Pan-Mitochondrial Genomic Analysis of Ginseng (Panax ginseng) Reveals Structural Variation, Phylogenetic Relationships, and Genetic Diversity

Pan-Mitochondrial Genomic Analysis of Ginseng (Panax ginseng) Reveals Structural Variation, Phylogenetic Relationships, and Genetic Diversity | 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 Article Pan-Mitochondrial Genomic Analysis of Ginseng (Panax ginseng) Reveals Structural Variation, Phylogenetic Relationships, and Genetic Diversity Yidan Xi, Xuejiao Liao, Haoyu Hu, Shuiming Xiao, Shuai Guo, Wei Shang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6622134/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Ginseng ( Panax ginseng ), a vital medicinal plant, faces challenges in genetic stability and directed breeding. This study aimed to elucidate mitochondrial genome structure, evolution, and phylogenetic relationships to guide germplasm conservation and molecular breeding. Using PacBio HiFi and Illumina sequencing, we assembled the complete mitochondrial genome of the BT cultivar (~ 465 kb, 55% A + T, encoding 80 functional genes). Repetitive sequences and codon usage patterns (preference for G/C at third codon positions) were characterized. Selective pressure analysis showed that most genes underwent purifying selection, but respiratory chain genes (nad4, cox2) exhibited positive selection signals. Phylogenetic analysis confirmed close relationships between ginseng and P. quinquefolius , with P. notoginseng forming a distinct clade. A pan-mitochondrial genome was constructed by integrating data from six ginseng populations. Analysis of this pan-genome revealed high genetic stability across populations, with SNPs, InDels, and structural variations identified. These findings provide insights into mitochondrial conservation, adaptive evolution, and population variation, supporting targeted breeding strategies for ginseng varieties. Biological sciences/Genetics Biological sciences/Plant sciences Panax ginseng mitochondrial genome phylogenetic analysis structural variation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Mitochondria are critical centers of energy metabolism in eukaryotic cells, providing adenosine triphosphate (ATP) through the process of oxidative phosphorylation. In plants, they also participate in complex physiological activities such as photorespiration, cell apoptosis regulation, and growth and development 1 . As semi-autonomous organelles, mitochondria possess an independent genetic system, including DNA, RNA, ribosomes, and transcription-translation mechanisms, enabling them to autonomously encode certain functional proteins and complete various steps of protein synthesis. The plant mitochondrial genome (mitogenome) originated from an endosymbiotic event involving primitive bacteria and is closely related to the Proteobacteria. Its structure exhibits significant complexity and dynamism 2 – 4 . The genome size varies greatly among different species, ranging from 66 kb in parasitic plants 5 to 11.3 Mb in Silene conoidea 6 . This variation is primarily due to the expansion of repetitive sequences in non-coding regions, gene migration between organelles, or horizontal gene transfer 3 , 6 , 7 . The genome contains conserved functional genes (such as those encoding respiratory chain complexes I-V, ribosomal RNA, and tRNA genes) and a large number of non-coding sequences 8 . For most higher plants, the nuclear genome follows a biparental inheritance pattern, while chloroplast and mitochondrial genomes are maternally inherited. This method eliminates the influence of paternal genes, significantly reducing the complexity of genetic research and facilitating the analysis of genetic mechanisms. Plant mitochondrial genomes, with their smaller size, faster evolutionary rate, lower recombination rate, and ease of sequencing, along with their rich genetic variation information, have become ideal molecular markers for studying plant origins, evolution, population genetic diversity, and systematics. With the innovation of high-throughput sequencing technologies, including second-generation Illumina sequencing, third-generation PacBio single-molecule real-time sequencing, and Nanopore long-read sequencing, the efficiency and accuracy of plant mitochondrial genome analysis have significantly improved. By 2025, the NCBI database had collected over 2240 complete mitochondrial genome sequences from plants, providing a data foundation for in-depth analysis of their structural characteristics and functions. These technological breakthroughs not only solved the assembly challenges posed by highly repetitive sequences in traditional sequencing but also provided a basis for revealing the complex dynamics of plant mtDNA recombination. For example, the mitochondrial genome of Zea mays exists as multiple linear molecules 9 , while the mitochondrial genome of the maintainer line in the "three-line hybrid system" of soybeans exists simultaneously in linear and circular forms 10 . Technological advancements have driven the application of mitochondrial genomes in plant research. For instance, the mitochondrial gene orf188 in Brassica napus promotes seed oil content by enhancing ATP synthesis, making it a potential target for oil crop improvement 11 . The mitochondrial genotype of Capsicum annuum is associated with fruit shape 12 . In systematics, combining mitochondrial and chloroplast data, researchers have analyzed the distribution of cytoplasmic diversity in wild soybeans in China 13 and the gene flow patterns between conifer species 14 . In molecular breeding, the maternal inheritance of mitochondrial genomes simplifies genetic analysis. By integrating genomic and phenotypic data, it is possible to precisely screen for desirable traits (such as stress resistance or high yield), accelerating the development of new varieties. Therefore, advancements in sequencing technology have not only deepened the understanding of the dynamic structure and functional evolution of mitochondrial genomes but also promoted their practical applications in the discovery of medicinal components, molecular breeding, and systematic evolution research, providing key tools for plant genetic improvement. Panax ginseng C.A. Mey., a perennial herb in the Araliaceae family, is native to the Himalayan region 15 . It primarily grows in high-altitude mountainous areas in East Asia and North America, with China being the world's largest producer of ginseng. Currently, ginseng cultivation in China is mainly concentrated in the provinces of Jilin, Liaoning, and Heilongjiang, with Jilin alone accounting for over 80% of the national ginseng production 16 . And ginseng is widely used for health and medical purposes in China, Japan, South Korea, and other countries 17 . Although the history of ginseng cultivation in China spans nearly 2000 years, systematic germplasm resource research only began in the past 30 years. Traditional ginseng breeding primarily relies on the selection of offspring with superior traits for hybridization and propagation. However, due to the diversity of ginseng origins, varieties, and traits, it is challenging to precisely screen and control target traits within a few generations 18 , 19 . Traditional genetic methods struggle to deeply analyze the genetic structure and differentiation mechanisms of ginseng germplasm resources. The maternal inheritance characteristics and dynamic structural variations of the ginseng mitochondrial genome make it a crucial tool for elucidating population evolutionary pathways and genetic backgrounds. Through high-throughput sequencing and phylogenetic analysis, high-resolution phylogenetic trees can be constructed, providing important data support for the protection of ginseng population genetic diversity and in-depth analysis of evolutionary mechanisms. In this study, we employed a hybrid sequencing approach combining PacBio HiFi and Illumina technologies to complete the assembly of the complete mitochondrial genome of the "biantiao" (BT) ginseng variety (total length 464,658 bp) and revealed its structural features, including gene repeats, non-canonical start codons, and codon usage preferences. Phylogenetic analysis with the mitochondrial genomes of 15 species revealed the evolutionary patterns and phylogenetic relationships of the Panax genus mitochondrial genome. Furthermore, we integrated the newly assembled mitochondrial genomes of four ginseng varieties from this study with two publicly available datasets to construct a ginseng pan-mitochondrial genome. Through whole-genome alignment and homologous gene family clustering analysis, we systematically identified and elucidated the distribution patterns of single nucleotide polymorphisms, small insertions/deletions, and structural variations. This research not only provides key data for the analysis of ginseng population variations, genetic diversity, and high-resolution analysis of Araliaceae phylogenetic relationships but also lays a theoretical foundation for the functional study of medicinal plant mitochondrial genomes, genetic improvement, and germplasm resource conservation. Results Assembly and structural characteristics of the ginseng mitochondrial genome Using a hybrid assembly strategy combining PacBio HiFi long-read sequencing and Illumina short-read correction, we successfully assembled the complete mitochondrial genome of the BT ginseng variety (Fig. 1 A). The genome exhibits a single circular structure with a total length of 464,658 bp (Fig. 1 B). Nucleotide composition analysis revealed a moderate A + T bias, accounting for 55.0%, with the base composition as follows: A (27.5%), T (27.5%), G (22.5%), and C (22.5%). Genome annotation identified a total of 80 functional genes, including 57 protein-coding genes (PCGs), 29 tRNA genes, and 4 rRNA genes (Table 1 , Fig. 1 C). Among these, 11 genes were found to be duplicated, with the trnM-CAU gene having 7 complete copies, the highest copy number among the tRNA genes. The cob (cytochrome b) and nad6 (NADH dehydrogenase subunit 6) genes were found to have 2 and 3 copies, respectively, indicating the widespread occurrence of gene duplication. Table 1 Gene content of P. ginseng mitogenome. The asterisks besides genes denotes intron-containing genes. The asterisks besides genes denotes intron-containing genes. Function Genes Complex I (NADH dehydrogenase) nad1, nad2 *, nad3, nad4 *, nad4L, nad5 *, nad6, nad7 *, nad95 Complex II (succinate dehydrogenase) sdh3, sdh4 Complex III (ubiquinol cytochrome c re-ductase) cob Complex IV (cytochrome c oxidase) cox1, cox2, cox3 Complex V (ATP synthase) atp1, atp4, atp6, atp8, atp9, atpE Cytochrome c biogenesis ccmB, ccmC, ccmFc *, ccmFn Ribosomal proteins (SSU) rpl10, rpl14, rpl16, rpl22 Ribosomal proteins (LSU) rps1, rps10, rps11, rps12, rps3, rps4, rps7, rps8 Maturases matK, matR rRNA rrn18, rrn26, rrn5 tRNA trnC-GCA, trnD-GUC, trnE-UUC, trnF-AAA, trnF-GAA trnG-GCC, trnH-GUG, trnK-CUU, trnK-UUU, trnM-CAU trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnS-GCU trnS-UGA, trnT-GGU, trnV-GAC, trnW-CCA, trnY-GUA Others infA, petD, psbA, psbD, rpoA, tatC Gene structure analysis revealed that 7 genes ( ccmFc , nad2 , nad4 , etc.) possess a multi-exon structure. Notably, the 5 exons of the nad2 gene are distributed at distant locations, making it the most dispersed gene in the genome. The start codons of the protein-coding genes exhibited diverse characteristics: in addition to the standard start codon ATG, non-canonical start