Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species

Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species Ruifeng Yang, Mengyue Wang, Mingliang Wang, Jun Li, Jinglong Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7230374/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Medicago is an economically important forage genus widely distributed across China, yet its mitochondrial genomes remain poorly characterized. Comprehensive analysis of mitochondrial genome structure, function, and evolution is essential for uncovering plant biological mechanisms, enhancing germplasm utilization, and supporting molecular breeding efforts. Results Here, we assembled and compared the mitochondrial genome of eight Medicago species, including six newly sequenced genomes. Our results revealed that five species possess typical single circular mitochondrial genome structures, while M. falcata , M. platycarpos , and M. sativa exhibit complex multipartite circular conFigureurations. These mitochondrial genome sizes ranged from 281,240 to 356,577 bp, containing 55–74 functional genes. Repetitive sequence analysis identified 141 simple sequence repeats (SSRs) and 76 tandem repeats (TSRs), dominated by A/T-rich mononucleotide motifs, while dispersed repeats were mainly 30–49 bp in length. Codon usage analysis showed strong A/T bias and a preference for leucine, serine, and isoleucine. RNA editing sites were predominantly C-to-U substitutions, primarily located at the first and second codon positions. Phylogenetic reconstruction based on 31 conserved mitochondrial protein-coding genes (PCGs) strongly supported the monophyly of Medicago and resolved interspecific relationships consistent with previous chloroplast and nuclear genome studies. Most PCGs were under purifying selection, whereas a few genes, such as matR , exhibited signals of positive selection, suggesting lineage-specific adaptive evolution. Conclusions Altogether, this study enriches the mitochondrial genomic resources of Medicago and deepens the understanding of its structural evolution and phylogenetic relationships, providing valuable insights for evolutionary and functional studies in Fabaceae plants. Medicago Mitochondrial genome Genome rearrangements Phylogenetic analysis Comparative analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Mitochondria are essential, semi-autonomous organelles in eukaryotic plant cells that play central roles in energy production and metabolic processes, such as oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid metabolism, iron–sulfur cluster assembly, and calcium homeostasis. These functions provide both energy support and regulatory support for plant growth, development, reproduction, and responses to environmental stresses [ 1 – 3 ]. In addition, mitochondria are actively involved in programmed cell death, autophagy, signal transduction, and the maintenance of redox homeostasis. They are also essential in biological processes such as pathogen defense, responses to abiotic stress responses, and cytoplasmic male sterility (CMS) [ 4 – 7 ]. It is widely accepted that mitochondria originated from an ancient endosymbiotic event involving an α-proteobacterium approximately 1.5 billion years ago. This origin was followed by extensive gene transfer to the nuclear genome, as well asintegration and loss of genes during plant evolution [ 8 ]. The plant mitochondrial genome (mitogenome) is physically and genetically independent of the nuclear genome and is typically maternally inherited, although cases of paternal or biparental inheritance have also been documented. This results in a semi-autonomous inheritance mode [ 9 ]. Compared to chloroplasts genomes and animal mitochondrial genomes, plant mitogenomes show much greater variability and complexity in terms of structural conFigureuration, genome size, abundance and types of repeat sequences, and incorporation of foreign DNA [ 10 ]. These features make plant mitogenomes a valuable model for studying adaptive evolution and the coordination between nuclear and organellar genomes. Although plant mitochondria encode a relatively small number of genes (typically 19–41 protein-coding genes), their gene content and sequences are relatively conserved [ 11 ]. However, the overall genome structure is highly dynamic, appearing in diverse forms including single circular, multi-circular, linear, branched, or even multipartite conFigureurations, with multiple structural types often coexisting within the same species [ 8 ]. The reported sizes of plant mitochondrial genomes range from 66 kb (e.g., Viscum scurruloideum ) to over 18 Mb (e.g., Cathaya argyrophylla ), representing more than a 200-fold difference [ 12 , 13 ]. This remarkable variation is primarily attributed to the proliferation of repetitive sequences, integration of foreign DNA, and frequent recombination events [ 14 ]. Despite having extremely low nucleotide substitution rates plant mitogenomes, undergo frequent structural rearrangements and exhibit widespread RNA editing, These RNA editing events not only contributes to the diversification at the protein level, but are also closely associated with CMS [ 15 , 16 ]. As of May 2025, only 688 plant mitochondrial genomes have been deposited in the NCBI database, a number far lower than the 15,396 publicly available chloroplast genomes, highlighting the technical challenges posed by their structural heterogeneity and recombination mediated by repetitive elements [ 13 ]. With the advancement of third-generation sequencing platforms such as PacBio HiFi and Oxford Nanopore Technologies (ONT), as well as specialized assembly and annotation tools like GSAT and PMAT, research on plant mitogenomes is progressing rapidly, These technologies offering tremendous potential for clarifying mitogenome structural complexity, evolutionary history, and their associations with CMS and phylogeny [ 17 ]. Therefore, comprehensive investigations into the structure, function, and evolution of plant mitochondrial genomes are of great theoretical and practical significance for uncovering plant biological mechanisms, mining elite germplasm resources, and supporting molecular breeding strategies. Medicago is an annual or perennial herbaceous genus in the Fabaceae family, originating from the Mediterranean coastal regions and Southwest Asia. In China, it is mainly distributed across the northern, northwestern, and northeastern regions. Medicago is rich in proteins, vitamins, minerals, polysaccharides, soybean flavonoids, isoflavones, and various identified growth-promoting factors (UGFs), earning it the title "King of Forage." It not only serves as a high-quality forage for herbivorous animals such as dairy cattle, but also enhances animal productivity, immunity, and meat quality, while modulating gut microbiota [ 18 , 19 ]. Currently, the cultivated area of Medicago in China exceeds one million hectares, with an annual yield of over two million tons. Its hay and derived products are easy to store and transport, and are widely used in feed, medicine, bioenergy, and ecological restoration. The cultivation of Medicago in China can be traced back to the Han Dynasty, as recorded in Records of the Grand Historian《Shiji·Dayuan Liezhuan》, where it was noted that “horses favor Medicago ” indicating its early integration into agricultural and pastoral systems. Medicago exhibits self-incompatibility and inter-subspecific hybridization, reflecting its high genetic diversity and strong ecological adaptability [ 20 ]. Despite its promising applications, the taxonomy and germplasm characterization of Medicago remain relatively underdeveloped in China. The number of species recorded varies across references—for instance, 《Illustrated Flora of Higher Plants in China》 (1972) listed six species, 《Flora of China》 (1998) reports 13 species and one variety, whereas Lu Xinshi and colleagues identified 46 taxonomic units in China, including 30 perennial wild species (comprising 12 varieties and one subspecies), five annual species, and 11 introduced species. In summary, Medicago holds significant importance in agriculture, animal husbandry, and ecosystems. Its taxonomic classification, germplasm resource exploration, and molecular breeding remain key focal points in current Medicago research. Although the mitochondrial genomes of several plant genera, such as Capsicum , Fragaria , and Morus Linn , have been successfully decoded [ 21 – 24 ], current research on the genus Medicago remains largely focused on single species or specific populations, with a lack of systematic comparative analyses across multiple representative species within the genus [ 25 , 26 ]. In this study, we performed de novo assembly and functional annotation of mitochondrial genomes from six Medicago species using the PacBio long-read sequencing platform. Combined with two previously published mitochondrial genomes, we constructed a comparative dataset encompassing eight representative Medicago species. Based on this dataset, we systematically analyzed gene content, repeat sequence characteristics, relative synonymous codon usage (RSCU), RNA editing sites, selective pressure (Ka/Ks), and synteny relationships among the Medicago mitochondrial genomes. In addition, we reconstructed a phylogenetic tree using 31 single-copy protein-coding genes (PCGs) to comprehensively elucidate the evolutionary relationships within the Medicago genus and between Medicago and other species in the Fabaceae family. This study provides a solid data foundation and theoretical basis for the taxonomic classification, germplasm exploration and utilization, and organellar genome evolution research of Medicago species. Results Characteristics of the mitochondrial genomes of eight Medicago species To comprehensively elucidate the structural characteristics and evolutionary divergence of mitochondrial genomes in Medicago species, we conducted a comparative analysis of the mitochondrial genomes from eight Medicago species. Among them, the mitochondrial genomes of six species were newly assembled de novo and functionally annotated (Fig. 1 A–F). In terms of structural conFigureuration, the mitochondrial genomes of M. lupulina , M. minima , M. edgeworthii , M. truncatula , and M. arabica exhibit a single circular structure, while M. falcata , M. platycarpos , and M. sativa display complex multichromosomal circular structures (Fig. 1 A–F). The genome sizes range from 281,240 ~ 356,577 bp, with a ~ 1.27-fold difference (Table S3), and the GC content ranges from 44.92%~45.58%, which is consistent with the typical features of angiosperm mitochondrial genomes. Functional annotation revealed that each genome contains 55–74 functional genes, comprising 33–41 PCGs, 18–24 rRNA genes, and 3–5 tRNA genes (Table S3–S5; Figure S1 –S6). The number of protein-coding genes was generally conserved across species, with the exception of M. falcata , which exhibited a notable increase. For instance, sdh4 was present as a single copy in M. edgeworthii, M. platycarpos , and M. arabica , but existed in multiple copies in the other species. Similarly, atp1 , atp4 , and nad4L appeared in multiple copies in both M. falcata and M. arabica , while multicopy ccmB , ccmFN , and cox3 were uniquely observed in M. falcata . Additionally, rps1 was only detected in M. truncatula and was absent in the other seven species (Fig. 1 G). Variations in gene copy number and presence or absence were also observed in rRNA and tRNA genes. For example, rrn26 was present in multiple copies in M. falcata ; trnQ-UUG was consistently found in multiple copies across all eight Medicago species; trnF-GAA appeared as a single copy only in M. arabica but as multiple copies in the remaining species; trnfM-CAU was present in multiple copies in M. falcata , M. platycarpos , and M. sativa ; trnM-CAU was present in multiple copies in M. lupulina and M. arabica ; and the multicopy presence of trnN-GUU and trnY-GUA was exclusive to M. falcata . These variations may be associated with structural duplications or potential functional redundancy within the mitochondrial genomes of certain species (Fig. 1 G). Repeat sequences analysis in the Medicago mitochondrial genomes To further investigate the distribution characteristics and evolutionary divergence of repetitive sequences in Medicago mitochondrial genomes, we identified and analyzed three types of repeats—SSRs, TSRs, and DSRs—across the mitochondrial genomes of eight Medicago species (Fig. 2 A). SSRs, composed of 1–6 nucleotide units, are short repetitive sequences widely present in plant organelle genomes and can contribute to genome rearrangement and length expansion [ 27 ]. A total of 141 SSRs were identified across the eight Medicago mitochondrial genomes, with the number per species ranging from 11 to 23 (Fig. 2 B, Table S6). Mononucleotide repeats represented the most prevalent type, accounting for 69.57–95.2% of all SSRs, followed by dinucleotide and trinucleotide repeats (4.8–30.43%). Tetranucleotide, pentanucleotide, and hexanucleotide repeats were not detected in any species. Among mononucleotide SSRs, A/T repeats were dominant (~ 92.9%), while C/G repeats were relatively rare (~ 7.1%) and were not detected in M. falcata , M. edgeworthii and M. sativa . For dinucleotide SSRs, the (AG/CT or AT/TA) motif was the most common (4.76–21.75%), though (AT/TA) repeats were absent in M. falcata and M. dgeworthii . Trinucleotide repeats were present in all species except M. edgeworthii . Overall, although differences in SSR types were observed among species, their distribution patterns remained relatively consistent. SSRs were generally evenly distributed throughout the genome, but in M. minima and M. arabica , clustering in specific chromosomal regions was observed, suggesting potential localized enrichment or selective retention (Fig. 2 A). TSRs consist of tandemly arranged repeat units longer than 6 bp [ 28 ]. A total of 76 TSRs were identified across the eight Medicago mitochondrial genomes, with 4 to 18 TSRs detected per species. Repeat unit lengths ranged from 6 to 33 bp, and all matches showed greater than 77% similarity. Notably, M. arabica exhibited a significantly higher number of TSRs compared to other species, which may reflect increased structural duplication or recombination activity (Fig. 2 C, Table S7). DSRs, also known as dispersed repeats, are sequences that can facilitate recombination and contribute to genome rearrangement and are widely found in plant mitochondrial genomes [ 29 ]. Three types of DSRs were detected in this study: forward (F) and palindromic (P) repeats were found in most species, while reverse (R) repeats were only observed in M. falcata (one instance), and complementary (C) repeats were not detected in any Medicago species (Fig. 2 C, Table S7). In M. arabica , only 126 F-type repeats were detected, with no P-type repeats found. In the other species, F-type and P-type repeats ranged from 64 to 138 and 46 to 135, respectively (Fig. 2 C, Table S8). Although the number of DSRs varied among species, their length distribution was largely consistent, with most repeats falling within the 30–49 bp range (~ 73.56%), followed by the 50–99 bp range (~ 17.26%). Additionally, M. falcata exhibited a significantly higher number of DSRs longer than 500 bp compared to other species, which may contribute to the larger size of its mitochondrial genome (Figure S7). Analysis of codon usage in Medicago To investigate codon usage patterns and preferences of mitochondrial PCGs in Medicago species, a comprehensive codon usage analysis using the mitochondrial genomes of eight Medicago species (Fig. 3 , Table S9). The total number of codons in PCGs ranged from 9,639 to 11,515 across species, encompassing 64 codons corresponding to 20 standard amino acids and stop codons. Overall, leucine (Leu) was the most frequently used amino acid in most species, followed by serine (Ser) and isoleucine (Ile), whereas tryptophan (Trp) and cysteine (Cys) were the least frequently used. As expected, most amino acids are encoded by multiple synonymous codons; for instance, arginine (Arg), leucine (Leu), and serine (Ser) each have six synonymous codons, while methionine (Met) and tryptophan (Trp) are each encoded by a single codon (ATG and TGG, respectively). Further analysis of the relative RSCU values for all 64 codons revealed that approximately 75% of the codons exhibited RSCU