Structural Features and Synteny Analysis of the Sea Buckthorn Mitochondrial Genome

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In order to gain a deeper understanding of the sea buckthorn mitochondrial genome, sequencing and assembly of the sea buckthorn mitochondrial genome was performed, and its substructures were explored in this study. Results The mitochondrial genome of seabuckthorn consists of two circular chromosomes, with lengths of 297,507 bp and 167,037 bp, respectively. It encompasses 36 annotated protein-coding genes, 3 rRNA genes, and 25 tRNA genes. In addition, 212 pairs of repeats were detected, including a pair that mediated homologous recombination of seabuckthorn mitochondrial chromosomes to form two conformations. The existence of these conformations was confirmed through PCR amplification and Sanger sequencing. A total of 162 SSR loci were identified in the sea buckthorn mitochondrial genome. There are 30 homologous fragments in chloroplast and mitochondrial genomes, with a total length of 44,950 bp, accounting for 9.89% of the total length of mitochondrial genomes. Conclusions In summary, this study conducted the assembly and annotation of the sea buckthorn mitochondrial genome, providing a comprehensive understanding of the genome of this plant. This knowledge is of great significance for effective utilization and genetic improvement of seabuckthorn, especially in breeding and evolutionary analysis of cytoplasmic male sterility. Sea buckthorn Mitochondrial genome Homologous recombination Phylogenetic analysis RNA editing events Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Background Mitochondria are essential semi-autonomous organelles in eukaryotic cells, possessing their own genetic material and genetic systems that operate independently of the plant nuclear genome [ 1 – 2 ]. In addition to providing energy to the cell, mitochondria are involved in various metabolic processes such as cell differentiation, cell signaling [ 3 ], and apoptosis [ 4 ]. Mitochondria are often referred to as the “cellular powerhouses” [ 5 – 6 ], and play a crucial role in the synthesis and metabolism of vital substances related to life activities, including vitamins, lipids, and amino acids [ 7 – 8 ]. The outer mitochondrial membrane serves as a signaling platform, providing scaffolding for many key proteins involved in cell signaling [ 9 ], and these functions are of paramount importance in the growth and development of plants [ 10 ]. An important characteristic of plant mitochondrial genomes is the presence of extensive RNA editing sites and the utilization of a universal genetic code [ 11 ], involving single base substitutions and the addition of bases to complete start or stop codons [ 12 ]. Furthermore, a phenomenon known as RNA editing occurs in both animal and plant mitochondria, where mRNA sequences generated by gene transcription are not perfectly identical to the template sequences. RNA editing is a post-transcriptional modification that alters mRNA produced by gene transcription [ 13 , 14 – 16 ]. Compared to animals, plant mitochondrial genomes are larger, more plastic [ 17 ], and may contain increased phylogenetic signals. One of the primary advantages of mitochondrial genome as a phylogenetic marker [ 18 ] is the differential substitution rates among its 36 genes, with some genes exhibiting higher rates of substitution than others, facilitating the tracking of relatively recent divergences [ 19 – 20 ]. Additionally, mitochondrial genome is associated with cytoplasmic male sterility (CMS), a condition controlled by genes within the mitochondria, expressed maternally, and can be restored by nuclear genes known as restorer of fertility (Rf) genes. In the absence of Rf genes in the nuclear genome, plants exhibit male gametophyte or pollen sterility while the female reproductive organs remain normal [ 21 – 22 ].At present, the study of plant mitochondrial genome has become a hot spot. Zeng et al. studied and analyzed the mitochondrial genome of Hippophae tibetana, which has 1 chromosome and is 464,208 bp long. It included 31 tRNA genes, 3 rRNA genes, 37 protein-coding genes and 3 pseudogenes [ 23 ]; Fajardo et al. found that the mitochondrial genome of American cranberry consisted of a single mitochondrial chromosome 459,678 bp in length, with 34 protein-coding genes, 3 rRNA genes, and 17 tRNA genes [ 24 ]; Liu et al. studied the mitochondrial genome of Phellodendron amurense, a total of two mitochondrial genomes with a length of 566,285 bp, and identified 32 protein-coding genes, 33 tRNA genes and 3 rRNA genes. There were 181 simple sequence repeats, 19 tandem repeats, and 310 scattered repeats, all with multiple conformations [ 25 ]. Sea buckthorn (Hippophae rhamnoides L.) is a deciduous shrub or small arbor belonging to the family Elaeagnaceae. It exhibits strong ecological adaptability and remarkable resilience, demonstrating tolerance to drought, cold, salinity, barrenness, and nitrogen fixation capabilities. Sea buckthorn can endure extreme temperatures ranging from − 40°C to 40°C [ 26 ]. Moreover, it is recognized for its soil improvement and water conservation functions, making it a pioneer tree species in afforestation of barren hills and slopes [ 27 ]. Sea buckthorn is also a plant with both medicinal and culinary uses. Its fruits are rich in nutrients and bioactive compounds, including proteins, vitamins, polysaccharides, flavonoids, and terpenes [ 28 ]. With advancements in molecular biology techniques, molecular biology studies of sea buckthorn have made progress, providing a foundation for elucidating the biosynthesis of secondary metabolites and the discovery of functional genes [ 29 ]. Therefore, in-depth study of its mitochondrial genome is of great significance for the effective utilization and genetic improvement of sea buckthorn. Currently, the NCBI database contains genomic information for 35,757 eukaryotic organisms, with the sea buckthorn complete genome submitted in October 2023 but remains unreported. Information on sea buckthorn mitochondrial genome, including its length, GC content, gene count, codon preferences, repetitive sequences, phylogeny, sequence migration, and RNA editing events, is still lacking. In this study, the sequencing, assembly, and annotation of the sea buckthorn mitochondrial genome were performed, and its genomic and structural characteristics were investigated. The existence of various conformations of the sea buckthorn mitochondrial genome was further confirmed through PCR experiments and Sanger sequencing, providing theoretical support for cytoplasmic male sterility breeding in higher plants and plant evolution. Results Structural Features of the Sea Buckthorn Mitochondrial Genome The sea buckthorn mitochondrial genome was sequenced, assembled, and annotated in this study. After eliminating duplicate regions from Nanopore data, two major circular overlapping contigs were obtained, which were used to construct the sea buckthorn mitochondrial genome as shown in Fig. 1. It was observed that the sea buckthorn mitochondrial genome consists of two chromosomes, as depicted in Fig. 2, with a total length of 454,544 bp and a GC content of 44.85%. Chromosome 1 and Chromosome 2 have lengths of 297,507 bp and 157,037 bp, respectively. The annotation of the sea buckthorn mitochondrial genome revealed the presence of 36 unique protein-coding genes, which could be categorized into 10 major classes, as shown in Table 1. Among these genes, there were 24 unique mitochondrial core genes and 12 non-core genes. The mitochondrial core genes included: 1. 5 ATP synthase genes ( atp 1 , atp 4 , atp 6 , atp 8 , and atp 9). 2. 9 NADH dehydrogenase genes ( nad 1 , nad 2 , nad 3 , nad 4 , nad 4L , nad 5 , nad 6 , nad 7 , and nad 9). 3. 4 Cytochrome C biogenesis genes ( ccm B, ccm C, ccm FC, and ccm FN). 4. 3 Cytochrome C oxidase genes ( cox 1 , cox 2 , and cox 3). 5. 1 Membrane transport protein gene ( mtt B). 6. 1 Maturation enzyme gene ( mat R). 7. 1 Ferrocytochrome C reductase gene ( cob ). The non-core genes included: 1. 4 Ribosomal large subunit genes ( rpl 2, rpl 5, rpl 10, rpl 16). 2. 6 Ribosomal small subunit genes ( rps 1, rps 3, rps 4, rps 7, rps 12, rps 19). 3. 