codons such as TTG (e.g., nad2 , T→A), ACG (e.g., rps10 , cox1 , rps1 , and nad4L , C→U), and ATC (e.g., sdh3 , C→G) were detected. Relative synonymous codon usage (RSCU) analysis showed a significant bias towards G/C in the third position, with an average of 71.79%. A total of 61 codons were identified (Fig. 2 ), with serine (Ser) and isoleucine (Ile) being the most abundant amino acids, accounting for 9.67% and 8.15% of the total codons, respectively, while cysteine (Cys) had the lowest proportion (1.44%). The size variation in plant mitochondrial genomes is primarily driven by dynamic changes in repetitive sequences, including dispersed repeats, simple sequence repeats (SSRs), and tandem repeats. The highly repetitive nature of angiosperm mitochondrial genomes has made them a focal point of research even before the widespread adoption of complete genome assembly technologies. In this study, a total of 50 dispersed repeats (≥ 30 bp) were identified, with a total length of 50,610 bp, accounting for 10.89% of the genome length. Among these, 29 were forward repeats, and 21 were palindromic repeats, with no inverted or complementary repeats detected. The length distribution of repeat sequences was skewed: 68.00% were in the 45–67 bp range, 82.00% were less than 98 bp, and only 9 exceeded 100 bp. SSR analysis showed that among the 26 SSRs identified, mononucleotide repeats dominated (69.23%), followed by trinucleotide (19.23%) and dinucleotide (7.69%) repeats. In the mononucleotide SSRs, A/T repeats accounted for 88.9% (A: 59.5%, T: 29.4%), with no dominant C/G repeats detected. Dinucleotide repeats were primarily AT/TA repeats. Additionally, 12 tandem repeats (5–39 bp) were detected, all located in the intergenic regions. Selective pressure analysis In genetic studies, the K a /K s ratio serves as a critical indicator for measuring the direction and intensity of natural selection on homologous protein-coding genes (PCGs) during species divergence, holding significant theoretical value 20 . Compared to other neutral evolution testing methods in population genetics, this ratio has the advantages of fewer assumptions and higher testing power. When K a /K s 1 reflects positive selection or Darwinian selection (promoting adaptive variations); and a ratio of exactly 1 conforms to the neutral evolution model. It is important to note that the K a /K s ratio can significantly exceed 1 only when there are significant beneficial mutations at the gene locus. In this study, based on the comparative analysis of the mitochondrial genomes of P. ginseng , Daucus carota , P. notoginseng , and Bupleurum chinense , we calculated the K a /K s evolutionary selection pressure for 34 PCGs (Fig. 3 ),. The results showed that approximately 88.1% of the genes had a K a /K s value less than 1.0 in cross-species comparisons, indicating that mitochondrial genes primarily undergo purifying selection during evolution, consistent with the conservative nature of plant mitochondrial genomes 21 . Notably, in comparisons within the same genus ( P. ginseng vs. P. notoginseng ), the core respiratory chain genes nad4 (K a /K s = 1.06) and cox2 (K a /K s = 1.05) showed signals of positive selection. In comparisons within the P. ginseng and B. chinense , the positive selection characteristics of nad4 (K a /K s = 1.21) and tatC (twin-arginine translocation protein, K a /K s = 1.25) were further extended to the membrane transport system. In another cross-family comparisons (ginseng vs. carrot), the strong positive selection signals of rpl10 (ribosomal large subunit protein, K a /K s = 3.05) and ccmC (cytochrome c maturation protein, K a /K s = 1.21) suggested adaptive differentiation in ribosomal translation and cytochrome c assembly pathways. Further functional association analysis indicates that the evolutionary drive of positively selected genes may be closely linked to the regulation of energy metabolism. As core components of respiratory chain complexes I and IV, nad4 and cox2 exhibit a conspecific positive selection pattern within the same genus ( P. ginseng and. P. notoginseng ), which may promote adaptive differentiation among closely related species by regulating the efficiency of oxidative phosphorylation. The positive selection characteristics of tatC in both P. ginseng and B. chinense suggest a co-evolutionary pressure between mitochondrial membrane transport systems and respiratory chain functions. In the species divergence between ginseng and carrot, the high K a /K s value of rpl10 suggests that the rapid evolution of ribosomal translation efficiency may be a response to the adaptive demands for translation accuracy among species with greater evolutionary distance. Additionally, other mitochondrial genes, including atp4 , atp8 , cob , and ccmB , have been reported to have K a /K s >1, indicating that mitochondrial genes in different plant species may be subject to varying selective pressures during evolution. The above results demonstrate that the evolution of the ginseng mitochondrial genome exhibits significant heterogeneity: respiratory chain genes ( nad4 , cox2 ) frequently experience positive selection during close species divergence, while ribosomal proteins ( rpl10 ) maintain the conservation of core metabolic pathways through functional constraints. This provides a new perspective for elucidating the molecular mechanisms of ecological adaptation in Panax species. Phylogenetic analysis With the rapid development of sequencing technologies and genome assembly methods, an increasing number of complete plant mitochondrial genomes have been successfully assembled, providing significant opportunities for phylogenetic analysis using mitochondrial genomes. This study aims to clarify the phylogenetic position of ginseng within the Araliaceae family and angiosperms. We constructed a phylogenetic tree using 20 universally presentPCGs ( atp1 , atp9 , ccmB , ccmC , ccmFn , cob , cox1 , cox2 , cox3 , matR , nad1 , nad2 , nad3 , nad4 , nad4L , nad5 , nad6 , nad7 , nad9 , rps3 ) from the mitochondrial genomes of 15 species, including P. ginseng , its close relatives P. quinquefolius and P. notoginseng , species from the Apiaceae family ( D. carota , B. falcatum , and Ageratum conyzoides ), Rosaceae family ( Malus domestic a, Fragaria vesca ), Asteraceae family ( Helianthus annuus , Lactuca sativa ), as well as Platycodon grandiflorum , Codonopsis pilosula , Lonicera japonica and etc (Fig. 4 ),. Arabidopsis thaliana and Oryza sativa were used as outgroups. The results show that all branches of the phylogenetic tree have bootstrap support values exceeding 85%, with six branches achieving 100% support. Based on the maximum likelihood (ML) phylogenetic tree, it was found that the Panax species form a monophyletic clade, and P. notoginseng , P. quinquefolius , and P. ginseng cluster together, with P. ginseng and P. quinquefolius showing the closest relationship. This result is consistent with previous studies 22 – 25 , providing mitochondrial evidence for the similar genome expansion and evolution experienced by P. ginseng and P. quinquefolius during their evolutionary history. The evolutionary pattern of the ginseng mitochondrial genome demonstrates a high degree of conservation of core energy metabolism genes and dynamic adaptability of non-core genes. As with the significant differences in gene composition and arrangement observed in the mitochondrial genomes of higher plants 13 , the mitochondrial genome evolution of ginseng and its close relatives also follows this rule: key genes for core energy metabolism functions (such as subunits of complexes I, III, and V, and genes related to cytochrome c biosynthesis) are highly conserved in angiosperms (Fig. 5 ),. For example, the nad series genes, atp1 , atp6 , and cob are all intact in P. ginseng , P. notoginseng , and P. quinquefolius , indicating their irreplaceable role in maintaining mitochondrial core functions. In contrast, some non-essential genes (such as ribosomal protein genes and specific complex subunits) show significant loss. For instance, the rpl2 gene is completely lost in P. ginseng but retained in its close relative P. notoginseng and D. carota , the rps19 gene is generally absent in P. ginseng and its close relatives but present in A. thaliana and O. sativa , suggesting that its function may be compensated by nuclear genes. Additionally, functionally replaceable subunits such as atpE are retained in P. ginseng and P. quinquefolius but lost in species from the Rosaceae and Apiaceae families. The genes sdh3 and sdh4 are present in Panax species but specifically lost in other families, reflecting the adaptive evolutionary differences between the Araliaceae and other dicotyledonous groups after divergence. This evolutionary pattern indicates that P. ginseng has achieved mitochondrial genome streamlining and functional balance by strictly conserving core metabolic genes, selectively losing non-essential genes, and relying on nuclear gene compensation mechanisms driven by gene transfer between the mitochondrial and nuclear genomes. This dynamic pattern may reflect the shaping effects of lineage-specific metabolic demands and adaptive evolution on gene retention and loss. Construction and comparative analysis of the pan-mitochondrial genome of ginseng from different origins To further understand the variations in the mitochondrial genomes of ginseng from different origins and types, we selected P. ginseng samples from Liaoning and Jilin provinces, specifically JA, FC, SZ, and BT, along with the previously published Korean ginseng samples Gumpoong (GU) and Jakyung (JY), for pan-mitochondrial genome construction and comparative analysis. Initially, we statistically analyzed the six ginseng mitochondrial genomes and found that the size variations ranged from 0.72–5.42%, with JY having the largest genome at 464,705 bp and the smallest genome at 431,475 bp. Further gene analysis of the six ginseng mitochondrial genomes revealed that, apart from JY, which had an additional rpl23 gene related to the synthesis of the ribosomal large subunit, the other five ginseng mitochondrial genomes contained 45 PCGs. Based on the pan-mitochondrial genome constructed from the six ginseng mitochondrial genomes, we conducted a comprehensive variation analysis after statistically analyzing their sizes and genes. We categorized the variations into six types: single nucleotide polymorphisms (SNPs), small insertions/deletions (InDels, < 50 bp), deletions (DELs), inversions (INVs), translocations (TRANSs), and copy number variations (CNVs). Using the haplotype genome of BT as a reference, we identified a total of 111 SNPs and 39 InDels (Fig. 6 A). The fewest SNPs and InDels were identified in JA, with only 1 SNP and 1 InDel. The count range of identified SNPs and InDels in the other four mitochondrial genomes was 2–62 and 3–19, respectively. Additionally, through further analysis of the distribution of SNPs and InDels, we found that 32.43% of SNPs and 61.54% of InDels were located in gene regions. Besides the small variations of SNPs and InDels mentioned above, we also identified a small number of structural variations (SVs) between individual samples and BT through the pan-mitochondrial genome variation analysis of ginseng. Integrating the identified SV data, we found a total of 193 SVs, including 75 insertions, 59 deletions, 9 inversions, and 50 translocations (Fig. 6 B). There were certain differences in the number and type of SVs among different