values greater than 1 in multiple species, indicating a significant usage preference. The remaining ~ 25% of codons had RSCU values less than 1, suggesting that their usage frequencies were significantly lower than expected under equal codon usage assumptions. Some codons, such as Asp (GAC) and Leu (CTC, CTG), had RSCU values close to 1, consistent with a "no preference" usage pattern. The overall RSCU patterns were highly consistent among the Medicago species, particularly for Phe (TTT), which consistently exhibited the highest RSCU value across all species, suggesting that such codons may be under similar selective pressures or mutation biases. In addition, analysis of GC content showed that the third codon positions of all species had relatively low GC content, averaging between 35.88% and 37.66%, reflecting a general A/T bias in the Medicago mitochondrial genomes. Collectively, the codon usage patterns of mitochondrial PCGs in Medicago species exhibit a high degree of consistency and a strong preference for A/T, indicating that natural selection may play a crucial role in shaping codon usage evolution in this genus. Prediction of RNA editing sites in Medicago To gain deeper insights into the post-transcriptional regulatory mechanisms of mitochondrial PCGs, we analyzed RNA editing sites and their characteristics in the mitochondrial genomes of eight Medicago species. We identified widespread C-to-U RNA editing events across the PCGs of all species, along with a few U-to-C type events. The total number of editing sites per species ranged from 452 to 568, showing limited variation, which suggests that RNA editing is a conserved post-transcriptional regulatory mechanism in higher plant mitochondria (Fig. 4 A, Table S10). Approximately 92.53–95.35% of these were cytidine-to-uridine (C-to-U) single-nucleotide substitutions. Further analysis of codon position distribution revealed that more than 50% of editing events occurred at the second codon position (approximately 59.40–67.77%), followed by the first position (approximately 32.23–40.60%), with no editing events detected at the third codon position (Fig. 4 B, Table S11). The distribution of editing sites varied significantly among different genes. Genes involved in functions such as the electron transport chain, cytochrome biosynthesis, and membrane transport—such as nad4 and ccmB —harbored a large number of editing sites (up to 46–89), whereas ribosomal protein-coding genes like rpsI ( rpsI16 , rpsI5 ) had relatively few editing sites (only 12–14). Common editing conversions included serine (Ser) to phenylalanine (Phe), leucine (Leu), or proline (Pro); arginine (Arg) to cysteine (Cys) or tryptophan (Trp); and proline (Pro) to leucine (Leu) or phenylalanine (Phe). These changes were predominantly transitions from hydrophilic to hydrophobic amino acids, which may contribute to enhancing protein structural stability (Fig. 4 C, Table S12). Additionally, editing events associated with stop codons were detected in genes such as atp6 , atp9 , and ccmFC , suggesting a potential regulatory role of RNA editing in translation initiation and termination processes. Phylogenetic relationship among the Medicago species To elucidate the mitochondrial genome-level phylogenetic relationships of Medicago species within the Fabaceae family, we performed a phylogenetic analysis using the complete mitochondrial PCG sequences from 25 Fabaceae species. A total of 31 conserved single-copy PCGs (including atp , cox , cob , nad , rps , etc.) were extracted and used to construct phylogenetic trees based on both the Maximum likelihood (ML) method (Fig. 5 A) and Bayesian inference (BI) method (Fig. 5 B). The results showed that both methods produced highly congruent tree topologies, with strong support for most branches (bootstrap ≥ 90%, posterior probability ≥ 0.95), indicating that the selected gene set provides a robust phylogenetic signal. In both resulting trees, species clustered into well-defined clades, and genera or species with close evolutionary relationships tended to group together, reflecting a high degree of phylogenetic consistency. All sampled Medicago species were resolved as a monophyletic clade, supporting their shared evolutionary origin within the genus. Further analysis revealed that M. minima and M. lupulina were recovered as sister species, forming a clade with M. truncatula . M. falcata and M. sativa were resolved as a strongly supported sister pair. Meanwhile, M. platycarpos , M. edgeworthii , and M. arabica were inferred as more distantly related lineages, positioned outside the two aforementioned clusters. It suggests possible differences in evolutionary rates or selective pressures among these species. The inferred phylogenetic relationships were highly consistent with those based on chloroplast genomes and conformed to the taxonomic framework of angiosperms as outlined in the APG-IV system [ 30 ], further supporting the scientific validity and reliability of constructing phylogenies based on conserved mitochondrial PCGs. Notably, some species, such as M. truncatula , showed relatively low support values in the tree, indicating potential uncertainty in their phylogenetic placement. This may be attributed to structural complexity or incomplete gene annotation in their mitochondrial genomes, warranting further investigation. Overall, the well-supported phylogenetic framework established in this study provides novel mitochondrial evidence for understanding evolutionary relationships in Fabaceae plants and offers fundamental data for taxonomic studies and investigations into the origin and evolution of species within the family. Synteny analysis of mitochondrial sequences of Medicago species To investigate the structural evolutionary characteristics of mitochondrial genomes in Medicago species, we conducted a comprehensive collinearity analysis of eight representative Medicago mitochondrial genomes. The results revealed that although these species are phylogenetically closely related, their mitochondrial genomes exhibit a certain degree of structural variation in terms of the arrangement, length, and order of homologous blocks. Using BLASTn comparisons, we identified 23–44 homologous collinear segments longer than 400 bp between each pair of genomes (Fig. 6 ). These segments include both forward collinear blocks and reverse complementary blocks, which are widely distributed across the circular chromosomes of the mitochondrial genomes. For example, between M. falcata and M. sativa , 24 collinear blocks were detected, with the longest segment reaching 74,236 bp and exhibiting over 95% sequence similarity, indicating a high degree of structural conservation (Table S13). Moreover, we observed that some conserved blocks span nearly the entire genome or are clustered near specific functional regions (such as nad , cox , and ccm gene loci). These structures may be closely associated with essencial mitochondrial energy metabolism functions and could have been selectively retained during evolution. Despite certain rearrangements in overall genome architecture, many PCG regions remain highly collinear among species, suggesting that these functional genes are subject to strong purifying selection. In conclusion, while the mitochondrial genomes of Medicago species exhibit a high level of sequence homology, they also display considerable structural rearrangement and diversity. These variations are likely linked to recombination events mediated by repetitive sequences during species divergence. This study provides important theoretical insights into the structural evolution of Medicago mitochondrial genomes and their phylogenetic relationships. Analysis of selection pressure of shared PCGs in Medicago species To evaluate the selective pressures acting on mitochondrial PCGs in Medicago species during evolution, we conducted a systematic analysis of the nonsynonymous to synonymous substitution rate ratios (Ka/Ks) for mitochondrial single-copy PCGs shared among eight representative species. Alignments of 27 common single-copy PCGs were performed, and the Ka, Ks, and Ka/Ks values for each gene were calculated across pairwise species comparisons to investigate the potential selection mechanisms (Fig. 7 A–C). The results showed that the Ka/Ks ratios for most PCGs were significantly less than 1, indicating that these genes have been predominantly subjected to purifying selection and are functionally conserved during evolution (Fig. 7 C). Notably, genes such as atp1 , nad1 , nad4L , nad5 , nad6 , nad7 , and rps7 exhibited Ka/Ks values approaching zero. These genes are mainly involved in energy metabolism, electron transport, and ribosomal function, suggesting their crucial roles in maintaining core mitochondrial precesses. Importantly, several genes displayed Ka/Ks values greater than 1 in certain species pairs, implying they may have undergone positive selection (Table S14). For instance, cox2 , matR , and rpl16 showed significantly elevated Ka/Ks ratios in some comparisons, particularly between M. arabica , M. edgeworthii , M. falcata , and M. truncatula , where the Ka/Ks value of matR reached as high as 3.34. This suggests that matR may have played a key role in the adaptive evolution of these species. In summary, mitochondrial PCGs in Medicago generally exhibit strong evolutionary conservation, although a subset of genes may have experienced positive selection in specific lineages. The Ka/Ks analysis provides theoretical insights into the adaptive evolutionary mechanisms of Medicago species and lays the groundwork for identifying candidate genes with accelerated evolutionary rates for further functional studies. Discussion Mitochondria, often referred to as the "powerhouses" of the cell, play a critical role in plant growth and development through their stable function. Plant mitochondrial genomes exhibit considerably greater complexity in both structure and regulatory mechanisms than those of animals and fungi, characterized by substantial variation in genome size, diverse gene content, abundant repetitive elements, frequent intron insertions, and highly plastic genome conformations [ 31 , 32 ]. In this study, we characterized the mitochondrial genomes of eight representative Medicago species, including six newly reported assemblies. Compared with other Fabaceae species, such as Glycine soja (402,545 bp) [ 33 ] and Vicia faba (588,000 bp) [ 34 ], the Medicago mitochondrial genomes ranging from 281,240 to 356,577 bp, exhibit relatively moderate genome sizes. This suggests a more constrained genome expansion during their evolutionary history, possibly due to limited acquisition of foreign DNA or less proliferation of repeat elements. In terms of gene content, certain species (e.g., M. falcata ) exhibited increased copy numbers of several PCGs, such as atp1 , atp4 , ccmB , ccmFN , cox3 , and nad4L , In contrast, rps1 was annotated only in M. truncatula and absent in the other species (Fig. 1 ). Similar lineage-specific gene duplication and loss patterns have been observed in Camellia duntsa [ 35 ] and Mangifera [ 36 ], suggesting that dynamic gene copy number variation is a common feature in angiosperm mitogenomes. These events may contribute to functional diversification, regulation of respiratory efficiency, or species-specific adaptation. The GC content of the Medicago mitochondrial genomes remained relatively stable (44.92–45.58%) (Table S3), similar to that observed in Phaseolus vulgaris (45.11%) [ 26 ] and Momordica charantia (45.60%) [ 36 ]. This stability in GC content may reflect evolutionary constraints related to replication fidelity, genome stability, and possibly the thermodynamic properties of the mitochondrial genome [ 37 ]. Moreover, notable structural differences were observed among Medicago species: M. lupulina and M. minima possessed typical single circular conformations, whereas M. falcata and M. sativa exhibited more complex multi-circular structures. Such structural heterogeneity has also been reported in species such as Michelia [ 38 ], Punica [ 39 ], and Astragalus membranaceus in Fabaceae [ 40 ], highlighting the high degree of evolutionary plasticity in angiosperm mitochondrial genome organization [ 41 ]. This diversity in genome conformation may arise from recombination between dispersed repeats or differential replication mechanisms. Repeat sequence analysis revealed that Medicago mitochondrial genomes are rich in diverse types of repeats, notably SSRs dominated by AT motifs, short repeats less than 200 bp, and large palindromic repeats exceeding 500 bp. These elements may facilitate genomic rearrangements and conformational evolution through homologous recombination (Fig. 2 ) [ 42 ]. Similar repeat profiles have also been identified in Broussonetia [ 24 ] and Camellia [ 35 ], further supporting the widespread role of repeat-mediated structural variation across plant mitochondrial genomes. Intron analysis showed that multiple intron-containing PCGs (e.g., nad1 , nad2 , nad5 , nad7 ) were conserved in Medicago mitochondrial genomes (Table S5). These patterns are consistent with those reported in other Fabaceae genera such as Glycine and Cicer [ 33 , 43 ]. Most introns were located within cis-splicing regions and exhibited highly conserved structures, suggesting strong evolutionary constraints. Their conservation across taxa also indicates potential utility in phylogenetics and population genetics [ 44 , 45 ]. Collectively, the Medicago mitochondrial genomes exhibit both interspecific variability and conserved features in genome size, gene content, structural conformation, and repeat composition, providing valuable insights into mitochondrial genome evolution in Fabaceae. Regarding codon usage, Medicago mitochondrial genomes exhibited pronounced bias toward synonymous codons ending in A/T. Leucine (Leu) was the most frequently encoded amino acid, while tryptophan (Trp) and cysteine (Cys) were used the least (Fig. 3 ). This pattern is consistent with that observed in Mangifera [ 23 ]and Broussonetia [ 24 ], supporting the idea that codon usage in plant mitochondria is influenced by a combination of natural selection, mutational bias, and genetic drift [ 46 ]. These features not only reflect the potential translational and transcriptional efficiency adaptations in Medicago , but also align with codon usage preferences widely observed in other angiosperm mitochondrial genomes. RNA editing analysis revealed the widespread presence of 452–568 potential C-to-U type RNA editing sites in the mitochondrial PCGs of Medicago species (Fig. 4 ). Over 92% of these editing events occurred at the first and second codon positions, typically resulting in nonsynonymous substitutions that alter amino acid properties. Notably, these changes often involved a transitions from hydrophilic to hydrophobic amino acids, potentially enhancing protein structural stability and functional efficiency. Editing sites were predominantly concentrated in core metabolic genes such as nad4 and ccmB , suggesting their crucial roles in maintaining mitochondrial respiratory chain function. Consistent with findings in species such as Acer truncatum and Phaseolus vulgaris [ 43 , 47 ], Medicago species also exhibited high conservation of RNA editing sites, further underscoring the fundamental role of this mechanism in the post-transcriptional regulation of mitochondrial gene expression regulation in angiosperms. Synteny analysis indicated that despite the close phylogenetic relationships among Medicago species, their mitochondrial genomes exhibited 23–44 homologous syntenic blocks longer than 400 bp. These blocks were primarily distributed across core functional regions such as nad , cox , and ccm , displaying high sequence conservation (Fig. 6 ). The presence of conserved syntenic structures suggests strong purifying selection acting on these regions. This pattern aligns with observations in other angiosperm families such as Theaceae and Lauraceae [ 42 , 48 ], indicating evolutionary stability of key mitochondrial genomic components. In phylogenetic analyses, ML and BI trees were constructed based on 31 single-copy mitochondrial PCGs from 25 Fabaceae species. All Medicago species were recovered as a monophyletic group. Within this clade, M. minima and M. lupulina were identified as sister species, M. falcata and M. sativa as another sister pair, while M. platycarpos was resolved as