2 Succinate dehydrogenase genes ( sdh 3, sdh 4). In addition to the protein-coding genes, the sea buckthorn mitochondrial genome also contained 25 tRNA genes, including 8 tRNA genes that had multiple copies, and 3 rRNA genes. This comprehensive annotation provided valuable insights into the gene composition and functional elements of the sea buckthorn mitochondrial genome. Repeat Sequence Analysis In Chromosome 1 of the sea buckthorn mitochondrial genome, a total of 111 SSRs were identified. Mononucleotide and dinucleotide SSRs accounted for 46.85% of the total SSRs. Among the mononucleotide SSRs, adenine (A) mononucleotide repeat sequences accounted for 60.00% (21 out of 35 mononucleotide SSRs). No hexanucleotide SSRs were detected. Chromosome 1 also contained 11 tandem repeat sequences with a match percentage greater than 69% and lengths ranging from 9 to 39 bp. In the analysis of scattered repeat sequences on Chromosome 1, a total of 41 pairs of repeat sequences were observed with a length of 50 or more, including 20 palindromic repeats, 10 forward repeats（1 reverse repeat, and no complementary repeats）. The longest palindromic repeat was 4,102 bp, while the longest forward repeat was 145 bp. In Chromosome 2 of the sea buckthorn mitochondrial genome, a total of 61 SSRs were found. Mononucleotide and dinucleotide SSRs constituted 55.74% of the total SSRs. Thymine (T) mononucleotide repeat sequences accounted for 52.38% (11 out of 21 mononucleotide SSRs). Chromosome 2 had 6 tandem repeat sequences with a match percentage greater than 83% and lengths ranging from 11 to 24 bp. Analysis of scattered repeat sequences on Chromosome 2 revealed a total of 9 pairs of repeat sequences with a length of 50 or more, including 2 palindromic repeats and 1 forward repeats. （No reverse repeats or complementary repeats were detected.） The longest palindromic repeat was 85 bp, while the longest forward repeat was 79 bp. And 8 pairs of interspersed repeated sequences between Chromosome 1 and Chromosome 2 were detected. Homologous Recombination Mediated by Repetitive Sequences Previous studies have shown that repetitive sequences can mediate homologous recombination in mitochondrial genomes [30]. We identified a total of two pairs of long repeat sequences that may mediate homologous recombination in the mitochondrial genome, namely R1 and R3. R1（coting 4） is 4102 bp long, with both repeat units located within contig1, potentially mediating an inversion of part of the sequence on contig1. There are 53 long-reads supporting our currently assembled configuration, while another 48 long-reads support the configuration after inversion, indicating that the inversion event occurs at a rate close to 50%. Notably, the two repeat units of R3 are located on two different circles, possibly acting as an important mediator for recombining the two small circles into a larger circular genome. However, our long-read data show that 93.79% of the long-reads (136 reads) support the independent existence of the two small circles, while only 6.21% of the long-reads (9 reads) support the recombination into a large circle. For the remaining short repeat sequences, we did not observe significant long-reads supporting recombination, but we cannot rule out the possibility of recombination as a lower frequency of recombination events might be difficult to detect. Hippophae tibetana (Xizang Sea Buckthorn) is a single ring structure, which is different from the sea buckthorn in this study. Table 1: Number of long-reads and recombination rates supporting different configurations mediated by the two pairs of repeat sequences To further validate whether the repetitive sequences can indeed mediate homologous recombination, PCR amplification and Sanger sequencing were conducted to confirm the presence of alternative conformations. The primer design strategy is depicted in Fig. 4. Identification of Sequence Transfer The sea buckthorn mitochondrial genome contains sequences known as mitochondrial plastid sequences (MTPTs). Based on sequence similarity analysis, a total of 30 segments were identified as homologous segments between the mitochondrial genome and the chloroplast genome (Fig. 7), with a cumulative length of 44,950 bp, constituting 9.89% of the total length of mitochondrial genome. Among them, MTPT6 was the longest, spanning 15,994 bp. Upon annotation of these homologous sequences, 28 complete genes were identified within the 30 homologous segments. These genes included 15 protein-coding genes ( cem A , clp P , ndh B , ndh J , pet A , pet B , psb C , psb D , psb H , psb N , psb T , rpl 23 , rps 4 , rps 7 , ycf 2) and 13 tRNA genes ( trn D-GUC , trn F-GAA , trn H-GUG , trn I-CAU , trn L-CAA , trn L-UAA , trn M-CAU , trn N-GUU , trn P-UGG , trn S-GGA , trn T-UGU , trn V-GAC , trn W-CCA). The fact that the 13 tRNA sequences are conserved between the chloroplast and mitochondrial genomes suggests that they may still function within the mitochondrial genome. Phylogenetic Analysis Based on the DNA sequences of 26 conserved mitochondrial Protein Coding Genes (PCGs), a phylogenetic tree was constructed for 33 species from four orders of angiosperms (Fig. 8). The protein-coding genes used for the analysis were atp 1 , atp 4 , atp 6 , atp 8 , ccm B , ccm C , ccm FC , ccm FN , cob, cox 3 , mat R , mtt B , nad 1 , nad 2 , nad 3 , nad 4 , nad 4L , nad 5 , nad 6 , nad 7 , nad 9 , rpl 2 , rpl 16 , rps 3 , rps 4 , and rps 19. Two mitochondrial genomes from the Fabales order were designated as outgroups. The topology of the phylogenetic tree based on mitochondrial DNA was consistent with the latest classification by the Angiosperm Phylogeny Group (APG). Sea buckthorn belonged to the order Rosales and the family Elaeagnaceae , and it clustered with Hippophae tibetana . We are annotated Hippophae tibetana and found that its protein coding gene is consistent with the sea buckthorn studied in this study, and the rps 19 also has two copies. We also analyzed the RNA editing and repeat sequences of Hippophae tibetana for comparison with Hippophae rhamnoides L . Sea Buckthorn ( Hippophae rhamnoides L. ) RNA Editing Events RNA editing events in the 36 unique PCGs from the sea buckthorn mitochondrial genome were identified. A total of 430 potential RNA editing sites were identified across the 36 mitochondrial PCGs (Fig. 9), all involving the conversion of base C to U. Among the mitochondrial genes, the ccm B gene was found to have 35 RNA editing sites, making it the most extensively edited gene. Following ccmB , the nad5 gene exhibited 31 RNA editing events, indicating the second-highest frequency of editing among all mitochondrial genes. The same RNA editing events were observed in reports of Tibetan sea buckthorn. RNA editing events were also identified in 35 unique PCGs of the mitochondria of Tibetan sea buckthorn, a close relative of sea buckthorn, and a total of 427 potential RNA editing sites were identified on the 35 mitochondrial PCGs (Fig. 10), all of which were base C to U editing, the same as those of sea buckthorn. In the mitochondrial genes, the occurrence times of ccm B, the gene with the highest number of gene edits, and the second nad 5 gene were also the same as those of Sea Buckthorn, However, there are differences between sdh 3 and sdh 4. There is no sdh 3 RNA editing site in Tibetan Sea Buckthorn, and sdh 4 RNA editing site is also less than sea buckthorn. Co-linearity Analysis Co-linearity analysis of sea buckthorn and closely related species (Fig. 11). The Chromosome 1 of sea buckthorn is highly coincident with the Tibetan sea buckthorn Chromosome Collinear Block and has a high degree of homology, while the Chromosome 2 of sea buckthorn is heavily inverted compared to the Tibetan sea buckthorn Chromosome. At the same time, homological collinear blocks were also detected in the near-origin species of sea buckthorn and Rosaceae, but the length of these collinear blocks was shorter. In addition, some blank areas were found, sequences that are unique to sea buckthorn and have no homology with the rest of the species. The results showed that the collinear block arrangement order among the mitochondrial genomes of these six species was inconsistent, and the mitochondrial genome of sea buckthorn underwent a large number of genome rearrangements between the mitochondrial genome and its relatives, and the mitochondrial genome was extremely unconserved in structure. Discussion Multiple Circular Configurations of Plant Mitochondrial Genomes Mitochondria play a crucial role in various metabolic processes related to the synthesis and degradation of intracellular substances, signal transduction, and energy production [ 30 ]. Plant mitochondrial genomes are more complex than animal mitochondrial genomes, exhibiting extensive differences in size, structure, sequence arrangement, and repetitive content [ 31 ]. Previous studies have shown that approximately 10% of plant mitochondrial genomes have a multipartite structure, with most mitochondrial genomes being circular, such as in Salvia miltiorrhiza [ 32 ]. However, some are non-circular, and the mitochondrial genome of okra is likely to have multiple conformations [ 33 ], while the oak mitochondrial genome displays various structural variations [ 34 ]. In eukaryotes, not only do the structural shapes differ, but the repetitive evolution of chromosomes also varies [ 31 ]. Sea buckthorn, Salvia miltiorrhiza [ 35 ], and sagebrush [ 36 ] share a similar pattern, cucumber mitochondrial genome has three independent chromosomes [ 37 ], and the mitochondrial genome of Paphiopedilum micranthum consists of 26 small