samples. The GU sample had relatively fewer structural variations, while the FC sample had the highest number of identified structural variations, with 28 insertions, 22 deletions, 1 inversion, and 29 translocations. Although the protein-coding genes of the mitochondria are relatively stable, the above results indicate that there may still be certain variations in the mitochondrial genomes of different ginseng samples. Discussion Plant mitochondrial genomes are larger and more structurally complex than those of animals, making the assembly and scaffolding of complete mitochondrial genomes challenging. The complexity of plant mitochondrial genomes is not only reflected in their larger genome sizes but also in their diverse repetitive sequences and structural variations, which make sequencing and assembly a challenging task. However, with the development of sequencing technologies, increased read lengths, and the development of mitochondrial assembly software such as GetOrganelle, NOVOPlasty, and PMAT, it has become possible to assemble complete circular mitochondrial genomes. Ginseng, an important medicinal plant in the Araliaceae family, faces many challenges in terms of genetic stability and directed breeding. Therefore, in-depth research on its mitochondrial genome is of great significance. Since the release of the mitochondrial genomes of Gumoong 26 and Jakyung 27 from South Korea, no studies have been conducted on the mitochondrial genomes of unique ginseng varieties from the main ginseng-producing regions of Jilin and Liaoning in China. This study is the first to perform mitochondrial genome assembly for four different ginseng varieties from Jilin and Liaoning. The size of the mitochondrial genomes of different ginseng varieties varies between 0.72% and 5.42%. The BT ginseng mitochondrial genome size is 464,658 bp, which is closest in size to GU and JY, differing by only 3 bp and 47 bp, respectively. However, the size of the JA mitochondrial genome from the same origin but a different variety differs by 11 kb from BT, indicating significant genetic variation among varieties. Further study of the BT genome structure revealed an A + T content of 55.0%. Further annotation of the BT mitochondrial genome identified a total of 80 genes, including 57 PCGs, 29 tRNA genes, and 4 rRNA genes. Comparing the 4 mitochondrial PCGs assembled in this study with GU and JY, it was found that, except for the addition of the rpl23 gene related to ribosomal large subunit synthesis in JY, the other 5 ginseng mitochondrial genomes all had 45 PCGs. The stability of PCGs not only indicates that ginseng mitochondrial genomes maintain relatively high conservation and stability in core genes but also lays a foundation for subsequent molecular breeding and functional gene research. Since the establishment of the genus Panax by Carl Linnaeus, taxonomic research on this genus has undergone a transformation from morphology to molecular systematics. Due to the convergent evolution of root and stem morphology and the complex arrangement of leaf sequences in Panax , traditional morphological methods are difficult to accurately distinguish species. Mitochondrial genomes, as maternally inherited genomes, are often used for species classification and phylogenetic studies and are important tools for elucidating the phylogenetic relationships between species. Based on comparative analysis of the mitochondrial genomes of P. ginseng , and other 3 plants, it was found that ginseng mitochondrial genes mainly experience purifying selection during evolution, with respiratory chain genes showing signals of positive selection in closely related species differentiation. This may be related to the regulation of energy metabolism and adaptive differentiation. These results reveal the heterogeneous selection pressure on ginseng mitochondrial genomes during evolution, providing a new perspective for elucidating the molecular mechanisms of ecological adaptation in Panax species. Additionally, by constructing a phylogenetic tree including 15 species such as P. ginseng , P. quinquefolius , P. notoginseng , and H. annuus , it was found that Panax species form a monophyletic clade. The close evolutionary relationship between ginseng and American ginseng mitochondrial genomes indicates their close phylogenetic relationship, consistent with the conclusions of previous genomic evolution studies of ginseng 23 , 25 . The concept of pan-mitochondrial genomes aims to comprehensively analyze genetic variations, functional genes, and adaptive evolution by integrating information from multiple mitochondrial genomes within a species. This research approach breaks the limitations of traditional reliance on a single reference genome, providing a new perspective for genetic diversity studies. Taking ginseng as an example, although two versions of the mitochondrial genome have been released, these data only partially reflect the diversity of ginseng mitochondrial genomes. Therefore, constructing a larger-scale pan-mitochondrial genome is crucial for capturing the variation in ginseng mitochondrial genomes. In this study, we conducted variation analysis on the mitochondrial genomes of six different ginseng varieties from China and Korea, revealing the distribution characteristics of genetic variations in ginseng. The results showed that the ginseng mitochondrial genome contains various types of variations, including insertions, deletions, inversions, translocations, and copy number variations. Specifically, the numbers of these variations were 75 insertions, 59 deletions, 9 inversions, and 50 translocations, but no copy number variations were identified. There were significant differences in the quantity and types of variations among different samples, indicating a certain degree of genetic diversity in ginseng mitochondrial genomes among different varieties. Although the ginseng mitochondrial genome overall exhibits high stability, these genetic variations provide rich resources for subsequent research and applications. Based on the variation sites on the mitochondrial genomes from different origins, it is possible to construct SNP chips targeting these variation sites, thereby achieving precise identification of different ginseng samples. This not only helps in the protection and utilization of ginseng genetic resources but also provides important molecular markers for ginseng molecular breeding and functional gene research. Methods Sample collection and sequencing In this study, based on the domestication history and distribution of Panax ginseng , four different tetraploid ginseng lines (2n = 4x = 48), including JA, SZ, BT, and FC, were selected for whole-genome sequencing. Fresh leaves were rapidly frozen in liquid nitrogen and stored in a -80°C ultra-low-temperature environment. Genomic DNA was extracted from fresh young leaves using a modified 3×CTAB method, and DNA concentration was quantified by measuring the A260 absorbance value with a ND-2000 spectrophotometer. For the JA, SZ, BT, and FC lines, Hi-Fi libraries with an insert size of 10 kb were constructed and sequenced using the Pacific Biosciences Sequel II platform for third-generation sequencing. In the whole-genome resequencing part, paired-end sequencing libraries with an insert size of 300–500 bp were constructed and resequenced on the MGI DNBSEQ T7 platform. Mitochondrial genome assembly and annotation Based on the PacBio HiFi sequencing raw data, the hifi raw data was first assembled using the PMAT2 v2.0.2 28 (-g 5G). The assembly results were visualized and manually corrected using Bandage v0.8.1 29 . To improve assembly accuracy, the original HiFi reads were further mapped back to the initial assembly sequence using minimap2, and iterative error correction optimization was performed using NextPolish. This resulted in a highly reliable mitochondrial genome sequence, and its graph framework file (GFA) was adjusted. The P. ginseng mitochondrial genome was annotated using the MITOFY online annotation platform ( http://dogma.ccbb.utexas.edu/mitofy/ ) and corrected by comparing with homologous genes in Araliaceae plant mitochondrial genomes. tRNAscan-SE 30 ( http://trna.ucsc.edu/tRNAscan-SE/ ) was used to annotate transfer RNAs, and Open Reading Frame Finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ) was used to annotate ORFs. The MEGA v7.0. 31 bioinformatics tool was used for codon usage analysis, calculating RSCU and amino acid composition characteristics. Finally, the OGDraw 32 program ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) was used to construct a circular visualization map of the mitochondrial genome. Repeat sequence prediction The REPuter 33 program ( https://bibiserv.cebitec.uni-bielefeld.de/reputer/ ) was used to identify dispersed repeat sequences, with parameters set as: repeat sequence consistency > 90%, minimum repeat unit length ≥ 30 bp, and Hamming distance of 3. This analysis effectively identified various types of dispersed repeats, including forward repeats, reverse repeats, complement repeats, and palindromic repeats. Additionally, SSRs were identified using MISA ( http://pgrc.ipk-gatersleben.de/misa/ ), analyzing six types of SSRs: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide, with repeat thresholds set at 10, 5, 4, 3, 3, and 3, respectively. Tandem Repeats Finder v4.09 ( http://tandem.bu.edu/trf/trf.html ) was used to detect tandem repeat sequences. Selection pressure analysis The natural selection pressure during the mitochondrial evolution of P. ginseng was inferred by calculating the non-synonymous substitution rate (K a ), synonymous substitution rate (K s ), and their ratio (K a /K s ) of PCGs, using the mitochondrial genomes of D. carota , P. notoginseng , and B. chinense as references. Homologous gene pairs were first aligned and formatted using ParaAT2.0 software. The K a , K s , and K a /K s values of each gene were then calculated using the KaKs_Calculator v.3.0 34 based on the YN algorithm, and the statistical significance of the substitution rates was verified using Fisher's exact test (P < 0.05). Phylogenetic analysis To accurately infer the phylogenetic relationship of P. ginseng within the Panax genus, this study conducted a phylogenetic analysis based on the PCGs of 15 higher plant mitochondrial genomes. The mitochondrial genome information of all species (except P. ginseng ) used in the phylogenetic analysis was obtained from the NCBI Organelle Genome Resources database ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ). Perl scripts were used to screen for single-copy orthologous PCGs common to the 15 analyzed species. All conserved mitochondrial PCG sequences were extracted from each mitochondrial genome, combined into a single dataset, and aligned using the Muscle software. The ML tree was then constructed using MEGA v7.0 31 . The bootstrap values displayed next to the branches in the phylogenetic tree represent the reliability of the clustering of related taxa, which were obtained through 1000 repetitions. Construction of the P. ginseng pan-mitochondrial genome Using the BT mitochondrial genome as a reference, this study compared it with the JA, FC, and SZ mitochondrial genomes assembled in this study, as well as two previously published P. ginseng mitochondrial genomes (GenBank accession no. MW029460.1, and MZ389476.1), to identify and statistically analyze SNPs and InDels. Initially, nucmer v4.0.0rc1 was used for alignment (parameters: --maxmatch -c 1000 -l 40), followed by filtering with delta-filter (parameters: -m -i 90 -l 100), and further filtering with delta-filter (parameters: -l -i 90 -l 100). The resulting files were then used with delta2vcf to identify SNPs and InDels. In the identification of structural variations in P. ginseng , nucmer was used to align with the reference genome, and the filtered alignment results were identified using the SyRI v1.7.0 35 software (parameters: --nc 10 --nosnp). This process allowed for the identification of collinear regions, structural rearrangements, and local variation regions. Declarations Author contributions statement Conceptualization, D.D.; methodology, J.X.; formal analysis, Y.X. and X.L.; investigation, Y.X. and X.L.; resources, Y.H. and D.D.; writing—original draft preparation, S.X. and X.L.; writing—review and editing, X.L. and S.G.; visualization, W.S., L.S.and T.Y.; supervision, S.C. and D.D.; project administration, J.X.; funding acquisition, D.D. