5. The NCBI nucleotide database has 136 mitochondrial genomes for Malpighiales, 2 for Celastrales, and 251 for Fabales. Phylogenetic relationships between T. wilfordii and nine additional species were inferred based on the sequences of 24 PCGs from their mitochondrial genomes. Phylogenetic trees were constructed using the maximum likelihood (ML) method. The numbers on the branches represent bootstrap values, indicating the support level for each node. GenBank accession numbers for the mitochondrial genomes are provided following the Latin names of the species, and the corresponding plant families are listed on the right side of the tree.
Figure 5. The NCBI nucleotide database has 136 mitochondrial genomes for Malpighiales, 2 for Celastrales, and 251 for Fabales. Phylogenetic relationships between T. wilfordii and nine additional species were inferred based on the sequences of 24 PCGs from their mitochondrial genomes. Phylogenetic trees were constructed using the maximum likelihood (ML) method. The numbers on the branches represent bootstrap values, indicating the support level for each node. GenBank accession numbers for the mitochondrial genomes are provided following the Latin names of the species, and the corresponding plant families are listed on the right side of the tree.
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Figure 6. Numbers of the RNA-editing sites within the T. wilfordii mitogenome. (a) The x-axis and y-axis represent the amino acids altered under RNA editing and the number of changed residues for each amino acid, respectively. (b) The x-axis and y-axis represent the number of genes affected by RNA editing and the number of RNA edits, respectively. Different colors indicate various families of genes.
Figure 6. Numbers of the RNA-editing sites within the T. wilfordii mitogenome. (a) The x-axis and y-axis represent the amino acids altered under RNA editing and the number of changed residues for each amino acid, respectively. (b) The x-axis and y-axis represent the number of genes affected by RNA editing and the number of RNA edits, respectively. Different colors indicate various families of genes.
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Table 1. Attribution of contig assembly within the T. wilfordii mitogenome. DBS: double-bifurcating structure.
Table 1. Attribution of contig assembly within the T. wilfordii mitogenome. DBS: double-bifurcating structure.
No.NameLength (bp)Bifurcation Structures
1contig171,533No
2contig2/DBS35483Yes
3contig3151,728No
4contig417,421No
5contig5/DBS15937Yes
6contig610,857No
7contig751,730No
8contig8/DBS41728Yes
9contig918,187No
10contig10140,923No
11contig1118,808No
12contig1286,897No
13contig13/DBS25600Yes
14contig1455,721No
15contig1549,130No
16contig169875Yes
Table 2. Genes predicted within the T. wilfordii mitogenome.
Table 2. Genes predicted within the T. wilfordii mitogenome.
Group of GenesNames of Genes
Subunit of ATPaseatp1, atp4, atp6, atp8, atp9
Cytochrome c biogenesisccmB, ccmC, ccmFN
Apocytochrome bcob
Subunit of cytochrome c oxidasecox1, cox2, cox3
Maturase RmatR
Transport membrane proteinmttB
Subunit of NADH dehydrogenasenad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, nad9
Small subunit of ribosomerps1, rps10, rps12, rps13, rps19, rps3, rps4
Large subunit of ribosomerpl10, rpl16, rpl2, rpl5
Subunit of succinate dehydrogenasesdh4
Transfer RNAstrnF–GAA(×2), trnN–GUU, trnQ–UUG, trnT–GGU, trnC–GCA, trnE–UUC, trnG–GCC(×2), trnK–UUU, tr nM–CAU, trnI–CAU, trnM–CAU (×2), trnP–UGG ×3), trnS–GCU, trnS–GGA, trnS–UGA, trnW–CCA, trnY–GUA
Ribosomal RNAsrrn18, rrn26(×2), rrn5
Table 3. Statistics of dispersed repetitive sequences in the T. wilfordii mitochondrial genome.
Table 3. Statistics of dispersed repetitive sequences in the T. wilfordii mitochondrial genome.
IDRepeat LengthRepeat TypeStart1End1Start2End2
DRS19875F565,608575,482605,947615,821
DRS25937F213,806219,742471,391477,327
DRS35600F86,89892,497141,628147,227
DRS45483P237,164242,646319,662314,180
DRS51728P529,058530,785550,700548,973
DRS6388P250,458250,845593,826593,439
DRS7295F289,376289,670341,859342,153
DRS8288F260,811261,098301,192301,479
DRS9241P73,19073,430252,355252,115
DRS10225P380,076380,300455,856455,632
DRS11200F338,753338,952536,718536,917
DRS12165F523,613523,777552,149552,313