circular chromosomes [ 38 ]. Therefore, in-depth studies on plant multipartite mitochondrial genomes will contribute to new discoveries in their genome evolution and molecular functions. Homologous Recombination Mediated by Repetitive Sequences DNA sequences in biological cells contain many repetitive elements, which can be broadly categorized into two main types: tandem repeats [ 39 ] and dispersed repeats [ 40 ]. Among tandem repeats, there is a special type known as Short Sequence Repeat (SSR) or microsatellite repeat sequences. Previous studies have indicated the significance of repetitive sequences in mitochondrial genomes for intermolecular recombination. In plum mitochondrial genomes, three pairs of repetitive sequences mediated genome recombination were discovered, resulting in eight different conformations [ 41 ]. In sweet potato, three pairs of direct repeat sequences were found to mediate homologous recombination, forming four independent circular molecules and leading to the formation of seven conformations [ 42 ]. Within tandem repeats, Simple Sequence Repeat (SSR), a particular type of microsatellite repeat sequence typically consisting of no more than 6 bp, is often used for molecular marker development due to its dominant genetic characteristics [ 43 ]. The presence of abundant repetitive sequences in the sea buckthorn mitochondrial genome (Fig. 3 , supplementary data) suggested the frequent occurrence of intermolecular recombination within the mitochondrial genome, resulting in dynamic changes in sequence and conformation during evolution. Repeat-mediated homologous recombination creates a high degree of diversity in plant mitochondrial plant genomes. For example, the mitochondrial genome of Panax notoginseng has various combinations of repeats that can mediate the recombination of seven subgenome ring molecules to form the main loop [ 44 ], high-frequency recombination of two large repeats in the mitochondrial genome of Ginkgo biloba led to the prediction of multipart structure [ 45 ]. The homologous recombination (HR) pathway, on the other hand, relies on the identification of sequences that are highly similar to the region to be repaired, usually other genomic copies or large repeats [ 46 ]. Double-strand break repair (DSBR) involves the recruitment of the second strand of the DSB and the formation of two Holliday nodes (HJ). HJs can be resolved by dissolution, intermigration of branches, and topoisomerase dechaining, or by analytic enzymatic cleavage, resulting in crossover or no crossing, depending on how they are resolved. The Break-Induced Replication (BIR) pathway uses homologous DNA to resynthesize DNA that has passed through DSB. BIR can lead to significant genomic rearrangements when the homologous sequence is not an allele, such as repetitive sequences [ 47 ]. In plant mitochondria, HR is the main DNA repair pathway, is also responsible for the rapid evolution of genomic organization, and plays an important role in the replication and isolation of mtDNA [ 48 ]. The mitochondrial genome of plants typically has very low mutation rates [ 49 ]. On the other hand, they exhibit excessively high rates of genomic rearrangements, resulting in poor homology conservation, even within the same species [ 50 ]. In the absence of stress, ectopic recombination mediated by medium-sized repeats is rare, and the smaller the repeat size, the lower the frequency and irreversibility of recombination [ 51 ] and seems to depend on the repeat length and its location in the genome [ 52 – 53 ], and whether they are in two different subgenomic molecules (intermolecular recombination) or in a single DNA molecule (intramolecular recombination). The distinction between single-ring and double-ring mitochondrial genome structures arises from extensive sequence rearrangements and recombination events evident in long-read sequencing data. Motivated by the hypothesis that additional substructures might exist beyond the canonical single-ring configuration, we performed hybrid assembly using Unicycler with default parameters. The algorithm successfully aligned long reads to repetitive elements, and subsequent analysis revealed reads spanning entire repeat regions, leading to the conclusion that the dimeric conformation represents the most plausible mitochondrial genome architecture. Experimental validation demonstrated that repeat element R1 (coting4) mediates dual structural conformations in Hippophae rhamnoides mitochondria (Fig. 6 ). We propose that repetitive sequence-mediated homologous recombination, potentially through double-strand break repair (DSBR) and break-induced replication (BIR) mechanisms, drives this structural polymorphism. Notably, the R3 repeat exhibited limited capacity to generate large circular conformations, likely attributable to its shorter length (287 bp vs. R1's 1,024 bp). Comparative analysis revealed significant structural divergence between Tibetan and reference sea buckthorn mitochondrial genomes, with Chromosome 2 demonstrating near-complete inversion (93% collinearity loss). While these findings provide mechanistic insights, definitive validation of repeat-mediated recombination in sea buckthorn mitochondria requires further experimental investigation. Phylogenetic Relationship of Homologous Species The rapid evolution of plant mitochondria has led to extensive gene recombination, genome heterogeneity, and gene fusion among mitochondria of different species [ 54 – 55 ]. While the size and structure of plant mitochondrial genomes vary significantly, functional genes remain conserved [ 56 ]. Here, we employed IQ-TREE to construct a sequence-based phylogenetic tree to explore the evolutionary relationships between sea buckthorn and representative taxa of angiosperms (Fig. 9 ). This tree clearly reflected distinct classification relationships among these groups, and the results also suggested a closer phylogenetic relationship between Hippophae rhamnoides L. and Hippophae tibetana . RNA Editing Site Prediction RNA editing is widely present in higher plant mitochondria, and it is an essential step in gene expression in plant mitochondrial genomes [ 57 – 58 ]. RNA editing falls under the category of post-transcriptional modification, with the most common type being C-to-U editing, which tends to occur at the second codon position [ 59 ], and the majority of these edits are complete. Predicting potential RNA editing sites can facilitate to understand the expression of genes in plant mitochondria. Liu J et al. reported 28 RNA editing sites in cannabis. Among these, 14 were in intergenic regions, and 14 were in the protein-coding regions of genes, involving both C-to-U and G-to-A editing events [ 60 ]. Liu Q et al. identified 269 RNA editing sites in Oat mitochondria , all of which were C-to-U edits [ 61 ]. In addition, RNA editing may contribute to the stability and quantity of proteins [ 62 ]. Although the average coverage is not high due to low expression levels or limited RNA sequencing data, it still provides a wealth of information to further understand the potential functional role of RNA editing in the mitogenome[ 63 ]. In this study, we predicted a total of 430 potential editing sites in 36 genes, all of which involved C-to-U editing. The prediction and characterization of potential RNA editing sites provide important clues for predicting gene functions using the new codons. Endosymbiotic Sequence Transfer Events in the Sea Buckthorn Mitochondrial Genome Endosymbiotic or intracellular gene transfer (EGT/IGT) occurs within plant cells [ 64 ]. For example, Stupar et al. discovered a 630 kb-long mitochondrial sequence on chromosome 2 of Arabidopsis thaliana, with sequence homology of over 99% [ 65 ]. In this study, 30 homologous segments that may be MTPT sequences were found, among which 15 protein-coding genes and 13 complete tRNA genes migrated from the chloroplast genome to the mitochondrial genome (see supplementary table). These sequences have a total length of 44,950 bp, accounting for 9.89% of the size of the mitochondrial genome. In this study, only three homologous fragments of mitochondria and chloroplasts were analyzed, which proved that two plastids had sequence migration, and found that 28 of them were complete, and 15 of them were protein-coding genes, and two genes were detected as incomplete. This phenomenon was also observed in other plant mitochondria [ 66 – 67 , 1 ]. Similar occurrences have been reported in species like Catharanthus roseus and Bupleurum chinense [ 68 ]. Conclusion In this study, the sea buckthorn mitochondrial genome was assembled successfully using a hybrid strategy involving Illumina and Nanopore sequencing. The sea buckthorn mitochondrial genome consists of two circular chromosomes with a total length of 454,544 bp. A total of 64 genes were annotated within the sea buckthorn mitochondrial genome, including 36 Protein Coding Genes (PCGs), 25 