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by Scientific and technological innovation project of China Academy of Chinese Medical Sciences (CI2023E002), the National Key Research and Development Program of China (2023YFC3504000), the Fundamental Research Funds for the Central Public Welfare Re-search Institutes (ZZ13-YQ-047), the special fund for Science and Technology Innovation Teams of Shanxi Province (202204051001030). Additional information Competing interests The authors declare no conflicts of interest. Data Availability Statement Data are contained within the article and Supplementary Materials. References Chevigny, N., Schatz-Daas, D., Lotfi, F. & Gualberto, J. M. DNA Repair and the Stability of the Plant Mitochondrial Genome. Int. J. Mol. Sci. 21 10.3390/ijms21010328 (2020). Fan, L. et al. Phylogenetic analyses with systematic taxon sampling show that mitochondria branch within Alphaproteobacteria. Nat. Ecol. Evol. 4 , 1213–1219. 10.1038/s41559-020-1239-x (2020). Alverson, A. 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MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33 , 1870–1874. 10.1093/molbev/msw054 (2016). Lohse, M., Drechsel, O. & Bock, R. OrganellarGenomeDRAW (OGDRAW): a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr. Genet. 52 , 267–274. 10.1007/s00294-007-0161-y (2007). Kurtz, S. et al. REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 29 , 4633–4642. 10.1093/nar/29.22.4633 (2001). Zhang, Z. KaKs_Calculator 3.0: Calculating Selective Pressure on Coding and Non-coding Sequences. Genom. Proteom. Bioinform. 20 , 536–540. 10.1016/j.gpb.2021.12.002 (2022). Goel, M., Sun, H., Jiao, W. B. & Schneeberger, K. SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. Genome Biol. 20 , 277. 10.1186/s13059-019-1911-0 (2019). Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiles.zip Cite Share Download PDF Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Jul, 2025 Reviews received at journal 15 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers agreed at journal 06 Jul, 2025 Reviewers invited by journal 06 Jul, 2025 Editor assigned by journal 27 Jun, 2025 Editor invited by journal 27 May, 2025 Submission checks completed at journal 23 May, 2025 First submitted to journal 08 May, 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-6622134","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":482079290,"identity":"245d8c38-00f2-4bcd-9354-6cc5a21be97c","order_by":0,"name":"Yidan Xi","email":"","orcid":"","institution":"Liaoning University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yidan","middleName":"","lastName":"Xi","suffix":""},{"id":482079291,"identity":"54b07a26-8ef0-496f-8df8-a3922784b1b5","order_by":1,"name":"Xuejiao Liao","email":"","orcid":"","institution":"Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xuejiao","middleName":"","lastName":"Liao","suffix":""},{"id":482079293,"identity":"24754ed7-0805-4523-98fa-e0e6d1cdaebd","order_by":2,"name":"Haoyu Hu","email":"","orcid":"","institution":"Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haoyu","middleName":"","lastName":"Hu","suffix":""},{"id":482079296,"identity":"cf688fe2-16f0-4b1b-a789-fe66c14ccc17","order_by":3,"name":"Shuiming Xiao","email":"","orcid":"","institution":"Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuiming","middleName":"","lastName":"Xiao","suffix":""},{"id":482079297,"identity":"53ed9daf-eb4f-4ddc-90d4-4291e0d637ef","order_by":4,"name":"Shuai Guo","email":"","orcid":"","institution":"Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Guo","suffix":""},{"id":482079298,"identity":"6a6b9e23-ca13-49f2-8611-197ebeb7ca05","order_by":5,"name":"Wei Shang","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, the Seventh Medical Center of PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Shang","suffix":""},{"id":482079299,"identity":"b48f8c7f-54c2-45d8-988e-13ab37d69f79","order_by":6,"name":"Tae-Jin Yang","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Tae-Jin","middleName":"","lastName":"Yang","suffix":""},{"id":482079300,"identity":"765e4cd5-5aab-4cd1-b1ce-d72c78cfa403","order_by":7,"name":"Shilin Chen","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shilin","middleName":"","lastName":"Chen","suffix":""},{"id":482079301,"identity":"efe3790e-1353-4642-9655-a639ea836458","order_by":8,"name":"Jiang Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBACxgYGxgcJpGphNiBNCxCwSZCmnnlGjlnFg5pt0Qzshx8w/NxBjMN6zpjdSDh2O7eBJ82AsfcMMVrae8xuJDYAtTDkMDAzthGjpZnHrACshf8NsVqAtjCAtUgQbUvPsWIJkF/aJJ4ZHOwlRovhjOSNH3/U3M7t509++OAnUVoaoAw2ID5AhAYGBnmiVI2CUTAKRsHIBgC+njUefddBewAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Xu","suffix":""},{"id":482079302,"identity":"dd9687a7-c99c-4047-802b-49dd53f7b50b","order_by":9,"name":"Deqiang Dou","email":"","orcid":"","institution":"Liaoning University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Deqiang","middleName":"","lastName":"Dou","suffix":""}],"badges":[],"createdAt":"2025-05-08 16:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6622134/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6622134/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-28721-z","type":"published","date":"2025-12-29T15:57:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86539946,"identity":"116b0ee1-466c-46a9-b171-bcf9bc01fc09","added_by":"auto","created_at":"2025-07-11 19:59:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8030840,"visible":true,"origin":"","legend":"\u003cp\u003eDe novo assembly and characterization of \u003cem\u003eP. ginseng\u003c/em\u003e mitochondrial genome. (A) Contig linkage topology of \u003cem\u003eP. ginseng\u003c/em\u003e mitogenome assembly. (B) Circular molecular architecture reconstructed from contig assemblies (C) Functional annotation map of mitochondrial genome.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/49c0805817ab99346d7453d4.png"},{"id":86540162,"identity":"f63f8240-8bba-4f3c-a35c-4575813df7d1","added_by":"auto","created_at":"2025-07-11 20:07:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9579046,"visible":true,"origin":"","legend":"\u003cp\u003eRSCU profiling across coding regions.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/9e21a832db0f7bf009ce7be7.png"},{"id":86539948,"identity":"83d54262-c410-4894-82fd-77468cec5af0","added_by":"auto","created_at":"2025-07-11 19:59:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":601487,"visible":true,"origin":"","legend":"\u003cp\u003eK\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e ratios for 34 protein coding genes of\u003cem\u003e P. ginseng\u003c/em\u003e, \u003cem\u003eD. carota\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e, and \u003cem\u003eB. chinense\u003c/em\u003e. The blue, orange, and purple boxes indicate K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e ratios of\u003cem\u003e D. carota\u003c/em\u003e vs \u003cem\u003eP. ginseng\u003c/em\u003e, P. no\u003cem\u003etoginseng\u003c/em\u003e vs \u003cem\u003eP. ginseng\u003c/em\u003e, and \u003cem\u003eB. chinense\u003c/em\u003e vs \u003cem\u003eP. ginseng\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/abe6b8cd456739a5120f960d.png"},{"id":86540164,"identity":"f1a90332-ba69-44c2-8395-21b679fc2d96","added_by":"auto","created_at":"2025-07-11 20:07:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5012361,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogeny of \u003cem\u003eP.ginseng\u003c/em\u003e. PCGs distribution in plant mitogenomes. Numbers on each node are bootstrap support values\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/90a908bf8e0a3dca88e416b2.png"},{"id":86539950,"identity":"e8bcb54b-7c37-4bff-8175-3b52342fd0a2","added_by":"auto","created_at":"2025-07-11 19:59:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2325370,"visible":true,"origin":"","legend":"\u003cp\u003ePCGs distribution in plant mitogenomes. Yellow cells denote the absence of genes in mitochondrial genomes, while purple cells indicate their presence.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/95fae7f9b7b5c2f27a9ec029.png"},{"id":86540163,"identity":"0920799e-2062-4ab8-9e7d-8b08b6c26a8e","added_by":"auto","created_at":"2025-07-11 20:07:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528284,"visible":true,"origin":"","legend":"\u003cp\u003eVariation analysis of the pan-mitochondrial genome of ginseng. (A) Distribution of snps and indels in ginseng mitochondrial genomes. (B) Proportions of different types of svs in ginseng mitochondrial genomes.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/86002b09550533d7ff3599e3.png"},{"id":99545331,"identity":"e3263b7c-089e-4f3e-9169-588bb2f61f67","added_by":"auto","created_at":"2026-01-05 16:05:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18653371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/d8dfb63c-1305-4837-90f2-66fc9b659e8b.pdf"},{"id":86539953,"identity":"d143b820-60c5-4cf0-8a3b-7d1fd9f6c5d9","added_by":"auto","created_at":"2025-07-11 19:59:15","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":529607,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.zip","url":"https://assets-eu.researchsquare.com/files/rs-6622134/v1/7aa1680dfae9efaad1c000fa.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pan-Mitochondrial Genomic Analysis of Ginseng (Panax ginseng) Reveals Structural Variation, Phylogenetic Relationships, and Genetic Diversity ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMitochondria are critical centers of energy metabolism in eukaryotic cells, providing adenosine triphosphate (ATP) through the process of oxidative phosphorylation. In plants, they also participate in complex physiological activities such as photorespiration, cell apoptosis regulation, and growth and development\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As semi-autonomous organelles, mitochondria possess an independent genetic system, including DNA, RNA, ribosomes, and transcription-translation mechanisms, enabling them to autonomously encode certain functional proteins and complete various steps of protein synthesis. The plant mitochondrial genome (mitogenome) originated from an endosymbiotic event involving primitive bacteria and is closely related to the Proteobacteria. Its structure exhibits significant complexity and dynamism\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The genome size varies greatly among different species, ranging from 66 kb in parasitic plants\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e to 11.3 Mb in \u003cem\u003eSilene conoidea\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This variation is primarily due to the expansion of repetitive sequences in non-coding regions, gene migration between organelles, or horizontal gene transfer\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The genome contains conserved functional genes (such as those encoding respiratory chain complexes I-V, ribosomal RNA, and tRNA genes) and a large number of non-coding sequences\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. For most higher plants, the nuclear genome follows a biparental inheritance pattern, while chloroplast and mitochondrial genomes are maternally inherited. This method eliminates the influence of paternal genes, significantly reducing the complexity of genetic research and facilitating the analysis of genetic mechanisms. Plant mitochondrial genomes, with their smaller size, faster evolutionary rate, lower recombination rate, and ease of sequencing, along with their rich genetic variation information, have become ideal molecular markers for studying plant origins, evolution, population genetic diversity, and systematics.