Assembly and analysis of the complete mitochondrial genome of an endemic Camellia species of China, Camellia tachangensis

Assembly and analysis of the complete mitochondrial genome of an endemic Camellia species of China, Camellia tachangensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assembly and analysis of the complete mitochondrial genome of an endemic Camellia species of China, Camellia tachangensis Dongzhen Jiang, Lei Zhou, Zhaohui Ran, Xu Xiao, Xuehang Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5634953/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 May, 2025 Read the published version in BMC Genomics → Version 1 posted 8 You are reading this latest preprint version Abstract Background Camellia tachangensis F. C. Zhang is an endemic Camellia species of the junction of Yunnan, Guizhou and Guangxi Provinces in China. It is characterized by a primitive five-chambered ovary morphology and serves as the botanical source of the renowned “Pu’an Red Tea”. Unfortunately, the populations of the species have declined due to the destruction of their habitats by human activities. The lack of mitochondrial genomic resources has hindered research into molecular breeding and phylogenetic evolution of C. tachangensis . Result In this study, we had sequenced, assembled, and annotated the mitochondrial genome of C. tachangensis to reveal its genetic characteristics and phylogenetic relation with other Camellia species. The assembly result indicated that the mitochondrial genome sequence of C. tachangensis was 746,931 bp (GC content = 45.86%). It consisted of one multibranched sequence (Chr1) and one circular sequence (Chr2), with Chr1 capable of producing 7 substructures. The comparative analysis of the mitochondrial and chloroplast DNA of C. tachangensis revealed 23 pairs of chloroplast homologous fragments, with 10 fully preserved tRNA genes within them. Comparison of interspecies Ka/Ks revealed that mutations in protein-coding genes (PCGs) of C. tachangensis were predominantly shaped by purifying selection throughout its evolution (Ka/Ks < 1). The phylogenetic tree constructed from mitochondrial CDS indicated that C. tachangensis and certain variants of C. sinensis were distinct from other Camellia species, forming a clade with relatively low support (BS = 22%, PP = 0.41). Meanwhile, the chloroplast genomes-based phylogenetic analyses revealed that C. tachangensis was most closely related to C. taliensis , C. makuanica , and C. gymnogyna , with strong statistical support (BS = 100, PP = 1.00). Conclusions Our study deciphered the mitochondrial genome and its multibranched structure of C. tachangensis. These findings not only enhanced our comprehension of the complexity and diversity of mitochondrial genome structures in Camellia species, but also established a foundational genetic data framework for future research on molecular breeding programs and phylogenetic relationship involving C. tachangensis and its related species. Camellia tachangensis Horizontal transfer Mitochondrial genome Evolution Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Camellia tachangensis F. C. Zhang, belonging to sect. Thea (L.) of the genus Camellia , is an endemic species native to the border regions of Yunnan, Guizhou, and Guangxi Provinces in China [ 1 ]. This species has a distinctive morphological features of a capsule with five-locular ovary and been recognized as the primitive plant within sect. Thea Dyer in genus Camellia [ 2 , 3 ]. Some studies on the systematic taxonomy of Camellia plants showed C. tachangensis has high scientific value for exploring the origin and evolution of Camellia in southwest China [ 4 – 7 ]. At the same time, as a member of sect. Thea , C. tachangensis was served as a processing raw material for “Pu’an red tea” with unique aroma and flavor that it is a product of China’s national geographical indication [ 8 ]. Due to environmental degradation and human interference, coupled with its relatively low reproductive capacity, the survival of C. tachangensis was facing a significant crisis [ 9 ]. To improve the survival status of C. tachangensis , researchers had conducted comprehensive and in-depth studies on its physiological, breeding, propagation, biochemical characteristics and so on [ 10 – 13 ]. However, there were few studies in genome biology, and most of them only focused on population genetics and chloroplast genomes [ 14 – 16 ]. Studies concerning the complete mitochondrial genomes were still lacking. Mitochondria in plant cells are essential organelles that not only participate in cellular respiration but also play crucial roles in regulating intracellular metabolic networks [ 17 , 18 ]. In botanical research, the mitochondrial genes of plants hold significant value for study. First, mitochondrial genes are involved in the synthesis of essential enzymatic components such as ATP synthase, cytochrome c oxidase, and NADH dehydrogenase, which are crucial for the respiratory metabolism of plants [ 19 – 21 ]. Investigating the composition and evolution of these genes will provide novel insights into the genetic improvement of C. tachangensis and its related species. Furthermore, most plant mitochondrial DNA exhibits maternal inheritance characteristics, with a lower level of heterozygosity than nuclear genes [ 22 , 23 ]. Consequently, the mitochondrial genome can be effectively utilized for the classification and identification of species within the genus Camellia , which display significant genetic heterozygosity and phenotypic diversity [ 24 – 26 ]. Additionally, structural variations and the insertion of chloroplast-derived fragments occur relatively frequently in mitochondrial DNA. These processes typically take place during different stages of plant lineage differentiation. By comparing the similarities and differences in mitochondrial genome structures and chloroplast-derived fragments across different species, we can gain deeper insights into the evolutionary history of the Camellia genus [ 27 – 29 ]. This study employed a combination of high-throughput sequencing and long-read sequencing to achieve the first complete sequencing, assembly, and annotation of mitochondrial genome of C. tachangensis while also exploring its substructure. Comprehensive analyses were performed to investigate multiple genomic features including codon usage patterns, chloroplast-derived homologous sequences, repetitive element distribution, RNA editing sites, evolutionary selection pressures through Ka/Ks ratio calculations, and phylogenetic relationships. The findings of this work established a foundational genetic data framework for future research on molecular breeding programs and evolutionary dynamics involving C. tachangensis and its related species. 2. Materials and methods 2.1. Material collection, DNA extraction and sequencing In this study, we selected the leaves of C. tachangensis from the forestry center in Longli County, Guizhou Province (N 26°24′49″–26°44′30″, E 106°48′12″–107°8′50″), as the research materials. Fresh leaves were collected and preserved in liquid nitrogen at -80°C. Total DNA was extracted by CTAB method [ 30 ]. The mitochondrial genome was sequenced and assembled via a combination of high-throughput sequencing (Illumina Novaseq 6000) and long-read sequencing (Oxford Nanopore R10.4). The high-throughput sequencing strictly conformed to the standard operating procedures provided by Illumina. First, DNA quality and concentration were assessed via 1% agarose gel electrophoresis and a NanoDrop 2000. The high-quality DNA samples were then fragmented through ultrasonic mechanical disruption. The fragmented DNA subsequently underwent purification, end repair, and ligation of sequencing adapters. Finally, the selected DNA was amplified via PCR to construct the sequencing library. The constructed library underwent quality control, and those that conformed with the quality assessment were sequenced via the Illumina Novaseq 6000 platform (Illumina, San Diego, California, United States) [ 31 ]. The raw data were subsequently filtered via fastp v0.23.4 software ( https://github.com/OpenGene/fastp ) [ 32 ]. In the process of the long-read sequencing, genomic DNA was first randomly fragmented. Subsequently, magnetic beads were utilized for enrichment and purification to obtain large DNA fragments. Following this step, gel extraction was performed on the large fragments, and damage repair was conducted on the fragmented DNA. The purified fragments subsequently underwent end repair and A-tailing. The SQK-LSK109 kit was used to ligate adapters, thereby constructing a DNA library for quantitative assessment. Next, an appropriate amount of the DNA library was loaded onto the flow cell and subjected to real-time single-molecule sequencing on the Oxford Nanopore PromethION sequencer [ 33 ]. Finally, the long-read sequencing data were filtered via Filtlong v0.24 software, and a Perl script was used for data analysis. 2.2. Assembly and annotation of mitochondrial genome First, the raw long-read sequencing data were aligned with the plant mitochondrial gene database via minimap2 v2.1. Subsequently, sequences with alignment lengths greater than 50 bp were selected as candidate sequences. Among these candidates, those exhibiting a greater number of aligned genes (a single sequencing read containing multiple core genes) and superior alignment quality (exhibiting a relatively complete coverage of core genes) were chosen as seed sequences. Minimap2 was subsequently employed to align the original sequencing data against the seed sequences, filtering for overlaps greater than 1 kb and similarity exceeding 70% to incorporate additional sequences into the seed sequences [ 34 ]. The third-generation assembly software Canu v2.2 was employed to correct the obtained third-generation data [ 35 ]. Subsequently, Bowtie2 v2.3.5.1 was utilized to align the second-generation data with the corrected sequences. The paired high-throughput data and the corrected long-read sequencing data were then assembled via the default parameters of Unicycler v0.4.8 [ 36 ]. Subsequently, Bandage software v0.8.1 was employed to visualize the assembly results and make manual adjustments as necessary. Owing to the presence of multiple subcircular structures or even non-circular complex physical configurations in mitochondrial genomes, the corrected third-generation sequencing data were aligned to the contigs generated by Unicycler via minimap2. The branch directions were subsequently manually determined to obtain the final assembly results. The annotation of the mitochondria genes was carried out through the following steps: utilizing the Basic Local Alignment Search Tool-Nucleotide (BLASTN) ( https://blast.ncbi.nlm.nih.gov/ ) [ 37 ], the protein-coding genes and rRNA genes were compared with publicly available reference mitochondrial genome sequences from plants. Manual adjustments were subsequently made using the closely related species C. sinensis var. sinensis (Genbank ID: PP212895) as a reference genome [ 2 – 7 , 29 ]. In addition, tRNA genes were annotated via tRNAscan-SE ( http://lowelab.ucsc.edu/tRNAscan-SE/ ) [ 38 ]. The Open Reading Frame Finder ( http://www.ncbi.nlm.nih.gov/gorf/gorf.html ) was used for ORF annotation [ 39 ]. The minimum length was set to 102 bp, with redundant sequences and overlapping known genes excluded. Sequences longer than 300 bp were aligned against the NR database for annotation. The mitochondrial genome map was constructed via OGDraw ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) [ 40 ]. 2.3. Analysis of repeat sequences and RNA editing prediction To clarify the 3 types of repetitive sequences in the mitochondrial genome of C. tachangensis , simple sequence repeat (SSR), tandem repeat, and dispersed repeat, MISA v1.0 was employed ( https://webblast.ipk-gatersleben.de/misa/ ) for the identification of SSRs [ 41 , 42 ]. Tandem repeats were identified via Tandem Repeats Finder v4.09 ( http://tandem.bu.edu/trf/trf.submit.options.html ) [ 43 ]. The identification of dispersed repeats was conducted via BLASTN software (v2.10.1, parameters: -word size 7, evalue 1e-5). During this process, redundant and tandem repeat sequences were removed [ 44 ]. Ultimately, all the identification results were visualized via Circos v0.69-5 [ 45 ]. To further investigate the RNA editing sites, we utilized the online tool PREPACT3 ( http://www.prepact.de/ ) to predict RNA editing events, setting a critical threshold of 0.001 [ 46 ]. In Excel 2021, the distribution of RNA editing sites for different genes and the number of amino acid variation types were visualized using bar charts; while the proportion of hydrophilic and hydrophobic group change types was presented through pie charts to show their ratio relationship. 2.4. Analysis of relative synonymous codon usage (RSCU) and mitochondrial plastid DNAs (MTPTs) in the mitogenome The protein-coding sequences were obtained via the default settings of Phylosuit v1.22 software [ 47 ]. The relative synonymous codon usage rates (RSCU) based on mitochondrial genome protein-coding genes were calculated via MEGA v7.0 software [ 48 ]. Mitochondrial plastid DNAs (MTPTs) refer to DNA fragments of plasmid origin present in the mitochondrial genome. In this study, chloroplast genome sequences from the same samples were extracted, and BLASTN software was used to identify homologous sequences between the chloroplast and mitochondrial genomes, with a similarity threshold set at 70% and an E value of 1e-5. To visualize the homologous segments between the chloroplast and mitochondrial genomes more intuitively, Circos v0.69-5 was used [ 49 ]. 2.5. Analysis of nucleotide diversity (Pi) and selection pressure To comprehensively analyze the diversity and the impact of selection on mitochondrial genomes in Camellia species, this study selected mitochondrial genomes from 9 representative species (including C. tachangensis ) within 4 related taxonomic groups of genus Camellia : sect. Thea , sect. Chrysantha , sect. Camellia , and sect. Heterogenea for comparison, and conducted Pi analysis and Ka/Ks analysis as follows: mitochondrial sequences of 8 Camellia species were downloaded from the NCBI database ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ): C. tianeensis (PP727208), C. nitidissima (NC_067639), C. chekiangoleosa (NC_086749), C. sinensis var. sinensis cv. Dahongpao (PP212895), C. sinensis var. sinensis cv. Rougui (PP212896), C. sinensis var. pubilimba (ON782577), C. sinensis var. assamica (NC_043914), and C. gigantocarpa (OP270590). The MAFFT v7.427 software was used for global alignment of these plant mitochondrial genomes along with that of C. tachangensis [ 50 , 51 ]. The resulting alignment file was used to calculate Pi values for each shared gene with DnaSP v6.12.03 and Ka/Ks ratios for shared PCGs with KaKs_Calculator v3.0 [ 52 , 53 ]. The Ka/Ks ratio data were visualized in the form of a box plot using Excel 2021. 