a more distantly related lineage within the genus. These phylogenetic relationships are highly consistent with results from chloroplast genome data [ 49 ], nuclear transcriptome analyses [ 50 ], and the APG IV classification system, further validating the effectiveness and reliability of mitochondrial PCGs in resolving evolutionary relationships in Fabaceae (Fig. 5 ). Ka/Ks ratio analysis further revealed that most PCGs in the Medicago mitochondrial genomes had Ka/Ks values significantly less than 1, indicative of widespread purifying selection (Fig. 7 ). This was particularly evident in genes associated with energy metabolism, such as members of the atp and nad gene families. These findings are consistent with patterns observed in other plant lineages like Michelia , Theaceae, and other plant lineages, where mitochondrial genes generally evolve slowly and are functionally conserved [ 35 , 51 ]. However, several genes—such as ccmB , ccmFc , ccmFn , nad3 , and nad9 —exhibited Ka/Ks values greater than 1 in certain species comparisons, suggesting potential roles in functional innovation during species divergence and adaptive evolution within Medicago . Particularly noteworthy is the matR gene, which showed a Ka/Ks value as high as 3.34 in certain species pairs, indicating strong positive selection and implicating its involvement in lineage-specific rapid evolutionary processes. In summary, the Medicago mitochondrial genomes exhibit overall conservation in gene content, genome structure, codon usage, and RNA editing patterns, while also demonstrating structural rearrangements and signatures of positive selection in specific genes. Comparative analyses with other Fabaceae and angiosperms further clarified the phylogenetic placement of Medicago within the family and highlight the dynamic nature of its adaptive evolution. These results not only enhanced our understanding of the evolutionary mechanisms shaping the Medicago mitochondrial genome, but also provide a theoretical foundation for future studies in functional genomics, genetic resource conservation, and molecular breeding. Integrative multi-omics approaches, incorporating nuclear and transcriptomic data, will be essential for further elucidating the complex regulatory and evolutionary dynamics of this economically and ecologically important legume genus. Conclusions We assembled and compared the mitochondrial genomes of eight Medicago species, including six sequenced for the first time. The results revealed that five species possess a single circular genome structure, while three exhibit multichromosomal configurations. Genome sizes ranged from 281,240 to 356,577 bp, with 55 to 74 PCGs identified. Repetitive sequences were dominated by A/T mononucleotide repeats, and both codon usage and RNA editing showed a strong A/T bias and C-to-U conversions, respectively. The phylogenetic tree supported the monophyly of the genus Medicago . Most genes were subject to purifying selection, while a few, such as matR , exhibited signs of positive selection. Our analysis enhances the understanding of mitochondrial genome structure and evolution in Medicago species and provides a valuable foundation for future studies on Fabaceae mitochondrial genome evolution. Materials and Methods Plant materials and Cultivation In this study, six representative species of the genus Medicago were selected, including Medicago lupulina ( M. lupulina ), Medicago falcata ( M. falcata ), Medicago minima ( M. minima ), Medicago edgeworthii ( M. edgeworthii ), Medicago platycarpos ( M. platycarpos ), and diploid Medicago sativa ( M. sativa ). Among them, seeds of M. minima were obtained from the Kunming Institute of Botany, Chinese Academy of Sciences. All seeds were thoroughly cleaned and surface-sterilized before being sown in pots filled with nutrient soil and cultivated under greenhouse conditions (14 h light/10 h dark photoperiod, temperature 25 ± 2°C, and relative humidity of 60–70%). After 35 days of cultivation, young and healthy mature leaves were collected, immediately flash-frozen in liquid nitrogen on-site, and then stored at − 80°C in the laboratory for subsequent genomic DNA extraction. In addition, the mitochondrial genome sequences of Medicago truncatula ( M. truncatula ) (NC_029641.1) and Medicago arabica ( M. arabica ) (DQ662798.1) were downloaded from the public database ( https://www.ncbi.nlm.nih.gov/ ) for use in subsequent comparative genomic analyses. DNA Extraction and Sequencing Total genomic DNA was extracted from each sample using a modified CTAB method [ 52 ]. DNA quality was assessed by 0.75% agarose gel electrophoresis to evaluate fragment size and potential degradation. DNA purity was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific), and DNA concentration was accurately quantified using a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA). For each sample, 5–10 µg of high-quality genomic DNA was selected for PacBio library construction. Library preparation was performed using the PCR-free SMRTbell method, followed by sequencing on the PacBio Revio platform. The library preparation process included DNA damage repair, end repair, adapter ligation, library purification, and quality control. Each sample yielded approximately 21 ~ 42.9 Gb of raw sequencing data (Table S1 ). The raw PacBio subreads.bam files were processed for quality control and data filtering using the official PacBio software SMRT Link (v12.0). The resulting high-quality data were used for subsequent mitochondrial genome assembly and analysis. Mitochondrial genome assembly and annotation To enrich mitochondrial sequences, PacBio long reads were first aligned to the conserved mitochondrial genome sequence of the model Medicago species M. truncatula using Minimap2 (v2.28) [ 53 ]. Candidate reads with alignment lengths greater than 6,000 bp and covering core gene regions were retained. Based on these seed reads, iterative alignment and extension were performed to aggregate all highly overlapping reads (length ≥ 6 kb, identity ≥ 70%), thereby extracting the complete set of mitochondrial-associated reads for each sample. The filtered mitochondrial reads were then subjected to de novo error correction and assembly using Flye (v2.9.1). Assembly graphs were visualized using Bandage (v0.8.1) to determine whether the mitochondrial genome exhibited a typical circular structure or a multi-branched conFigureuration. Mitochondrial genome annotation was conducted using a combination of tools. General gene annotation was performed using the online platforms PMGA [ 54 ] and CPGview [ 55 ]. tRNA genes were annotated with tRNAscan-SE [ 56 ], and open reading frames (ORFs) were predicted using the Open Reading Frame Finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ). ORFs shorter than 102 bp or overlapping with known genes were excluded. ORFs longer than 300 bp were annotated against the NR database based on alignment results. All annotation results were manually inspected and curated to ensure accuracy. Circular genome maps were automatically generated using the IPMGA platform. To ensure consistency in annotation formats for downstream comparative genomic analyses, the two Medicago mitochondrial genomes downloaded from the public database and the six newly assembled genomes from this study were all re-annotated using the same pipeline and software. Analysis of repeated sequences To investigate the types and distribution characteristics of repetitive sequences in the mitochondrial genomes of Medicago species, three categories of repeats were systematically identified and analyzed in this study: simple sequence repeats (SSRs), tandem repeats (TSRs), and dispersed repeats (DSRs). SSRs were identified using the MISA tool (Misa-web-IPK Gatersleben, ( https://webblast.ipk-gatersleben.de/misa/ ) [ 27 ]. TSRs (with repeat unit lengths greater than 6 bp) were detected using Tandem Repeats Finder ( https://tandem.bu.edu/trf/basic_submit ) with default parameters [ 57 ]. DSRs were identified using the REPuter program on the BiBiserv2 platform ( https://bibiserv.cebitec.uni-bielefeld.de/reputer?id=reputer_view_submission ), with the Hamming distance set to 3, a minimum repeat length of 30 bp, and a maximum repeat number of 5000 [ 29 ].The distribution patterns of all identified repetitive sequences were visualized using the Circos module in TBtools [ 58 ]. Analysis of codon usage and Prediction of RNA editing sites Differences in codon usage frequency among organisms are considered the result of an evolutionary equilibrium shaped by long-term natural selection and mutational biases. In this study, relative synonymous codon usage (RSCU) analysis of mitochondrial genomes from eight Medicago species was performed using Perl scripts. Representative and unique CDS sequences were selected, and the codon usage counts for each gene were tabulated [ 59 ]. To predict RNA editing sites in the mitochondrial genomes of these eight Medicago species, the plant RNA editing prediction tool PREPACT ( http://www.prepact.de/prepact-main.php ) was employed for analysis [ 60 ]. Phylogenetic Analysis To clarify the phylogenetic position of Medicago species within the Fabaceae family, a phylogenetic analysis based on mitochondrial genomes was conducted in this study. Mitochondrial genome data of 17 Fabaceae species related to Medicago were downloaded from the NCBI database and re-annotated using the same annotation software, PMGA [ 54 ], to ensure consistent annotation formats for subsequent analyses. Suriana maritima was selected as the outgroup in the phylogenetic analysis. All analyzed species and their corresponding GenBank accession numbers are listed in Table S2 . Using PhyloSuite (v1.2.3) [ 61 ], conserved mitochondrial protein-coding gene (CDS) sequences shared among the 25 Fabaceae species were extracted. After manual alignment and curation, 31 shared coding genes were retained for further analysis. Multiple sequence alignment of these nucleotide sequences was performed using MAFFT [ 62 ] with default parameters. The aligned sequences were then concatenated into a single matrix using the "Concatenate Sequence" function in PhyloSuite for phylogenetic tree construction. Phylogenetic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was conducted with IQ-TREE [ 63 ] using the TVM + F + I + G4 nucleotide substitution model, and node support was evaluated with 1,000 bootstrap replicates. BI analysis was performed using MrBayes (v3.2) [ 64 ]. The resulting phylogenetic trees were visualized using the ITOL web platform [ 65 ]. Colinear analysis To investigate structural conservation and rearrangement characteristics among the mitochondrial genomes of Medicago species, a comprehensive synteny analysis was performed on eight Medicago mitochondrial genomes. First, pairwise genome alignments were conducted using BLASTn (v2.14.0+) [ 66 ] to identify conserved homologous sequence fragments. The BLASTn parameters were set as follows: -evalue 1e-10, with low-quality alignments filtered out. Only homologous regions longer than 400 bp and with sequence identity greater than 90% were retained as syntenic units for subsequent comparison. To further illustrate the structural similarities and variations of mitochondrial genomes among different Medicago species, the extracted syntenic blocks were visualized using LINKVIEW2 (v1.0.5) software . Analysis of Ka/Ks values To investigate the natural selection pressures during the evolutionary process of Medicago species, eight representative species were selected for analysis. Shared PCGs were aligned using MAFFT (v11) [ 67 ], and the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions (Ka/Ks) was calculated using KaKs_Calculator (v2.0) [ 68 ]. Abbreviations CMS Cytoplasmic male sterility ONT Oxford Nanopore Technologies RSCU Relative synonymous codon usage PCGs Protein-coding genes M. lupulina Medicago lupulina M. minima Medicago minima M. edgeworthii Medicago edgeworthii M. truncatula Medicago truncatula M. arabica Medicago arabica M. falcata Medicago falcata M. platycarpos Medicago platycarpos M. sativa Medicago sativa rRNA Ribosomal RNA tRNA Transfer RNA SSRs Simple Sequence Repeats TSRs Tandem Sequence Repeats DSRs Dispersed Sequence Repeats F Forward P Palindromic R Reverse C Complementary Leu Leucine Ser Serine Ile Isoleucine Trp Tryptophan Cys Cysteine Arg Arginine Leu Leucine Ser Serine Met Methionine Trp Tryptophan GC Guanine-Cytosine content ML Maximum likelihood BI Bayesian inference Declarations Acknowledgements Not applicable. CRediT authorship contribution statement R-F Y was responsible for visualization, methodology, formal analysis, data curation, conceptualization, and drafting of the manuscript. M-Y W contributed to conceptualization and methodology. M-L W participated in data curation, visualization, and methodological development. J L : methodology. J-L L and C-H H jointly oversaw conceptualization, supervision, project administration, and secured funding for the study. Funding Declaration This work was supported by funds from the National Natural Science Foundation of China (32270232, 31970224, and 31770242) and State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, Key Laboratory of Herbage & Endemic Crop Biology, Ministry of Education (Inner Mongolia University). Also supported by State Key Laboratory of Genetics and Development of Complex Phenotypes, State Key Laboratory of Wetland Conservation and Restoration, National Observations and Research Station for Wetland Ecosystems of the Yangtze Estuary, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Human Phenome Data Center (Fudan university). Data Availability The raw data of resequencing genome have been deposited in NCBI Sequence Read Archive (SRA) under BioProject accession PV916040, PV916041, PV916042, PV882493–PV882495, PV892890–PV892892, PV892893–PV892894. All data supporting this research result can be obtained in the paper. Ethics approval and consent to participate Experimental research studies on plants, including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteom Bioinf. 2010;8(1):77–80. Additional Declarations No competing interests reported. Supplementary Files SupplementalFigure.pdf Supplementary Figure 1. Circular map of the mitochondrial genome of Medicago lupulina Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 2. Circular map of the mitochondrial genome of Medicago falcata Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 3. Circular map of the mitochondrial genome of Medicago minima Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 4. Circular map of the mitochondrial genome of Medicago edgeworthii Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 5. Circular map of the mitochondrial genome of Medicago platycarpos Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 6. Circular map of the mitochondrial genome of Medicago sativa Genomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions. Supplementary Figure 7. Length distribution of dispersed repeats in the mitochondrial genomes of Medicago species The figure shows the classification and abundance of dispersed repeats by length across different species. The x-axis represents repeat length intervals, the y-axis indicates the corresponding number of repeats, and different colors denote different species. Exact counts are labeled above each bar. Teal Blue: M. lupulina ; Olive Green: M. falcata ; Golden Yellow: M. minima ; Honey Gold: M. edgeworthii ; Rosewood: M. platycarpos ; Rose Beige: M. sativa ; Turquoise Blue: M. truncatula ; Royal Blue: M. arabica . Supplementary Figure 8. Statistics of the length and number of syntenic blocks The figure displays the length distribution and quantity of syntenic blocks among Medicago species. Different colors represent homologous regions between specific species pairs. Soft Rose: M. lupulina VS M. minima ; Mint Green: M. truncatula VS M. lupulina ; Sky Blue: M. sativa VS M. truncatula ; Warm Apricot: M. falcata VS M. sativa ; Light Apricot: M. arabica VS M. falcata ; Pastel Green: M. edgeworthii VS M. arabica ; Pale Sky Blue: M. platycarpos VS M. edgeworthii . SupplementalTable.xlsx Table S1. Statistical Summary of Third-Generation HiFi Sequencing Data in the six Medicago species. Table S2. Summary of species information and sequence sources used for phylogenetic tree construction. Table S3. Summary of basic annotation information for the mitochondrial genomes of eight Medicago species. Table S4. Genetic composition of mitochondrial genome of eight Medicago species. Table S5. The Lengths of Introns and Exons in the mitochondrion Genome of eight Medicago species. Table S6. SSRs in the mitochondrion genome of eight Medicago species. Table S7. Tandem repeat （TSRs）sequences in the mitochondrion genome of eight Medicago species. Table S8. Dispersed repeat （DSRs） sequences in the mitochondrion genome of eight Medicago species. Table S9. Codon usage and relative synonymous codon usage (RSCU) of protein-coding genes in the mitochondrial genomes of eight Medicago species. Table S10. Statistics of RNA editing types and the number of corresponding sites in 31 protein-coding genes across eight Medicago species. Table S11. Statistics of the codon positions where RNA editing events occur in the mitochondrial genomes of eight Medicago species. Table S12. Statistics of amino acid changes caused by RNA editing events in the mitochondrial genomes of eight Medicago species. Table S13. Comparative synteny statistics of the mitochondrial genomes of eight Medicago species, including the number, length, and distribution of homologous blocks between species pairs. Table S14. Ka, Ks, and Ka/Ks values of 27 mitochondrial protein-coding genes in eight Medicago species. 