tRNA genes, and 3 rRNA genes. One repetitive sequence with a length of 410.2 bp that mediated homologous recombination and led to the formation of two possible conformations, was identified in the sea buckthorn mitochondrial genome. This research provides valuable insights for a better understanding of the genetics of sea buckthorn and other higher plants. Materials and Methods Experimental Materials The experimental samples were collected from a 405 years old sea buckthorn orchard located in Liupingdi Village, Changsheng Town, Aohan Banner, Chifeng City, and fresh young leaves were collected. The material used in the experiment is the high yield variety "Yulu 1" recognized by the provincial level of sea buckthorn. The collected leaves were rapidly frozen using liquid nitrogen and stored in a -80°C freezer. Later, DNA was extracted from the leaf samples through liquid nitrogen grinding. Experimental Determination Indicators Mitochondrial Genome Assembly The sea buckthorn mitochondrial genome was assembled using long-read sequencing data. The Flye software [ 69 ] was employed with default parameters to perform direct assembly of the long-read sequencing data, resulting in a graphical assembly in GFA format. For all contigs assembled in fasta format, a database was constructed using the make BLASTb tool. Subsequently, the BLASTn program was used with the conserved mitochondrial genes from Arabidopsis thaliana as query sequences to identify contig fragments containing mitochondrial genome sequences. The parameters used for BLASTn were "-evalue le-5 -outfmt 6 -max_hsps 10-word_size 7-task blastn-short." The Bandage software (version 0.8.1) [ 70 ] was used to visualize the GFA file, and contigs containing the sea buckthorn mitochondrial genome were selected based on the BLASTn results, resulting in a draft mitochondrial genome map for sea buckthorn. The sequencing strategy of this study is Illumina (second generation) + Nanopore (third generation). We used DNBSEQ and Nanopore GridION (Oxford Nanopore Technology, Oxford Science Park) sequencing platforms for sequencing and library construction to obtain raw sequence data. Low quality sequences are then removed using Trimmomatic. removing sequences with a mass value of Q < 19. Validation and Correction of Mitochondrial Genome The long-reads and short-reads data were aligned to the mitochondrial contigs using the BWA software (version 0.7.17) [ 71 ]. Reads that aligned to the mitochondrial contigs were filtered and exported for further use in the assembly. These filtered reads were saved for subsequent hybrid assembly. A hybrid assembly strategy was employed to assemble the sea buckthorn mitochondrial genome, utilizing both the short-reads and long-reads data. The Unicycler software (version 60) [ 72 ] was used with default parameters to perform the hybrid assembly, resulting in the final assembly of the sea buckthorn mitochondrial genome. The Bandage software (version 0.8.1) [ 70 ] was used for the visualization of this mitochondrial genome. Mitochondrial Genome Annotation The protein-coding genes of the mitochondrial genome were annotated using Geseq software (version 2.03) [ 73 ], with Arabidopsis thaliana (NC_037304) and Liriodendron tulipifera (NC_021152.1) as reference genomes. The tRNA genes within the mitochondrial genome were annotated using tRNA scan-SE software (version 2.0.11) [ 74 ], while the rRNA genes were annotated using BLASTN software (version 2.13.0) [ 75 ]. Any annotation errors in the mitochondrial genome were manually corrected using Apollo software (version 1.11.8) [ 76 ]. Mitochondrial Genome Codon Usage Bias Analysis The protein-coding sequences of the mitochondrial genome were extracted using Phylosuite software (version 1.1.16) [ 77 ]. Codon usage bias analysis of the protein-coding genes in the mitochondrial genome was performed using Mega software (version 7.0) [ 78 ], and the Relative Synonymous Codon Usage (RSCU) values were calculated. Mitochondrial Genome Repeat Sequence Analysis We identified dispersed repetitive sequences in the mitochondrial genome using Rousfinder with default parameters, with a minimum repeat unit length of 50 bp. Repeat sequences, including microsatellite sequence repeats, tandem repeats, and dispersed repeats, were identified using the following tools: MISA (v2.1): Microsatellite sequences were identified using the MISA software, accessible at https://webblast.ipk-gatersleben.de/misa/ [ 79 ]. TRF (v4.09): Tandem repeats were identified using the Tandem Repeats Finder (TRF) software, available at https://tandem.bu.edu/trf/trf.unix.help.html [ 80 ]. Rousfinder web server: Dispersed repeats were identified using the REPuter web server, which can be found at https://bibiserv.cebitec.uni-bielefeld.de/reputer/ [ 81 ]. The results were visualized using Excel (2021) software and the Circos package (v0.69-9) [ 82 ]. Mitochondrial Genome Structure Analysis and PCR Amplification Validation The mixed assembly was performed using default parameters with Unicycler [ 73 ]. Based on the sea buckthorn mitochondrial genome data, sequences of 500 bp upstream and downstream of the junction sites were extracted and recombined to form two additional configurations.Primers were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) and synthesized by Genewiz (Shanghai) Co., Ltd. The PCR reaction system had a total volume of 50 µL, consisting of 1 µL template DNA, 2 µL forward primer, 2 µL reverse primer, 25 µL 2× Phanta Max Master Mix, and 20 µL ddH2O. The PCR program included an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s. The final extension was performed at 72°C for 15 min. Phylogenetic Analysis Based on phylogenetic relationships, 32 closely related species were selected, and their mitochondrial genomes were downloaded. Vigna radiata (mitochondrial: NC_015121.1) and Glycine max (mitochondrial: NC_020455.1) were chosen as outgroups. Common genes were extracted using PhyloSuite software [ 77 ], followed by multiple sequence alignment analysis using MAFFT software (v7.505) [ 83 ]. Systematic phylogenetic analysis was conducted using IQ-TREE software (v1.6.12) [ 84 ]. The results of the phylogenetic analysis were visualized using ITOL software (v6) [ 85 ]. RNA Editing Event Analysis The sequences of all Protein Coding Genes (PCGs) encoded by the mitochondrial genome of this species were used as input files for the prediction of C to U RNA editing sites in mitochondrial PCGs using Deepred-mt [ 86 ]. This tool is based on a Convolutional Neural Network (CNN) model for prediction and offers higher accuracy compared to previous prediction tools. All results with a probability value greater than 0.9 were retained. PhyloSuite software was used to extract shared genes, multiple sequence alignment analysis was performed using MAFFT software (v7.505), then phylogenetic analysis was performed using IQ-TREE software (v1.6.12), and the results of phylogenetic analysis were visualized using ITOL software (v6). Co-linearity Analysis BLAST program was used to obtain pairwise comparisons of mitochondrial genomes for all species, and sequences with a length exceeding 500 bp were retained as conserved syntenic blocks for generating a Multiple Synteny Plot. Based on sequence similarity, we utilized the source code of MCscanX [ 87 ] to create a Multiple Synteny Plot comparing the mitochondrial genome of the sea buckthorn with its closely related species. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data availability Data is provided within the manuscript. Competing interests The authors declare no competing interests. Funding This study was supported by grants from the demonstration and popularization of rapid propagation and intensive management technology of seabuckthorn （2022ZY0094）. Author Contributions L.D. and Y.N.T. contributed equally to this work. X.J. M. and X.Y. L. designed the study. L.D., Y.N. T.,T.L. and X.J L. performed the work, analyzed data,and wrote the manuscript. J.M.Z. and X.S.W. provided advice on interpretation of data, and editing of the manuscript. All authors contributed to and approved the manuscript. Acknowledgements We sincerely thank all researchers who unselfishly. References You C, Cui T, Zhang C, et al. Assembly of the Complete Mitochondrial Genome of Gelsemium elegans Revealed the Existence of Homologous Conformations Generated by a Repeat Mediated Recombination[J]. Int J Mol Sci. 2022;24(1):527. Spinelli JB, Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism[J]. Nat Cell Biol. 2018;20(7):745–54. Abate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence[C]//Seminars in cell & developmental biology. Acad Press. 2020;98:139–53. Maréchal A, Brisson N. Recombination and the maintenance of plant organelle genome stability[J]. New Phytol. 2010;186(2):299–317. Tao M, Zhu W, Han H, et al. Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment[J]. J Proteom. 