2.6. Phylogenetic analysis In the phylogenetic analysis of mitochondrial and chloroplast genomes, while we both primarily focused on species within the genus Camellia , the study adopted differentiated phylogenetic tree construction strategies due to the data imbalance between the 2 organelle genomes in the NCBI database ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ): For mitochondrial genomes with relatively scarce data, the mitochondrial CDS based phylogenetic tree incorporated 15 represented Camellia species (including C. sinensis and its varieties, C. oleifera , C. chekiangoleosa , etc.) to investigate the phylogenetic position of C. tachangensis. It also included 14 species from different angiosperm families (e.g., Ericaceae, Solanaceae and Apiaceae) and the gymnosperm Taxus wallichiana as outgroups. Tbtools software ( https://github.com/CJ-Chen/TBtools/releases ) was utilized to extract 24 conserved mitochondrial protein-coding genes (PCGs) among these species [ 54 ], including atp 1, atp 4, atp 6, atp 8, atp 9, ccm B, ccm C, ccm Fc, ccm Fn, cob , cox 1, cox 2, cox 3, mat R, mtt B, nad 1, nad 2, nad 3, nad 4, nad 4L, nad 5, nad 6, nad 7, and nad 9. The coding sequences (CDSs) of these genes within the mitochondrial genomes of 28 species were aligned via MAFFT v7.427 for interspecies sequence comparison [ 50 , 51 ]. The aligned sequences were concatenated and trimmed via trimAl v1.4. Model prediction was subsequently conducted with jmodeltest v2.1.10 to identify the GTR model. The maximum likelihood phylogenetic tree was then constructed via RAxML v8.2.10 with the GTRGAMMA model and a bootstrap value of 1000 [ 55 ]. The Bayesian phylogenetic tree was constructed via MrBayes v3.2.7 with the Markov chain Monte Carlo method for 1,000,000 generations, and sampling trees every 100 generations[ 56 ]. In the phylogenetic analysis based on chloroplast PCGs, 23 representative Camellia species (covering 10 significant sections including sect. Thea , sect. Chrysantha , and sect. Oleifera ), due to they are closely related in Camellia genus. Meanwhile, the sister group ( Polyspora axillaris and Schima superba ) of Camellia genus was selected as the outgroup. The maximum likelihood phylogenetic tree and the Bayesian phylogenetic tree were constructed based on 53 conserve d chloroplast PCGs among these species: acc D, atp A, atp E, atp F, atp H, atp I, mat K, pet A, pet B, pet D, pet G, pet L, pet N, psa A, psa B, psa C, psb A, psb C, psb D, psb E, psb F, psb H, The analysis methods employed were identical to those used for mitochondrial genomes. Finally, visualization was performed via Interactive Tree Of Life (ITOL) software v4.0 ( https://itol.embl.de/ ) [ 57 ]. 3. Results 3.1. Genomic features of C. tachangensis mitochondrial genome The total mitochondrial DNA of C. tachangensis was sequenced, and the raw data were prepared for assembly, resulting in 16.34 Gb Illumina sequencing data (Q20 = 96.36%,Q30 = 90.75%) and 20.5 Gb Nanopore PromethION sequencing data with a N50 read length of 21,799 bp. The assembly results indicated that the mitochondrial genome sequence of C. tachangensis was 746,931 bp (GC content = 45.86%), consisting of one multibranched sequence and one circular sequence, which were designated chromosome 1 (Chr1) and chromosome 2 (Chr2), with lengths of 525,875 bp and 221,056 bp, respectively (Fig. 1 ). The results of the read mapping indicated that there were no reads present between Chr1 and Chr2 (Fig. S1 ), suggesting that Chr1 and Chr2 were relatively independent. Additionally, Chr1 was capable of producing 7 kinds of substructures, whereas Chr2 didn’t exhibit any substructures. A total of 24 core protein-coding genes, 16 variable protein-coding genes, 3 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes were identified. The core protein-coding genes could be categorized into 7 functional groups: ATP synthase ( atp 1, atp 4, atp 6, atp 8, and atp 9), Cytochrome c maturation proteins ( ccm B, ccm C, ccm Fc, and ccm Fn), Ubiquinol cytochrome c reductase ( cob ), Cytochrome c oxidases ( cox 1, cox 2, and cox 3), Maturases ( mat R), Transport membrane proteins ( mtt B), and NADH dehydrogenases ( nad 1, nad 2, and nad 3) (Table 1 ). Notably, the exons of nad1 and nad2 were distributed on both Chr1 and Chr2; these segments require post-transcriptional RNA splicing to assemble into complete gene sequences. The analysis of 14 variable protein-coding genes revealed 8 types of small subunit ribosomal proteins ( rps 1, rps 13, rps 14, rps 3, rps 4, rps 7, rps 12, and rps 20), 4 types of large subunit ribosomal proteins ( rpl 10, rpl 16, rpl 2, and rpl 5), and 2 types of succinate dehydrogenases ( sdh 3 and sdh 4). Notably, both sdh 3 and rps 19 appeared twice in the genome: once as functional genes and another as pseudogenes. A total of 30 tRNA genes were annotated. Among these annotations, trn M-CAU was recorded 5 times on Chr1 and once on Chr2. Additionally, trn S-UGA was annotated twice on Chr1, trn I-GAU was annotated twice on Chr2, and trn P-UGG was noted once on both Chr1 and Chr2. Furthermore, 14 genes contained introns. Among these genes, ten possess one intron each ( ccm Fc, rpl 2, rps 1, rps 3, trn A-UGC, trn F-AAA, trn I-GAU (2), trn S-UGA, and trn T-UGU); one gene contained 2 introns ( nad 4); and 4 genes had 4 introns ( nad 1, nad 2, nad 5, and nad 7). Table 1 List of genes in the mitochondrial genome of C. tachangensis . Group of genes Gene name (Chr1) Gene name (Chr2) Core genes ATP synthase atp 1, atp 6, atp 8, atp 9 atp 4 Cytohrome c biogenesis ccm B, ccm Fc ccm C, ccm Fn Ubichinol cytochrome c reductase cob Cytochrome c oxidase cox 1, cox 2, cox 3 Maturases mat R Transport membrane protein mtt B NADH dehydrogenase nad 1-TS, nad 2-TS, nad 4, nad 5-TS, nad 6 nad 1-TS, nad 2-TS, nad 3, nad 4L, nad 7, nad 9 Variable genes Ribosomal proteins (LSU) rpl 10, rpl 16, rpl 2, rpl 5 Ribosomal proteins (SSU) rps 1, rps 13, rps 14, rps 3, rps 4, rps 7, # rps 19 rps 12, rps 19 Succinate dehydrogenase # sdh 3, sdh 3, sdh 4 Ribosomal RNAs rrn 18, rrn 26, rrn 5 Transfer RNAs trn C-GCA, trn D-GTC, trn F-GAA, trn K-TTT, trn M-CAT(5), trn N-GTT, trn P-TGG, trn S-CGA, trn S-GCT, trn S-TGA(2), trn T-TGT, trn Y-GTA trn A-TGC, trn E-TTC, trn F-AAA, trn G-GCC, trn H-GTG, trn I-GAT(2), trn M-CAT, trn N-ATT, trn P-TGG, trn Q-TTG, trn V-GAC, trn W-CCA Note: Numbers after gene names are the number of copies. Genes preceded by the # symbol represent pseudogenes. Genes followed by -TS need to be spliced into complete genes by RNA splicing after transcription. 3.2. Different configurations of the C. tachangensis mitogenome The phenomenon of recombination mediated by homologous fragments was commonly observed in the mitochondrial genome of cells. On Chr1, 3 pairs of dispersed repeats (homologous fragments), designated R7, R8, and R10, with lengths ranging from 1,190 to 8,440 bp, were identified. The similarity between the paired repeat units reached as high as 99.965–100%. Among these sequences, R7 and R8 were classified as direct repeats, whereas R10 was categorized as a palindromic repeat. Collectively, these 3 pairs of repeats facilitated the formation of 7 substructures within Chr1 (Fig. 2 ). The homologous fragments of the mitochondrial genome in C. tachangensis mediated recombination through 2 distinct mechanisms, which were primarily determined by the orientation differences between these segments: (1) In M1, the arrangement directions of the homologous segments R7 and R8 within their respective groups were identical. When either R7 or R8 broke, the 2 homologous segments recombined in a head-to-tail manner, resulting in M1 splitting from a large ring into 2 smaller rings, producing either M8 or M6. If both R7 and R8 break simultaneously, M1 could recombine to form a new large ring designated M5. At this point, in a clockwise direction, the sequence order of the non-homologous fragments changes from C1→C5→C9→C4→C6→C3 to C1→C6→C3→C9→C4→C5. The distinction between M1 and M5 lies in the reciprocal positioning of C6 and C3 relative to C5. However, there is no alteration in the arrangement direction of each sequence. (2) In contrast, within M1, owing to opposing orientations between the 2 homologous segments associated with R10, an inversion phenomenon occurs among adjacent non-homologous fragments. Specifically, during the recombination from M1 to M2 mediated by R10, segment C6→C4 experiences a 180° inversion in its clockwise orientation, thus altering the sequence order of the non-homologous fragments from C1→C5→C9→C6→C4→C3 to C1→C5→C9→C4→C6→C3. Furthermore, one homologous segment of R8 is located between fragments C4 and C6. Consequently, this inversion involving fragment pairings from C4 to C6 resulted in an inverse arrangement for that particular homologous segment: it is transformed from R + 8 to R-8. Both homologs of R8 now exhibit opposite orientations, leading to an inverted recombination mechanism for R8 that allows for the transformation of M2 into M3. A similar outcome is observed during the transition from M5 to M4 through recombination processes. The above hypothesis can be observed in the coverage validation map aligned to the assembly results of long reads, confirming the presence of 7 potential substructures on Chr1 (Fig. S1 ). This phenomenon revealed that recombination mediated by specific pairs of homologous segments within mitochondrial genomes could influence alternative pairs’ modes of recombination, thereby enriching DNA with diverse substructures. 3.3. Analysis of repeat sequences A total of 223 SSRs were identified in the mitochondrial genome of C. tachangensis (Fig. 3 , Table S1 A), with 160 located on Chr1 and 63 on Chr2. On Chr1 and Chr2, there are 19 and 12 mononucleotide (mono-), 48 and 12 dinucleotide (di-), 22 and 7 trinucleotide (tri-), and 61 and 25 tetranucleotide (tetra-) SSRs, respectively. There is 1 hexanucleotide (hexa-) SSR present on both Chr1 and Chr2. The highest proportion of SSRs on both chromosomes were found to be tetranucleotides, accounting for approximately 38.125% on Chr1 and 39.682% on Chr2. Furthermore, we observed that A/T is the most prevalent type among the mononucleotide SSRs. In total, there were also 28 tandem repeat sequences within the mitochondrial genome; the longest sequence was located on Chr1, with a copy number of 2, measuring 78 bp in length, whereas the shortest sequence resided on Chr2, with a copy number of 2, measuring only 24 bp in length (Table S1 B). The mitochondrial genome contains a substantial number of dispersed repetitive sequences, totaling 479. This dataset included 266 palindromic repeat sequences and 213 forward repeat sequences, with lengths ranging from 29 to 8,452 bp (Table S1 C). Notably, 88.28% of these sequences were shorter than 100 bp, with the most common lengths falling between 29 and 49 bp. Furthermore, the analysis of dispersed repetitive sequences indicated that transposon exchange between Chr1 and Chr2 occurred quite frequently; 161 sequences were copied from Chr1 to Chr2, and only 28 sequences were transferred in the opposite direction (from Chr2 to Chr1). Among all the homologous fragments identified, only 3 segments exceeded a length of 1,000 bp, each exhibiting greater than or equal to 99.96% similarity. Of these longer segments, 2 were classified as direct repeat sequences, whereas one was categorized as a palindromic repeat sequence; these specific sequences played a role in mediating recombination within the mitochondrial genome. 3.4. Prediction of RNA editing sites In this study, we predicted RNA editing sites in the mitochondrial genome of C. tachangensis , focusing on 38 protein-coding genes. A total of 537 non-synonymous editing sites were identified (Fig. 4 A, Table S2 A), involving changes in 14 amino acids, including H(His)→Y(Tyr), R(Arg)→C(Cys), T(Thr)→I(Ile), T(Thr)→M(Met), R(Arg)→W(Trp), S(Ser)→L(Leu), S(Ser)→F(Phe), P(Pro)→S(Ser), P(Pro)→L(Leu), P(Pro)→F(Phe), L(Leu)→F(Phe), A(Ala)→V(Val), Q(Gln)→*, and Arg(R)→* (* represents a stop codon). Among these changes, the most common alteration was Ser to Leu, with a total of 128 RNA editing sites accounting for 23.84% of the total. Among all the amino acid changes observed, 259 (48.23%) of the hydrophilic amino acids were converted to hydrophobic ones; conversely, 39 (7.26%) of the hydrophobic amino acids were transformed into hydrophilic ones, whereas 235 (43.76%) amino acids exhibited no change in hydrophobicity. Additionally, 4 (0.74%) codons encoding hydrophilic amino acids were converted into stop codons (Fig. 4 B). Specifically, 3 instances of CGA(R)—UGA(*) conversion occurred at the last codon positions of the ccm Fc, atp 9, and sdh 4 genes; one instance of CAG(Q)—TAG(*) conversion was found at the 13 codon position of the rpl 16 gene, which might lead to premature termination of mRNA translation. In terms of genetic analysis, the ccm Fn gene presented the highest frequency of RNA editing sites, with a total of 39 occurrences. This was followed by the ccm B and ccm C genes, which had 34 and 32 instances, respectively. In contrast, the sdh 3 gene had the lowest frequency of RNA editing sites, with only 2 identified editing locations (Fig. 4 C, Table S2 B). 3.5 Codon usage analysis of protein-coding genes (PCGs) Relative synonymous codon usage frequency (RSCU) analysis was conducted on 64 codons of the mitochondrial genome of C. tachangensis . The results indicated that all 64 codons are expressed in PCGs (Fig. 5 , Table S3 ). Among these, the GCU (encoding alanine) exhibited the highest RSCU value of 1.5743, whereas the CAC (encoding histidine) had the lowest RSCU value of 0.4586. In the PCGs, the start codon was consistently ATG, with no codon usage bias (RSCU = 1). The stop codons included UAA, UAG, and UGA, among which only UAA had an RSCU value greater than 1. Among the 61 coding amino acid codons analyzed, 29 had RSCU values exceeding 1, indicating a strong preference for their use. There were 10 A-ending codons and 17 U-ending codons; conversely, there was only one C-ending or G-ending codon each. The proportion of high-frequency codons (RSCU > 1) ending with A or U reached 93.103%, whereas those ending with C or G accounted for only 6.897%. Therefore, it could be concluded that in the mitochondrial genome of C. tachangensis , there was a notable preference for the use of A- or U-ending codons. 3.6 Mitochondrial plastid DNAs (MTPTs) in the mitogenome To investigate the sequence transfer between the mitochondrial and chloroplast genomes of C. tachangensis , we conducted a comparative analysis of both organellar genomes. The results indicated that there were a total of 23 groups of chloroplast homologous fragments within the C. tachangensis mitochondrial genome, with MTPT 1–7 located on Chr1 and MTPT 8–23 located on Chr2 (Fig. 6 , Table S4 ). These fragments collectively spanned a length of 16,396 bp, accounting for approximately 2.1951% of the total length of the mitochondrial genome. Among these fragments, MTPT1 was the longest at 9,556 bp and was located within the range of 221,056–211,509 bp on Chr2. In contrast, MTPT23 was the shortest fragment, with a length of only 32 bp, and was found within the range of 216,692–216,661 bp on Chr1. The annotation results indicate that these fragments originate from protein-coding genes, rRNA genes, tRNA genes, and intergenic regions of the chloroplast genome. However, all the chloroplast protein-coding genes and rRNA coding genes were not retained after the insertion of the mitochondrial sequences, whereas the tRNA coding genes were preserved relatively intact within the mitochondria. A total of 7 completed tRNA genes were distributed across 7 homologous sequences: trn A-UGC, trn I-GAU, trn V-GAC, trn W-CCA, trn P-UGG, trn M-CAU, and trn N-GUU. 3.7. Analysis of Pi and Ka/Ks To analyze the sequence differences between C. tachangensis and its related species, we calculated the nucleotide diversity (Pi) values for 41 common genes across 9 species of the genus Camellia (Table S5 ). The data indicate significant differences in nucleotide diversity (Pi values) among the mitochondrial genomes of 9 Camellia species across different genes, ranging from 0 to 0.06872. The highest Pi value was observed in rrn 18 at (0.06872), followed by nad 5 (0.01673) and cox 2 (0.0172). While, 15 genes (e.g., nad 6, cox 1,and nad 4L) exhibited Pi values of 0, indicating their high conservation. Some shorter regions showed a higher number of mutations despite their limited length (e.g., sdh 3 [length: 321 bp; mutations: 6; Pi = 0.00744]), while longer regions like rrn 26 (length: 3614 bp; mutations: 48; Pi = 0.00564) displayed lower mutation density. To further investigate the impact of environmental stress on mitochondrial PCG mutations in the aforementioned species, we conducted Ka/Ks analysis and screened 20 genes with Ka/Ks values. The results (Fig. 7 , Table S6 ) showed that nearly all Ka/Ks values of mitochondrial PCGs in C. tachangensis were less than 1 when compared with Camellia species (only the cox2 gene showed Ka/Ks = 1.01438 in C. tachangensis vs. C. pubilimba (ON782577) and displayed the ratio of 1.41208 in C. tachangensis vs C. assamica (NC_043914). This indicated that C. tachangensis , as a endemic species of China, had undergone predominantly purifying selection during evolution. Notably, some genes exhibited a wide range of Ka/Ks values due to their higher mutation rates. For example, the protein-coding gene nad 2, which had the highest Pi value, yielded 16 distinct Ka/Ks values, primarily ranging between 0.3 and 0.65. In contrast, certain genes such as rpl 2, cox 2, and sdh 3 demonstrated both conservation and heterogeneity across species comparisons, resulting in relatively limited Ka/Ks variations. Taking rpl 2 as an example, its sequence exhibited either complete identity or divergence across different species comparisons, yielding only 3 distinct Ka/Ks values: 1.03902 ( C. tachangensis / C. chekiangoleosa vs. C. gigantocarpa ), 0.783174 ( C. tachangensis / C. chekiangoleosa vs. C. sinensis variants PP212895/ PP212896/ ON782577), and 0.304929 ( C. sinensis variants PP212895/ PP212896/ ON782577 vs. C. gigantocarpa ). 