\u003c/p\u003e\u003cp\u003eWith the innovation of high-throughput sequencing technologies, including second-generation Illumina sequencing, third-generation PacBio single-molecule real-time sequencing, and Nanopore long-read sequencing, the efficiency and accuracy of plant mitochondrial genome analysis have significantly improved. By 2025, the NCBI database had collected over 2240 complete mitochondrial genome sequences from plants, providing a data foundation for in-depth analysis of their structural characteristics and functions. These technological breakthroughs not only solved the assembly challenges posed by highly repetitive sequences in traditional sequencing but also provided a basis for revealing the complex dynamics of plant mtDNA recombination. For example, the mitochondrial genome of \u003cem\u003eZea mays\u003c/em\u003e exists as multiple linear molecules\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while the mitochondrial genome of the maintainer line in the \"three-line hybrid system\" of soybeans exists simultaneously in linear and circular forms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Technological advancements have driven the application of mitochondrial genomes in plant research. For instance, the mitochondrial gene \u003cem\u003eorf188\u003c/em\u003e in \u003cem\u003eBrassica napus\u003c/em\u003e promotes seed oil content by enhancing ATP synthesis, making it a potential target for oil crop improvement\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The mitochondrial genotype of \u003cem\u003eCapsicum annuum\u003c/em\u003e is associated with fruit shape\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In systematics, combining mitochondrial and chloroplast data, researchers have analyzed the distribution of cytoplasmic diversity in wild soybeans in China\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and the gene flow patterns between conifer species \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In molecular breeding, the maternal inheritance of mitochondrial genomes simplifies genetic analysis. By integrating genomic and phenotypic data, it is possible to precisely screen for desirable traits (such as stress resistance or high yield), accelerating the development of new varieties. Therefore, advancements in sequencing technology have not only deepened the understanding of the dynamic structure and functional evolution of mitochondrial genomes but also promoted their practical applications in the discovery of medicinal components, molecular breeding, and systematic evolution research, providing key tools for plant genetic improvement.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Mey., a perennial herb in the Araliaceae family, is native to the Himalayan region\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. It primarily grows in high-altitude mountainous areas in East Asia and North America, with China being the world's largest producer of ginseng. Currently, ginseng cultivation in China is mainly concentrated in the provinces of Jilin, Liaoning, and Heilongjiang, with Jilin alone accounting for over 80% of the national ginseng production\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. And ginseng is widely used for health and medical purposes in China, Japan, South Korea, and other countries \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Although the history of ginseng cultivation in China spans nearly 2000 years, systematic germplasm resource research only began in the past 30 years. Traditional ginseng breeding primarily relies on the selection of offspring with superior traits for hybridization and propagation. However, due to the diversity of ginseng origins, varieties, and traits, it is challenging to precisely screen and control target traits within a few generations \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Traditional genetic methods struggle to deeply analyze the genetic structure and differentiation mechanisms of ginseng germplasm resources. The maternal inheritance characteristics and dynamic structural variations of the ginseng mitochondrial genome make it a crucial tool for elucidating population evolutionary pathways and genetic backgrounds. Through high-throughput sequencing and phylogenetic analysis, high-resolution phylogenetic trees can be constructed, providing important data support for the protection of ginseng population genetic diversity and in-depth analysis of evolutionary mechanisms.\u003c/p\u003e\u003cp\u003eIn this study, we employed a hybrid sequencing approach combining PacBio HiFi and Illumina technologies to complete the assembly of the complete mitochondrial genome of the \"biantiao\" (BT) ginseng variety (total length 464,658 bp) and revealed its structural features, including gene repeats, non-canonical start codons, and codon usage preferences. Phylogenetic analysis with the mitochondrial genomes of 15 species revealed the evolutionary patterns and phylogenetic relationships of the \u003cem\u003ePanax\u003c/em\u003e genus mitochondrial genome. Furthermore, we integrated the newly assembled mitochondrial genomes of four ginseng varieties from this study with two publicly available datasets to construct a ginseng pan-mitochondrial genome. Through whole-genome alignment and homologous gene family clustering analysis, we systematically identified and elucidated the distribution patterns of single nucleotide polymorphisms, small insertions/deletions, and structural variations. This research not only provides key data for the analysis of ginseng population variations, genetic diversity, and high-resolution analysis of Araliaceae phylogenetic relationships but also lays a theoretical foundation for the functional study of medicinal plant mitochondrial genomes, genetic improvement, and germplasm resource conservation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAssembly and structural characteristics of the ginseng mitochondrial genome\u003c/h2\u003e\u003cp\u003eUsing a hybrid assembly strategy combining PacBio HiFi long-read sequencing and Illumina short-read correction, we successfully assembled the complete mitochondrial genome of the BT ginseng variety (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The genome exhibits a single circular structure with a total length of 464,658 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Nucleotide composition analysis revealed a moderate A\u0026thinsp;+\u0026thinsp;T bias, accounting for 55.0%, with the base composition as follows: A (27.5%), T (27.5%), G (22.5%), and C (22.5%). Genome annotation identified a total of 80 functional genes, including 57 protein-coding genes (PCGs), 29 tRNA genes, and 4 rRNA genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Among these, 11 genes were found to be duplicated, with the \u003cem\u003etrnM-CAU\u003c/em\u003e gene having 7 complete copies, the highest copy number among the tRNA genes. The \u003cem\u003ecob\u003c/em\u003e (cytochrome b) and \u003cem\u003enad6\u003c/em\u003e (NADH dehydrogenase subunit 6) genes were found to have 2 and 3 copies, respectively, indicating the widespread occurrence of gene duplication.\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\u003eGene content of \u003cem\u003eP. ginseng\u003c/em\u003e mitogenome. The asterisks besides genes denotes intron-containing genes. The asterisks besides genes denotes intron-containing genes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex I (NADH dehydrogenase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003enad1, nad2 *, nad3, nad4 *, nad4L, nad5 *, nad6, nad7 *, nad95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex II (succinate dehydrogenase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esdh3, sdh4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex III (ubiquinol cytochrome c re-ductase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecob\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex IV (cytochrome c oxidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecox1, cox2, cox3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex V (ATP synthase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eatp1, atp4, atp6, atp8, atp9, atpE\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytochrome c biogenesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eccmB, ccmC, ccmFc *, ccmFn\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRibosomal proteins (SSU)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003erpl10, rpl14, rpl16, rpl22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRibosomal proteins (LSU)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003erps1, rps10, rps11, rps12, rps3, rps4, rps7, rps8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaturases\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ematK, matR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003erRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003errn18, rrn26, rrn5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003etrnC-GCA, trnD-GUC, trnE-UUC, trnF-AAA, trnF-GAA\u003c/p\u003e\u003cp\u003etrnG-GCC, trnH-GUG, trnK-CUU, trnK-UUU, trnM-CAU\u003c/p\u003e\u003cp\u003etrnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnS-GCU\u003c/p\u003e\u003cp\u003etrnS-UGA, trnT-GGU, trnV-GAC, trnW-CCA, trnY-GUA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003einfA, petD, psbA, psbD, rpoA, tatC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene structure analysis revealed that 7 genes (\u003cem\u003eccmFc\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e, etc.) possess a multi-exon structure. Notably, the 5 exons of the nad2 gene are distributed at distant locations, making it the most dispersed gene in the genome. The start codons of the protein-coding genes exhibited diverse characteristics: in addition to the standard start codon ATG, non-canonical start codons such as TTG (e.g., \u003cem\u003enad2\u003c/em\u003e, T\u0026rarr;A), ACG (e.g., \u003cem\u003erps10\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003erps1\u003c/em\u003e, and \u003cem\u003enad4L\u003c/em\u003e, C\u0026rarr;U), and ATC (e.g., \u003cem\u003esdh3\u003c/em\u003e, C\u0026rarr;G) were detected. Relative synonymous codon usage (RSCU) analysis showed a significant bias towards G/C in the third position, with an average of 71.79%. A total of 61 codons were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with serine (Ser) and isoleucine (Ile) being the most abundant amino acids, accounting for 9.67% and 8.15% of the total codons, respectively, while cysteine (Cys) had the lowest proportion (1.44%).