2022;254:104410. Li F, Xiang R, Liu Y et al. Approaches and challenges in identifying, quantifying, and manipulating dynamic mitochondrial genome variations[J]. Cell Signal, 2024: 111123. Shtolz N, Mishmar D. The mitochondrial genome–on selective constraints and signatures at the organism, cell, and single mitochondrion levels. Front Ecol Evol. 2019;7:342. Gras DE, Mansilla N, Rodríguez C, Welchen E, Gonzalez DH. Arabidopsis thaliana SURFEIT1-like genes link mitochondrial function to early plant development and hormonal growth responses. Plant J Cell Mol Biol. 2020;103:690–704. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[J]. Cell. 1996;86(1):147–57. Tang J, Luo Z, Zhang J, et al. Multi-Chromosomal mitochondrial genome of medicinal plant Acorus tatarinowii (Acoraceae): Firstly reported from Acorales Order[J]. Gene. 2024;892:147847–. Moller IM, Rasmusson AG, Van Aken O. Plant mitochondria–past, present and future[J]. Plant J. 2021;108(4):912–59. Baluška F, Lyons S. Archaeal origins of eukaryotic cell and nucleus[J]. BioSystems. 2021;203:104375. Takenaka M. Quantification of mitochondrial RNA editing efficiency using sanger sequencing data[J]. Plant Mitochondria: Methods Protocols, 2022: 263–78. Petersen G, Anderson B, Braun HP, et al. Mitochondria in parasitic plants[J]. Mitochondrion. 2020;52:173–82. Alverson AJ, Wei XX, Rice DW, et al. Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)[J]. Volume 27. Molecular biology and evolution; 2010. pp. 1436–48. 6. Bi C, Lu N, Xu Y, et al. Characterization and analysis of the mitochondrial genome of common bean (Phaseolus vulgaris) by comparative genomic approaches[J]. Int J Mol Sci. 2020;21(11):3778. Kobayashi Y, Odahara M, Sekine Y, et al. Holliday junction resolvase MOC1 maintains plastid and mitochondrial genome integrity in algae and bryophytes[J]. Plant Physiol. 2020;184(4):1870–83. Cheng L, Wang W, Yao Y, et al. Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis[J]. PLoS Biol. 2021;19(8):e3001357. Odahara M, Nakamura K, Sekine Y, et al. Ultra-deep sequencing reveals dramatic alteration of organellar genomes in Physcomitrella patens due to biased asymmetric recombination[J]. Commun Biology. 2021;4(1):633. Wang S, Li D, Yao X, et al. Evolution and diversification of kiwifruit mitogenomes through extensive whole-genome rearrangement and mosaic loss of intergenic sequences in a highly variable region[J]. Genome Biol Evol. 2019;11(4):1192–206. Chustecki JM, Johnston IG. Collective mitochondrial dynamics resolve conflicting cellular tensions: From plants to general principles[C]//Seminars in Cell & Developmental Biology. Volume 156. Academic; 2024. pp. 253–65. Gâtlan AM, Gutt G. Sea buckthorn in plant based diets. An analytical approach of sea buckthorn fruits composition: Nutritional value, applications, and health benefits[J]. Int J Environ Res Public Health. 2021;18(17):8986. Zeng Z, Zhang Z, Tso N, et al. Complete mitochondrial genome of Hippophae tibetana: insights into adaptation to high-altitude environments[J]. Front Plant Sci. 2024;15:1449606. Fajardo D, Schlautman B, Steffan S, et al. The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants[J]. Gene. 2014;536(2):336–43. Liu J, Yu S, Lü P, et al. De novo assembly and characterization of the complete mitochondrial genome of Phellodendron amurense reveals three repeat-mediated recombination[J]. Gene. 2025;935:149031. Zhang C, Zhang Q, Jiang J, et al. Pullout properties of Hippophae rhamnoides L. roots in the loess area[J]. Arch Agron Soil Sci. 2023;69(11):2170–86. Yang X, Liu Y, Tang Z, et al. Total flavonoids of Hippophae rhamnoides L. improves type 2 diabetes symptoms in rats through down-regulating of the DAG/PRKCA/MAPK10/p65/TNF-α signalling pathway[J]. J Ethnopharmacol. 2024;318:116962. Yao Y, Dong L, Fu X, et al. HrTCP20 dramatically enhance drought tolerance of sea buckthorn ( Hippophae rhamnoides L ). by mediating the JA signaling pathway[J]. Plant Physiol Biochem. 2022;174:51–62. He ZS, Zhu A, Yang JB, et al. Organelle genomes and transcriptomes of Nymphaea reveal the interplay between intron splicing and RNA editing[J]. Int J Mol Sci. 2021;22(18):9842. Van Aken O. Mitochondrial redox systems as central hubs in plant metabolism and signaling[J]. Plant Physiol. 2021;186(1):36–52. Kozik A, Rowan BA, Lavelle D, Berke L, Schranz ME, Michelmore RW, Christensen AC. The alternative reality of plant mitochondrial DNA: One ring does not rule them all. PLoS Genet. 2019, 15, e1008373. Yang H, Chen H, Ni Y, et al. De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species[J]. Int J Mol Sci. 2022;23(22):14267. Li J, Li J, Ma Y, et al. The complete mitochondrial genome of okra (Abelmoschus esculentus): Using nanopore long reads to investigate gene transfer from chloroplast genomes and rearrangements of mitochondrial DNA molecules[J]. BMC Genomics. 2022;23(1):481. Liu D, Guo H, Zhu J, et al. Complex physical structure of complete mitochondrial genome of Quercus acutissima (Fagaceae): A significant energy plant[J]. Genes. 2022;13(8):1321. Wu ZQ, Liao XZ, Zhang XN, et al. Genomic architectural variation of plant mitochondria—A review of multichromosomal structuring[J]. J Syst Evol. 2022;60(1):160–8. Yang H, Chen H, Ni Y, et al. Mitochondrial Genome Sequence of Salvia officinalis (Lamiales: Lamiaceae) Suggests Diverse Genome Structures in Cogeneric Species and Finds the Stop Gain of Genes through RNA Editing Events[J]. Int J Mol Sci. 2023;24(6):5372. Alverson AJ, Rice DW, Dickinson S, et al. Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber[J]. Plant Cell. 2011;23(7):2499–513. Yang JX, Dierckxsens N, Bai MZ, et al. Multichromosomal mitochondrial genome of Paphiopedilum micranthum: Compact and fragmented genome, and rampant intracellular gene transfer[J]. Int J Mol Sci. 2023;24(4):3976. Debrauwere H, Gendrel CG, Lechat S, et al. Differences and similarities between various tandem repeat sequences: minisatellites and microsatellites[J]. Biochimie. 1997;79(9–10):577–86. Mehrotra S, Goyal V. Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function[J]. Genomics Proteom Bioinf. 2014;12(4):164–71. Fang B, Li J, Zhao Q et al. Assembly of the complete mitochondrial genome of Chinese plum (Prunus salicina): Characterization of genome recombination and RNA editing sites[J]. Genes, 2021, 12(12): 1970. Yang Z, Ni Y, Lin Z, et al. De novo assembly of the complete mitochondrial genome of sweet potato (Ipomoea batatas [L.] Lam) revealed the existence of homologous conformations generated by the repeat-mediated recombination[J]. BMC Plant Biol. 2022;22(1):285. Li YC, Korol AB, Fahima T, et al. Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review[J]. Mol Ecol. 2002;11(12):2453–65. Guo W, Grewe F, Fan W, et al. Ginkgo and Welwitschia mitogenomes reveal extreme contrasts in gymnosperm mitochondrial evolution[J]. Mol Biol Evol. 2016;33(6):1448–60. Yu X, Ma Z, Liu S, et al. Analysis of the Rhodomyrtus tomentosa mitochondrial genome: Insights into repeat-mediated recombination and intra-cellular DNA transfer[J]. Gene. 2024;909:148288. Mileshina D, Ibrahim N, Boesch P, et al. Mitochondrial transfection for studying organellar DNA repair, genome maintenance and). aging[J]. Mech Ageing Dev. 2011;132(8–9):412–23. Chevigny N, Schatz-Daas D, Lotfi F, Gualberto JM. DNA Repair and the Stability of the Plant Mitochondrial Genome. Int J Mol Sci. 2020;21(1):328. 10.3390/ijms21010328 . PMID: 31947741; PMCID: PMC6981420. Wolfe KH, Li WH, Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc. Natl. Acad. Sci. USA. 1987; 84:9054–9058. 10.1073/pnas.84.24.9054 Palmer JD, Herbon LA. Plant mitochondrial DNA evolved rapidly in structure, but slowly in sequence[J]. J Mol Evol. 1988;28:87–97. Lovett ST, Hurley RL, Sutera VA Jr., Aubuchon RH, Lebedeva MA. Crossing over between regions of limited). homology in E. coli. RecA-dependent and RecA-independent pathways. Genetics. 2002;160:851–9. Lovett ST, Hurley RL, Sutera VA Jr., Aubuchon RH, Lebedeva MA. Crossing over between regions of limited). homology in E. coli. RecA-dependent and RecA-independent pathways. Genetics. 2002;160:851–9. Davila JI, Arrieta-Montiel MP, Wamboldt Y, et al. Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis[J]. BMC Biol. 2011;9:1–14. Miller-Messmer M, Kühn K, Bichara M, et al. RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome[J]. Plant Physiol. 2012;159(1):211–26. Sun M, Zhang M, Chen X, et al. Rearrangement and domestication as drivers of Rosaceae mitogenome plasticity[J]. BMC Biol. 2022;20(1):181. Wynn EL, Christensen AC. Repeats of unusual size in plant mitochondrial genomes: identification, incidence and evolution. G3 (Bethesda). 2019;9:549–J559. Hong Z, Liao X, Ye Y, et al. A complete mitochondrial genome for fragrant Chinese rosewood (Dalbergia odorifera, Fabaceae) with comparative analyses of genome structure and intergenomic sequence transfers[J]. BMC Genomics. 