3.8. Phylogenetic analysis The mitochondrial CDS-based phylogenetic tree was constructed using Taxus wallichiana as the outgroup. It revealed that Diospyros kaki (NC_082859) and Rhododendron simsii (NC_053763), both belonging to the order Ericales, formed a well-supported clade with Camellia species (BS = 100, PP = 1.00). Within the Camellia lineage, C. tachangensis was phylogenetically distinct from species in the sections Oleifera , Camellia , Heterogenea and Chrysantha . It occupied a basal position within the Clade Ⅰ that includes 5 variants of C. sinensis . However, this phylogenetic grouping exhibited weak nodal support (BS = 22, PP = 0.41) (Fig. 8). The chloroplast PCG-based phylogenetic tree utilizing Polyspora axillaris (NC_035709) and Schima superba (NC_035545) as outgroups demonstrated high phylogenetic resolution for C. tachangensis : Among the Camellia species analyzed, This species formed a strongly supported basal group in Clade II (BS = 100, PP = 1), alongside C. makuanica (NC_087766), C. taliensis (NC_022264), and C. gymnogyna (NC_039626) (Fig. 9 ). 4. Discussion Although branched structures had been widely reported in plant mitochondrial genomes [ 58 – 60 ], the mitochondrial DNA of most Camellia species (e.g., C. sinensis, C. assamica , and C. nitidissima ) were traditionally presented as circular structures due to differing assembly strategies [ 29 , 61 – 64 ]. Only 3 species of sect. Oleifera ( C. drupifera , C. oleifera , and C. lanceoleosa ) exhibit mitochondrial genomes with multibranched configurations [ 27 , 28 ]. In this study, we achieved the first complete mitochondrial genome sequencing and assembly for C. tachangensis , which revealed its unique dual-component architecture comprising a multibranched sequence and a circular sequence. Subsequent structural analysis further identified the multibranched sequence could form 7 substructures via 3 pairs of dispersed repeats over 1000 bp. These findings not only enhanced our comprehension of the complexity and diversity of mitochondrial genome structures in Camellia species, but also established a foundational genetic data framework for future research on molecular breeding programs targeting C. tachangensis. Beyond structural variations, the mitochondrial genome length of C. tachangensis significantly differed from other Camellia species. The total mtDNA length of C. tachangensis was 746,931 bp, while other Camellia species range from 1,082,025 bp in C. sinensis var. sinensis cv. Dahongpao (CSSDHP, PP212895) (longest) to 707,441 bp in C. assamica (shortest), representing a 374,584 bp difference [ 27 – 29 , 61 – 64 ]. Among these, C. huana (733,752 bp) showed the closest genome length to C. tachangensis , differing by only 13,179 bp. However, the length of repetitive sequences identified in the studies cannot directly explain the differences in mitochondrial DNA length between these Camellia species. This is evidenced by the contrasting repetitive sequence lengths between CSSDHP and C. tachangensis : SSR (4,548 vs. 2,688 bp, including tandem repeats) and dispersed repeats (33,871 vs. 46,509 bp). Even when combining the total lengths of simple sequence repeats and dispersed repeats, CSSDHP still exhibits a reverse correlation in total repetitive sequence content compared to C. tachangensis (38,419 bp vs. 49,197 bp). This paradoxical phenomenon may be attributed to 3 possible factors: first, differential loss and transfer of mitochondrial DNA fragments between the 2 species; second, frequent recombination and mutation events in intergenic regions of plant mitochondrial DNA that obscure detection of original repetitive sequences; third, CSSDHP's mitochondrial DNA had acquired longer chloroplast-derived homologous sequences compared to C. tachangensis (20,733 vs. 16,448 bp) [ 64 – 66 ]. However, the first 2 factors still require further exploration and validation specifically for Camellia species. Although C. tachangensis possessed complete mitochondrial PCG composition, it only retained pseudogene copies for rps 19 and sdh 3. In contrast, other Camellia species exhibited varying PCG duplications. For Instance, CSSDHP contained 8 duplicated PCGs (e.g., atp 8, atp 9, nad 6, cox 3), C. nitidissima shows 2 duplicated genes ( rps 12 and rps 16), and C. oleifera retained 4 duplicated genes ( cox 1, rpl 16, rps 3, and rpl 2) [ 28 , 29 , 64 ]. These differences likely resulted from combined effects of transposon activity and environmental adaptation [ 67 ], offering new perspectives for exploring plant mitochondrial genome evolution through further investigation. While other tRNA gene copy numbers vary among Camellia species, all of them (including C. tachangensis ) exhibited high copies of the trn M-CAU gene. This might relate to its role in transporting the initiation codon AUG. trn M-CAU likely enhances its expression to competitively occupy ribosomal P-sites, preventing non-initiator tRNA misbinding and ensuring protein synthesis fidelity [ 68 ]. The GC content (45.86%) and codon usage bias of C. tachangensis showed remarkable conservation, being highly similar to both Camellia and other species, reflecting the evolutionary stability of these genetic features in Angiosperms [ 27 – 29 , 58 – 64 ]. RNA editing is a widely occurring post-transcriptional mechanism that modified RNA by altering the types of nucleotides present within it [ 69 ]. To determine the final protein sequences of the mitochondrial genes in C. tachangensis , it is essential to predict RNA editing events for each gene. In this study, 537 RNA editing sites across 38 genes in C. tachangensis were identified. Previous indicated that these editing sites played a crucial role in gene expression. RNA editing in plants could restore codons altered by mutations, thereby ensuring that mRNAs encode proteins with normal functionality [ 70 ]. In addition, RNA editing is a prerequisite for the proper translation of certain mRNAs. In the mitochondrial genome of C. tachangensis , the initial codons for cox1 and nad4L are ACG. Through RNA editing, these start codons could be converted from ACG to ATG, thereby ensuring the proper function of mRNA translation. More importantly, RNA editing had been demonstrated to play a crucial role in regulating responses to environmental stress in certain plant species. Research indicated that specific RNA editing modifications—such as enhanced editing of mitochondrial genes nad 3, nad 7, and ccm Fn in Oryza sativa , alongside deficient editing of nad 4 and cox 3 in Arabidopsis thaliana —are correlated with improved tolerance to salt and drought stress, respectively [ 71 , 72 ]. However, although previous studies had described possible interactions between PLS-CsPPR proteins and target sequences of RNA editing sites in mitochondrial and chloroplast genes in Camellia species ( C. sinensis ) [ 73 ], a direct link between mitochondrial RNA editing and environmental stress adaptation in Camellia plants had not yet been established. To address this research gap, future studies could employ multi-omics correlation analysis methods, integrating the predicted RNA editing site data from this study, to investigate the potential mechanisms and roles of mitochondrial gene RNA editing in stress physiological responses of C. tachangensis and other Camellia species. This approach would provide theoretical support for developing precise conservation strategies. DNA could be transferred between the mitochondrial and chloroplast genomes within cells [ 74 ]. This process was accompanied by the insertion of exogenous tRNA genes to support the translation of mitochondrial PCGs [ 75 – 77 ]. In this study, we identified a total of 23 MTPTs in the mitochondrial genome of C. tachangensis , among which 7 MTPTs contained 1–3 tRNA genes. By comparing these fragments with those from other species in the sect. Thea and sect. Oleifera , we found partial MTPTs shared similarities across these species: these fragments were highly similar in length, and their tRNA gene compositions were entirely consistent (e.g., trnM-CAT, trnA-UGC–trnI-GAU–trnV-GAC, and trnD-GUC). Therefore, we hypothesized that the transfer events of these fragments could be traced back to before the divergence of these 2 taxonomic groups. In contrast, MTPTs identified in 4 species of the sect. Chrysantha were relatively scarce, with only 5–14 MTPTs per species. Moreover, only 1–2 MTPTs in each species contained tRNA genes, which might reflect the unique evolutionary trajectory of sect. Chrysantha . The Ka/Ks analysis indicated that the mitochondrial PCGs of C. tachangensis had primarily undergone purifying selection during the course of evolution (Ka/Ks < 1), which aligned with previous Ka/Ks analysis results between C. drupifera (sect. Oleifera ) and species from sect. Thea and sect. Chrysantha [ 27 ]. These phenomena suggest that purifying selection may play a dominant role in the evolution of mitochondrial PCGs in Camellia plants. It might stem from mitochondrial genes predominantly functioning in core metabolic pathways like oxidative phosphorylation. Non-synonymous mutations in these genes were often deleterious, resulting in their persistent purging through natural selection to maintain functional evolutionary conservation [ 78 ]. Furthermore, sequences of rpl 2 and sdh 3 genes detected in C. tachangensis completely match those of C. chekiangoleosa (sect. Camellia ) but differed from cultivated varieties (CSDHP, CSSRG) within the sect. Thea . This pattern might arise because C. tachangensis , as an early-diverged species of sect. Thea , retained primitive sequence characteristics of rpl 2 and sdh 3 genes shared with C. chekiangoleosa from the initial differentiation stage between sect. Thea and Sect. Camellia groups. In contrast, later-diverged Camellia species like CSDHP and CSSRG had accumulated mutations in these genes during evolution, ultimately resulting in sequence divergence from their corresponding genes in C. tachangensis . Although the species selected for the 2 phylogenetic trees were not entirely consistent, we can still observe that the chloroplast PCG phylogenetic tree exhibits higher resolution compared to the mitochondrial CDS-based phylogenetic tree. Additionally, the phylogenetic position of C. tachangensis in the mitochondrial CDS-based tree did not appear at the base of Camellia species as observed in the chloroplast PCG-based phylogenetic tree, but instead clustered with some C. sinensis variants with extremely low support values (BS = 22, PP = 0.41). This discrepancy may be related to several factors: First, the lack of available mitochondrial genome data for closely related species such as C. taliensis and C. gymnogyna likely reduced the phylogenetic support for the branch containing C. tachangensis . Future studies should prioritize generating mitochondrial genome data for these species to resolve the phylogenetic placement of C. tachangensis . Additionally, studies had shown that mitochondrial genomes evolved at a slower rate compared to chloroplast genomes, resulting in smaller genetic distances among related species in mitochondrial genomes [ 79 , 80 ]. Furthermore, although mitochondrial and chloroplast genomes were predominantly maternally inherited, both might undergo paternal leakage during inheritance, resulting in discrepancies in the genetic lineages of these 2 organellar genomes within the same species [ 81 ] 5. Conclusion This study reported the first sequencing and annotation of the mitochondrial genome of C. tachangensis , which exhibited a multichromosomal structure, comprising a 585,875 bp branched molecule (resolvable into 7 substructures) and a 221,056 bp circular molecule. A total of 63 functional elements were annotated, including 30 protein-coding genes (PCGs), 30 tRNAs, and 3 rRNAs. Comparative analysis identified 23 homologous chloroplast-derived fragments in the mitochondrial genome, introducing 10 intact tRNA genes. Ka/Ks analysis indicated that PCGs evolved predominantly under purifying selection (Ka/Ks < 1). Phylogenetic analysis based on chloroplast genome analysis strongly supported C. tachangensis close relationship with C. makuanica , C. taliensis , and C. gymnogyna (BS = 100, PP = 1.00). However, the phylogenetic tree based on mitochondrial CDS failed to identify species closely related to C. tachangensis due to the current lack of comprehensive mitochondrial genome data for the genus Camellia . Despite this limitation, our study filled a critical gap in organelle genomics of Camellia , offering valuable genomic resources for elucidating evolutionary mechanisms, advancing genetic improvement programs, and informing conservation strategies for this ecologically and economically important genus. Abbreviations PCGs Protein-coding genes mtDNA Mitochondrial genome cpDNA Chloroplast genome Ka/Ks Non-synonymous/synonymous mutation ratio RSCU Relative synonymous codon usage MTPT Mitochondrial plastid DNA sequence tRNA Transfer RNA rRNA Ribosomal RNA SSR simple sequence repeat Pi nucleotide diversity BS bootstrap support value PP posterior probabilities PPR Pentatricopeptide repeat. Declarations Acknowledgments We thank the Editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript. Authors’ contributions Z.L: Conceptualization, D.Z.J: Writing - original draft, Data curation, Formal analysis, Software. Z.L: Funding acquisition, Resources, Review & editing, Investigation. L.Z: Investigation, Methodology. Z.H.R: Resources, Supervision. X.X: Visualization, Investigation. X.H.Y: Methodology, Validation. Funding This research was supported by the National Natural Science Foundation of China (Grant No. 32400179), the Guizhou Provincial Basic Research Program (Natural Science) 2022 (072), the Guizhou University Student Innovation Project 2024 (302), and the 2024 Guizhou Science and Technology Innovation Talent Team Construction Project: Wildlife Innovation Team of the Forestry college of Guizhou University (Qiankeherencai CXTD[2025]053). Availability of data and materials The mitogenome sequences supporting the conclusions of this article are available in GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers: PQ658231 and PQ658232. Declaration of competing interest The authors declare that they have no competing or conflicting interests. Ethics approval and consent to participate All materials used in this study comply with international and national legal standards. 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Dietrich A, Small I, Cosset A, Weil JH, Maréchal-Drouard L. Editing and import: Strategies for providing plant mitochondria with a complete set of functional transfer RNAs. Biochimie. 