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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-7230374","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499017395,"identity":"a0a1de3f-874a-4e1d-b995-22d42e40f39c","order_by":0,"name":"Ruifeng Yang","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Ruifeng","middleName":"","lastName":"Yang","suffix":""},{"id":499017397,"identity":"fcef7af3-c1c1-407a-bb63-f91ea456d848","order_by":1,"name":"Mengyue Wang","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Mengyue","middleName":"","lastName":"Wang","suffix":""},{"id":499017399,"identity":"e57d845f-301c-439b-acc4-9bc3f0629359","order_by":2,"name":"Mingliang Wang","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Mingliang","middleName":"","lastName":"Wang","suffix":""},{"id":499017400,"identity":"5e647a7c-f4ad-4300-bacf-9a1a51627f6e","order_by":3,"name":"Jun Li","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":499017401,"identity":"4d679caa-819a-4845-a128-627e0bd629c8","order_by":4,"name":"Jinglong Li","email":"","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":false,"prefix":"","firstName":"Jinglong","middleName":"","lastName":"Li","suffix":""},{"id":499017402,"identity":"a32def0a-43de-45f9-9ecf-90ae219ed262","order_by":5,"name":"Chien-Hsun Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACCR4wxcPP3tj4IOGHDfFaZCR7DjcbPOxJI16LjcGN9DbJB2yHCevgn917TOLnjloegzMH2yoSeA4z8Ld3J+C35M65NMneM8d5JI83tt1IsEhnkDhzdgNeLQYSOWYSvG3HePiAttxI4LEGiuQS1iL5F6iF4UZiW0ECGzNxWqR522p4BIBaGBLYnAlrkbiRY2wt23aAR7LnYLNEYk8aD0G/8M/IMbz5tq3Onp+9/eHHHz9s5Pjbe/FrgQJEdPAQoxwE6ohVOApGwSgYBSMRAABMrkom1Mi6kgAAAABJRU5ErkJggg==","orcid":"","institution":"Inner Mongolia University","correspondingAuthor":true,"prefix":"","firstName":"Chien-Hsun","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-07-28 06:23:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7230374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7230374/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07650-z","type":"published","date":"2025-11-28T15:58:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88886372,"identity":"01fc234c-9b92-497b-a3ea-660436a13fbc","added_by":"auto","created_at":"2025-08-12 11:57:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5445040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBasic characteristics of the mitochondrial genomes of eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–F) Branch-structured assembly graphs of the mitochondrial genomes from eight Medicago species, visualized using Bandage, with chromosome number and length shown for each species: A, \u003cem\u003eM. lupulina\u003c/em\u003e; B, \u003cem\u003eM. falcata\u003c/em\u003e; C, \u003cem\u003eM. minima\u003c/em\u003e; D, \u003cem\u003eM. edgeworthii\u003c/em\u003e; E, \u003cem\u003eM. platycarpos\u003c/em\u003e; F, \u003cem\u003eM. sativa\u003c/em\u003e. (G) Gene content distribution in the mitochondrial genomes of the eight \u003cem\u003eMedicago\u003c/em\u003especies. Different colors indicate gene absence or copy number variation across species. Bright Teal: Missing gene; Very Pale Pink: Singleton gene; Soft Purple: Duplicate gene; Muted Salmon: Triple copy gene.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/b4aa8b93aefb0f2944d04112.png"},{"id":88886740,"identity":"971d9030-a5b0-46f5-acb2-67f969ec21f7","added_by":"auto","created_at":"2025-08-12 12:05:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5439224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution characteristics of repeat sequences in the mitochondrial genomes of eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Genome-wide distribution of repeat sequences in the eight \u003cem\u003eMedicago\u003c/em\u003e species. From innermost to outermost rings: genome circle, tandem repeats, simple sequence repeats (SSRs), and connecting lines of dispersed repeats.\u003c/p\u003e\n\u003cp\u003e(B) Statistical summary of different SSR types. The x-axis represents species, and the y-axis shows the number of identified SSRs. Different colors denote SSR types, with exact counts labeled above each bar. Soft Pink: Monomeric; Pastel Green: Dimeric; Light Sky Blue: Trimeric; Pastel Yellow-Green: Tetrameric; Cool Gray-Blue: Pentameric; Muted Olive: Hexameric.\u003c/p\u003e\n\u003cp\u003e(C) Statistics of tandem and dispersed repeat types. Dispersed repeats are classified into four types: forward, reverse, palindromic, and complementary. Different colors indicate the number of each type, with corresponding values displayed. Soft Pink: Tandem repeats; Pastel Green: Palindromic repeats; Light Sky Blue: Forward repeats; Pastel Yellow-Green: Reverse repeats; Cool Gray-Blue: Complementary repeats.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/c1219778513cc36d9d33da55.png"},{"id":88885060,"identity":"6dd17446-4f1b-4b9f-ad38-69bd57956119","added_by":"auto","created_at":"2025-08-12 11:49:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative synonymous codon usage (RSCU) of mitochondrial protein-coding genes in eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRSCU values of codons corresponding to different amino acids are shown to indicate codon usage bias. The species (from left to right) are \u003cem\u003eM. lupulina\u003c/em\u003e, \u003cem\u003eM. falcata\u003c/em\u003e, \u003cem\u003eM. minima\u003c/em\u003e, \u003cem\u003eM. edgeworthii\u003c/em\u003e, \u003cem\u003eM. platycarpos\u003c/em\u003e, \u003cem\u003eM. sativa\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, and \u003cem\u003eM. arabica\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/4a8da97668534260ee79028f.png"},{"id":88886374,"identity":"f3e02ccd-ff9a-4ba6-83f2-ee5c3e2f8830","added_by":"auto","created_at":"2025-08-12 11:57:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1671727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of RNA editing features in mitochondrial protein-coding genes among eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Distribution of identified RNA editing sites in protein-coding genes, with different colors representing different species. Soft Pink: \u003cem\u003eM. lupulina\u003c/em\u003e; Pastel Green: \u003cem\u003eM. falcata\u003c/em\u003e; Powder Blue: \u003cem\u003eM. minima\u003c/em\u003e; Pastel Yellow-Green: \u003cem\u003eM. edgeworthii\u003c/em\u003e; Cool Gray Blue: \u003cem\u003eM. platycarpos\u003c/em\u003e; Muted Olive Green: \u003cem\u003eM. sativa\u003c/em\u003e; Pale Lilac: \u003cem\u003eM. truncatula\u003c/em\u003e; Light Aqua Green: \u003cem\u003eM. arabica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(B) Proportional distribution of editing sites across the three codon positions, with colors distinguishing the first positions (1st), second positions (2st), and third positions (3st). Soft Pink: 1st; Pastel Green: 2st; Powder Blue: 3st.\u003c/p\u003e\n\u003cp\u003e(C) Number of RNA editing events associated with different amino acid conversions. Colors represent various editing types. P: Proline; L: Leucine; S: Serine; F: Phenylalanine; R: Arginine; C: Cysteine; W: Tryptophan. Soft Pink: Others; Pastel Green: P→L/S/F; Powder Blue: R→C/W; Pastel Yellow-Green:S→F/L/P.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/f055ee076534c77aaf98becf.png"},{"id":88885066,"identity":"7bc83f6f-b332-41fd-8904-b2b71dd1a936","added_by":"auto","created_at":"2025-08-12 11:49:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1762668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species and 17 additional plant species based on mitochondrial protein-coding genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Maximum likelihood (ML) phylogenetic tree showing branch lengths.\u003c/p\u003e\n\u003cp\u003e(B) Majority-rule consensus tree constructed using MrBayes, with branch lengths also indicated. Suriana maritima was used as the outgroup. Support values are shown at all nodes: ML bootstrap values (maximum of 100) and Bayesian posterior probabilities (maximum of 1.0). Pink: \u003cem\u003eMedicago\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/5e317d920a9b3b3c4279af5d.png"},{"id":88885072,"identity":"0768e5c8-fbb1-4c27-ab1b-7ae5b6f4e371","added_by":"auto","created_at":"2025-08-12 11:49:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1164723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynteny analysis of the mitochondrial genomes of eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach row represents the mitochondrial genome of one species. Connecting lines indicate syntenic regions among different species. Blush Pink: \u003cem\u003eM. minima\u003c/em\u003e; Medium Mint Green: \u003cem\u003eM. lupulina\u003c/em\u003e; Sky Blue: \u003cem\u003eM. truncatula\u003c/em\u003e; Soft Terracotta: \u003cem\u003eM. sativa\u003c/em\u003e; Light Apricot: \u003cem\u003eM. falcata\u003c/em\u003e; Pale Mint: \u003cem\u003eM. arabica\u003c/em\u003e; Pale Sky Blue: \u003cem\u003eM. edgeworthii\u003c/em\u003e; Pale Lilac: \u003cem\u003eM. platycarpos\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/d53319632d5c8dbdc7c0bc04.png"},{"id":88885067,"identity":"bc7c2e9c-0917-438b-8ce9-11e0c5e534ef","added_by":"auto","created_at":"2025-08-12 11:49:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":905563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of Ka, Ks, and Ka/Ks values for 27 mitochondrial protein-coding genes in eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the Ka, Ks, and Ka/Ks values of each PCG across species, reflecting differences in selective pressure during evolution.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/c07246702b6e4e98efc3fee5.png"},{"id":97178743,"identity":"0bbb305b-99fd-4eea-8262-cccca1056595","added_by":"auto","created_at":"2025-12-01 16:13:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18416982,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/1914d23b-715d-4666-9084-18b4a16b98df.pdf"},{"id":88885059,"identity":"89f5e584-31f4-4d23-9168-afdd8d0c680a","added_by":"auto","created_at":"2025-08-12 11:49:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":635730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elupulina\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago falcata\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago minima\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago edgeworthii\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago platycarpos\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 6. Circular map of the mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago sativa\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenomic features are mapped on the inner and outer sides of the circular genome according to functional categories, with different colors used to distinguish gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 7. Length distribution of dispersed repeats in the mitochondrial genomes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMedicago\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe figure shows the classification and abundance of dispersed repeats by length across different species. The x-axis represents repeat length intervals, the y-axis indicates the corresponding number of repeats, and different colors denote different species. Exact counts are labeled above each bar. Teal Blue: \u003cem\u003eM. lupulina\u003c/em\u003e; Olive Green: \u003cem\u003eM. falcata\u003c/em\u003e; Golden Yellow: \u003cem\u003eM. minima\u003c/em\u003e; Honey Gold: \u003cem\u003eM. edgeworthii\u003c/em\u003e; Rosewood: \u003cem\u003eM. platycarpos\u003c/em\u003e; Rose Beige: \u003cem\u003eM. sativa\u003c/em\u003e; Turquoise Blue: \u003cem\u003eM. truncatula\u003c/em\u003e; Royal Blue: \u003cem\u003eM. arabica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 8. Statistics of the length and number of syntenic blocks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe figure displays the length distribution and quantity of syntenic blocks among Medicago species. Different colors represent homologous regions between specific species pairs. Soft Rose: \u003cem\u003eM. lupulina\u003c/em\u003e VS \u003cem\u003eM. minima\u003c/em\u003e; Mint Green: \u003cem\u003eM. truncatula\u003c/em\u003e VS \u003cem\u003eM. lupulina\u003c/em\u003e; Sky Blue: \u003cem\u003eM. sativa\u003c/em\u003eVS \u003cem\u003eM. truncatula\u003c/em\u003e; Warm Apricot: \u003cem\u003eM. falcata\u003c/em\u003e VS \u003cem\u003eM. sativa\u003c/em\u003e; Light Apricot: \u003cem\u003eM. arabica\u003c/em\u003e VS \u003cem\u003eM. falcata\u003c/em\u003e; Pastel Green: \u003cem\u003eM. edgeworthii\u003c/em\u003e VS \u003cem\u003eM. arabica\u003c/em\u003e; Pale Sky Blue: \u003cem\u003eM. platycarpos\u003c/em\u003e VS \u003cem\u003eM. edgeworthii\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SupplementalFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/4ce61741fc3f2a52a93863bf.pdf"},{"id":88886739,"identity":"022a4705-bf92-4bb4-b91e-9d82221c5d39","added_by":"auto","created_at":"2025-08-12 12:05:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":178725,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. Statistical Summary of Third-Generation HiFi Sequencing Data in the six \u003cem\u003eMedicago \u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eTable S2. Summary of species information and sequence sources used for phylogenetic tree construction.\u003c/p\u003e\n\u003cp\u003eTable S3. Summary of basic annotation information for the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S4. Genetic composition of mitochondrial genome of eight \u003cem\u003eMedicago\u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eTable S5. The Lengths of Introns and Exons in the mitochondrion Genome of eight \u003cem\u003eMedicago\u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eTable S6. SSRs in the mitochondrion genome of eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S7. Tandem repeat （TSRs）sequences in the mitochondrion genome of eight \u003cem\u003eMedicago\u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eTable S8. Dispersed repeat （DSRs） sequences in the mitochondrion genome of eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S9. Codon usage and relative synonymous codon usage (RSCU) of protein-coding genes in the mitochondrial genomes of eight\u003cem\u003e Medicago \u003c/em\u003especies.\u003c/p\u003e\n\u003cp\u003eTable S10. Statistics of RNA editing types and the number of corresponding sites in 31 protein-coding genes across eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S11. Statistics of the codon positions where RNA editing events occur in the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S12. Statistics of amino acid changes caused by RNA editing events in the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eTable S13. Comparative synteny statistics of the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003especies, including the number, length, and distribution of homologous blocks between species pairs.\u003c/p\u003e\n\u003cp\u003eTable S14. Ka, Ks, and Ka/Ks values of 27 mitochondrial protein-coding genes in eight \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"SupplementalTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7230374/v1/3e29200cc7e048125ceafd61.