2021;22(1):1–13. Chen Z, Zhao N, Li S, et al. Plant mitochondrial genome evolution and cytoplasmic male sterility[J]. CRC Crit Rev Plant Sci. 2017;36(1):55–69. Guo W, Grewe F, Mower JP. Variable frequency of plastid RNA editing among ferns and repeated loss of uridine-to-cytidine editing from vascular plants[J]. PLoS ONE. 2015;10(1):e0117075. Wang M, Liu H, Ge L, et al. Identification and analysis of RNA editing sites in the chloroplast transcripts of Aegilops tauschii L[J]. Genes. 2016;8(1):13. Liu J, Ni Y, Liu C. Polymeric structure of the Cannabis sativa L. mitochondrial genome identified with an assembly graph model[J]. Gene. 2023;853:147081. Liu Q, Yuan H, Xu J, et al. The mitochondrial genome of the diploid oat Avena longiglumis[J]. BMC Plant Biol. 2023;23(1):218. Jiang H, Lu Q, Qiu S et al. Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice[J]. Proceedings of the National Academy of Sciences, 2022, 119(34): e2208759119. Lai C, Wang J, Kan S, et al. Comparative analysis of mitochondrial genomes of Broussonetia spp.(Moraceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing[J]. Front Plant Sci. 2022;13:1052151. Timmis JN, Ayliffe MA, Huang CY, et al. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[J]. Nat Rev Genet. 2004;5(2):123–35. Stupar RM, Lilly JW, Town CD et al. Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: implication of potential sequencing errors caused by large-unit repeats[J]. Proceedings of the National Academy of Sciences, 2001, 98(9): 5099–5103. Cao P, Huang Y, Zong M, et al. De Novo Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Chaenomeles speciosa (Sweet) Nakai Revealed the Existence of Two Structural Isomers[J]. Genes. 2023;14(2):526. Bi C, Paterson AH, Wang X et al. Analysis of the complete mitochondrial genome sequence of the diploid cotton Gossypium raimondii by comparative genomics approaches[J]. BioMed Research International, 2016, 2016. Cheng Y, He X, Priyadarshani S, et al. Assembly and comparative analysis of the complete mitochondrial genome of Suaeda glauca[J]. BMC Genomics. 2021;22(1):1–15. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019;37(5):540–6. Wick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015;31(20):3350–2. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. Michael T, Pascal L, Tommaso P, Ulbricht-Jones ES, Axel F, Ralph B, Stephan G. GeSeq –versatile and accurate annotation of organelle genomes. Nucleic Acids Res 2017(W1):W1. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25(5):955–64. Chen Y, Ye W, Zhang Y, Xu Y. High speed BLASTN: an accelerated MegaBLAST search tool. Nucleic Acids Res. 2015;43(16):7762–8. Lewis SE, Searle S, Harris N, Gibson M, Iyer V, Richter J, Wiel C, Bayraktaroglu L, Birney E, Crosby M. Apollo: a sequence annotation editor. Genome Biol. 2002;3(12):1–14. Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT. PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Resour. 2020;20(1):348–55. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol biology Evol. 2016;33(7):1870–4. Beier S, Thiel T, Münch T, Scholz U, Mascher M. MISA-web: a web server for microsatellite prediction. Bioinformatics. 2017;33(16):2583–5. Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999;27(2):573–80. Wynn EL, Christensen AC. Repeats of unusual size in plant mitochondrial genomes: identification, incidence and evolution[J]. Volume G3. Genes, Genomes, Genetics; 2019. pp. 549–59. 2. Zhang H, Meltzer P, Davis S. RCircos: an R package for Circos 2D track plots. BMC Bioinformatics. 2013;14(1):1–5. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268–74. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47(W1):W256–9. Edera AA, Small I, Milone DH, Sanchez-Puerta MV. Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria. Comput Biol Med. 2021;136:104682. Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, Lee T-h, Jin H, Marler B, Guo H. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49–49. Additional Declarations No competing interests reported. Supplementary Files Seabuckthorndata.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5607252","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445799784,"identity":"d0b9995c-9090-4052-9dba-d2ab0d9b52d1","order_by":0,"name":"Lei Ding","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Ding","suffix":""},{"id":445799785,"identity":"2ab9da63-30c2-4149-b587-de7a10db0be6","order_by":1,"name":"Yana Tong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yana","middleName":"","lastName":"Tong","suffix":""},{"id":445799786,"identity":"71b041e4-31af-4904-b825-ca05261ec987","order_by":2,"name":"JiaMin Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"JiaMin","middleName":"","lastName":"Zhang","suffix":""},{"id":445799787,"identity":"31f122e5-96b7-405f-819e-8e5ce5780663","order_by":3,"name":"te Lu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"te","middleName":"","lastName":"Lu","suffix":""},{"id":445799788,"identity":"b731c7c6-7438-4062-b381-1f81ec898eaa","order_by":4,"name":"Xuesong Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuesong","middleName":"","lastName":"Wang","suffix":""},{"id":445799790,"identity":"141eca56-e722-4c70-83f4-c53cf523ce20","order_by":5,"name":"Xinjing Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xinjing","middleName":"","lastName":"Liu","suffix":""},{"id":445799792,"identity":"216a45e3-f2a4-48d2-8cd7-e9731f1f1377","order_by":6,"name":"Xiaojun Ma","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Ma","suffix":""},{"id":445799793,"identity":"f4962d47-42e0-4b3b-9056-7b142a6f8fec","order_by":7,"name":"Xianyu Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCcaGD0CKh5/h8MEHCRU1RGlpnAGkZCQbjyUbPDhzjBgtDIwgLTYGh8+YST5sYSasg392c2PDxx21PAbHjqVVJDawMfC3dyfgt+TOwcbGmWeO80ieOXzsRuIOGQaJM2c34NViIJHY/pi37RgP341jaTcSz7ABRXIJamls/gvUwnD/jVlBYhszkVoY22p4BA6cMWMgSovEjcTGxt62AzySDceSJRLOHOMh6Bf+GekPG3621dmDovLjj4oaOf72XvxaoOAwnMVDjHIQqCNW4SgYBaNgFIxEAAAxbVP9Mk7Q0QAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Xianyu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-12-09 08:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5607252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5607252/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81233738,"identity":"78a5de4c-19ca-47c4-a851-d4c12db6d8f5","added_by":"auto","created_at":"2025-04-23 18:38:49","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30701,"visible":true,"origin":"","legend":"\u003cp\u003erepresents a schematic diagram of the sea buckthorn mitochondrial genome. The diagram consists of four nodes, each labeled with a name, and each node represents a contig obtained through assembly. If two nodes are connected by a black line, it indicates the presence of an overlapping region between the two sequences. All of these sequences together form a complex, multi-branch closed genome structure, representing the complete mitochondrial genome sequence of this species.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/ba8be060730194bab36d4c86.jpeg"},{"id":81233970,"identity":"b2a16086-55f5-4796-ad6d-d6a1cc47884b","added_by":"auto","created_at":"2025-04-23 18:46:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99377,"visible":true,"origin":"","legend":"\u003cp\u003eCircular Map of the Sea Buckthorn Mitochondrial Genome. Genomic features of genes transcribed in the clockwise and counterclockwise directions are represented on the inner and outer circles, respectively. Color cod-ing is used to differentiate genes belonging to different functional groups\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/28050d0783fc8e0ffa6fec29.jpeg"},{"id":81234409,"identity":"cf02c5b1-4674-4f5a-8ec3-2c398754c994","added_by":"auto","created_at":"2025-04-23 18:54:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68663,"visible":true,"origin":"","legend":"\u003cp\u003e(A) displays the relationship between mitochondrial molecules and the number of repetitive segments. The horizontal axis represents different mitochondrial molecules, while the vertical axis represents the corresponding number of repetitive segments. In the figure, blue represents mononucleotide SSRs, orange represents dinucleotide SSRs, gray represents trinucleotide SSRs, green represents tetranucleotide SSRs, purple represents pentanucleotide SSRs, and red represents hexanucleotide SSRs.\u003cstrong\u003e Fig. 3 \u003c/strong\u003e(B) illustrates the association between mitochondrial molecules and the number of different types of repetitive segments. The horizontal axis represents different mitochondrial molecules, while the vertical axis represents the corresponding number of repetitive segments. In the figure, purple represents tandem repeats, green represents palindromic repeats, yellow represents forward repeats, red represents reverse repeats, and blue represents complementary repeats.