1996;78(6):518–29. https://doi.org/10.1016/0300-9084(96)84758-4 . Ceci LR, Veronico P, Callerani R. Identification and mapping of tRNA genes on the Helianthus annuus mitochondrial genome. DNA Seq. 1996;6(3):159–66. https://doi.org/10.3109/10425179609010203 . Sangaré A, Weil JH, Grienenberger JM, Fauron C, Lonsdale D. Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize. Mol Gen Genet. 1990;223(2):224–32. https://doi.org/10.1007/BF00265058 . Unseld M, Marienfeld JR, Brandt P, Brennicke A. The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nat Genet. 1997;15(1):57–61. https://doi.org/10.1038/ng0197-57 . Mower JP, Sloan DB, Alverson AJ. Plant Mitochondrial Genome Diversity: The Genomics Revolution. In: Wendel J, Greilhuber J, Dolezel J, Leitch I, editors. Plant Genome Diversity. Volume 1. Vienna: Springer; 2012. pp. 123–44. https://doi.org/10.1007/978-3-7091-1130-7_9 . Wolfe KH, Li WH, Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc Natl Acad Sci USA. 1987;84(24):9054–8. https://doi.org/10.1073/pnas.84.24.9054 . Palmer JD, Herbon LA. Plant mitochondrial DNA evolves rapidly in structure, but slowly in sequence. J Mol Evol. 1988;28(1–2):87–97. https://doi.org/10.1007/BF02143500 . Bentley KE, Mandel JR, McCauley DE. Paternal leakage and heteroplasmy of mitochondrial genomes in Silene vulgaris: evidence from experimental crosses. Genetics. 2010;185(3):961–8. https://doi.org/10.1534/genetics.110.115360 . Additional Declarations No competing interests reported. Supplementary Files Fig.S1.pdf TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx TableS6.xlsx Cite Share Download PDF Status: Published Journal Publication published 15 May, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 09 Apr, 2025 Reviews received at journal 09 Apr, 2025 Reviews received at journal 29 Mar, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 29 Mar, 2025 Submission checks completed at journal 27 Mar, 2025 First submitted to journal 20 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5634953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435826330,"identity":"ed63e9ef-b7db-4a60-a3fa-f47154957000","order_by":0,"name":"Dongzhen Jiang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Dongzhen","middleName":"","lastName":"Jiang","suffix":""},{"id":435826331,"identity":"c74f3799-2f3d-4763-8c03-bc9890e5c7e5","order_by":1,"name":"Lei Zhou","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhou","suffix":""},{"id":435826332,"identity":"cb3512f9-1e26-4b58-9cc5-522afec3ca84","order_by":2,"name":"Zhaohui Ran","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhaohui","middleName":"","lastName":"Ran","suffix":""},{"id":435826333,"identity":"c88ab2db-e877-42fc-9a7b-49367be82406","order_by":3,"name":"Xu Xiao","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Xiao","suffix":""},{"id":435826334,"identity":"8dbcb239-8feb-4d65-a064-4bb883a3c394","order_by":4,"name":"Xuehang Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xuehang","middleName":"","lastName":"Yang","suffix":""},{"id":435826335,"identity":"58b0817e-028b-478e-98c4-eb27d2f6bbba","order_by":5,"name":"zhi li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDACdsb2Hx8gTAMitTAzNkjOIFELA4M0D0la5JuZG4xtftUlNrA3b5NgqLlDWAtjM2NDcm4fW2IDz7EyCYZjz4hwF9Avh3N7eBIbJHLMJIBswlrYmBkbmy17JBIb5N8QqYWHmbGZmeGHAdAWHiK1SDAztjH2NiQYt/GkFVskHCNCi3x7+zOGH3/qZPvZD2+88aGGCC1gwNgG9BSIkUCkBiD4Q7zSUTAKRsEoGIEAACHmMckoAG0gAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"zhi","middleName":"","lastName":"li","suffix":""}],"badges":[],"createdAt":"2024-12-13 02:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5634953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5634953/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11673-z","type":"published","date":"2025-05-15T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79692814,"identity":"244a22c5-13f5-4d11-90e8-c44176eb9a58","added_by":"auto","created_at":"2025-04-01 14:56:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3550997,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of the mitochondrial genome of \u003cem\u003eC. tachangensis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/945a9d8b6ba19da9e8dac5bc.jpeg"},{"id":79691675,"identity":"ae114682-7abc-41eb-a913-b97079353330","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2916242,"visible":true,"origin":"","legend":"\u003cp\u003eHypothetical products generated by recombination mediated by R7, R8, and R10. The black arrows indicate the repetitive sequences R7, R8, and R10 (simply written as 7, 8, and 10) involved in recombination, with the arrow direction showing their orientation. The colored segments represent the DNA fragments C1, C3, C4, C5, C6, C9, and C10 (simply written as 1, 3, 4, 5, 6, 9, and 10) located between these repetitive sequences.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/e72e068926563dca73dbe073.jpeg"},{"id":79691682,"identity":"6369801e-a7b8-425f-b643-f016ba8d669f","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6822435,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat analysis of the mitochondrial genome in \u003cem\u003eC. tachangensis\u003c/em\u003e. The arc represents Chr1 (green) and Chr2 (purple).The ticks inner circles are SSR (Blue) and tandem repeat (red). The ribbons represents dispersed repeat.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/1391d9b67871e0f41754221e.jpeg"},{"id":79693376,"identity":"917a7788-cc8b-4e94-9cbb-08414e541ec8","added_by":"auto","created_at":"2025-04-01 15:04:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":305513,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of RNA editing sites in the \u003cem\u003eC. tachangensis \u003c/em\u003emitochondrial genome. (A: Characterization of RNA-editing sites; B: Proportion of different RNA-editing types; C: Numbers of RNA-editing sites in the mtDNA)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/cc25d8be81e10f2535f0e121.png"},{"id":79693375,"identity":"e994f482-9415-4410-bead-15852fcdfbf3","added_by":"auto","created_at":"2025-04-01 15:04:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101084,"visible":true,"origin":"","legend":"\u003cp\u003eRelative synonymous codon usage in the \u003cem\u003eC.\u003c/em\u003e \u003cem\u003etachangensis\u003c/em\u003e mitochondrial genome\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/cd6117f3a53d73dfbca682e9.png"},{"id":79693377,"identity":"7626ddc2-cad8-4441-a2e0-8fd4976ed405","added_by":"auto","created_at":"2025-04-01 15:04:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2433276,"visible":true,"origin":"","legend":"\u003cp\u003eCovariance analysis of the homologous fragments of \u003cem\u003eC. tachangensis\u003c/em\u003emitochondrial genomes. The blue arc represents Chr1. The light green represents Chr2. The dark green arc represents chloroplast DNA. The homologous fragments are indicated by the connecting ribbons between the blue (light green) and dark green arcs.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/f5bdf11940ebb9cfd9b623c0.png"},{"id":79691685,"identity":"19f75351-7f32-4999-afda-c3ad47f673cf","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":123510,"visible":true,"origin":"","legend":"\u003cp\u003eSubstitution rates of conserved PCGs in the mitogenomes of 9 species of the genus \u003cem\u003eCamellia\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/7d832aaa41adee30530f1af1.png"},{"id":79691694,"identity":"df90d187-971b-418a-a619-7e124b6d43e0","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1739312,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on mitochondrial CDS. (Maximum likelihood (ML) and Bayesian (BI) trees; BS and PP are indicated above the branches as BS/PP)\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/aa91ac91accb6ef9defc31c3.jpeg"},{"id":79691689,"identity":"404da9bc-2b2e-4862-93a0-f8e9849e23dc","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1056877,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on chloroplast PCG. (Maximum likelihood (ML) and Bayesian (BI) trees; BS and PP are indicated above the branches as BS/PP)\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/36aa0fec75ad77aa4f065b18.jpeg"},{"id":83067970,"identity":"ac0592c3-4cbc-410e-8641-ad0057d348d6","added_by":"auto","created_at":"2025-05-19 16:08:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19790843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/7de3d0e8-a799-485a-885a-9b17a72bf5de.pdf"},{"id":79692818,"identity":"fb27019b-db10-48fc-8048-f59f0a017c88","added_by":"auto","created_at":"2025-04-01 14:56:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":220873,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/58d1c97415324c8b5a8d9b2d.pdf"},{"id":79691683,"identity":"fab75cbf-62ca-409a-a27c-c1d6db958912","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":55674,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/51225fbfd35d36edc5a731ba.xlsx"},{"id":79691676,"identity":"5c2677a9-a969-482f-89fb-cb4f26a248f3","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":35576,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/796fb4c9185bfb880338bf31.xlsx"},{"id":79691678,"identity":"71f36aa1-6fa6-4c40-af3b-fb3b7fc16f7e","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12212,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/9885214389c2738d336a41e1.xlsx"},{"id":79691686,"identity":"2f245267-80f0-4d59-bb82-618dfe7637bb","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11502,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/c7b1776800b73d29eb55125d.xlsx"},{"id":79692823,"identity":"e3ec287f-9c8e-4f4c-8668-2e4848b03a53","added_by":"auto","created_at":"2025-04-01 14:56:22","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11359,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/0a1284e012bd3fa476be6b6a.xlsx"},{"id":79691690,"identity":"db341af2-6b01-4eeb-afd4-2850f23d5df8","added_by":"auto","created_at":"2025-04-01 14:48:22","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14936,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5634953/v1/60860df4db76c2afdb6be58e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assembly and analysis of the complete mitochondrial genome of an endemic Camellia species of China, Camellia tachangensis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eCamellia tachangensis\u003c/em\u003e F. C. Zhang, belonging to sect. \u003cem\u003eThea\u003c/em\u003e (L.) of the genus \u003cem\u003eCamellia\u003c/em\u003e, is an endemic species native to the border regions of Yunnan, Guizhou, and Guangxi Provinces in China [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This species has a distinctive morphological features of a capsule with five-locular ovary and been recognized as the primitive plant within sect. \u003cem\u003eThea\u003c/em\u003e Dyer in genus \u003cem\u003eCamellia\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Some studies on the systematic taxonomy of \u003cem\u003eCamellia\u003c/em\u003e plants showed \u003cem\u003eC. tachangensis\u003c/em\u003e has high scientific value for exploring the origin and evolution of \u003cem\u003eCamellia\u003c/em\u003e in southwest China [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the same time, as a member of sect. \u003cem\u003eThea\u003c/em\u003e, \u003cem\u003eC. tachangensis\u003c/em\u003e was served as a processing raw material for \u0026ldquo;Pu\u0026rsquo;an red tea\u0026rdquo; with unique aroma and flavor that it is a product of China\u0026rsquo;s national geographical indication [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Due to environmental degradation and human interference, coupled with its relatively low reproductive capacity, the survival of \u003cem\u003eC. tachangensis\u003c/em\u003e was facing a significant crisis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To improve the survival status of \u003cem\u003eC. tachangensis\u003c/em\u003e, researchers had conducted comprehensive and in-depth studies on its physiological, breeding, propagation, biochemical characteristics and so on [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, there were few studies in genome biology, and most of them only focused on population genetics and chloroplast genomes [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Studies concerning the complete mitochondrial genomes were still lacking.\u003c/p\u003e \u003cp\u003eMitochondria in plant cells are essential organelles that not only participate in cellular respiration but also play crucial roles in regulating intracellular metabolic networks [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In botanical research, the mitochondrial genes of plants hold significant value for study. First, mitochondrial genes are involved in the synthesis of essential enzymatic components such as ATP synthase, cytochrome c oxidase, and NADH dehydrogenase, which are crucial for the respiratory metabolism of plants [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Investigating the composition and evolution of these genes will provide novel insights into the genetic improvement of \u003cem\u003eC. tachangensis\u003c/em\u003e and its related species. Furthermore, most plant mitochondrial DNA exhibits maternal inheritance characteristics, with a lower level of heterozygosity than nuclear genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consequently, the mitochondrial genome can be effectively utilized for the classification and identification of species within the genus \u003cem\u003eCamellia\u003c/em\u003e, which display significant genetic heterozygosity and phenotypic diversity [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, structural variations and the insertion of chloroplast-derived fragments occur relatively frequently in mitochondrial DNA. These processes typically take place during different stages of plant lineage differentiation. By comparing the similarities and differences in mitochondrial genome structures and chloroplast-derived fragments across different species, we can gain deeper insights into the evolutionary history of the \u003cem\u003eCamellia\u003c/em\u003e genus [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study employed a combination of high-throughput sequencing and long-read sequencing to achieve the first complete sequencing, assembly, and annotation of mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e while also exploring its substructure. Comprehensive analyses were performed to investigate multiple genomic features including codon usage patterns, chloroplast-derived homologous sequences, repetitive element distribution, RNA editing sites, evolutionary selection pressures through Ka/Ks ratio calculations, and phylogenetic relationships. The findings of this work established a foundational genetic data framework for future research on molecular breeding programs and evolutionary dynamics involving \u003cem\u003eC. tachangensis\u003c/em\u003e and its related species.