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species","fulltext":[{"header":"Background","content":"\u003cp\u003eMitochondria are essential, semi-autonomous organelles in eukaryotic plant cells that play central roles in energy production and metabolic processes, such as oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid metabolism, iron–sulfur cluster assembly, and calcium homeostasis. These functions provide both energy support and regulatory support for plant growth, development, reproduction, and responses to environmental stresses [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, mitochondria are actively involved in programmed cell death, autophagy, signal transduction, and the maintenance of redox homeostasis. They are also essential in biological processes such as pathogen defense, responses to abiotic stress responses, and cytoplasmic male sterility (CMS) [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e–\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is widely accepted that mitochondria originated from an ancient endosymbiotic event involving an α-proteobacterium approximately 1.5\u0026nbsp;billion years ago. This origin was followed by extensive gene transfer to the nuclear genome, as well asintegration and loss of genes during plant evolution [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The plant mitochondrial genome (mitogenome) is physically and genetically independent of the nuclear genome and is typically maternally inherited, although cases of paternal or biparental inheritance have also been documented. This results in a semi-autonomous inheritance mode [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared to chloroplasts genomes and animal mitochondrial genomes, plant mitogenomes show much greater variability and complexity in terms of structural conFigureuration, genome size, abundance and types of repeat sequences, and incorporation of foreign DNA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These features make plant mitogenomes a valuable model for studying adaptive evolution and the coordination between nuclear and organellar genomes.\u003c/p\u003e\u003cp\u003eAlthough plant mitochondria encode a relatively small number of genes (typically 19–41 protein-coding genes), their gene content and sequences are relatively conserved [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the overall genome structure is highly dynamic, appearing in diverse forms including single circular, multi-circular, linear, branched, or even multipartite conFigureurations, with multiple structural types often coexisting within the same species [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The reported sizes of plant mitochondrial genomes range from 66 kb (e.g., \u003cem\u003eViscum scurruloideum\u003c/em\u003e) to over 18 Mb (e.g., \u003cem\u003eCathaya argyrophylla\u003c/em\u003e), representing more than a 200-fold difference [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This remarkable variation is primarily attributed to the proliferation of repetitive sequences, integration of foreign DNA, and frequent recombination events [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Despite having extremely low nucleotide substitution rates plant mitogenomes, undergo frequent structural rearrangements and exhibit widespread RNA editing, These RNA editing events not only contributes to the diversification at the protein level, but are also closely associated with CMS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As of May 2025, only 688 plant mitochondrial genomes have been deposited in the NCBI database, a number far lower than the 15,396 publicly available chloroplast genomes, highlighting the technical challenges posed by their structural heterogeneity and recombination mediated by repetitive elements [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. With the advancement of third-generation sequencing platforms such as PacBio HiFi and Oxford Nanopore Technologies (ONT), as well as specialized assembly and annotation tools like GSAT and PMAT, research on plant mitogenomes is progressing rapidly, These technologies offering tremendous potential for clarifying mitogenome structural complexity, evolutionary history, and their associations with CMS and phylogeny [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, comprehensive investigations into the structure, function, and evolution of plant mitochondrial genomes are of great theoretical and practical significance for uncovering plant biological mechanisms, mining elite germplasm resources, and supporting molecular breeding strategies.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMedicago\u003c/em\u003e is an annual or perennial herbaceous genus in the Fabaceae family, originating from the Mediterranean coastal regions and Southwest Asia. In China, it is mainly distributed across the northern, northwestern, and northeastern regions. \u003cem\u003eMedicago\u003c/em\u003e is rich in proteins, vitamins, minerals, polysaccharides, soybean flavonoids, isoflavones, and various identified growth-promoting factors (UGFs), earning it the title \"King of Forage.\" It not only serves as a high-quality forage for herbivorous animals such as dairy cattle, but also enhances animal productivity, immunity, and meat quality, while modulating gut microbiota [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Currently, the cultivated area of \u003cem\u003eMedicago\u003c/em\u003e in China exceeds one million hectares, with an annual yield of over two million tons. Its hay and derived products are easy to store and transport, and are widely used in feed, medicine, bioenergy, and ecological restoration. The cultivation of \u003cem\u003eMedicago\u003c/em\u003e in China can be traced back to the Han Dynasty, as recorded in Records of the Grand Historian《Shiji·Dayuan Liezhuan》, where it was noted that “horses favor \u003cem\u003eMedicago\u003c/em\u003e” indicating its early integration into agricultural and pastoral systems. \u003cem\u003eMedicago\u003c/em\u003e exhibits self-incompatibility and inter-subspecific hybridization, reflecting its high genetic diversity and strong ecological adaptability [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite its promising applications, the taxonomy and germplasm characterization of \u003cem\u003eMedicago\u003c/em\u003e remain relatively underdeveloped in China. The number of species recorded varies across references—for instance, 《Illustrated Flora of Higher Plants in China》 (1972) listed six species, 《Flora of China》 (1998) reports 13 species and one variety, whereas Lu Xinshi and colleagues identified 46 taxonomic units in China, including 30 perennial wild species (comprising 12 varieties and one subspecies), five annual species, and 11 introduced species. In summary, \u003cem\u003eMedicago\u003c/em\u003e holds significant importance in agriculture, animal husbandry, and ecosystems. Its taxonomic classification, germplasm resource exploration, and molecular breeding remain key focal points in current Medicago research.\u003c/p\u003e\u003cp\u003eAlthough the mitochondrial genomes of several plant genera, such as \u003cem\u003eCapsicum\u003c/em\u003e, \u003cem\u003eFragaria\u003c/em\u003e, and \u003cem\u003eMorus Linn\u003c/em\u003e, have been successfully decoded [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], current research on the genus \u003cem\u003eMedicago\u003c/em\u003e remains largely focused on single species or specific populations, with a lack of systematic comparative analyses across multiple representative species within the genus [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, we performed \u003cem\u003ede novo\u003c/em\u003e assembly and functional annotation of mitochondrial genomes from six \u003cem\u003eMedicago\u003c/em\u003e species using the PacBio long-read sequencing platform. Combined with two previously published mitochondrial genomes, we constructed a comparative dataset encompassing eight representative \u003cem\u003eMedicago\u003c/em\u003e species. Based on this dataset, we systematically analyzed gene content, repeat sequence characteristics, relative synonymous codon usage (RSCU), RNA editing sites, selective pressure (Ka/Ks), and synteny relationships among the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes. In addition, we reconstructed a phylogenetic tree using 31 single-copy protein-coding genes (PCGs) to comprehensively elucidate the evolutionary relationships within the \u003cem\u003eMedicago\u003c/em\u003e genus and between \u003cem\u003eMedicago\u003c/em\u003e and other species in the Fabaceae family. This study provides a solid data foundation and theoretical basis for the taxonomic classification, germplasm exploration and utilization, and organellar genome evolution research of \u003cem\u003eMedicago\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCharacteristics of the mitochondrial genomes of eight\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo comprehensively elucidate the structural characteristics and evolutionary divergence of mitochondrial genomes in \u003cem\u003eMedicago\u003c/em\u003e species, we conducted a comparative analysis of the mitochondrial genomes from eight \u003cem\u003eMedicago\u003c/em\u003e species. Among them, the mitochondrial genomes of six species were newly assembled \u003cem\u003ede novo\u003c/em\u003e and functionally annotated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn terms of structural conFigureuration, the mitochondrial genomes of \u003cem\u003eM. lupulina\u003c/em\u003e, \u003cem\u003eM. minima\u003c/em\u003e, \u003cem\u003eM. edgeworthii\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e, and \u003cem\u003eM. arabica\u003c/em\u003e exhibit a single circular structure, while \u003cem\u003eM. falcata\u003c/em\u003e, \u003cem\u003eM. platycarpos\u003c/em\u003e, and \u003cem\u003eM. sativa\u003c/em\u003e display complex multichromosomal circular structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;F). The genome sizes range from 281,240\u0026thinsp;~\u0026thinsp;356,577 bp, with a\u0026thinsp;~\u0026thinsp;1.27-fold difference (Table S3), and the GC content ranges from 44.92%~45.58%, which is consistent with the typical features of angiosperm mitochondrial genomes.\u003c/p\u003e\u003cp\u003eFunctional annotation revealed that each genome contains 55\u0026ndash;74 functional genes, comprising 33\u0026ndash;41 PCGs, 18\u0026ndash;24 rRNA genes, and 3\u0026ndash;5 tRNA genes (Table S3\u0026ndash;S5; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;S6). The number of protein-coding genes was generally conserved across species, with the exception of \u003cem\u003eM. falcata\u003c/em\u003e, which exhibited a notable increase. For instance, \u003cem\u003esdh4\u003c/em\u003e was present as a single copy in \u003cem\u003eM. edgeworthii, M. platycarpos\u003c/em\u003e, and \u003cem\u003eM. arabica\u003c/em\u003e, but existed in multiple copies in the other species. Similarly, \u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp4\u003c/em\u003e, and \u003cem\u003enad4L\u003c/em\u003e appeared in multiple copies in both \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. arabica\u003c/em\u003e, while multicopy \u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmFN\u003c/em\u003e, and \u003cem\u003ecox3\u003c/em\u003e were uniquely observed in \u003cem\u003eM. falcata\u003c/em\u003e. Additionally, \u003cem\u003erps1\u003c/em\u003e was only detected in \u003cem\u003eM. truncatula\u003c/em\u003e and was absent in the other seven species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eVariations in gene copy number and presence or absence were also observed in rRNA and tRNA genes. For example, \u003cem\u003errn26\u003c/em\u003e was present in multiple copies in \u003cem\u003eM. falcata\u003c/em\u003e; \u003cem\u003etrnQ-UUG\u003c/em\u003e was consistently found in multiple copies across all eight \u003cem\u003eMedicago\u003c/em\u003e species; \u003cem\u003etrnF-GAA\u003c/em\u003e appeared as a single copy only in \u003cem\u003eM. arabica\u003c/em\u003e but as multiple copies in the remaining species; \u003cem\u003etrnfM-CAU\u003c/em\u003e was present in multiple copies in \u003cem\u003eM. falcata\u003c/em\u003e, \u003cem\u003eM. platycarpos\u003c/em\u003e, and \u003cem\u003eM. sativa\u003c/em\u003e; \u003cem\u003etrnM-CAU\u003c/em\u003e was present in multiple copies in \u003cem\u003eM. lupulina\u003c/em\u003e and \u003cem\u003eM. arabica\u003c/em\u003e; and the multicopy presence of \u003cem\u003etrnN-GUU\u003c/em\u003e and \u003cem\u003etrnY-GUA\u003c/em\u003e was exclusive to \u003cem\u003eM. falcata\u003c/em\u003e. These variations may be associated with structural duplications or potential functional redundancy within the mitochondrial genomes of certain species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRepeat sequences analysis in the\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e \u003cb\u003emitochondrial genomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the distribution characteristics and evolutionary divergence of repetitive sequences in \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes, we identified and analyzed three types of repeats\u0026mdash;SSRs, TSRs, and DSRs\u0026mdash;across the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSSRs, composed of 1\u0026ndash;6 nucleotide units, are short repetitive sequences widely present in plant organelle genomes and can contribute to genome rearrangement and length expansion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A total of 141 SSRs were identified across the eight \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes, with the number per species ranging from 11 to 23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table S6). Mononucleotide repeats represented the most prevalent type, accounting for 69.57\u0026ndash;95.2% of all SSRs, followed by dinucleotide and trinucleotide repeats (4.8\u0026ndash;30.43%). Tetranucleotide, pentanucleotide, and hexanucleotide repeats were not detected in any species. Among mononucleotide SSRs, A/T repeats were dominant (~\u0026thinsp;92.9%), while C/G repeats were relatively rare (~\u0026thinsp;7.1%) and were not detected in \u003cem\u003eM. falcata\u003c/em\u003e, \u003cem\u003eM. edgeworthii\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e. For dinucleotide SSRs, the (AG/CT or AT/TA) motif was the most common (4.76\u0026ndash;21.75%), though (AT/TA) repeats were absent in \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. dgeworthii\u003c/em\u003e. Trinucleotide repeats were present in all species except \u003cem\u003eM. edgeworthii\u003c/em\u003e. Overall, although differences in SSR types were observed among species, their distribution patterns remained relatively consistent. SSRs were generally evenly distributed throughout the genome, but in \u003cem\u003eM. minima\u003c/em\u003e and \u003cem\u003eM. arabica\u003c/em\u003e, clustering in specific chromosomal regions was observed, suggesting potential localized enrichment or selective retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTSRs consist of tandemly arranged repeat units longer than 6 bp [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A total of 76 TSRs were identified across the eight \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes, with 4 to 18 TSRs detected per species. Repeat unit lengths ranged from 6 to 33 bp, and all matches showed greater than 77% similarity. Notably, \u003cem\u003eM. arabica\u003c/em\u003e exhibited a significantly higher number of TSRs compared to other species, which may reflect increased structural duplication or recombination activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S7).