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/1516723dbfbf892f70dcc79d.jpeg"},{"id":81234406,"identity":"056440ca-063a-4293-8a47-478357c9471d","added_by":"auto","created_at":"2025-04-23 18:54:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27663,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic Diagram of Primer Design for Repetitive Sequence-Mediated Homologous Recombination Based on the sea buckthorn mitochondrial genome data, sequences of 500 bp upstream and downstream of the junction sites were extracted. Four pairs of verification primers were designed to validate the junction sites between contigs in the sea buckthorn mitochondrial genome, as shown in Fig. 5. All junction sites between contigs could successfully amplify bands of the expected size. Sanger sequencing results demonstrated that the PCR products were consistent with the template sequences, confirming that the repetitive sequence contig4 in the sea buckthorn mitochondrial genome could mediate chromosomal recombination, resulting in the two conformations depicted in Fig.6. The two conformations could be interconverted through contig4.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/ffe71fa5c2d819580fd8ff1e.png"},{"id":81234408,"identity":"8457d44f-db25-4741-a6eb-40fb592f9c33","added_by":"auto","created_at":"2025-04-23 18:54:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":365399,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of Homologous Recombination Mediated by Repetitive Sequences. A represents the con-figurations of the sea buckthorn mitochondrial genome. B represents the validation of the mitochondrial genome junction points in sea buckthorn. The experiments from left to right include: Marker, Primer F1/R1 PCR results, Primer F2/R2 PCR results, Primer F3/R3 PCR results, Primer F4/R4 PCR results.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/647cad8b877c816850380fe8.png"},{"id":81233746,"identity":"ab6893c6-f19e-4775-a617-954f1a0b2491","added_by":"auto","created_at":"2025-04-23 18:38:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":275729,"visible":true,"origin":"","legend":"\u003cp\u003eGenome Configurations Generated by Homologous Recombination Mediated by the Repetitive Sequence contig4. In the figure, the red section on the chromosome represents ctg4, which represents the repetitive sequence. The circles represent configurations of the mitochondrial genome.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/dee64e1fa3afdcc85d4359ea.png"},{"id":81233751,"identity":"2b563025-b07a-4681-9bf3-801e101b57a0","added_by":"auto","created_at":"2025-04-23 18:38:50","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":126659,"visible":true,"origin":"","legend":"\u003cp\u003eHomologous Segments between Sea Buckthorn Mitochondrial and Chloroplast Genomes. In the figure, the purple arc represents the mitochondrial genome, the green arc represents the chloroplast genome, and the pink lines connecting the arcs correspond to homologous segments between the two genomes.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/6f474cd044155bcbc14cefb6.jpeg"},{"id":81233973,"identity":"ce6ba28e-cf37-44d7-a381-25a403f87e20","added_by":"auto","created_at":"2025-04-23 18:46:49","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":184334,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic Analysis of Sea Buckthorn (\u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/a26ecd38a2e51540a95ef6b4.jpeg"},{"id":81233977,"identity":"6e763d0f-2c3e-4041-b9d1-f09ea966516f","added_by":"auto","created_at":"2025-04-23 18:46:49","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":169567,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of RNA Editing Events in the Sea Buckthorn (\u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e) Mitochondrial Genome. The horizontal axis represents gene names, and the vertical axis represents the number of RNA editing events.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/87726af77616c079a6910939.jpeg"},{"id":81233975,"identity":"bd1eb6ab-561a-4ee4-b24f-031fa3783434","added_by":"auto","created_at":"2025-04-23 18:46:49","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":86096,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of RNA Editing Events in the Tibetan Sea Buckthorn \u003cem\u003e(Hippophae tibetana\u003c/em\u003e) Mitochondrial Genome. The horizontal axis represents gene names, and the vertical axis represents the number of RNA editing events.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/ce94363343097e25289135bc.jpeg"},{"id":81233759,"identity":"ea784d89-fa95-42cc-bad4-ad60a7e34664","added_by":"auto","created_at":"2025-04-23 18:38:50","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":107359,"visible":true,"origin":"","legend":"\u003cp\u003eCo-linearity Analysis between Sea Buckthorn and Closely Related Species. The red curved regions represent inverted regions, and the gray regions represent regions with good homology. To better visualize the results, co-linearity blocks with lengths less than 0.5 kb are not retained in the results.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/9e46def2fae957878f4f3d72.jpeg"},{"id":109615489,"identity":"a5454a76-52fe-4e86-92d0-8be0964bd7d3","added_by":"auto","created_at":"2026-05-20 08:26:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1825935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/a178648f-4e39-4bfc-9539-c70e9ed98d01.pdf"},{"id":81233739,"identity":"d3c05499-4875-4166-8a26-ace7dbec330b","added_by":"auto","created_at":"2025-04-23 18:38:49","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17809,"visible":true,"origin":"","legend":"","description":"","filename":"Seabuckthorndata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5607252/v1/9d3f03723a62824d1b666f4a.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural Features and Synteny Analysis of the Sea Buckthorn Mitochondrial Genome","fulltext":[{"header":"Background","content":"\u003cp\u003eMitochondria are essential semi-autonomous organelles in eukaryotic cells, possessing their own genetic material and genetic systems that operate independently of the plant nuclear genome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition to providing energy to the cell, mitochondria are involved in various metabolic processes such as cell differentiation, cell signaling [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and apoptosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Mitochondria are often referred to as the \u0026ldquo;cellular powerhouses\u0026rdquo; [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and play a crucial role in the synthesis and metabolism of vital substances related to life activities, including vitamins, lipids, and amino acids [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The outer mitochondrial membrane serves as a signaling platform, providing scaffolding for many key proteins involved in cell signaling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and these functions are of paramount importance in the growth and development of plants [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn important characteristic of plant mitochondrial genomes is the presence of extensive RNA editing sites and the utilization of a universal genetic code [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], involving single base substitutions and the addition of bases to complete start or stop codons [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, a phenomenon known as RNA editing occurs in both animal and plant mitochondria, where mRNA sequences generated by gene transcription are not perfectly identical to the template sequences. RNA editing is a post-transcriptional modification that alters mRNA produced by gene transcription [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Compared to animals, plant mitochondrial genomes are larger, more plastic [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and may contain increased phylogenetic signals. One of the primary advantages of mitochondrial genome as a phylogenetic marker [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] is the differential substitution rates among its 36 genes, with some genes exhibiting higher rates of substitution than others, facilitating the tracking of relatively recent divergences [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, mitochondrial genome is associated with cytoplasmic male sterility (CMS), a condition controlled by genes within the mitochondria, expressed maternally, and can be restored by nuclear genes known as restorer of fertility (Rf) genes. In the absence of Rf genes in the nuclear genome, plants exhibit male gametophyte or pollen sterility while the female reproductive organs remain normal [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].At present, the study of plant mitochondrial genome has become a hot spot. Zeng et al. studied and analyzed the mitochondrial genome of Hippophae tibetana, which has 1 chromosome and is 464,208 bp long. It included 31 tRNA genes, 3 rRNA genes, 37 protein-coding genes and 3 