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Material collection, DNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eIn this study, we selected the leaves of \u003cem\u003eC. tachangensis\u003c/em\u003e from the forestry center in Longli County, Guizhou Province (N 26\u0026deg;24\u0026prime;49\u0026Prime;\u0026ndash;26\u0026deg;44\u0026prime;30\u0026Prime;, E 106\u0026deg;48\u0026prime;12\u0026Prime;\u0026ndash;107\u0026deg;8\u0026prime;50\u0026Prime;), as the research materials. Fresh leaves were collected and preserved in liquid nitrogen at -80\u0026deg;C. Total DNA was extracted by CTAB method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mitochondrial genome was sequenced and assembled via a combination of high-throughput sequencing (Illumina Novaseq 6000) and long-read sequencing (Oxford Nanopore R10.4). The high-throughput sequencing strictly conformed to the standard operating procedures provided by Illumina. First, DNA quality and concentration were assessed via 1% agarose gel electrophoresis and a NanoDrop 2000. The high-quality DNA samples were then fragmented through ultrasonic mechanical disruption. The fragmented DNA subsequently underwent purification, end repair, and ligation of sequencing adapters. Finally, the selected DNA was amplified via PCR to construct the sequencing library. The constructed library underwent quality control, and those that conformed with the quality assessment were sequenced via the Illumina Novaseq 6000 platform (Illumina, San Diego, California, United States) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The raw data were subsequently filtered via fastp v0.23.4 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the process of the long-read sequencing, genomic DNA was first randomly fragmented. Subsequently, magnetic beads were utilized for enrichment and purification to obtain large DNA fragments. Following this step, gel extraction was performed on the large fragments, and damage repair was conducted on the fragmented DNA. The purified fragments subsequently underwent end repair and A-tailing. The SQK-LSK109 kit was used to ligate adapters, thereby constructing a DNA library for quantitative assessment. Next, an appropriate amount of the DNA library was loaded onto the flow cell and subjected to real-time single-molecule sequencing on the Oxford Nanopore PromethION sequencer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Finally, the long-read sequencing data were filtered via Filtlong v0.24 software, and a Perl script was used for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Assembly and annotation of mitochondrial genome\u003c/h2\u003e \u003cp\u003eFirst, the raw long-read sequencing data were aligned with the plant mitochondrial gene database via minimap2 v2.1. Subsequently, sequences with alignment lengths greater than 50 bp were selected as candidate sequences. Among these candidates, those exhibiting a greater number of aligned genes (a single sequencing read containing multiple core genes) and superior alignment quality (exhibiting a relatively complete coverage of core genes) were chosen as seed sequences. Minimap2 was subsequently employed to align the original sequencing data against the seed sequences, filtering for overlaps greater than 1 kb and similarity exceeding 70% to incorporate additional sequences into the seed sequences [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The third-generation assembly software Canu v2.2 was employed to correct the obtained third-generation data [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Subsequently, Bowtie2 v2.3.5.1 was utilized to align the second-generation data with the corrected sequences. The paired high-throughput data and the corrected long-read sequencing data were then assembled via the default parameters of Unicycler v0.4.8 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Subsequently, Bandage software v0.8.1 was employed to visualize the assembly results and make manual adjustments as necessary. Owing to the presence of multiple subcircular structures or even non-circular complex physical configurations in mitochondrial genomes, the corrected third-generation sequencing data were aligned to the contigs generated by Unicycler via minimap2. The branch directions were subsequently manually determined to obtain the final assembly results.\u003c/p\u003e \u003cp\u003eThe annotation of the mitochondria genes was carried out through the following steps: utilizing the Basic Local Alignment Search Tool-Nucleotide (BLASTN) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the protein-coding genes and rRNA genes were compared with publicly available reference mitochondrial genome sequences from plants. Manual adjustments were subsequently made using the closely related species \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e (Genbank ID: PP212895) as a reference genome [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, tRNA genes were annotated via tRNAscan-SE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lowelab.ucsc.edu/tRNAscan-SE/\u003c/span\u003e\u003cspan address=\"http://lowelab.ucsc.edu/tRNAscan-SE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The Open Reading Frame Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/gorf/gorf.html\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/gorf/gorf.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for ORF annotation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The minimum length was set to 102 bp, with redundant sequences and overlapping known genes excluded. Sequences longer than 300 bp were aligned against the NR database for annotation. The mitochondrial genome map was constructed via OGDraw (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Analysis of repeat sequences and RNA editing prediction\u003c/h2\u003e \u003cp\u003eTo clarify the 3 types of repetitive sequences in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, simple sequence repeat (SSR), tandem repeat, and dispersed repeat, MISA v1.0 was employed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webblast.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"https://webblast.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the identification of SSRs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Tandem repeats were identified via Tandem Repeats Finder v4.09 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tandem.bu.edu/trf/trf.submit.options.html\u003c/span\u003e\u003cspan address=\"http://tandem.bu.edu/trf/trf.submit.options.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The identification of dispersed repeats was conducted via BLASTN software (v2.10.1, parameters: -word size 7, evalue 1e-5). During this process, redundant and tandem repeat sequences were removed [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Ultimately, all the identification results were visualized via Circos v0.69-5 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To further investigate the RNA editing sites, we utilized the online tool PREPACT3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.prepact.de/\u003c/span\u003e\u003cspan address=\"http://www.prepact.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict RNA editing events, setting a critical threshold of 0.001 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In Excel 2021, the distribution of RNA editing sites for different genes and the number of amino acid variation types were visualized using bar charts; while the proportion of hydrophilic and hydrophobic group change types was presented through pie charts to show their ratio relationship.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Analysis of relative synonymous codon usage (RSCU) and mitochondrial plastid DNAs (MTPTs) in the mitogenome\u003c/h2\u003e \u003cp\u003eThe protein-coding sequences were obtained via the default settings of Phylosuit v1.22 software [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The relative synonymous codon usage rates (RSCU) based on mitochondrial genome protein-coding genes were calculated via MEGA v7.0 software [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondrial plastid DNAs (MTPTs) refer to DNA fragments of plasmid origin present in the mitochondrial genome. In this study, chloroplast genome sequences from the same samples were extracted, and BLASTN software was used to identify homologous sequences between the chloroplast and mitochondrial genomes, with a similarity threshold set at 70% and an E value of 1e-5. To visualize the homologous segments between the chloroplast and mitochondrial genomes more intuitively, Circos v0.69-5 was used [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Analysis of nucleotide diversity (Pi) and selection pressure\u003c/h2\u003e \u003cp\u003eTo comprehensively analyze the diversity and the impact of selection on mitochondrial genomes in \u003cem\u003eCamellia\u003c/em\u003e species, this study selected mitochondrial genomes from 9 representative species (including \u003cem\u003eC. tachangensis\u003c/em\u003e) within 4 related taxonomic groups of genus \u003cem\u003eCamellia\u003c/em\u003e: sect. \u003cem\u003eThea\u003c/em\u003e, sect. \u003cem\u003eChrysantha\u003c/em\u003e, sect. \u003cem\u003eCamellia\u003c/em\u003e, and sect. \u003cem\u003eHeterogenea\u003c/em\u003e for comparison, and conducted Pi analysis and Ka/Ks analysis as follows: mitochondrial sequences of 8 \u003cem\u003eCamellia\u003c/em\u003e species were downloaded from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome/organelle/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome/organelle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): \u003cem\u003eC. tianeensis\u003c/em\u003e (PP727208), \u003cem\u003eC. nitidissima\u003c/em\u003e (NC_067639), \u003cem\u003eC. chekiangoleosa\u003c/em\u003e (NC_086749), \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e cv. Dahongpao (PP212895), \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e cv. Rougui (PP212896), \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003epubilimba\u003c/em\u003e (ON782577), \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003eassamica\u003c/em\u003e (NC_043914), and \u003cem\u003eC. gigantocarpa\u003c/em\u003e (OP270590). The MAFFT v7.427 software was used for global alignment of these plant mitochondrial genomes along with that of \u003cem\u003eC. tachangensis\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The resulting alignment file was used to calculate Pi values for each shared gene with DnaSP v6.12.03 and Ka/Ks ratios for shared PCGs with KaKs_Calculator v3.0 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The Ka/Ks ratio data were visualized in the form of a box plot using Excel 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eIn the phylogenetic analysis of mitochondrial and chloroplast genomes, while we both primarily focused on species within the genus \u003cem\u003eCamellia\u003c/em\u003e, the study adopted differentiated phylogenetic tree construction strategies due to the data imbalance between the 2 organelle genomes in the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome/organelle/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome/organelle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e): For mitochondrial genomes with relatively scarce data, the mitochondrial CDS based phylogenetic tree incorporated 15 represented \u003cem\u003eCamellia\u003c/em\u003e species (including \u003cem\u003eC. sinensis\u003c/em\u003e and its varieties, \u003cem\u003eC. oleifera\u003c/em\u003e, \u003cem\u003eC. chekiangoleosa\u003c/em\u003e, etc.) to investigate the phylogenetic position of \u003cem\u003eC. tachangensis.\u003c/em\u003e It also included 14 species from different angiosperm families (e.g., Ericaceae, Solanaceae and Apiaceae) and the gymnosperm \u003cem\u003eTaxus wallichiana\u003c/em\u003e as outgroups. Tbtools software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/CJ-Chen/TBtools/releases\u003c/span\u003e\u003cspan address=\"https://github.com/CJ-Chen/TBtools/releases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to extract 24 conserved mitochondrial protein-coding genes (PCGs) among these species [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], including \u003cem\u003eatp\u003c/em\u003e1, \u003cem\u003eatp\u003c/em\u003e4, \u003cem\u003eatp\u003c/em\u003e6, \u003cem\u003eatp\u003c/em\u003e8, \u003cem\u003eatp\u003c/em\u003e9, \u003cem\u003eccm\u003c/em\u003eB, \u003cem\u003eccm\u003c/em\u003eC, \u003cem\u003eccm\u003c/em\u003eFc, \u003cem\u003eccm\u003c/em\u003eFn, \u003cem\u003ecob\u003c/em\u003e, \u003cem\u003ecox\u003c/em\u003e1, \u003cem\u003ecox\u003c/em\u003e2, \u003cem\u003ecox\u003c/em\u003e3, \u003cem\u003emat\u003c/em\u003eR, \u003cem\u003emtt\u003c/em\u003eB, \u003cem\u003enad\u003c/em\u003e1, \u003cem\u003enad\u003c/em\u003e2, \u003cem\u003enad\u003c/em\u003e3, \u003cem\u003enad\u003c/em\u003e4, \u003cem\u003enad\u003c/em\u003e4L, \u003cem\u003enad\u003c/em\u003e5, \u003cem\u003enad\u003c/em\u003e6, \u003cem\u003enad\u003c/em\u003e7, and \u003cem\u003enad\u003c/em\u003e9. The coding sequences (CDSs) of these genes within the mitochondrial genomes of 28 species were aligned via MAFFT v7.427 for interspecies sequence comparison [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The aligned sequences were concatenated and trimmed via trimAl v1.4. Model prediction was subsequently conducted with jmodeltest v2.1.10 to identify the GTR model. The maximum likelihood phylogenetic tree was then constructed via RAxML v8.2.10 with the GTRGAMMA model and a bootstrap value of 1000 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The Bayesian phylogenetic tree was constructed via MrBayes v3.2.7 with the Markov chain Monte Carlo method for 1,000,000 generations, and sampling trees every 100 generations[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the phylogenetic analysis based on chloroplast PCGs, 23 representative \u003cem\u003eCamellia\u003c/em\u003e species (covering 10 significant sections including sect. \u003cem\u003eThea\u003c/em\u003e, sect. \u003cem\u003eChrysantha\u003c/em\u003e, and sect. \u003cem\u003eOleifera\u003c/em\u003e), due to they are closely related in \u003cem\u003eCamellia\u003c/em\u003e genus. Meanwhile, the sister group (\u003cem\u003ePolyspora axillaris\u003c/em\u003e and \u003cem\u003eSchima superba\u003c/em\u003e) of \u003cem\u003eCamellia\u003c/em\u003e genus was selected as the outgroup. The maximum likelihood phylogenetic tree and the Bayesian phylogenetic tree were constructed based on 53 conserve\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ed\u003c/span\u003e chloroplast PCGs among these species: \u003cem\u003eacc\u003c/em\u003eD, \u003cem\u003eatp\u003c/em\u003eA, \u003cem\u003eatp\u003c/em\u003eE, \u003cem\u003eatp\u003c/em\u003eF, \u003cem\u003eatp\u003c/em\u003eH, \u003cem\u003eatp\u003c/em\u003eI, \u003cem\u003emat\u003c/em\u003eK, \u003cem\u003epet\u003c/em\u003eA, \u003cem\u003epet\u003c/em\u003eB, \u003cem\u003epet\u003c/em\u003eD, \u003cem\u003epet\u003c/em\u003eG, \u003cem\u003epet\u003c/em\u003eL, \u003cem\u003epet\u003c/em\u003eN, \u003cem\u003epsa\u003c/em\u003eA, \u003cem\u003epsa\u003c/em\u003eB, \u003cem\u003epsa\u003c/em\u003eC, \u003cem\u003epsb\u003c/em\u003eA, \u003cem\u003epsb\u003c/em\u003eC, \u003cem\u003epsb\u003c/em\u003eD, \u003cem\u003epsb\u003c/em\u003eE, \u003cem\u003epsb\u003c/em\u003eF, \u003cem\u003epsb\u003c/em\u003eH, The analysis methods employed were identical to those used for mitochondrial genomes. Finally, visualization was performed via Interactive Tree Of Life (ITOL) software v4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Genomic features of \u003cem\u003eC. tachangensis\u003c/em\u003e mitochondrial genome\u003c/h2\u003e \u003cp\u003eThe total mitochondrial DNA of \u003cem\u003eC. tachangensis\u003c/em\u003e was sequenced, and the raw data were prepared for assembly, resulting in 16.34 Gb Illumina sequencing data (Q20\u0026thinsp;=\u0026thinsp;96.36%,Q30\u0026thinsp;=\u0026thinsp;90.75%) and 20.5 Gb Nanopore PromethION sequencing data with a N50 read length of 21,799 bp. The assembly results indicated that the mitochondrial genome sequence of \u003cem\u003eC. tachangensis\u003c/em\u003e was 746,931 bp (GC content\u0026thinsp;=\u0026thinsp;45.86%), consisting of one multibranched sequence and one circular sequence, which were designated chromosome 1 (Chr1) and chromosome 2 (Chr2), with lengths of 525,875 bp and 221,056 bp, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results of the read mapping indicated that there were no reads present between Chr1 and Chr2 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), suggesting that Chr1 and Chr2 were relatively independent. Additionally, Chr1 was capable of producing 7 kinds of