\u003c/p\u003e\u003cp\u003eThe size variation in plant mitochondrial genomes is primarily driven by dynamic changes in repetitive sequences, including dispersed repeats, simple sequence repeats (SSRs), and tandem repeats. The highly repetitive nature of angiosperm mitochondrial genomes has made them a focal point of research even before the widespread adoption of complete genome assembly technologies. In this study, a total of 50 dispersed repeats (\u0026ge;\u0026thinsp;30 bp) were identified, with a total length of 50,610 bp, accounting for 10.89% of the genome length. Among these, 29 were forward repeats, and 21 were palindromic repeats, with no inverted or complementary repeats detected. The length distribution of repeat sequences was skewed: 68.00% were in the 45\u0026ndash;67 bp range, 82.00% were less than 98 bp, and only 9 exceeded 100 bp. SSR analysis showed that among the 26 SSRs identified, mononucleotide repeats dominated (69.23%), followed by trinucleotide (19.23%) and dinucleotide (7.69%) repeats. In the mononucleotide SSRs, A/T repeats accounted for 88.9% (A: 59.5%, T: 29.4%), with no dominant C/G repeats detected. Dinucleotide repeats were primarily AT/TA repeats. Additionally, 12 tandem repeats (5\u0026ndash;39 bp) were detected, all located in the intergenic regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSelective pressure analysis\u003c/h3\u003e\n\u003cp\u003eIn genetic studies, the K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e ratio serves as a critical indicator for measuring the direction and intensity of natural selection on homologous protein-coding genes (PCGs) during species divergence, holding significant theoretical value \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Compared to other neutral evolution testing methods in population genetics, this ratio has the advantages of fewer assumptions and higher testing power. When K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e \u0026lt; 1, it indicates that the gene is under purifying selection or stabilizing selection (suppressing the accumulation of variations); K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e \u0026gt;1 reflects positive selection or Darwinian selection (promoting adaptive variations); and a ratio of exactly 1 conforms to the neutral evolution model. It is important to note that the K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e ratio can significantly exceed 1 only when there are significant beneficial mutations at the gene locus. In this study, based on the comparative analysis of the mitochondrial genomes of \u003cem\u003eP. ginseng\u003c/em\u003e, \u003cem\u003eDaucus carota\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e, and \u003cem\u003eBupleurum chinense\u003c/em\u003e, we calculated the K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e evolutionary selection pressure for 34 PCGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e),. The results showed that approximately 88.1% of the genes had a K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e value less than 1.0 in cross-species comparisons, indicating that mitochondrial genes primarily undergo purifying selection during evolution, consistent with the conservative nature of plant mitochondrial genomes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Notably, in comparisons within the same genus (\u003cem\u003eP. ginseng\u003c/em\u003e vs. \u003cem\u003eP. notoginseng\u003c/em\u003e), the core respiratory chain genes \u003cem\u003enad4\u003c/em\u003e (K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 1.06) and cox2 (K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 1.05) showed signals of positive selection. In comparisons within the \u003cem\u003eP. ginseng\u003c/em\u003e and \u003cem\u003eB. chinense\u003c/em\u003e, the positive selection characteristics of \u003cem\u003enad4\u003c/em\u003e (K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 1.21) and \u003cem\u003etatC\u003c/em\u003e (twin-arginine translocation protein, K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 1.25) were further extended to the membrane transport system. In another cross-family comparisons (ginseng vs. carrot), the strong positive selection signals of \u003cem\u003erpl10\u003c/em\u003e (ribosomal large subunit protein, K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 3.05) and \u003cem\u003eccmC\u003c/em\u003e (cytochrome c maturation protein, K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e = 1.21) suggested adaptive differentiation in ribosomal translation and cytochrome c assembly pathways.\u003c/p\u003e\u003cp\u003eFurther functional association analysis indicates that the evolutionary drive of positively selected genes may be closely linked to the regulation of energy metabolism. As core components of respiratory chain complexes I and IV, \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003ecox2\u003c/em\u003e exhibit a conspecific positive selection pattern within the same genus (\u003cem\u003eP. ginseng\u003c/em\u003e and. \u003cem\u003eP. notoginseng\u003c/em\u003e), which may promote adaptive differentiation among closely related species by regulating the efficiency of oxidative phosphorylation. The positive selection characteristics of \u003cem\u003etatC\u003c/em\u003e in both \u003cem\u003eP. ginseng\u003c/em\u003e and \u003cem\u003eB. chinense\u003c/em\u003e suggest a co-evolutionary pressure between mitochondrial membrane transport systems and respiratory chain functions. In the species divergence between ginseng and carrot, the high K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e value of rpl10 suggests that the rapid evolution of ribosomal translation efficiency may be a response to the adaptive demands for translation accuracy among species with greater evolutionary distance.\u003c/p\u003e\u003cp\u003eAdditionally, other mitochondrial genes, including \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003eccmB\u003c/em\u003e, have been reported to have K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e \u0026gt;1, indicating that mitochondrial genes in different plant species may be subject to varying selective pressures during evolution. The above results demonstrate that the evolution of the ginseng mitochondrial genome exhibits significant heterogeneity: respiratory chain genes (\u003cem\u003enad4\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e) frequently experience positive selection during close species divergence, while ribosomal proteins (\u003cem\u003erpl10\u003c/em\u003e) maintain the conservation of core metabolic pathways through functional constraints. This provides a new perspective for elucidating the molecular mechanisms of ecological adaptation in \u003cem\u003ePanax\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eWith the rapid development of sequencing technologies and genome assembly methods, an increasing number of complete plant mitochondrial genomes have been successfully assembled, providing significant opportunities for phylogenetic analysis using mitochondrial genomes. This study aims to clarify the phylogenetic position of ginseng within the Araliaceae family and angiosperms. We constructed a phylogenetic tree using 20 universally presentPCGs (\u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp9\u003c/em\u003e, \u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmC\u003c/em\u003e, \u003cem\u003eccmFn\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, \u003cem\u003ematR\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e, \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e, \u003cem\u003enad9\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e) from the mitochondrial genomes of 15 species, including \u003cem\u003eP. ginseng\u003c/em\u003e, its close relatives \u003cem\u003eP. quinquefolius\u003c/em\u003e and \u003cem\u003eP. notoginseng\u003c/em\u003e, species from the Apiaceae family (\u003cem\u003eD. carota\u003c/em\u003e, \u003cem\u003eB. falcatum\u003c/em\u003e, and \u003cem\u003eAgeratum conyzoides\u003c/em\u003e), Rosaceae family (\u003cem\u003eMalus domestic\u003c/em\u003ea, \u003cem\u003eFragaria vesca\u003c/em\u003e), Asteraceae family (\u003cem\u003eHelianthus annuus\u003c/em\u003e, \u003cem\u003eLactuca sativa\u003c/em\u003e), as well as \u003cem\u003ePlatycodon grandiflorum\u003c/em\u003e, \u003cem\u003eCodonopsis pilosula\u003c/em\u003e, \u003cem\u003eLonicera japonica\u003c/em\u003e and etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e),. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e were used as outgroups. The results show that all branches of the phylogenetic tree have bootstrap support values exceeding 85%, with six branches achieving 100% support. Based on the maximum likelihood (ML) phylogenetic tree, it was found that the \u003cem\u003ePanax\u003c/em\u003e species form a monophyletic clade, and \u003cem\u003eP. notoginseng\u003c/em\u003e, \u003cem\u003eP. quinquefolius\u003c/em\u003e, and \u003cem\u003eP. ginseng\u003c/em\u003e cluster together, with \u003cem\u003eP. ginseng\u003c/em\u003e and \u003cem\u003eP. quinquefolius\u003c/em\u003e showing the closest relationship. This result is consistent with previous studies \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, providing mitochondrial evidence for the similar genome expansion and evolution experienced by \u003cem\u003eP. ginseng\u003c/em\u003e and \u003cem\u003eP. quinquefolius\u003c/em\u003e during their evolutionary history.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe evolutionary pattern of the ginseng mitochondrial genome demonstrates a high degree of conservation of core energy metabolism genes and dynamic adaptability of non-core genes. As with the significant differences in gene composition and arrangement observed in the mitochondrial genomes of higher plants \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, the mitochondrial genome evolution of ginseng and its close relatives also follows this rule: key genes for core energy metabolism functions (such as subunits of complexes I, III, and V, and genes related to cytochrome c biosynthesis) are highly conserved in angiosperms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e),. For example, the \u003cem\u003enad\u003c/em\u003e series genes, \u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, and \u003cem\u003ecob\u003c/em\u003e are all intact in \u003cem\u003eP. ginseng\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e, and \u003cem\u003eP. quinquefolius\u003c/em\u003e, indicating their irreplaceable role in maintaining mitochondrial core functions. In contrast, some non-essential genes (such as ribosomal protein genes and specific complex subunits) show significant loss. For instance, the \u003cem\u003erpl2\u003c/em\u003e gene is completely lost in \u003cem\u003eP. ginseng\u003c/em\u003e but retained in its close relative \u003cem\u003eP. notoginseng\u003c/em\u003e and \u003cem\u003eD. carota\u003c/em\u003e, the \u003cem\u003erps19\u003c/em\u003e gene is generally absent in \u003cem\u003eP. ginseng\u003c/em\u003e and its close relatives but present in \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eO. sativa\u003c/em\u003e, suggesting that its function may be compensated by nuclear genes. Additionally, functionally replaceable subunits such as \u003cem\u003eatpE\u003c/em\u003e are retained in \u003cem\u003eP. ginseng\u003c/em\u003e and \u003cem\u003eP. quinquefolius\u003c/em\u003e but lost in species from the Rosaceae and Apiaceae families. The genes \u003cem\u003esdh3\u003c/em\u003e and \u003cem\u003esdh4\u003c/em\u003e are present in \u003cem\u003ePanax\u003c/em\u003e species but specifically lost in other families, reflecting the adaptive evolutionary differences between the Araliaceae and other dicotyledonous groups after divergence. This evolutionary pattern indicates that \u003cem\u003eP. ginseng\u003c/em\u003e has achieved mitochondrial genome streamlining and functional balance by strictly conserving core metabolic genes, selectively losing non-essential genes, and relying on nuclear gene compensation mechanisms driven by gene transfer between the mitochondrial and nuclear genomes. This dynamic pattern may reflect the shaping effects of lineage-specific metabolic demands and adaptive evolution on gene retention and loss.