\u003c/p\u003e\u003cp\u003eDSRs, also known as dispersed repeats, are sequences that can facilitate recombination and contribute to genome rearrangement and are widely found in plant mitochondrial genomes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Three types of DSRs were detected in this study: forward (F) and palindromic (P) repeats were found in most species, while reverse (R) repeats were only observed in \u003cem\u003eM. falcata\u003c/em\u003e (one instance), and complementary (C) repeats were not detected in any \u003cem\u003eMedicago\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S7). In \u003cem\u003eM. arabica\u003c/em\u003e, only 126 F-type repeats were detected, with no P-type repeats found. In the other species, F-type and P-type repeats ranged from 64 to 138 and 46 to 135, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S8). Although the number of DSRs varied among species, their length distribution was largely consistent, with most repeats falling within the 30\u0026ndash;49 bp range (~\u0026thinsp;73.56%), followed by the 50\u0026ndash;99 bp range (~\u0026thinsp;17.26%). Additionally, \u003cem\u003eM. falcata\u003c/em\u003e exhibited a significantly higher number of DSRs longer than 500 bp compared to other species, which may contribute to the larger size of its mitochondrial genome (Figure S7).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of codon usage in\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate codon usage patterns and preferences of mitochondrial PCGs in \u003cem\u003eMedicago\u003c/em\u003e species, a comprehensive codon usage analysis using the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table S9). The total number of codons in PCGs ranged from 9,639 to 11,515 across species, encompassing 64 codons corresponding to 20 standard amino acids and stop codons. Overall, leucine (Leu) was the most frequently used amino acid in most species, followed by serine (Ser) and isoleucine (Ile), whereas tryptophan (Trp) and cysteine (Cys) were the least frequently used. As expected, most amino acids are encoded by multiple synonymous codons; for instance, arginine (Arg), leucine (Leu), and serine (Ser) each have six synonymous codons, while methionine (Met) and tryptophan (Trp) are each encoded by a single codon (ATG and TGG, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther analysis of the relative RSCU values for all 64 codons revealed that approximately 75% of the codons exhibited RSCU values greater than 1 in multiple species, indicating a significant usage preference. The remaining\u0026thinsp;~\u0026thinsp;25% of codons had RSCU values less than 1, suggesting that their usage frequencies were significantly lower than expected under equal codon usage assumptions. Some codons, such as Asp (GAC) and Leu (CTC, CTG), had RSCU values close to 1, consistent with a \"no preference\" usage pattern. The overall RSCU patterns were highly consistent among the \u003cem\u003eMedicago\u003c/em\u003e species, particularly for Phe (TTT), which consistently exhibited the highest RSCU value across all species, suggesting that such codons may be under similar selective pressures or mutation biases.\u003c/p\u003e\u003cp\u003eIn addition, analysis of GC content showed that the third codon positions of all species had relatively low GC content, averaging between 35.88% and 37.66%, reflecting a general A/T bias in the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes. Collectively, the codon usage patterns of mitochondrial PCGs in \u003cem\u003eMedicago\u003c/em\u003e species exhibit a high degree of consistency and a strong preference for A/T, indicating that natural selection may play a crucial role in shaping codon usage evolution in this genus.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePrediction of RNA editing sites in\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo gain deeper insights into the post-transcriptional regulatory mechanisms of mitochondrial PCGs, we analyzed RNA editing sites and their characteristics in the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species. We identified widespread C-to-U RNA editing events across the PCGs of all species, along with a few U-to-C type events. The total number of editing sites per species ranged from 452 to 568, showing limited variation, which suggests that RNA editing is a conserved post-transcriptional regulatory mechanism in higher plant mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table S10).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApproximately 92.53\u0026ndash;95.35% of these were cytidine-to-uridine (C-to-U) single-nucleotide substitutions. Further analysis of codon position distribution revealed that more than 50% of editing events occurred at the second codon position (approximately 59.40\u0026ndash;67.77%), followed by the first position (approximately 32.23\u0026ndash;40.60%), with no editing events detected at the third codon position (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table S11).\u003c/p\u003e\u003cp\u003eThe distribution of editing sites varied significantly among different genes. Genes involved in functions such as the electron transport chain, cytochrome biosynthesis, and membrane transport\u0026mdash;such as \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003eccmB\u003c/em\u003e\u0026mdash;harbored a large number of editing sites (up to 46\u0026ndash;89), whereas ribosomal protein-coding genes like \u003cem\u003erpsI\u003c/em\u003e (\u003cem\u003erpsI16\u003c/em\u003e, \u003cem\u003erpsI5\u003c/em\u003e) had relatively few editing sites (only 12\u0026ndash;14). Common editing conversions included serine (Ser) to phenylalanine (Phe), leucine (Leu), or proline (Pro); arginine (Arg) to cysteine (Cys) or tryptophan (Trp); and proline (Pro) to leucine (Leu) or phenylalanine (Phe). These changes were predominantly transitions from hydrophilic to hydrophobic amino acids, which may contribute to enhancing protein structural stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table S12). Additionally, editing events associated with stop codons were detected in genes such as \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003eatp9\u003c/em\u003e, and \u003cem\u003eccmFC\u003c/em\u003e, suggesting a potential regulatory role of RNA editing in translation initiation and termination processes.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic relationship among the\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the mitochondrial genome-level phylogenetic relationships of \u003cem\u003eMedicago\u003c/em\u003e species within the Fabaceae family, we performed a phylogenetic analysis using the complete mitochondrial PCG sequences from 25 Fabaceae species. A total of 31 conserved single-copy PCGs (including \u003cem\u003eatp\u003c/em\u003e, \u003cem\u003ecox\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, \u003cem\u003enad\u003c/em\u003e, \u003cem\u003erps\u003c/em\u003e, etc.) were extracted and used to construct phylogenetic trees based on both the Maximum likelihood (ML) method (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and Bayesian inference (BI) method (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results showed that both methods produced highly congruent tree topologies, with strong support for most branches (bootstrap\u0026thinsp;\u0026ge;\u0026thinsp;90%, posterior probability\u0026thinsp;\u0026ge;\u0026thinsp;0.95), indicating that the selected gene set provides a robust phylogenetic signal. In both resulting trees, species clustered into well-defined clades, and genera or species with close evolutionary relationships tended to group together, reflecting a high degree of phylogenetic consistency. All sampled \u003cem\u003eMedicago\u003c/em\u003e species were resolved as a monophyletic clade, supporting their shared evolutionary origin within the genus.\u003c/p\u003e\u003cp\u003eFurther analysis revealed that \u003cem\u003eM. minima\u003c/em\u003e and \u003cem\u003eM. lupulina\u003c/em\u003e were recovered as sister species, forming a clade with \u003cem\u003eM. truncatula\u003c/em\u003e. \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e were resolved as a strongly supported sister pair. Meanwhile, \u003cem\u003eM. platycarpos\u003c/em\u003e, \u003cem\u003eM. edgeworthii\u003c/em\u003e, and \u003cem\u003eM. arabica\u003c/em\u003e were inferred as more distantly related lineages, positioned outside the two aforementioned clusters. It suggests possible differences in evolutionary rates or selective pressures among these species.\u003c/p\u003e\u003cp\u003eThe inferred phylogenetic relationships were highly consistent with those based on chloroplast genomes and conformed to the taxonomic framework of angiosperms as outlined in the APG-IV system [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], further supporting the scientific validity and reliability of constructing phylogenies based on conserved mitochondrial PCGs. Notably, some species, such as \u003cem\u003eM. truncatula\u003c/em\u003e, showed relatively low support values in the tree, indicating potential uncertainty in their phylogenetic placement. This may be attributed to structural complexity or incomplete gene annotation in their mitochondrial genomes, warranting further investigation.\u003c/p\u003e\u003cp\u003eOverall, the well-supported phylogenetic framework established in this study provides novel mitochondrial evidence for understanding evolutionary relationships in Fabaceae plants and offers fundamental data for taxonomic studies and investigations into the origin and evolution of species within the family.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynteny analysis of mitochondrial sequences of\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the structural evolutionary characteristics of mitochondrial genomes in \u003cem\u003eMedicago\u003c/em\u003e species, we conducted a comprehensive collinearity analysis of eight representative \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes. The results revealed that although these species are phylogenetically closely related, their mitochondrial genomes exhibit a certain degree of structural variation in terms of the arrangement, length, and order of homologous blocks.\u003c/p\u003e\u003cp\u003eUsing BLASTn comparisons, we identified 23\u0026ndash;44 homologous collinear segments longer than 400 bp between each pair of genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These segments include both forward collinear blocks and reverse complementary blocks, which are widely distributed across the circular chromosomes of the mitochondrial genomes. For example, between \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e, 24 collinear blocks were detected, with the longest segment reaching 74,236 bp and exhibiting over 95% sequence similarity, indicating a high degree of structural conservation (Table S13).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, we observed that some conserved blocks span nearly the entire genome or are clustered near specific functional regions (such as \u003cem\u003enad\u003c/em\u003e, \u003cem\u003ecox\u003c/em\u003e, and \u003cem\u003eccm\u003c/em\u003e gene loci). These structures may be closely associated with essencial mitochondrial energy metabolism functions and could have been selectively retained during evolution. Despite certain rearrangements in overall genome architecture, many PCG regions remain highly collinear among species, suggesting that these functional genes are subject to strong purifying selection.\u003c/p\u003e\u003cp\u003eIn conclusion, while the mitochondrial genomes of \u003cem\u003eMedicago\u003c/em\u003e species exhibit a high level of sequence homology, they also display considerable structural rearrangement and diversity. These variations are likely linked to recombination events mediated by repetitive sequences during species divergence. This study provides important theoretical insights into the structural evolution of \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes and their phylogenetic relationships.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of selection pressure of shared PCGs in\u003c/b\u003e \u003cb\u003eMedicago\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the selective pressures acting on mitochondrial PCGs in \u003cem\u003eMedicago\u003c/em\u003e species during evolution, we conducted a systematic analysis of the nonsynonymous to synonymous substitution rate ratios (Ka/Ks) for mitochondrial single-copy PCGs shared among eight representative species. Alignments of 27 common single-copy PCGs were performed, and the Ka, Ks, and Ka/Ks values for each gene were calculated across pairwise species comparisons to investigate the potential selection mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results showed that the Ka/Ks ratios for most PCGs were significantly less than 1, indicating that these genes have been predominantly subjected to purifying selection and are functionally conserved during evolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Notably, genes such as \u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e, \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e, and \u003cem\u003erps7\u003c/em\u003e exhibited Ka/Ks values approaching zero. These genes are mainly involved in energy metabolism, electron transport, and ribosomal function, suggesting their crucial roles in maintaining core mitochondrial precesses.\u003c/p\u003e\u003cp\u003eImportantly, several genes displayed Ka/Ks values greater than 1 in certain species pairs, implying they may have undergone positive selection (Table S14). For instance, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003ematR\u003c/em\u003e, and \u003cem\u003erpl16\u003c/em\u003e showed significantly elevated Ka/Ks ratios in some comparisons, particularly between \u003cem\u003eM. arabica\u003c/em\u003e, \u003cem\u003eM. edgeworthii\u003c/em\u003e, \u003cem\u003eM. falcata\u003c/em\u003e, and \u003cem\u003eM. truncatula\u003c/em\u003e, where the Ka/Ks value of \u003cem\u003ematR\u003c/em\u003e reached as high as 3.34. This suggests that \u003cem\u003ematR\u003c/em\u003e may have played a key role in the adaptive evolution of these species.\u003c/p\u003e\u003cp\u003eIn summary, mitochondrial PCGs in \u003cem\u003eMedicago\u003c/em\u003e generally exhibit strong evolutionary conservation, although a subset of genes may have experienced positive selection in specific lineages. The Ka/Ks analysis provides theoretical insights into the adaptive evolutionary mechanisms of \u003cem\u003eMedicago\u003c/em\u003e species and lays the groundwork for identifying candidate genes with accelerated evolutionary rates for further functional studies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMitochondria, often referred to as the \"powerhouses\" of the cell, play a critical role in plant growth and development through their stable function. Plant mitochondrial genomes exhibit considerably greater complexity in both structure and regulatory mechanisms than those of animals and fungi, characterized by substantial variation in genome size, diverse gene content, abundant repetitive elements, frequent intron insertions, and highly plastic genome conformations [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we characterized the mitochondrial genomes of eight representative \u003cem\u003eMedicago\u003c/em\u003e species, including six newly reported assemblies. Compared with other Fabaceae species, such as \u003cem\u003eGlycine soja\u003c/em\u003e (402,545 bp) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and \u003cem\u003eVicia faba\u003c/em\u003e (588,000 bp) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes ranging from 281,240 to 356,577 bp, exhibit relatively moderate genome sizes. This suggests a more constrained genome expansion during their evolutionary history, possibly due to limited acquisition of foreign DNA or less proliferation of repeat elements. In terms of gene content, certain species (e.g., \u003cem\u003eM. falcata\u003c/em\u003e) exhibited increased copy numbers of several PCGs, such as \u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmFN\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, and \u003cem\u003enad4L\u003c/em\u003e, In contrast, \u003cem\u003erps1\u003c/em\u003e was annotated only in \u003cem\u003eM. truncatula\u003c/em\u003e and absent in the other species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar lineage-specific gene duplication and loss patterns have been observed in \u003cem\u003eCamellia duntsa\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and \u003cem\u003eMangifera\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], suggesting that dynamic gene copy number variation is a common feature in angiosperm mitogenomes. These events may contribute to functional diversification, regulation of respiratory efficiency, or species-specific adaptation.