substructures, whereas Chr2 didn\u0026rsquo;t exhibit any substructures. A total of 24 core protein-coding genes, 16 variable protein-coding genes, 3 ribosomal RNA (rRNA) genes, and 30 transfer RNA (tRNA) genes were identified. The core protein-coding genes could be categorized into 7 functional groups: ATP synthase (\u003cem\u003eatp\u003c/em\u003e1, \u003cem\u003eatp\u003c/em\u003e4, \u003cem\u003eatp\u003c/em\u003e6, \u003cem\u003eatp\u003c/em\u003e8, and \u003cem\u003eatp\u003c/em\u003e9), Cytochrome c maturation proteins (\u003cem\u003eccm\u003c/em\u003eB, \u003cem\u003eccm\u003c/em\u003eC, \u003cem\u003eccm\u003c/em\u003eFc, and \u003cem\u003eccm\u003c/em\u003eFn), Ubiquinol cytochrome c reductase (\u003cem\u003ecob\u003c/em\u003e), Cytochrome c oxidases (\u003cem\u003ecox\u003c/em\u003e1, \u003cem\u003ecox\u003c/em\u003e2, and \u003cem\u003ecox\u003c/em\u003e3), Maturases (\u003cem\u003emat\u003c/em\u003eR), Transport membrane proteins (\u003cem\u003emtt\u003c/em\u003eB), and NADH dehydrogenases (\u003cem\u003enad\u003c/em\u003e1, \u003cem\u003enad\u003c/em\u003e2, and \u003cem\u003enad\u003c/em\u003e3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, the exons of nad1 and nad2 were distributed on both Chr1 and Chr2; these segments require post-transcriptional RNA splicing to assemble into complete gene sequences. The analysis of 14 variable protein-coding genes revealed 8 types of small subunit ribosomal proteins (\u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e13, \u003cem\u003erps\u003c/em\u003e14, \u003cem\u003erps\u003c/em\u003e3, \u003cem\u003erps\u003c/em\u003e4, \u003cem\u003erps\u003c/em\u003e7, \u003cem\u003erps\u003c/em\u003e12, and \u003cem\u003erps\u003c/em\u003e20), 4 types of large subunit ribosomal proteins (\u003cem\u003erpl\u003c/em\u003e10, \u003cem\u003erpl\u003c/em\u003e16, \u003cem\u003erpl\u003c/em\u003e2, and \u003cem\u003erpl\u003c/em\u003e5), and 2 types of succinate dehydrogenases (\u003cem\u003esdh\u003c/em\u003e3 and \u003cem\u003esdh\u003c/em\u003e4). Notably, both \u003cem\u003esdh\u003c/em\u003e3 and \u003cem\u003erps\u003c/em\u003e19 appeared twice in the genome: once as functional genes and another as pseudogenes. A total of 30 tRNA genes were annotated. Among these annotations, \u003cem\u003etrn\u003c/em\u003eM-CAU was recorded 5 times on Chr1 and once on Chr2. Additionally, \u003cem\u003etrn\u003c/em\u003eS-UGA was annotated twice on Chr1, \u003cem\u003etrn\u003c/em\u003eI-GAU was annotated twice on Chr2, and \u003cem\u003etrn\u003c/em\u003eP-UGG was noted once on both Chr1 and Chr2. Furthermore, 14 genes contained introns. Among these genes, ten possess one intron each (\u003cem\u003eccm\u003c/em\u003eFc, \u003cem\u003erpl\u003c/em\u003e2, \u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e3, \u003cem\u003etrn\u003c/em\u003eA-UGC, \u003cem\u003etrn\u003c/em\u003eF-AAA, \u003cem\u003etrn\u003c/em\u003eI-GAU (2), \u003cem\u003etrn\u003c/em\u003eS-UGA, and \u003cem\u003etrn\u003c/em\u003eT-UGU); one gene contained 2 introns (\u003cem\u003enad\u003c/em\u003e4); and 4 genes had 4 introns (\u003cem\u003enad\u003c/em\u003e1, \u003cem\u003enad\u003c/em\u003e2, \u003cem\u003enad\u003c/em\u003e5, and \u003cem\u003enad\u003c/em\u003e7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of genes in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene name (Chr1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene name (Chr2)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003eCore genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatp\u003c/em\u003e1, \u003cem\u003eatp\u003c/em\u003e6, \u003cem\u003eatp\u003c/em\u003e8, \u003cem\u003eatp\u003c/em\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eatp\u003c/em\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytohrome c biogenesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccm\u003c/em\u003eB, \u003cem\u003eccm\u003c/em\u003eFc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eccm\u003c/em\u003eC, \u003cem\u003eccm\u003c/em\u003eFn\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUbichinol cytochrome c reductase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecob\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCytochrome c oxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecox\u003c/em\u003e1,\u003cem\u003ecox\u003c/em\u003e2,\u003cem\u003ecox\u003c/em\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003emat\u003c/em\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransport membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003emtt\u003c/em\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNADH dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003enad\u003c/em\u003e1-TS, \u003cem\u003enad\u003c/em\u003e2-TS, \u003cem\u003enad\u003c/em\u003e4, \u003cem\u003enad\u003c/em\u003e5-TS, \u003cem\u003enad\u003c/em\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003enad\u003c/em\u003e1-TS, \u003cem\u003enad\u003c/em\u003e2-TS, \u003cem\u003enad\u003c/em\u003e3, \u003cem\u003enad\u003c/em\u003e4L, \u003cem\u003enad\u003c/em\u003e7, \u003cem\u003enad\u003c/em\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eVariable genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal proteins (LSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl\u003c/em\u003e10, \u003cem\u003erpl\u003c/em\u003e16, \u003cem\u003erpl\u003c/em\u003e2, \u003cem\u003erpl\u003c/em\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal proteins (SSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e13, \u003cem\u003erps\u003c/em\u003e14, \u003cem\u003erps\u003c/em\u003e3, \u003cem\u003erps\u003c/em\u003e4, \u003cem\u003erps\u003c/em\u003e7, #\u003cem\u003erps\u003c/em\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003erps\u003c/em\u003e12, \u003cem\u003erps\u003c/em\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuccinate dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#\u003cem\u003esdh\u003c/em\u003e3, \u003cem\u003esdh\u003c/em\u003e3, \u003cem\u003esdh\u003c/em\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRibosomal RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn\u003c/em\u003e18, \u003cem\u003errn\u003c/em\u003e26, \u003cem\u003errn\u003c/em\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransfer RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eC-GCA, \u003cem\u003etrn\u003c/em\u003eD-GTC,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eF-GAA, \u003cem\u003etrn\u003c/em\u003eK-TTT,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eM-CAT(5), \u003cem\u003etrn\u003c/em\u003eN-GTT, \u003cem\u003etrn\u003c/em\u003eP-TGG, \u003cem\u003etrn\u003c/em\u003eS-CGA,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eS-GCT, \u003cem\u003etrn\u003c/em\u003eS-TGA(2),\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eT-TGT, \u003cem\u003etrn\u003c/em\u003eY-GTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eA-TGC, \u003cem\u003etrn\u003c/em\u003eE-TTC,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eF-AAA, \u003cem\u003etrn\u003c/em\u003eG-GCC,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eH-GTG, \u003cem\u003etrn\u003c/em\u003eI-GAT(2),\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eM-CAT, \u003cem\u003etrn\u003c/em\u003eN-ATT,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eP-TGG, \u003cem\u003etrn\u003c/em\u003eQ-TTG,\u003c/p\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eV-GAC, \u003cem\u003etrn\u003c/em\u003eW-CCA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: Numbers after gene names are the number of copies. Genes preceded by the # symbol represent pseudogenes. Genes followed by -TS need to be spliced into complete genes by RNA splicing after transcription.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Different configurations of the \u003cem\u003eC. tachangensis\u003c/em\u003e mitogenome\u003c/h2\u003e \u003cp\u003eThe phenomenon of recombination mediated by homologous fragments was commonly observed in the mitochondrial genome of cells. On Chr1, 3 pairs of dispersed repeats (homologous fragments), designated R7, R8, and R10, with lengths ranging from 1,190 to 8,440 bp, were identified. The similarity between the paired repeat units reached as high as 99.965\u0026ndash;100%. Among these sequences, R7 and R8 were classified as direct repeats, whereas R10 was categorized as a palindromic repeat. Collectively, these 3 pairs of repeats facilitated the formation of 7 substructures within Chr1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe homologous fragments of the mitochondrial genome in \u003cem\u003eC. tachangensis\u003c/em\u003e mediated recombination through 2 distinct mechanisms, which were primarily determined by the orientation differences between these segments: (1) In M1, the arrangement directions of the homologous segments R7 and R8 within their respective groups were identical. When either R7 or R8 broke, the 2 homologous segments recombined in a head-to-tail manner, resulting in M1 splitting from a large ring into 2 smaller rings, producing either M8 or M6. If both R7 and R8 break simultaneously, M1 could recombine to form a new large ring designated M5. At this point, in a clockwise direction, the sequence order of the non-homologous fragments changes from C1\u0026rarr;C5\u0026rarr;C9\u0026rarr;C4\u0026rarr;C6\u0026rarr;C3 to C1\u0026rarr;C6\u0026rarr;C3\u0026rarr;C9\u0026rarr;C4\u0026rarr;C5. The distinction between M1 and M5 lies in the reciprocal positioning of C6 and C3 relative to C5. However, there is no alteration in the arrangement direction of each sequence. (2) In contrast, within M1, owing to opposing orientations between the 2 homologous segments associated with R10, an inversion phenomenon occurs among adjacent non-homologous fragments. Specifically, during the recombination from M1 to M2 mediated by R10, segment C6\u0026rarr;C4 experiences a 180\u0026deg; inversion in its clockwise orientation, thus altering the sequence order of the non-homologous fragments from C1\u0026rarr;C5\u0026rarr;C9\u0026rarr;C6\u0026rarr;C4\u0026rarr;C3 to C1\u0026rarr;C5\u0026rarr;C9\u0026rarr;C4\u0026rarr;C6\u0026rarr;C3. Furthermore, one homologous segment of R8 is located between fragments C4 and C6. Consequently, this inversion involving fragment pairings from C4 to C6 resulted in an inverse arrangement for that particular homologous segment: it is transformed from R\u0026thinsp;+\u0026thinsp;8 to R-8. Both homologs of R8 now exhibit opposite orientations, leading to an inverted recombination mechanism for R8 that allows for the transformation of M2 into M3. A similar outcome is observed during the transition from M5 to M4 through recombination processes. The above hypothesis can be observed in the coverage validation map aligned to the assembly results of long reads, confirming the presence of 7 potential substructures on Chr1 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis phenomenon revealed that recombination mediated by specific pairs of homologous segments within mitochondrial genomes could influence alternative pairs\u0026rsquo; modes of recombination, thereby enriching DNA with diverse substructures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Analysis of repeat sequences\u003c/h2\u003e \u003cp\u003eA total of 223 SSRs were identified in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), with 160 located on Chr1 and 63 on Chr2. On Chr1 and Chr2, there are 19 and 12 mononucleotide (mono-), 48 and 12 dinucleotide (di-), 22 and 7 trinucleotide (tri-), and 61 and 25 tetranucleotide (tetra-) SSRs, respectively. There is 1 hexanucleotide (hexa-) SSR present on both Chr1 and Chr2. The highest proportion of SSRs on both chromosomes were found to be tetranucleotides, accounting for approximately 38.125% on Chr1 and 39.682% on Chr2. Furthermore, we observed that A/T is the most prevalent type among the mononucleotide SSRs. In total, there were also 28 tandem repeat sequences within the mitochondrial genome; the longest sequence was located on Chr1, with a copy number of 2, measuring 78 bp in length, whereas the shortest sequence resided on Chr2, with a copy number of 2, measuring only 24 bp in length (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The mitochondrial genome contains a substantial number of dispersed repetitive sequences, totaling 479. This dataset included 266 palindromic repeat sequences and 213 forward repeat sequences, with lengths ranging from 29 to 8,452 bp (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Notably, 88.28% of these sequences were shorter than 100 bp, with the most common lengths falling between 29 and 49 bp. Furthermore, the analysis of dispersed repetitive sequences indicated that transposon exchange between Chr1 and Chr2 occurred quite frequently; 161 sequences were copied from Chr1 to Chr2, and only 28 sequences were transferred in the opposite direction (from Chr2 to Chr1). Among all the homologous fragments identified, only 3 segments exceeded a length of 1,000 bp, each exhibiting greater than or equal to 99.96% similarity. Of these longer segments, 2 were classified as direct repeat sequences, whereas one was categorized as a palindromic repeat sequence; these specific sequences played a role in mediating recombination within the mitochondrial genome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Prediction of RNA editing sites\u003c/h2\u003e \u003cp\u003eIn this study, we predicted RNA editing sites in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, focusing on 38 protein-coding genes. A total of 537 non-synonymous editing sites were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), involving changes in 14 amino acids, including H(His)\u0026rarr;Y(Tyr), R(Arg)\u0026rarr;C(Cys), T(Thr)\u0026rarr;I(Ile), T(Thr)\u0026rarr;M(Met), R(Arg)\u0026rarr;W(Trp), S(Ser)\u0026rarr;L(Leu), S(Ser)\u0026rarr;F(Phe), P(Pro)\u0026rarr;S(Ser), P(Pro)\u0026rarr;L(Leu), P(Pro)\u0026rarr;F(Phe), L(Leu)\u0026rarr;F(Phe), A(Ala)\u0026rarr;V(Val), Q(Gln)\u0026rarr;*, and Arg(R)\u0026rarr;* (* represents a stop codon). Among these changes, the most common alteration was Ser to Leu, with a total of 128 RNA editing sites accounting for 23.84% of the total. Among all the amino acid changes observed, 259 (48.23%) of the hydrophilic amino acids were converted to hydrophobic ones; conversely, 39 (7.26%) of the hydrophobic amino acids were transformed into hydrophilic ones, whereas 235 (43.76%) amino acids exhibited no change in hydrophobicity. Additionally, 4 (0.74%) codons encoding hydrophilic amino acids were converted into stop codons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Specifically, 3 instances of CGA(R)\u0026mdash;UGA(*) conversion occurred at the last codon positions of the \u003cem\u003eccm\u003c/em\u003eFc, \u003cem\u003eatp\u003c/em\u003e9, and \u003cem\u003esdh\u003c/em\u003e4 genes; one instance of CAG(Q)\u0026mdash;TAG(*) conversion was found at the 13 codon position of the \u003cem\u003erpl\u003c/em\u003e16 gene, which might lead to premature termination of mRNA translation. In terms of genetic analysis, the \u003cem\u003eccm\u003c/em\u003eFn gene presented the highest frequency of RNA editing sites, with a total of 39 occurrences. This was followed by the \u003cem\u003eccm\u003c/em\u003eB and \u003cem\u003eccm\u003c/em\u003eC genes, which had 34 and 32 instances, respectively. In contrast, the \u003cem\u003esdh\u003c/em\u003e3 gene had the lowest frequency of RNA editing sites, with only 2 identified editing locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Codon usage analysis of protein-coding genes (PCGs)\u003c/h2\u003e \u003cp\u003eRelative synonymous codon usage frequency (RSCU) analysis was conducted on 64 codons of the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e. The results indicated that all 64 codons are expressed in PCGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Among these, the GCU (encoding alanine) exhibited the