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eConstruction and comparative analysis of the pan-mitochondrial genome of ginseng from different origins\u003c/h3\u003e\n\u003cp\u003eTo further understand the variations in the mitochondrial genomes of ginseng from different origins and types, we selected \u003cem\u003eP. ginseng\u003c/em\u003e samples from Liaoning and Jilin provinces, specifically JA, FC, SZ, and BT, along with the previously published Korean ginseng samples Gumpoong (GU) and Jakyung (JY), for pan-mitochondrial genome construction and comparative analysis. Initially, we statistically analyzed the six ginseng mitochondrial genomes and found that the size variations ranged from 0.72\u0026ndash;5.42%, with JY having the largest genome at 464,705 bp and the smallest genome at 431,475 bp. Further gene analysis of the six ginseng mitochondrial genomes revealed that, apart from JY, which had an additional \u003cem\u003erpl23\u003c/em\u003e gene related to the synthesis of the ribosomal large subunit, the other five ginseng mitochondrial genomes contained 45 PCGs.\u003c/p\u003e\u003cp\u003eBased on the pan-mitochondrial genome constructed from the six ginseng mitochondrial genomes, we conducted a comprehensive variation analysis after statistically analyzing their sizes and genes. We categorized the variations into six types: single nucleotide polymorphisms (SNPs), small insertions/deletions (InDels, \u0026lt; 50 bp), deletions (DELs), inversions (INVs), translocations (TRANSs), and copy number variations (CNVs). Using the haplotype genome of BT as a reference, we identified a total of 111 SNPs and 39 InDels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The fewest SNPs and InDels were identified in JA, with only 1 SNP and 1 InDel. The count range of identified SNPs and InDels in the other four mitochondrial genomes was 2\u0026ndash;62 and 3\u0026ndash;19, respectively. Additionally, through further analysis of the distribution of SNPs and InDels, we found that 32.43% of SNPs and 61.54% of InDels were located in gene regions. Besides the small variations of SNPs and InDels mentioned above, we also identified a small number of structural variations (SVs) between individual samples and BT through the pan-mitochondrial genome variation analysis of ginseng. Integrating the identified SV data, we found a total of 193 SVs, including 75 insertions, 59 deletions, 9 inversions, and 50 translocations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). There were certain differences in the number and type of SVs among different samples. The GU sample had relatively fewer structural variations, while the FC sample had the highest number of identified structural variations, with 28 insertions, 22 deletions, 1 inversion, and 29 translocations. Although the protein-coding genes of the mitochondria are relatively stable, the above results indicate that there may still be certain variations in the mitochondrial genomes of different ginseng samples.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant mitochondrial genomes are larger and more structurally complex than those of animals, making the assembly and scaffolding of complete mitochondrial genomes challenging. The complexity of plant mitochondrial genomes is not only reflected in their larger genome sizes but also in their diverse repetitive sequences and structural variations, which make sequencing and assembly a challenging task. However, with the development of sequencing technologies, increased read lengths, and the development of mitochondrial assembly software such as GetOrganelle, NOVOPlasty, and PMAT, it has become possible to assemble complete circular mitochondrial genomes. Ginseng, an important medicinal plant in the Araliaceae family, faces many challenges in terms of genetic stability and directed breeding. Therefore, in-depth research on its mitochondrial genome is of great significance. Since the release of the mitochondrial genomes of Gumoong\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and Jakyung\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e from South Korea, no studies have been conducted on the mitochondrial genomes of unique ginseng varieties from the main ginseng-producing regions of Jilin and Liaoning in China.\u003c/p\u003e\u003cp\u003eThis study is the first to perform mitochondrial genome assembly for four different ginseng varieties from Jilin and Liaoning. The size of the mitochondrial genomes of different ginseng varieties varies between 0.72% and 5.42%. The BT ginseng mitochondrial genome size is 464,658 bp, which is closest in size to GU and JY, differing by only 3 bp and 47 bp, respectively. However, the size of the JA mitochondrial genome from the same origin but a different variety differs by 11 kb from BT, indicating significant genetic variation among varieties. Further study of the BT genome structure revealed an A\u0026thinsp;+\u0026thinsp;T content of 55.0%. Further annotation of the BT mitochondrial genome identified a total of 80 genes, including 57 PCGs, 29 tRNA genes, and 4 rRNA genes. Comparing the 4 mitochondrial PCGs assembled in this study with GU and JY, it was found that, except for the addition of the rpl23 gene related to ribosomal large subunit synthesis in JY, the other 5 ginseng mitochondrial genomes all had 45 PCGs. The stability of PCGs not only indicates that ginseng mitochondrial genomes maintain relatively high conservation and stability in core genes but also lays a foundation for subsequent molecular breeding and functional gene research.\u003c/p\u003e\u003cp\u003eSince the establishment of the genus \u003cem\u003ePanax\u003c/em\u003e by Carl Linnaeus, taxonomic research on this genus has undergone a transformation from morphology to molecular systematics. Due to the convergent evolution of root and stem morphology and the complex arrangement of leaf sequences in \u003cem\u003ePanax\u003c/em\u003e, traditional morphological methods are difficult to accurately distinguish species. Mitochondrial genomes, as maternally inherited genomes, are often used for species classification and phylogenetic studies and are important tools for elucidating the phylogenetic relationships between species. Based on comparative analysis of the mitochondrial genomes of \u003cem\u003eP. ginseng\u003c/em\u003e, and other 3 plants, it was found that ginseng mitochondrial genes mainly experience purifying selection during evolution, with respiratory chain genes showing signals of positive selection in closely related species differentiation. This may be related to the regulation of energy metabolism and adaptive differentiation. These results reveal the heterogeneous selection pressure on ginseng mitochondrial genomes during evolution, providing a new perspective for elucidating the molecular mechanisms of ecological adaptation in \u003cem\u003ePanax\u003c/em\u003e species. Additionally, by constructing a phylogenetic tree including 15 species such as \u003cem\u003eP. ginseng\u003c/em\u003e, \u003cem\u003eP. quinquefolius\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e, and \u003cem\u003eH. annuus\u003c/em\u003e, it was found that \u003cem\u003ePanax\u003c/em\u003e species form a monophyletic clade. The close evolutionary relationship between ginseng and American ginseng mitochondrial genomes indicates their close phylogenetic relationship, consistent with the conclusions of previous genomic evolution studies of ginseng \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe concept of pan-mitochondrial genomes aims to comprehensively analyze genetic variations, functional genes, and adaptive evolution by integrating information from multiple mitochondrial genomes within a species. This research approach breaks the limitations of traditional reliance on a single reference genome, providing a new perspective for genetic diversity studies. Taking ginseng as an example, although two versions of the mitochondrial genome have been released, these data only partially reflect the diversity of ginseng mitochondrial genomes. Therefore, constructing a larger-scale pan-mitochondrial genome is crucial for capturing the variation in ginseng mitochondrial genomes.\u003c/p\u003e\u003cp\u003eIn this study, we conducted variation analysis on the mitochondrial genomes of six different ginseng varieties from China and Korea, revealing the distribution characteristics of genetic variations in ginseng. The results showed that the ginseng mitochondrial genome contains various types of variations, including insertions, deletions, inversions, translocations, and copy number variations. Specifically, the numbers of these variations were 75 insertions, 59 deletions, 9 inversions, and 50 translocations, but no copy number variations were identified. There were significant differences in the quantity and types of variations among different samples, indicating a certain degree of genetic diversity in ginseng mitochondrial genomes among different varieties. Although the ginseng mitochondrial genome overall exhibits high stability, these genetic variations provide rich resources for subsequent research and applications. Based on the variation sites on the mitochondrial genomes from different origins, it is possible to construct SNP chips targeting these variation sites, thereby achieving precise identification of different ginseng samples. This not only helps in the protection and utilization of ginseng genetic resources but also provides important molecular markers for ginseng molecular breeding and functional gene research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eSample collection and sequencing\u003c/h2\u003e\u003cp\u003eIn this study, based on the domestication history and distribution of \u003cem\u003ePanax ginseng\u003c/em\u003e, four different tetraploid ginseng lines (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48), including JA, SZ, BT, and FC, were selected for whole-genome sequencing. Fresh leaves were rapidly frozen in liquid nitrogen and stored in a -80\u0026deg;C ultra-low-temperature environment. Genomic DNA was extracted from fresh young leaves using a modified 3\u0026times;CTAB method, and DNA concentration was quantified by measuring the A260 absorbance value with a ND-2000 spectrophotometer. For the JA, SZ, BT, and FC lines, Hi-Fi libraries with an insert size of 10 kb were constructed and sequenced using the Pacific Biosciences Sequel II platform for third-generation sequencing. In the whole-genome resequencing part, paired-end sequencing libraries with an insert size of 300\u0026ndash;500 bp were constructed and resequenced on the MGI DNBSEQ T7 platform.