\u003c/p\u003e\u003cp\u003eThe GC content of the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes remained relatively stable (44.92\u0026ndash;45.58%) (Table S3), similar to that observed in \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (45.11%) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and \u003cem\u003eMomordica charantia\u003c/em\u003e (45.60%) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This stability in GC content may reflect evolutionary constraints related to replication fidelity, genome stability, and possibly the thermodynamic properties of the mitochondrial genome [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, notable structural differences were observed among \u003cem\u003eMedicago\u003c/em\u003e species: \u003cem\u003eM. lupulina\u003c/em\u003e and \u003cem\u003eM. minima\u003c/em\u003e possessed typical single circular conformations, whereas \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e exhibited more complex multi-circular structures. Such structural heterogeneity has also been reported in species such as \u003cem\u003eMichelia\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], \u003cem\u003ePunica\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and \u003cem\u003eAstragalus membranaceus\u003c/em\u003e in Fabaceae [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], highlighting the high degree of evolutionary plasticity in angiosperm mitochondrial genome organization [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This diversity in genome conformation may arise from recombination between dispersed repeats or differential replication mechanisms.\u003c/p\u003e\u003cp\u003eRepeat sequence analysis revealed that \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes are rich in diverse types of repeats, notably SSRs dominated by AT motifs, short repeats less than 200 bp, and large palindromic repeats exceeding 500 bp. These elements may facilitate genomic rearrangements and conformational evolution through homologous recombination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similar repeat profiles have also been identified in \u003cem\u003eBroussonetia\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and \u003cem\u003eCamellia\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], further supporting the widespread role of repeat-mediated structural variation across plant mitochondrial genomes.\u003c/p\u003e\u003cp\u003eIntron analysis showed that multiple intron-containing PCGs (e.g., \u003cem\u003enad1\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e) were conserved in \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes (Table S5). These patterns are consistent with those reported in other Fabaceae genera such as \u003cem\u003eGlycine\u003c/em\u003e and \u003cem\u003eCicer\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Most introns were located within cis-splicing regions and exhibited highly conserved structures, suggesting strong evolutionary constraints. Their conservation across taxa also indicates potential utility in phylogenetics and population genetics [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Collectively, the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes exhibit both interspecific variability and conserved features in genome size, gene content, structural conformation, and repeat composition, providing valuable insights into mitochondrial genome evolution in Fabaceae.\u003c/p\u003e\u003cp\u003eRegarding codon usage, \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes exhibited pronounced bias toward synonymous codons ending in A/T. Leucine (Leu) was the most frequently encoded amino acid, while tryptophan (Trp) and cysteine (Cys) were used the least (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This pattern is consistent with that observed in \u003cem\u003eMangifera\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]and \u003cem\u003eBroussonetia\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], supporting the idea that codon usage in plant mitochondria is influenced by a combination of natural selection, mutational bias, and genetic drift [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These features not only reflect the potential translational and transcriptional efficiency adaptations in \u003cem\u003eMedicago\u003c/em\u003e, but also align with codon usage preferences widely observed in other angiosperm mitochondrial genomes.\u003c/p\u003e\u003cp\u003eRNA editing analysis revealed the widespread presence of 452\u0026ndash;568 potential C-to-U type RNA editing sites in the mitochondrial PCGs of \u003cem\u003eMedicago\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Over 92% of these editing events occurred at the first and second codon positions, typically resulting in nonsynonymous substitutions that alter amino acid properties. Notably, these changes often involved a transitions from hydrophilic to hydrophobic amino acids, potentially enhancing protein structural stability and functional efficiency. Editing sites were predominantly concentrated in core metabolic genes such as \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003eccmB\u003c/em\u003e, suggesting their crucial roles in maintaining mitochondrial respiratory chain function. Consistent with findings in species such as \u003cem\u003eAcer truncatum\u003c/em\u003e and \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eMedicago\u003c/em\u003e species also exhibited high conservation of RNA editing sites, further underscoring the fundamental role of this mechanism in the post-transcriptional regulation of mitochondrial gene expression regulation in angiosperms.\u003c/p\u003e\u003cp\u003eSynteny analysis indicated that despite the close phylogenetic relationships among Medicago species, their mitochondrial genomes exhibited 23\u0026ndash;44 homologous syntenic blocks longer than 400 bp. These blocks were primarily distributed across core functional regions such as \u003cem\u003enad\u003c/em\u003e, \u003cem\u003ecox\u003c/em\u003e, and \u003cem\u003eccm\u003c/em\u003e, displaying high sequence conservation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The presence of conserved syntenic structures suggests strong purifying selection acting on these regions. This pattern aligns with observations in other angiosperm families such as Theaceae and Lauraceae [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], indicating evolutionary stability of key mitochondrial genomic components. In phylogenetic analyses, ML and BI trees were constructed based on 31 single-copy mitochondrial PCGs from 25 Fabaceae species. All Medicago species were recovered as a monophyletic group. Within this clade, \u003cem\u003eM. minima\u003c/em\u003e and \u003cem\u003eM. lupulina\u003c/em\u003e were identified as sister species, \u003cem\u003eM. falcata\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e as another sister pair, while \u003cem\u003eM. platycarpos\u003c/em\u003e was resolved as a more distantly related lineage within the genus. These phylogenetic relationships are highly consistent with results from chloroplast genome data [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], nuclear transcriptome analyses [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and the APG IV classification system, further validating the effectiveness and reliability of mitochondrial PCGs in resolving evolutionary relationships in Fabaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eKa/Ks ratio analysis further revealed that most PCGs in the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes had Ka/Ks values significantly less than 1, indicative of widespread purifying selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This was particularly evident in genes associated with energy metabolism, such as members of the \u003cem\u003eatp\u003c/em\u003e and \u003cem\u003enad\u003c/em\u003e gene families. These findings are consistent with patterns observed in other plant lineages like \u003cem\u003eMichelia\u003c/em\u003e, Theaceae, and other plant lineages, where mitochondrial genes generally evolve slowly and are functionally conserved [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, several genes\u0026mdash;such as \u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmFc\u003c/em\u003e, \u003cem\u003eccmFn\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, and \u003cem\u003enad9\u003c/em\u003e\u0026mdash;exhibited Ka/Ks values greater than 1 in certain species comparisons, suggesting potential roles in functional innovation during species divergence and adaptive evolution within \u003cem\u003eMedicago\u003c/em\u003e. Particularly noteworthy is the \u003cem\u003ematR\u003c/em\u003e gene, which showed a Ka/Ks value as high as 3.34 in certain species pairs, indicating strong positive selection and implicating its involvement in lineage-specific rapid evolutionary processes.\u003c/p\u003e\u003cp\u003eIn summary, the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes exhibit overall conservation in gene content, genome structure, codon usage, and RNA editing patterns, while also demonstrating structural rearrangements and signatures of positive selection in specific genes. Comparative analyses with other Fabaceae and angiosperms further clarified the phylogenetic placement of \u003cem\u003eMedicago\u003c/em\u003e within the family and highlight the dynamic nature of its adaptive evolution. These results not only enhanced our understanding of the evolutionary mechanisms shaping the \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genome, but also provide a theoretical foundation for future studies in functional genomics, genetic resource conservation, and molecular breeding. Integrative multi-omics approaches, incorporating nuclear and transcriptomic data, will be essential for further elucidating the complex regulatory and evolutionary dynamics of this economically and ecologically important legume genus.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe assembled and compared the mitochondrial genomes of eight \u003cem\u003eMedicago\u003c/em\u003e species, including six sequenced for the first time. The results revealed that five species possess a single circular genome structure, while three exhibit multichromosomal configurations. Genome sizes ranged from 281,240 to 356,577 bp, with 55 to 74 PCGs identified. Repetitive sequences were dominated by A/T mononucleotide repeats, and both codon usage and RNA editing showed a strong A/T bias and C-to-U conversions, respectively. The phylogenetic tree supported the monophyly of the genus \u003cem\u003eMedicago\u003c/em\u003e. Most genes were subject to purifying selection, while a few, such as \u003cem\u003ematR\u003c/em\u003e, exhibited signs of positive selection. Our analysis enhances the understanding of mitochondrial genome structure and evolution in \u003cem\u003eMedicago\u003c/em\u003e species and provides a valuable foundation for future studies on \u003cem\u003eFabaceae\u003c/em\u003e mitochondrial genome evolution.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePlant materials and Cultivation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, six representative species of the genus \u003cem\u003eMedicago\u003c/em\u003e were selected, including \u003cem\u003eMedicago lupulina\u003c/em\u003e (\u003cem\u003eM. lupulina\u003c/em\u003e), \u003cem\u003eMedicago falcata\u003c/em\u003e (\u003cem\u003eM. falcata\u003c/em\u003e), \u003cem\u003eMedicago minima\u003c/em\u003e (\u003cem\u003eM. minima\u003c/em\u003e), \u003cem\u003eMedicago edgeworthii\u003c/em\u003e (\u003cem\u003eM. edgeworthii\u003c/em\u003e), Medicago platycarpos (\u003cem\u003eM. platycarpos\u003c/em\u003e), and diploid \u003cem\u003eMedicago sativa\u003c/em\u003e (\u003cem\u003eM. sativa\u003c/em\u003e). Among them, seeds of \u003cem\u003eM. minima\u003c/em\u003e were obtained from the Kunming Institute of Botany, Chinese Academy of Sciences. All seeds were thoroughly cleaned and surface-sterilized before being sown in pots filled with nutrient soil and cultivated under greenhouse conditions (14 h light/10 h dark photoperiod, temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and relative humidity of 60\u0026ndash;70%). After 35 days of cultivation, young and healthy mature leaves were collected, immediately flash-frozen in liquid nitrogen on-site, and then stored at \u0026minus;\u0026thinsp;80\u0026deg;C in the laboratory for subsequent genomic DNA extraction. In addition, the mitochondrial genome sequences of \u003cem\u003eMedicago truncatula\u003c/em\u003e (\u003cem\u003eM. truncatula\u003c/em\u003e) (NC_029641.1) and \u003cem\u003eMedicago arabica\u003c/em\u003e (\u003cem\u003eM. arabica\u003c/em\u003e) (DQ662798.1) were downloaded from the public database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for use in subsequent comparative genomic analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA Extraction and Sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal genomic DNA was extracted from each sample using a modified CTAB method [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. DNA quality was assessed by 0.75% agarose gel electrophoresis to evaluate fragment size and potential degradation. DNA purity was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific), and DNA concentration was accurately quantified using a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA). For each sample, 5\u0026ndash;10 \u0026micro;g of high-quality genomic DNA was selected for PacBio library construction. Library preparation was performed using the PCR-free SMRTbell method, followed by sequencing on the PacBio Revio platform. The library preparation process included DNA damage repair, end repair, adapter ligation, library purification, and quality control. Each sample yielded approximately 21\u0026thinsp;~\u0026thinsp;42.9 Gb of raw sequencing data (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The raw PacBio subreads.bam files were processed for quality control and data filtering using the official PacBio software SMRT Link (v12.0). The resulting high-quality data were used for subsequent mitochondrial genome assembly and analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMitochondrial genome assembly and annotation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo enrich mitochondrial sequences, PacBio long reads were first aligned to the conserved mitochondrial genome sequence of the model \u003cem\u003eMedicago\u003c/em\u003e species \u003cem\u003eM. truncatula\u003c/em\u003e using Minimap2 (v2.28) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Candidate reads with alignment lengths greater than 6,000 bp and covering core gene regions were retained. Based on these seed reads, iterative alignment and extension were performed to aggregate all highly overlapping reads (length\u0026thinsp;\u0026ge;\u0026thinsp;6 kb, identity\u0026thinsp;\u0026ge;\u0026thinsp;70%), thereby extracting the complete set of mitochondrial-associated reads for each sample.\u003c/p\u003e\u003cp\u003eThe filtered mitochondrial reads were then subjected to \u003cem\u003ede novo\u003c/em\u003e error correction and assembly using Flye (v2.9.1). Assembly graphs were visualized using Bandage (v0.8.1) to determine whether the mitochondrial genome exhibited a typical circular structure or a multi-branched conFigureuration.\u003c/p\u003e\u003cp\u003eMitochondrial genome annotation was conducted using a combination of tools. General gene annotation was performed using the online platforms PMGA [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and CPGview [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. tRNA genes were annotated with tRNAscan-SE [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], and open reading frames (ORFs) were predicted using the 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). ORFs shorter than 102 bp or overlapping with known genes were excluded. ORFs longer than 300 bp were annotated against the NR database based on alignment results. All annotation results were manually inspected and curated to ensure accuracy. Circular genome maps were automatically generated using the IPMGA platform.