highest RSCU value of 1.5743, whereas the CAC (encoding histidine) had the lowest RSCU value of 0.4586. In the PCGs, the start codon was consistently ATG, with no codon usage bias (RSCU\u0026thinsp;=\u0026thinsp;1). The stop codons included UAA, UAG, and UGA, among which only UAA had an RSCU value greater than 1. Among the 61 coding amino acid codons analyzed, 29 had RSCU values exceeding 1, indicating a strong preference for their use. There were 10 A-ending codons and 17 U-ending codons; conversely, there was only one C-ending or G-ending codon each. The proportion of high-frequency codons (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1) ending with A or U reached 93.103%, whereas those ending with C or G accounted for only 6.897%. Therefore, it could be concluded that in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, there was a notable preference for the use of A- or U-ending codons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Mitochondrial plastid DNAs (MTPTs) in the mitogenome\u003c/h2\u003e \u003cp\u003eTo investigate the sequence transfer between the mitochondrial and chloroplast genomes of \u003cem\u003eC. tachangensis\u003c/em\u003e, we conducted a comparative analysis of both organellar genomes. The results indicated that there were a total of 23 groups of chloroplast homologous fragments within the \u003cem\u003eC. tachangensis\u003c/em\u003e mitochondrial genome, with MTPT 1\u0026ndash;7 located on Chr1 and MTPT 8\u0026ndash;23 located on Chr2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). These fragments collectively spanned a length of 16,396 bp, accounting for approximately 2.1951% of the total length of the mitochondrial genome. Among these fragments, MTPT1 was the longest at 9,556 bp and was located within the range of 221,056\u0026ndash;211,509 bp on Chr2. In contrast, MTPT23 was the shortest fragment, with a length of only 32 bp, and was found within the range of 216,692\u0026ndash;216,661 bp on Chr1. The annotation results indicate that these fragments originate from protein-coding genes, rRNA genes, tRNA genes, and intergenic regions of the chloroplast genome. However, all the chloroplast protein-coding genes and rRNA coding genes were not retained after the insertion of the mitochondrial sequences, whereas the tRNA coding genes were preserved relatively intact within the mitochondria. A total of 7 completed tRNA genes were distributed across 7 homologous sequences: \u003cem\u003etrn\u003c/em\u003eA-UGC, \u003cem\u003etrn\u003c/em\u003eI-GAU, \u003cem\u003etrn\u003c/em\u003eV-GAC, \u003cem\u003etrn\u003c/em\u003eW-CCA, \u003cem\u003etrn\u003c/em\u003eP-UGG, \u003cem\u003etrn\u003c/em\u003eM-CAU, and \u003cem\u003etrn\u003c/em\u003eN-GUU.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Analysis of Pi and Ka/Ks\u003c/h2\u003e \u003cp\u003eTo analyze the sequence differences between \u003cem\u003eC. tachangensis\u003c/em\u003e and its related species, we calculated the nucleotide diversity (Pi) values for 41 common genes across 9 species of the genus \u003cem\u003eCamellia\u003c/em\u003e (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The data indicate significant differences in nucleotide diversity (Pi values) among the mitochondrial genomes of 9 \u003cem\u003eCamellia\u003c/em\u003e species across different genes, ranging from 0 to 0.06872. The highest Pi value was observed in \u003cem\u003errn\u003c/em\u003e18 at (0.06872), followed by \u003cem\u003enad\u003c/em\u003e5 (0.01673) and \u003cem\u003ecox\u003c/em\u003e2 (0.0172). While, 15 genes (e.g., \u003cem\u003enad\u003c/em\u003e6, \u003cem\u003ecox\u003c/em\u003e1,and \u003cem\u003enad\u003c/em\u003e4L) exhibited Pi values of 0, indicating their high conservation. Some shorter regions showed a higher number of mutations despite their limited length (e.g., \u003cem\u003esdh\u003c/em\u003e3 [length: 321 bp; mutations: 6; Pi\u0026thinsp;=\u0026thinsp;0.00744]), while longer regions like \u003cem\u003errn\u003c/em\u003e26 (length: 3614 bp; mutations: 48; Pi\u0026thinsp;=\u0026thinsp;0.00564) displayed lower mutation density.\u003c/p\u003e \u003cp\u003eTo further investigate the impact of environmental stress on mitochondrial PCG mutations in the aforementioned species, we conducted Ka/Ks analysis and screened 20 genes with Ka/Ks values. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e) showed that nearly all Ka/Ks values of mitochondrial PCGs in \u003cem\u003eC. tachangensis\u003c/em\u003e were less than 1 when compared with \u003cem\u003eCamellia\u003c/em\u003e species (only the cox2 gene showed Ka/Ks\u0026thinsp;=\u0026thinsp;1.01438 in \u003cem\u003eC. tachangensis\u003c/em\u003e vs. \u003cem\u003eC. pubilimba\u003c/em\u003e (ON782577) and displayed the ratio of 1.41208 in \u003cem\u003eC. tachangensis\u003c/em\u003e vs \u003cem\u003eC. assamica\u003c/em\u003e (NC_043914). This indicated that \u003cem\u003eC. tachangensis\u003c/em\u003e, as a endemic species of China, had undergone predominantly purifying selection during evolution. Notably, some genes exhibited a wide range of Ka/Ks values due to their higher mutation rates. For example, the protein-coding gene \u003cem\u003enad\u003c/em\u003e2, which had the highest Pi value, yielded 16 distinct Ka/Ks values, primarily ranging between 0.3 and 0.65. In contrast, certain genes such as \u003cem\u003erpl\u003c/em\u003e2, \u003cem\u003ecox\u003c/em\u003e2, and \u003cem\u003esdh\u003c/em\u003e3 demonstrated both conservation and heterogeneity across species comparisons, resulting in relatively limited Ka/Ks variations. Taking \u003cem\u003erpl\u003c/em\u003e2 as an example, its sequence exhibited either complete identity or divergence across different species comparisons, yielding only 3 distinct Ka/Ks values: 1.03902 (\u003cem\u003eC. tachangensis\u003c/em\u003e/ \u003cem\u003eC. chekiangoleosa\u003c/em\u003e vs. \u003cem\u003eC. gigantocarpa\u003c/em\u003e), 0.783174 (\u003cem\u003eC. tachangensis\u003c/em\u003e/ \u003cem\u003eC. chekiangoleosa\u003c/em\u003e vs. \u003cem\u003eC. sinensis\u003c/em\u003e variants PP212895/ PP212896/ ON782577), and 0.304929 (\u003cem\u003eC. sinensis\u003c/em\u003e variants PP212895/ PP212896/ ON782577 vs. \u003cem\u003eC. gigantocarpa\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe mitochondrial CDS-based phylogenetic tree was constructed using \u003cem\u003eTaxus wallichiana\u003c/em\u003e as the outgroup. It revealed that \u003cem\u003eDiospyros kaki\u003c/em\u003e (NC_082859) and \u003cem\u003eRhododendron simsii\u003c/em\u003e (NC_053763), both belonging to the order Ericales, formed a well-supported clade with \u003cem\u003eCamellia\u003c/em\u003e species (BS\u0026thinsp;=\u0026thinsp;100, PP\u0026thinsp;=\u0026thinsp;1.00). Within the \u003cem\u003eCamellia\u003c/em\u003e lineage, \u003cem\u003eC. tachangensis\u003c/em\u003e was phylogenetically distinct from species in the sections \u003cem\u003eOleifera\u003c/em\u003e, \u003cem\u003eCamellia\u003c/em\u003e, \u003cem\u003eHeterogenea\u003c/em\u003e and \u003cem\u003eChrysantha\u003c/em\u003e. It occupied a basal position within the Clade Ⅰ that includes 5 variants of \u003cem\u003eC. sinensis\u003c/em\u003e. However, this phylogenetic grouping exhibited weak nodal support (BS\u0026thinsp;=\u0026thinsp;22, PP\u0026thinsp;=\u0026thinsp;0.41) (Fig.\u0026nbsp;8). The chloroplast PCG-based phylogenetic tree utilizing \u003cem\u003ePolyspora axillaris\u003c/em\u003e (NC_035709) and \u003cem\u003eSchima superba\u003c/em\u003e (NC_035545) as outgroups demonstrated high phylogenetic resolution for \u003cem\u003eC. tachangensis\u003c/em\u003e: Among the \u003cem\u003eCamellia\u003c/em\u003e species analyzed, This species formed a strongly supported basal group in Clade II (BS\u0026thinsp;=\u0026thinsp;100, PP\u0026thinsp;=\u0026thinsp;1), alongside \u003cem\u003eC. makuanica\u003c/em\u003e (NC_087766), \u003cem\u003eC. taliensis\u003c/em\u003e (NC_022264), and \u003cem\u003eC. gymnogyna\u003c/em\u003e (NC_039626) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAlthough branched structures had been widely reported in plant mitochondrial genomes [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], the mitochondrial DNA of most \u003cem\u003eCamellia\u003c/em\u003e species (e.g., \u003cem\u003eC. sinensis, C. assamica\u003c/em\u003e, and \u003cem\u003eC. nitidissima\u003c/em\u003e) were traditionally presented as circular structures due to differing assembly strategies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Only 3 species of sect. \u003cem\u003eOleifera\u003c/em\u003e (\u003cem\u003eC. drupifera\u003c/em\u003e, \u003cem\u003eC. oleifera\u003c/em\u003e, and \u003cem\u003eC. lanceoleosa\u003c/em\u003e) exhibit mitochondrial genomes with multibranched configurations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, we achieved the first complete mitochondrial genome sequencing and assembly for \u003cem\u003eC. tachangensis\u003c/em\u003e, which revealed its unique dual-component architecture comprising a multibranched sequence and a circular sequence. Subsequent structural analysis further identified the multibranched sequence could form 7 substructures via 3 pairs of dispersed repeats over 1000 bp. These findings not only enhanced our comprehension of the complexity and diversity of mitochondrial genome structures in \u003cem\u003eCamellia\u003c/em\u003e species, but also established a foundational genetic data framework for future research on molecular breeding programs targeting \u003cem\u003eC. tachangensis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBeyond structural variations, the mitochondrial genome length of \u003cem\u003eC. tachangensis\u003c/em\u003e significantly differed from other \u003cem\u003eCamellia\u003c/em\u003e species. The total mtDNA length of \u003cem\u003eC. tachangensis\u003c/em\u003e was 746,931 bp, while other \u003cem\u003eCamellia\u003c/em\u003e species range from 1,082,025 bp in \u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e cv. Dahongpao (CSSDHP, PP212895) (longest) to 707,441 bp in \u003cem\u003eC. assamica\u003c/em\u003e (shortest), representing a 374,584 bp difference [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Among these, \u003cem\u003eC. huana\u003c/em\u003e (733,752 bp) showed the closest genome length to \u003cem\u003eC. tachangensis\u003c/em\u003e, differing by only 13,179 bp. However, the length of repetitive sequences identified in the studies cannot directly explain the differences in mitochondrial DNA length between these \u003cem\u003eCamellia\u003c/em\u003e species. This is evidenced by the contrasting repetitive sequence lengths between CSSDHP and \u003cem\u003eC. tachangensis\u003c/em\u003e: SSR (4,548 vs. 2,688 bp, including tandem repeats) and dispersed repeats (33,871 vs. 46,509 bp). Even when combining the total lengths of simple sequence repeats and dispersed repeats, CSSDHP still exhibits a reverse correlation in total repetitive sequence content compared to \u003cem\u003eC. tachangensis\u003c/em\u003e (38,419 bp vs. 49,197 bp). This paradoxical phenomenon may be attributed to 3 possible factors: first, differential loss and transfer of mitochondrial DNA fragments between the 2 species; second, frequent recombination and mutation events in intergenic regions of plant mitochondrial DNA that obscure detection of original repetitive sequences; third, CSSDHP's mitochondrial DNA had acquired longer chloroplast-derived homologous sequences compared to \u003cem\u003eC. tachangensis\u003c/em\u003e (20,733 vs. 16,448 bp) [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, the first 2 factors still require further exploration and validation specifically for \u003cem\u003eCamellia\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eC. tachangensis\u003c/em\u003e possessed complete mitochondrial PCG composition, it only retained pseudogene copies for \u003cem\u003erps\u003c/em\u003e19 and \u003cem\u003esdh\u003c/em\u003e3. In contrast, other \u003cem\u003eCamellia\u003c/em\u003e species exhibited varying PCG duplications. For Instance, CSSDHP contained 8 duplicated PCGs (e.g., \u003cem\u003eatp\u003c/em\u003e8, \u003cem\u003eatp\u003c/em\u003e9, \u003cem\u003enad\u003c/em\u003e6, \u003cem\u003ecox\u003c/em\u003e3), \u003cem\u003eC. nitidissima\u003c/em\u003e shows 2 duplicated genes (\u003cem\u003erps\u003c/em\u003e12 and \u003cem\u003erps\u003c/em\u003e16), and \u003cem\u003eC. oleifera\u003c/em\u003e retained 4 duplicated genes (\u003cem\u003ecox\u003c/em\u003e1, \u003cem\u003erpl\u003c/em\u003e16, \u003cem\u003erps\u003c/em\u003e3, and \u003cem\u003erpl\u003c/em\u003e2) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. These differences likely resulted from combined effects of transposon activity and environmental adaptation [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], offering new perspectives for exploring plant mitochondrial genome evolution through further investigation. While other tRNA gene copy numbers vary among \u003cem\u003eCamellia\u003c/em\u003e species, all of them (including \u003cem\u003eC. tachangensis\u003c/em\u003e) exhibited high copies of the \u003cem\u003etrn\u003c/em\u003eM-CAU gene. This might relate to its role in transporting the initiation codon AUG. \u003cem\u003etrn\u003c/em\u003eM-CAU likely enhances its expression to competitively occupy ribosomal P-sites, preventing non-initiator tRNA misbinding and ensuring protein synthesis fidelity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The GC content (45.86%) and codon usage bias of \u003cem\u003eC. tachangensis\u003c/em\u003e showed remarkable conservation, being highly similar to both \u003cem\u003eCamellia\u003c/em\u003e and other species, reflecting the evolutionary stability of these genetic features in Angiosperms [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR59 CR60 CR61 CR62 CR63\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRNA editing is a widely occurring post-transcriptional mechanism that modified RNA by altering the types of nucleotides present within it [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. To determine the final protein sequences of the mitochondrial genes in \u003cem\u003eC. tachangensis\u003c/em\u003e, it is essential to predict RNA editing events for each gene. In this study, 537 RNA editing sites across 38 genes in \u003cem\u003eC. tachangensis\u003c/em\u003e were identified. Previous indicated that these editing sites played a crucial role in gene expression. RNA editing in plants could restore codons altered by mutations, thereby ensuring that mRNAs encode proteins with normal functionality [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In addition, RNA editing is a prerequisite for the proper translation of certain mRNAs. In the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, the initial codons for cox1 and nad4L are ACG. Through RNA editing, these start codons could be converted from ACG to ATG, thereby ensuring the proper function of mRNA translation. More importantly, RNA editing had been demonstrated to play a crucial role in regulating responses to environmental stress in certain plant species. Research indicated that specific RNA editing modifications\u0026mdash;such as enhanced editing of mitochondrial genes \u003cem\u003enad\u003c/em\u003e3, \u003cem\u003enad\u003c/em\u003e7, and \u003cem\u003eccm\u003c/em\u003eFn in \u003cem\u003eOryza sativa\u003c/em\u003e, alongside deficient editing of \u003cem\u003enad\u003c/em\u003e4 and \u003cem\u003ecox\u003c/em\u003e3 in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u0026mdash;are correlated with improved tolerance to salt and drought stress, respectively [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. However, although previous studies had described possible interactions between PLS-CsPPR proteins and target sequences of RNA editing sites in mitochondrial and chloroplast genes in \u003cem\u003eCamellia\u003c/em\u003e species (\u003cem\u003eC. sinensis\u003c/em\u003e) [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], a direct link between mitochondrial RNA editing and environmental stress adaptation in \u003cem\u003eCamellia\u003c/em\u003e plants had not yet been established. To address this research gap, future studies could employ multi-omics correlation analysis methods, integrating the predicted RNA editing site data from this study, to investigate the potential mechanisms and roles of mitochondrial gene RNA editing in stress physiological responses of \u003cem\u003eC. tachangensis\u003c/em\u003e and other \u003cem\u003eCamellia\u003c/em\u003e species. This approach would provide theoretical support for developing precise conservation strategies.