DRS13138F444,909445,046682,863683,000
DRS14130F11,917120,46197,372197,501
DRS15125F19,46819,592163,717163,841
DRS16120P409,541409,422250,339250,458
DRS17111F503,030503,140716,487716,377
DRS18106F362,472362,577394,369394,474
DRS19101F159,986160,086700,671700,771
DRS2098P227,118227,215716,057715,960
DRS2185P701,099701,115300,221300,305
DRS2285F162,454162,538413,199413,283
DRS2373P553,929554,001586,127586,055
DRS2473P226,954227,026716,254716,182
DRS2573P61,72661,798590,179590,107
DRS2671F410,796410,866700,746700,816
DRS2770F248,538248,607482,163482,232
DRS2870P244,244244,313576,606576,537
DRS2967F87,18687,252141,916141,982
DRS3067F87,18687,252339,733339,799
DRS3167F141,916141,982339,733339,799
DRS3265P195,478195,542657,450657,386
DRS3364F91,72091,783146,450146,513
DRS3464F91,72091,783179,993180,056
DRS3564F146,450146,513179,993180,056
DRS3663F182,246182,308216,152216,214
DRS3763F182,246182,308473,737473,799
DRS3863F216,152216,214473,737473,799
DRS3961P291,156291,216504,253504,193
DRS4061F57,62257,68230,607530,6135
DRS4159F495,093495,151528,531528,589
DRS4258F47,03147,088693,744693,801
DRS4355P167,131167,185710,277710,223
DRS4453F195,516195,568312,661312,713
DRS4551F244,667244,717576,983577,033
DRS4650P442,609442,560248,466248,515
DRS4750P226,067226,116441,070441,021
Table 4. List of single-nucleotide polymorphisms (SNPs) detected in the PCGs of the T. jasminoides mitochondrial genome. “Position” means the position of the SNPs in the nucleotide sequences of the PCGs.
Table 4. List of single-nucleotide polymorphisms (SNPs) detected in the PCGs of the T. jasminoides mitochondrial genome. “Position” means the position of the SNPs in the nucleotide sequences of the PCGs.
RegionPositionReferenceCoverageBase Count [A, C, G, T]AllSubsFrequency
atp169G439[56, 0, 383, 0]GA0.13
atp171A415[352, 63, 0, 0]AC0.15
atp174T469[0, 62, 0, 407]TC0.13
atp175G472[0, 0, 410, 62]GT0.13
atp176A448[386, 62, 0, 0]AC0.14
atp179G453[59, 0, 394, 0]GA0.13
cox2698C302[0, 255, 0, 47]CT0.16
cox2721C205[0, 141, 0, 64]CT0.31
matR1753G450[43, 0, 407, 0]GA0.1
matR1760A438[396, 0, 42, 0]AG0.1
nad583C253[0, 225, 28, 0]CG0.11
nad584C254[28, 226, 0, 0]CA0.11
nad51295A441[374, 0, 0, 67]AT0.15
nad51301C428[62, 366, 0, 0]CA0.14
nad51312C417[0, 366, 0, 51]CT0.12
nad51351C376[0, 322, 0, 54]CT0.14
nad51353G378[53, 0, 325, 0]GA0.14
nad51361T356[0, 49, 0, 307]TC0.14
nad51363C353[0, 304, 0, 49]CT0.14
nad51377G349[58, 0, 291, 0]GA0.17
nad51509T279[0, 0, 40, 239]TG0.14
nad51510G278[40, 3, 235, 0]GA GC0.15
nad626C425[0, 333, 0, 92]CT0.22
nad655C520[0, 376, 0, 144]CT0.28
nad6154A727[487, 0, 240, 0]AG0.33
nad6185C764[0, 502, 261, 1]CG CT0.34
rps374A1[0, 0, 0, 1]AT1
rps463G570[0, 0, 448, 122]GT0.21
rps475T550[0, 102, 0, 448]TC0.19
rps478A560[467, 0, 0, 93]AT0.17
rps487A525[437, 88, 0, 0]AC0.17
rps493C553[0, 472, 0, 81]CT0.15
rps4100C540[75, 465, 0, 0]CA0.14
rps4159T646[0, 0, 121, 525]TG0.19
rps4185C652[109, 542, 0, 1]CA CT0.17
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Cai, Y.; Yang, S.; Chen, H.; Ni, Y.; Li, J.; Zhang, J.; Liu, C. De Novo Hybrid Assembly of the Tripterygium wilfordii Mitochondrial Genome Provides the Chromosomal Mitochondrial DNA Structure and RNA Editing Events. Int. J. Mol. Sci. 2025, 26, 7093. https://doi.org/10.3390/ijms26157093

AMA Style

Cai Y, Yang S, Chen H, Ni Y, Li J, Zhang J, Liu C. De Novo Hybrid Assembly of the Tripterygium wilfordii Mitochondrial Genome Provides the Chromosomal Mitochondrial DNA Structure and RNA Editing Events. International Journal of Molecular Sciences. 2025; 26(15):7093. https://doi.org/10.3390/ijms26157093

Chicago/Turabian Style

Cai, Yisha, Suxin Yang, Haimei Chen, Yang Ni, Jingling Li, Jinghong Zhang, and Chang Liu. 2025. "De Novo Hybrid Assembly of the Tripterygium wilfordii Mitochondrial Genome Provides the Chromosomal Mitochondrial DNA Structure and RNA Editing Events" International Journal of Molecular Sciences 26, no. 15: 7093. https://doi.org/10.3390/ijms26157093

APA Style

Cai, Y., Yang, S., Chen, H., Ni, Y., Li, J., Zhang, J., & Liu, C. (2025). De Novo Hybrid Assembly of the Tripterygium wilfordii Mitochondrial Genome Provides the Chromosomal Mitochondrial DNA Structure and RNA Editing Events. International Journal of Molecular Sciences, 26(15), 7093. https://doi.org/10.3390/ijms26157093

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