\u003c/p\u003e\u003cp\u003eTo ensure consistency in annotation formats for downstream comparative genomic analyses, the two \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes downloaded from the public database and the six newly assembled genomes from this study were all re-annotated using the same pipeline and software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of repeated sequences\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the types and distribution characteristics of repetitive sequences in the mitochondrial genomes of \u003cem\u003eMedicago\u003c/em\u003e species, three categories of repeats were systematically identified and analyzed in this study: simple sequence repeats (SSRs), tandem repeats (TSRs), and dispersed repeats (DSRs). SSRs were identified using the MISA tool (Misa-web-IPK Gatersleben, (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webblast.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"https://webblast.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. TSRs (with repeat unit lengths greater than 6 bp) were detected using Tandem Repeats Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tandem.bu.edu/trf/basic_submit\u003c/span\u003e\u003cspan address=\"https://tandem.bu.edu/trf/basic_submit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. DSRs were identified using the REPuter program on the BiBiserv2 platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bibiserv.cebitec.uni-bielefeld.de/reputer?id=reputer_view_submission\u003c/span\u003e\u003cspan address=\"https://bibiserv.cebitec.uni-bielefeld.de/reputer?id=reputer_view_submission\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with the Hamming distance set to 3, a minimum repeat length of 30 bp, and a maximum repeat number of 5000 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].The distribution patterns of all identified repetitive sequences were visualized using the Circos module in TBtools [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of codon usage and Prediction of RNA editing sites\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDifferences in codon usage frequency among organisms are considered the result of an evolutionary equilibrium shaped by long-term natural selection and mutational biases. In this study, relative synonymous codon usage (RSCU) analysis of mitochondrial genomes from eight \u003cem\u003eMedicago\u003c/em\u003e species was performed using Perl scripts. Representative and unique CDS sequences were selected, and the codon usage counts for each gene were tabulated [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. To predict RNA editing sites in the mitochondrial genomes of these eight \u003cem\u003eMedicago\u003c/em\u003e species, the plant RNA editing prediction tool PREPACT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.prepact.de/prepact-main.php\u003c/span\u003e\u003cspan address=\"http://www.prepact.de/prepact-main.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed for analysis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the phylogenetic position of \u003cem\u003eMedicago\u003c/em\u003e species within the Fabaceae family, a phylogenetic analysis based on mitochondrial genomes was conducted in this study. Mitochondrial genome data of 17 Fabaceae species related to \u003cem\u003eMedicago\u003c/em\u003e were downloaded from the NCBI database and re-annotated using the same annotation software, PMGA [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], to ensure consistent annotation formats for subsequent analyses. \u003cem\u003eSuriana maritima\u003c/em\u003e was selected as the outgroup in the phylogenetic analysis. All analyzed species and their corresponding GenBank accession numbers are listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eUsing PhyloSuite (v1.2.3) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], conserved mitochondrial protein-coding gene (CDS) sequences shared among the 25 Fabaceae species were extracted. After manual alignment and curation, 31 shared coding genes were retained for further analysis. Multiple sequence alignment of these nucleotide sequences was performed using MAFFT [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] with default parameters. The aligned sequences were then concatenated into a single matrix using the \"Concatenate Sequence\" function in PhyloSuite for phylogenetic tree construction.\u003c/p\u003e\u003cp\u003ePhylogenetic trees were constructed using both maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was conducted with IQ-TREE [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] using the TVM\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 nucleotide substitution model, and node support was evaluated with 1,000 bootstrap replicates. BI analysis was performed using MrBayes (v3.2) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The resulting phylogenetic trees were visualized using the ITOL web platform [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eColinear analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate structural conservation and rearrangement characteristics among the mitochondrial genomes of \u003cem\u003eMedicago\u003c/em\u003e species, a comprehensive synteny analysis was performed on eight \u003cem\u003eMedicago\u003c/em\u003e mitochondrial genomes. First, pairwise genome alignments were conducted using BLASTn (v2.14.0+) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] to identify conserved homologous sequence fragments. The BLASTn parameters were set as follows: -evalue 1e-10, with low-quality alignments filtered out. Only homologous regions longer than 400 bp and with sequence identity greater than 90% were retained as syntenic units for subsequent comparison. To further illustrate the structural similarities and variations of mitochondrial genomes among different \u003cem\u003eMedicago\u003c/em\u003e species, the extracted syntenic blocks were visualized using LINKVIEW2 (v1.0.5) software .\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of Ka/Ks values\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the natural selection pressures during the evolutionary process of \u003cem\u003eMedicago\u003c/em\u003e species, eight representative species were selected for analysis. Shared PCGs were aligned using MAFFT (v11) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions (Ka/Ks) was calculated using KaKs_Calculator (v2.0) [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCMS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cytoplasmic male sterility\u003c/p\u003e\n\u003cp\u003eONT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Oxford Nanopore Technologies\u003c/p\u003e\n\u003cp\u003eRSCU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Relative synonymous codon usage\u003c/p\u003e\n\u003cp\u003ePCGs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Protein-coding genes\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. lupulina\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medicago lupulina\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. minima\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medicago minima\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. edgeworthii\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medicago edgeworthii\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. truncatula\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Medicago truncatula\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. arabica\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Medicago arabica\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. falcata\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medicago falcata\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. platycarpos\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Medicago platycarpos\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM. sativa\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Medicago sativa\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003erRNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ribosomal RNA\u003c/p\u003e\n\u003cp\u003etRNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Transfer RNA\u003c/p\u003e\n\u003cp\u003eSSRs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Simple Sequence Repeats\u003c/p\u003e\n\u003cp\u003eTSRs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tandem Sequence Repeats\u003c/p\u003e\n\u003cp\u003eDSRs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dispersed Sequence Repeats\u003c/p\u003e\n\u003cp\u003eF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Forward\u003c/p\u003e\n\u003cp\u003eP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Palindromic\u003c/p\u003e\n\u003cp\u003eR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reverse\u003c/p\u003e\n\u003cp\u003eC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Complementary\u003c/p\u003e\n\u003cp\u003eLeu\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Leucine\u003c/p\u003e\n\u003cp\u003eSer\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Serine\u003c/p\u003e\n\u003cp\u003eIle\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Isoleucine\u003c/p\u003e\n\u003cp\u003eTrp\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Tryptophan\u003c/p\u003e\n\u003cp\u003eCys\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cysteine\u003c/p\u003e\n\u003cp\u003eArg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Arginine\u003c/p\u003e\n\u003cp\u003eLeu\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Leucine\u003c/p\u003e\n\u003cp\u003eSer\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Serine\u003c/p\u003e\n\u003cp\u003eMet\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Methionine\u003c/p\u003e\n\u003cp\u003eTrp\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Tryptophan\u003c/p\u003e\n\u003cp\u003eGC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Guanine-Cytosine content\u003c/p\u003e\n\u003cp\u003eML\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Maximum likelihood\u003c/p\u003e\n\u003cp\u003eBI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bayesian inference\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eR-F Y\u003c/strong\u003e was responsible for visualization, methodology, formal analysis, data curation, conceptualization, and drafting of the manuscript. \u003cstrong\u003eM-Y W\u003c/strong\u003e contributed to conceptualization and methodology. \u003cstrong\u003eM-L W\u003c/strong\u003e participated in data curation, visualization, and methodological development. \u003cstrong\u003eJ L\u003c/strong\u003e: methodology. \u0026nbsp;\u003cstrong\u003eJ-L L\u003c/strong\u003e and \u003cstrong\u003eC-H H\u003c/strong\u003e jointly oversaw conceptualization, supervision, project administration, and secured funding for the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funds from the National Natural Science Foundation of China (32270232, 31970224, and 31770242) and State Key Laboratory of Reproductive Regulation \u0026amp; Breeding of Grassland Livestock, Key Laboratory of Herbage \u0026amp; Endemic Crop Biology, Ministry of Education (Inner Mongolia University). Also supported by State Key Laboratory of Genetics and Development of Complex Phenotypes, State Key Laboratory of Wetland Conservation and Restoration, National Observations and Research Station for Wetland Ecosystems of the Yangtze Estuary, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Human Phenome Data Center (Fudan university).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data of resequencing genome have been deposited in NCBI Sequence Read Archive (SRA) under BioProject accession PV916040, PV916041, PV916042, PV882493\u0026ndash;PV882495, PV892890\u0026ndash;PV892892, PV892893\u0026ndash;PV892894. All data supporting this research result can be obtained in the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental research studies on plants, including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNiu Y, Zhang T, Chen M, et al. Analysis of the complete mitochondrial genome of the bitter gourd (Momordica charantia)[J]. 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Genomics Proteom Bioinf. 2010;8(1):77\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Medicago, Mitochondrial genome, Genome rearrangements, Phylogenetic analysis, Comparative analysis","lastPublishedDoi":"10.21203/rs.3.rs-7230374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7230374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003cem\u003e Medicago\u003c/em\u003e is an economically important forage genus widely distributed across China, yet its mitochondrial genomes remain poorly characterized. Comprehensive analysis of mitochondrial genome structure, function, and evolution is essential for uncovering plant biological mechanisms, enhancing germplasm utilization, and supporting molecular breeding efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Here, we assembled and compared the mitochondrial genome of eight \u003cem\u003eMedicago\u003c/em\u003e species, including six newly sequenced genomes. Our results revealed that five species possess typical single circular mitochondrial genome structures, while \u003cem\u003eM. falcata\u003c/em\u003e, \u003cem\u003eM. platycarpos\u003c/em\u003e, and \u003cem\u003eM. sativa\u003c/em\u003e exhibit complex multipartite circular conFigureurations. These mitochondrial genome sizes ranged from 281,240 to 356,577 bp, containing 55–74 functional genes. Repetitive sequence analysis identified 141 simple sequence repeats (SSRs) and 76 tandem repeats (TSRs), dominated by A/T-rich mononucleotide motifs, while dispersed repeats were mainly 30–49 bp in length. Codon usage analysis showed strong A/T bias and a preference for leucine, serine, and isoleucine. RNA editing sites were predominantly C-to-U substitutions, primarily located at the first and second codon positions. Phylogenetic reconstruction based on 31 conserved mitochondrial protein-coding genes (PCGs) strongly supported the monophyly of \u003cem\u003eMedicago\u003c/em\u003e and resolved interspecific relationships consistent with previous chloroplast and nuclear genome studies. Most PCGs were under purifying selection, whereas a few genes, such as \u003cem\u003ematR\u003c/em\u003e, exhibited signals of positive selection, suggesting lineage-specific adaptive evolution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Altogether, this study enriches the mitochondrial genomic resources of \u003cem\u003eMedicago\u003c/em\u003e and deepens the understanding of its structural evolution and phylogenetic relationships, providing valuable insights for evolutionary and functional studies in Fabaceae plants.\u003c/p\u003e","manuscriptTitle":"Assembly and Comparative Analysis of Chromosomal Mitochondrial Genomes in Multiple Medicago Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 11:49:54","doi":"10.21203/rs.3.rs-7230374/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T07:06:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T21:06:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T03:34:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-12T08:19:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311854497815471638218564230671286675741","date":"2025-08-10T05:58:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32266002640386609887683038999681784365","date":"2025-08-09T16:03:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56029614926064248022521670494311148654","date":"2025-08-09T07:42:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-07T07:55:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T06:21:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-05T13:36:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-05T13:34:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-28T06:19:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c94af4d-e418-46f2-a1fc-287b95b44e4c","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:05:55+00:00","versionOfRecord":{"articleIdentity":"rs-7230374","link":"https://doi.org/10.1186/s12870-025-07650-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-11-28 15:58:50","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-08-12 11:49:54","video":"","vorDoi":"10.1186/s12870-025-07650-z","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07650-z","workflowStages":[]},"version":"v1","identity":"rs-7230374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7230374","identity":"rs-7230374","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}