\u003c/p\u003e \u003cp\u003eDNA could be transferred between the mitochondrial and chloroplast genomes within cells [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. This process was accompanied by the insertion of exogenous tRNA genes to support the translation of mitochondrial PCGs [\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In this study, we identified a total of 23 MTPTs in the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, among which 7 MTPTs contained 1\u0026ndash;3 tRNA genes. By comparing these fragments with those from other species in the sect. \u003cem\u003eThea\u003c/em\u003e and sect. \u003cem\u003eOleifera\u003c/em\u003e, we found partial MTPTs shared similarities across these species: these fragments were highly similar in length, and their tRNA gene compositions were entirely consistent (e.g., trnM-CAT, trnA-UGC\u0026ndash;trnI-GAU\u0026ndash;trnV-GAC, and trnD-GUC). Therefore, we hypothesized that the transfer events of these fragments could be traced back to before the divergence of these 2 taxonomic groups. In contrast, MTPTs identified in 4 species of the \u003cem\u003esect. Chrysantha\u003c/em\u003e were relatively scarce, with only 5\u0026ndash;14 MTPTs per species. Moreover, only 1\u0026ndash;2 MTPTs in each species contained tRNA genes, which might reflect the unique evolutionary trajectory of sect. \u003cem\u003eChrysantha\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe Ka/Ks analysis indicated that the mitochondrial PCGs of \u003cem\u003eC. tachangensis\u003c/em\u003e had primarily undergone purifying selection during the course of evolution (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1), which aligned with previous Ka/Ks analysis results between \u003cem\u003eC. drupifera\u003c/em\u003e (sect. \u003cem\u003eOleifera\u003c/em\u003e) and species from \u003cem\u003esect. Thea\u003c/em\u003e and \u003cem\u003esect. Chrysantha\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These phenomena suggest that purifying selection may play a dominant role in the evolution of mitochondrial PCGs in \u003cem\u003eCamellia\u003c/em\u003e plants. It might stem from mitochondrial genes predominantly functioning in core metabolic pathways like oxidative phosphorylation. Non-synonymous mutations in these genes were often deleterious, resulting in their persistent purging through natural selection to maintain functional evolutionary conservation [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Furthermore, sequences of \u003cem\u003erpl\u003c/em\u003e2 and \u003cem\u003esdh\u003c/em\u003e3 genes detected in \u003cem\u003eC. tachangensis\u003c/em\u003e completely match those of \u003cem\u003eC. chekiangoleosa\u003c/em\u003e (sect. \u003cem\u003eCamellia\u003c/em\u003e) but differed from cultivated varieties (CSDHP, CSSRG) within the sect. \u003cem\u003eThea\u003c/em\u003e. This pattern might arise because \u003cem\u003eC. tachangensis\u003c/em\u003e, as an early-diverged species of sect. \u003cem\u003eThea\u003c/em\u003e, retained primitive sequence characteristics of \u003cem\u003erpl\u003c/em\u003e2 and \u003cem\u003esdh\u003c/em\u003e3 genes shared with \u003cem\u003eC. chekiangoleosa\u003c/em\u003e from the initial differentiation stage between \u003cem\u003esect. Thea\u003c/em\u003e and \u003cem\u003eSect. Camellia\u003c/em\u003e groups. In contrast, later-diverged \u003cem\u003eCamellia\u003c/em\u003e species like CSDHP and CSSRG had accumulated mutations in these genes during evolution, ultimately resulting in sequence divergence from their corresponding genes in \u003cem\u003eC. tachangensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough the species selected for the 2 phylogenetic trees were not entirely consistent, we can still observe that the chloroplast PCG phylogenetic tree exhibits higher resolution compared to the mitochondrial CDS-based phylogenetic tree. Additionally, the phylogenetic position of \u003cem\u003eC. tachangensis\u003c/em\u003e in the mitochondrial CDS-based tree did not appear at the base of \u003cem\u003eCamellia\u003c/em\u003e species as observed in the chloroplast PCG-based phylogenetic tree, but instead clustered with some \u003cem\u003eC. sinensis\u003c/em\u003e variants with extremely low support values (BS\u0026thinsp;=\u0026thinsp;22, PP\u0026thinsp;=\u0026thinsp;0.41). This discrepancy may be related to several factors: First, the lack of available mitochondrial genome data for closely related species such as \u003cem\u003eC. taliensis\u003c/em\u003e and \u003cem\u003eC. gymnogyna\u003c/em\u003e likely reduced the phylogenetic support for the branch containing \u003cem\u003eC. tachangensis\u003c/em\u003e. Future studies should prioritize generating mitochondrial genome data for these species to resolve the phylogenetic placement of \u003cem\u003eC. tachangensis\u003c/em\u003e. Additionally, studies had shown that mitochondrial genomes evolved at a slower rate compared to chloroplast genomes, resulting in smaller genetic distances among related species in mitochondrial genomes [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Furthermore, although mitochondrial and chloroplast genomes were predominantly maternally inherited, both might undergo paternal leakage during inheritance, resulting in discrepancies in the genetic lineages of these 2 organellar genomes within the same species [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study reported the first sequencing and annotation of the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e, which exhibited a multichromosomal structure, comprising a 585,875 bp branched molecule (resolvable into 7 substructures) and a 221,056 bp circular molecule. A total of 63 functional elements were annotated, including 30 protein-coding genes (PCGs), 30 tRNAs, and 3 rRNAs. Comparative analysis identified 23 homologous chloroplast-derived fragments in the mitochondrial genome, introducing 10 intact tRNA genes. Ka/Ks analysis indicated that PCGs evolved predominantly under purifying selection (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1). Phylogenetic analysis based on chloroplast genome analysis strongly supported \u003cem\u003eC. tachangensis\u003c/em\u003e close relationship with \u003cem\u003eC. makuanica\u003c/em\u003e, \u003cem\u003eC. taliensis\u003c/em\u003e, and \u003cem\u003eC. gymnogyna\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;100, PP\u0026thinsp;=\u0026thinsp;1.00). However, the phylogenetic tree based on mitochondrial CDS failed to identify species closely related to \u003cem\u003eC. tachangensis\u003c/em\u003e due to the current lack of comprehensive mitochondrial genome data for the genus \u003cem\u003eCamellia\u003c/em\u003e. Despite this limitation, our study filled a critical gap in organelle genomics of \u003cem\u003eCamellia\u003c/em\u003e, offering valuable genomic resources for elucidating evolutionary mechanisms, advancing genetic improvement programs, and informing conservation strategies for this ecologically and economically important genus.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein-coding genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emtDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial genome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecpDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChloroplast genome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKa/Ks\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-synonymous/synonymous mutation ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRSCU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative synonymous codon usage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTPT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial plastid DNA sequence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003etRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransfer RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibosomal RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esimple sequence repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enucleotide diversity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebootstrap support value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eposterior probabilities\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePentatricopeptide repeat.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.L: Conceptualization, D.Z.J: Writing - original draft, Data curation, Formal analysis, Software. Z.L: Funding acquisition, Resources, Review \u0026amp; editing, Investigation. L.Z: Investigation, Methodology. Z.H.R: Resources, Supervision. X.X: Visualization, Investigation. X.H.Y: Methodology, Validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (Grant No. 32400179), the Guizhou Provincial Basic Research Program (Natural Science) 2022 (072), the Guizhou University Student Innovation Project 2024 (302), and the 2024 Guizhou Science and Technology Innovation Talent Team Construction Project: Wildlife Innovation Team of the Forestry college of Guizhou University (Qiankeherencai CXTD[2025]053).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitogenome sequences supporting the conclusions of this article are available in GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers: PQ658231 and PQ658232.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing or conflicting interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials used in this study comply with international and national legal standards. The collected species material does not pose a threat to other species, and the collection of the species is recognized by the relevant authorities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChang H. \u003cem\u003eThea\u003c/em\u003e\u0026mdash;A Section of Beveragial Tea-Trees of the Genus \u003cem\u003eCamellia\u003c/em\u003e. Acta Scientiarum Naturalium Universitatis SunYatseni. 1981;20(1):89\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Yu F, Tong Q. Discussions on Phylogenetic Classification and Evolution of Sect. 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Genetics. 2010;185(3):961\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1534/genetics.110.115360\u003c/span\u003e\u003cspan address=\"10.1534/genetics.110.115360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Camellia tachangensis, Horizontal transfer, Mitochondrial genome, Evolution, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-5634953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5634953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCamellia tachangensis\u003c/em\u003e F. C. Zhang is an endemic \u003cem\u003eCamellia\u003c/em\u003e species of the junction of Yunnan, Guizhou and Guangxi Provinces in China. It is characterized by a primitive five-chambered ovary morphology and serves as the botanical source of the renowned \u0026ldquo;Pu\u0026rsquo;an Red Tea\u0026rdquo;. Unfortunately, the populations of the species have declined due to the destruction of their habitats by human activities. The lack of mitochondrial genomic resources has hindered research into molecular breeding and phylogenetic evolution of \u003cem\u003eC. tachangensis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eIn this study, we had sequenced, assembled, and annotated the mitochondrial genome of \u003cem\u003eC. tachangensis\u003c/em\u003e to reveal its genetic characteristics and phylogenetic relation with other \u003cem\u003eCamellia\u003c/em\u003e species. The assembly result indicated that the mitochondrial genome sequence of \u003cem\u003eC. tachangensis\u003c/em\u003e was 746,931 bp (GC content\u0026thinsp;=\u0026thinsp;45.86%). It consisted of one multibranched sequence (Chr1) and one circular sequence (Chr2), with Chr1 capable of producing 7 substructures. The comparative analysis of the mitochondrial and chloroplast DNA of \u003cem\u003eC. tachangensis\u003c/em\u003e revealed 23 pairs of chloroplast homologous fragments, with 10 fully preserved tRNA genes within them. Comparison of interspecies Ka/Ks revealed that mutations in protein-coding genes (PCGs) of \u003cem\u003eC. tachangensis\u003c/em\u003e were predominantly shaped by purifying selection throughout its evolution (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1). The phylogenetic tree constructed from mitochondrial CDS indicated that \u003cem\u003eC. tachangensis\u003c/em\u003e and certain variants of \u003cem\u003eC. sinensis\u003c/em\u003e were distinct from other \u003cem\u003eCamellia\u003c/em\u003e species, forming a clade with relatively low support (BS\u0026thinsp;=\u0026thinsp;22%, PP\u0026thinsp;=\u0026thinsp;0.41). Meanwhile, the chloroplast genomes-based phylogenetic analyses revealed that \u003cem\u003eC. tachangensis\u003c/em\u003e was most closely related to \u003cem\u003eC. taliensis\u003c/em\u003e, \u003cem\u003eC. makuanica\u003c/em\u003e, and \u003cem\u003eC. gymnogyna\u003c/em\u003e, with strong statistical support (BS\u0026thinsp;=\u0026thinsp;100, PP\u0026thinsp;=\u0026thinsp;1.00).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study deciphered the mitochondrial genome and its multibranched structure of \u003cem\u003eC. tachangensis.\u003c/em\u003e These findings not only enhanced our comprehension of the complexity and diversity of mitochondrial genome structures in \u003cem\u003eCamellia\u003c/em\u003e species, but also established a foundational genetic data framework for future research on molecular breeding programs and phylogenetic relationship involving \u003cem\u003eC. tachangensis\u003c/em\u003e and its related species.\u003c/p\u003e","manuscriptTitle":"Assembly and analysis of the complete mitochondrial genome of an endemic Camellia species of China, Camellia tachangensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 14:48:17","doi":"10.21203/rs.3.rs-5634953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-09T16:38:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-09T05:48:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-30T02:07:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276764548000637652699901554805376062951","date":"2025-03-30T00:44:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307175044632703975313298903118280040133","date":"2025-03-29T14:54:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T04:17:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-28T03:55:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-03-20T11:43:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bb085c9-6873-46dd-a7eb-1b042bf8d39e","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:04:09+00:00","versionOfRecord":{"articleIdentity":"rs-5634953","link":"https://doi.org/10.1186/s12864-025-11673-z","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-05-15 15:56:52","publishedOnDateReadable":"May 15th, 2025"},"versionCreatedAt":"2025-04-01 14:48:17","video":"","vorDoi":"10.1186/s12864-025-11673-z","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11673-z","workflowStages":[]},"version":"v1","identity":"rs-5634953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5634953","identity":"rs-5634953","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}