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eMitochondrial genome assembly and annotation\u003c/h3\u003e\n\u003cp\u003eBased on the PacBio HiFi sequencing raw data, the hifi raw data was first assembled using the PMAT2 v2.0.2 \u003csup\u003e28\u003c/sup\u003e (-g 5G). The assembly results were visualized and manually corrected using Bandage v0.8.1\u003csup\u003e29\u003c/sup\u003e. To improve assembly accuracy, the original HiFi reads were further mapped back to the initial assembly sequence using minimap2, and iterative error correction optimization was performed using NextPolish. This resulted in a highly reliable mitochondrial genome sequence, and its graph framework file (GFA) was adjusted. The \u003cem\u003eP. ginseng\u003c/em\u003e mitochondrial genome was annotated using the MITOFY online annotation platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dogma.ccbb.utexas.edu/mitofy/\u003c/span\u003e\u003cspan address=\"http://dogma.ccbb.utexas.edu/mitofy/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and corrected by comparing with homologous genes in Araliaceae plant mitochondrial genomes. tRNAscan-SE \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trna.ucsc.edu/tRNAscan-SE/\u003c/span\u003e\u003cspan address=\"http://trna.ucsc.edu/tRNAscan-SE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to annotate transfer RNAs, and Open Reading Frame Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to annotate ORFs. The MEGA v7.0.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e bioinformatics tool was used for codon usage analysis, calculating RSCU and amino acid composition characteristics. Finally, the OGDraw \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e program (\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) was used to construct a circular visualization map of the mitochondrial genome.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRepeat sequence prediction\u003c/h2\u003e\u003cp\u003eThe REPuter\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bibiserv.cebitec.uni-bielefeld.de/reputer/\u003c/span\u003e\u003cspan address=\"https://bibiserv.cebitec.uni-bielefeld.de/reputer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to identify dispersed repeat sequences, with parameters set as: repeat sequence consistency\u0026thinsp;\u0026gt;\u0026thinsp;90%, minimum repeat unit length\u0026thinsp;\u0026ge;\u0026thinsp;30 bp, and Hamming distance of 3. This analysis effectively identified various types of dispersed repeats, including forward repeats, reverse repeats, complement repeats, and palindromic repeats. Additionally, SSRs were identified using MISA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pgrc.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"http://pgrc.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), analyzing six types of SSRs: mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide, with repeat thresholds set at 10, 5, 4, 3, 3, and 3, respectively. Tandem Repeats Finder v4.09 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tandem.bu.edu/trf/trf.html\u003c/span\u003e\u003cspan address=\"http://tandem.bu.edu/trf/trf.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to detect tandem repeat sequences.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSelection pressure analysis\u003c/h2\u003e\u003cp\u003eThe natural selection pressure during the mitochondrial evolution of \u003cem\u003eP. ginseng\u003c/em\u003e was inferred by calculating the non-synonymous substitution rate (K\u003csub\u003ea\u003c/sub\u003e), synonymous substitution rate (K\u003csub\u003es\u003c/sub\u003e), and their ratio (K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e) of PCGs, using the mitochondrial genomes of \u003cem\u003eD. carota\u003c/em\u003e, \u003cem\u003eP. notoginseng\u003c/em\u003e, and \u003cem\u003eB. chinense\u003c/em\u003e as references. Homologous gene pairs were first aligned and formatted using ParaAT2.0 software. The K\u003csub\u003ea\u003c/sub\u003e, K\u003csub\u003es\u003c/sub\u003e, and K\u003csub\u003ea\u003c/sub\u003e/K\u003csub\u003es\u003c/sub\u003e values of each gene were then calculated using the KaKs_Calculator v.3.0 \u003csup\u003e34\u003c/sup\u003e based on the YN algorithm, and the statistical significance of the substitution rates was verified using Fisher's exact test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e\u003cp\u003eTo accurately infer the phylogenetic relationship of \u003cem\u003eP. ginseng\u003c/em\u003e within the \u003cem\u003ePanax\u003c/em\u003e genus, this study conducted a phylogenetic analysis based on the PCGs of 15 higher plant mitochondrial genomes. The mitochondrial genome information of all species (except \u003cem\u003eP. ginseng\u003c/em\u003e) used in the phylogenetic analysis was obtained from the NCBI Organelle Genome Resources 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). Perl scripts were used to screen for single-copy orthologous PCGs common to the 15 analyzed species. All conserved mitochondrial PCG sequences were extracted from each mitochondrial genome, combined into a single dataset, and aligned using the Muscle software. The ML tree was then constructed using MEGA v7.0\u003csup\u003e31\u003c/sup\u003e. The bootstrap values displayed next to the branches in the phylogenetic tree represent the reliability of the clustering of related taxa, which were obtained through 1000 repetitions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of the P. ginseng pan-mitochondrial genome\u003c/h2\u003e\u003cp\u003eUsing the BT mitochondrial genome as a reference, this study compared it with the JA, FC, and SZ mitochondrial genomes assembled in this study, as well as two previously published \u003cem\u003eP. ginseng\u003c/em\u003e mitochondrial genomes (GenBank accession no. MW029460.1, and MZ389476.1), to identify and statistically analyze SNPs and InDels. Initially, nucmer v4.0.0rc1 was used for alignment (parameters: --maxmatch -c 1000 -l 40), followed by filtering with delta-filter (parameters: -m -i 90 -l 100), and further filtering with delta-filter (parameters: -l -i 90 -l 100). The resulting files were then used with delta2vcf to identify SNPs and InDels. In the identification of structural variations in \u003cem\u003eP. ginseng\u003c/em\u003e, nucmer was used to align with the reference genome, and the filtered alignment results were identified using the SyRI v1.7.0\u003csup\u003e35\u003c/sup\u003e software (parameters: --nc 10 --nosnp). This process allowed for the identification of collinear regions, structural rearrangements, and local variation regions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions statement\u003c/p\u003e\n\u003cp\u003eConceptualization,\u0026nbsp;D.D.; methodology, J.X.; formal analysis, Y.X. and X.L.; investigation, Y.X. and X.L.; resources, Y.H. and D.D.; writing\u0026mdash;original draft preparation, S.X. and X.L.; writing\u0026mdash;review and editing, X.L. and S.G.; visualization, W.S., L.S.and T.Y.; supervision, S.C. and D.D.; project administration, J.X.; funding acquisition, D.D. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by Scientific and technological innovation project of China Academy of Chinese Medical Sciences (CI2023E002), the National Key Research and Development Program of China (2023YFC3504000), the Fundamental Research Funds for the Central Public Welfare Re-search Institutes (ZZ13-YQ-047),\u0026nbsp;the special fund for Science and Technology Innovation Teams of Shanxi Province (202204051001030).\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article and Supplementary Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChevigny, N., Schatz-Daas, D., Lotfi, F. \u0026amp; Gualberto, J. 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SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13059-019-1911-0\u003c/span\u003e\u003cspan address=\"10.1186/s13059-019-1911-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Panax ginseng, mitochondrial genome, phylogenetic analysis, structural variation","lastPublishedDoi":"10.21203/rs.3.rs-6622134/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6622134/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGinseng (\u003cem\u003ePanax ginseng\u003c/em\u003e), a vital medicinal plant, faces challenges in genetic stability and directed breeding. This study aimed to elucidate mitochondrial genome structure, evolution, and phylogenetic relationships to guide germplasm conservation and molecular breeding. Using PacBio HiFi and Illumina sequencing, we assembled the complete mitochondrial genome of the BT cultivar (~\u0026thinsp;465 kb, 55% A\u0026thinsp;+\u0026thinsp;T, encoding 80 functional genes). Repetitive sequences and codon usage patterns (preference for G/C at third codon positions) were characterized. Selective pressure analysis showed that most genes underwent purifying selection, but respiratory chain genes (nad4, cox2) exhibited positive selection signals. Phylogenetic analysis confirmed close relationships between ginseng and \u003cem\u003eP. quinquefolius\u003c/em\u003e, with \u003cem\u003eP. notoginseng\u003c/em\u003e forming a distinct clade. A pan-mitochondrial genome was constructed by integrating data from six ginseng populations. Analysis of this pan-genome revealed high genetic stability across populations, with SNPs, InDels, and structural variations identified. These findings provide insights into mitochondrial conservation, adaptive evolution, and population variation, supporting targeted breeding strategies for ginseng varieties.\u003c/p\u003e","manuscriptTitle":"Pan-Mitochondrial Genomic Analysis of Ginseng (Panax ginseng) Reveals Structural Variation, Phylogenetic Relationships, and Genetic Diversity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 19:59:10","doi":"10.21203/rs.3.rs-6622134/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T03:01:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T05:04:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T02:38:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206624378899030353794188122989560120903","date":"2025-07-07T06:29:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125362733724695895967908218581225035563","date":"2025-07-06T14:01:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-06T13:52:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-27T12:43:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-27T06:20:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-23T06:10:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-08T16:00:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b4de7b8-28a6-4b40-a53e-51058ca5396b","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51184076,"name":"Biological sciences/Genetics"},{"id":51184077,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-01-05T16:00:51+00:00","versionOfRecord":{"articleIdentity":"rs-6622134","link":"https://doi.org/10.1038/s41598-025-28721-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-29 15:57:53","publishedOnDateReadable":"December 29th, 2025"},"versionCreatedAt":"2025-07-11 19:59:10","video":"","vorDoi":"10.1038/s41598-025-28721-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-28721-z","workflowStages":[]},"version":"v1","identity":"rs-6622134","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6622134","identity":"rs-6622134","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}