pseudogenes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; Fajardo et al. found that the mitochondrial genome of American cranberry consisted of a single mitochondrial chromosome 459,678 bp in length, with 34 protein-coding genes, 3 rRNA genes, and 17 tRNA genes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; Liu et al. studied the mitochondrial genome of Phellodendron amurense, a total of two mitochondrial genomes with a length of 566,285 bp, and identified 32 protein-coding genes, 33 tRNA genes and 3 rRNA genes. There were 181 simple sequence repeats, 19 tandem repeats, and 310 scattered repeats, all with multiple conformations [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSea buckthorn \u003cem\u003e(Hippophae rhamnoides\u003c/em\u003e L.) is a deciduous shrub or small arbor belonging to the family Elaeagnaceae. It exhibits strong ecological adaptability and remarkable resilience, demonstrating tolerance to drought, cold, salinity, barrenness, and nitrogen fixation capabilities. Sea buckthorn can endure extreme temperatures ranging from \u0026minus;\u0026thinsp;40\u0026deg;C to 40\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, it is recognized for its soil improvement and water conservation functions, making it a pioneer tree species in afforestation of barren hills and slopes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Sea buckthorn is also a plant with both medicinal and culinary uses. Its fruits are rich in nutrients and bioactive compounds, including proteins, vitamins, polysaccharides, flavonoids, and terpenes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. With advancements in molecular biology techniques, molecular biology studies of sea buckthorn have made progress, providing a foundation for elucidating the biosynthesis of secondary metabolites and the discovery of functional genes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, in-depth study of its mitochondrial genome is of great significance for the effective utilization and genetic improvement of sea buckthorn.\u003c/p\u003e \u003cp\u003eCurrently, the NCBI database contains genomic information for 35,757 eukaryotic organisms, with the sea buckthorn complete genome submitted in October 2023 but remains unreported. Information on sea buckthorn mitochondrial genome, including its length, GC content, gene count, codon preferences, repetitive sequences, phylogeny, sequence migration, and RNA editing events, is still lacking. In this study, the sequencing, assembly, and annotation of the sea buckthorn mitochondrial genome were performed, and its genomic and structural characteristics were investigated. The existence of various conformations of the sea buckthorn mitochondrial genome was further confirmed through PCR experiments and Sanger sequencing, providing theoretical support for cytoplasmic male sterility breeding in higher plants and plant evolution.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStructural Features of the Sea Buckthorn Mitochondrial Genome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sea buckthorn mitochondrial genome was sequenced, assembled, and annotated in this study. After eliminating duplicate regions from Nanopore data, two major circular overlapping contigs were obtained, which were used to construct the sea buckthorn mitochondrial genome as shown in Fig. 1. It was observed that the sea buckthorn mitochondrial genome consists of two chromosomes, as depicted in Fig. 2, with a total length of 454,544 bp and a GC content of 44.85%. Chromosome 1 and Chromosome 2 have lengths of 297,507 bp and 157,037 bp, respectively.\u003c/p\u003e\n\u003cp\u003eThe annotation of the sea buckthorn mitochondrial genome revealed the presence of 36 unique protein-coding genes, which could be categorized into 10 major classes, as shown in Table 1. Among these genes, there were 24 unique mitochondrial core genes and 12 non-core genes.\u003c/p\u003e\n\u003cp\u003eThe mitochondrial core genes included:\u003c/p\u003e\n\u003cp\u003e1. 5 ATP synthase genes (\u003cem\u003eatp\u003c/em\u003e1\u003cem\u003e, atp\u003c/em\u003e4\u003cem\u003e, atp\u003c/em\u003e6\u003cem\u003e, atp\u003c/em\u003e8\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;atp\u003c/em\u003e9).\u003c/p\u003e\n\u003cp\u003e2. 9 NADH dehydrogenase genes (\u003cem\u003enad\u003c/em\u003e1\u003cem\u003e, nad\u003c/em\u003e2\u003cem\u003e, nad\u003c/em\u003e3\u003cem\u003e, nad\u003c/em\u003e4\u003cem\u003e, nad\u003c/em\u003e4L\u003cem\u003e, nad\u003c/em\u003e5\u003cem\u003e, nad\u003c/em\u003e6\u003cem\u003e, nad\u003c/em\u003e7\u003cem\u003e,\u003c/em\u003e and\u003cem\u003e\u0026nbsp;nad\u003c/em\u003e9).\u003c/p\u003e\n\u003cp\u003e3. 4 Cytochrome C biogenesis genes (\u003cem\u003eccm\u003c/em\u003eB, \u003cem\u003eccm\u003c/em\u003eC, \u003cem\u003eccm\u003c/em\u003eFC, and \u003cem\u003eccm\u003c/em\u003eFN).\u003c/p\u003e\n\u003cp\u003e4. 3 Cytochrome C oxidase genes (\u003cem\u003ecox\u003c/em\u003e1\u003cem\u003e, cox\u003c/em\u003e2\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;cox\u003c/em\u003e3).\u003c/p\u003e\n\u003cp\u003e5. 1 Membrane transport protein gene (\u003cem\u003emtt\u003c/em\u003eB).\u003c/p\u003e\n\u003cp\u003e6. 1 Maturation enzyme gene (\u003cem\u003emat\u003c/em\u003eR).\u003c/p\u003e\n\u003cp\u003e7. 1 Ferrocytochrome C reductase gene (\u003cem\u003ecob\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThe non-core genes included:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1. 4 Ribosomal large subunit genes (\u003cem\u003erpl\u003c/em\u003e2, \u003cem\u003erpl\u003c/em\u003e5, \u003cem\u003erpl\u003c/em\u003e10, \u003cem\u003erpl\u003c/em\u003e16).\u003c/p\u003e\n\u003cp\u003e2. 6 Ribosomal small subunit genes (\u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e3, \u003cem\u003erps\u003c/em\u003e4, \u003cem\u003erps\u003c/em\u003e7, \u003cem\u003erps\u003c/em\u003e12, \u003cem\u003erps\u003c/em\u003e19).\u003c/p\u003e\n\u003cp\u003e3. 2 Succinate dehydrogenase genes (\u003cem\u003esdh\u003c/em\u003e3, \u003cem\u003esdh\u003c/em\u003e4).\u003c/p\u003e\n\u003cp\u003eIn addition to the protein-coding genes, the sea buckthorn mitochondrial genome also contained 25 tRNA genes, including 8 tRNA genes that had multiple copies, and 3 rRNA genes. This comprehensive annotation provided valuable insights into the gene composition and functional elements of the sea buckthorn mitochondrial genome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepeat Sequence Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Chromosome 1 of the sea buckthorn mitochondrial genome, a total of 111 SSRs were identified. Mononucleotide and dinucleotide SSRs accounted for 46.85% of the total SSRs. Among the mononucleotide SSRs, adenine (A) mononucleotide repeat sequences accounted for 60.00% (21 out of 35 mononucleotide SSRs). No hexanucleotide SSRs were detected. Chromosome 1 also contained 11 tandem repeat sequences with a match percentage greater than 69% and lengths ranging from 9 to 39 bp. In the analysis of scattered repeat sequences on Chromosome 1, a total of 41 pairs of repeat sequences were observed with a length of 50 or more, including 20 palindromic repeats, 10 forward repeats（1 reverse repeat, and no complementary repeats）. The longest palindromic repeat was 4,102 bp, while the longest forward repeat was 145 bp.\u003c/p\u003e\n\u003cp\u003eIn Chromosome 2 of the sea buckthorn mitochondrial genome, a total of 61 SSRs were found. Mononucleotide and dinucleotide SSRs constituted 55.74% of the total SSRs. Thymine (T) mononucleotide repeat sequences accounted for 52.38% (11 out of 21 mononucleotide SSRs). Chromosome 2 had 6 tandem repeat sequences with a match percentage greater than 83% and lengths ranging from 11 to 24 bp. Analysis of scattered repeat sequences on Chromosome 2 revealed a total of 9 pairs of repeat sequences with a length of 50 or more, including 2 palindromic repeats and 1 forward repeats. （No reverse repeats or complementary repeats were detected.） The longest palindromic repeat was 85 bp, while the longest forward repeat was 79 bp.\u003c/p\u003e\n\u003cp\u003eAnd 8 pairs of interspersed repeated sequences between Chromosome 1 and Chromosome 2 were detected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHomologous Recombination Mediated by Repetitive Sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that repetitive sequences can mediate homologous recombination in mitochondrial genomes [30]. We identified a total of two pairs of long repeat sequences that may mediate homologous recombination in the mitochondrial genome, namely R1 and R3. R1（coting 4） is 4102 bp long, with both repeat units located within contig1, potentially mediating an inversion of part of the sequence on contig1. There are 53 long-reads supporting our currently assembled configuration, while another 48 long-reads support the configuration after inversion, indicating that the inversion event occurs at a rate close to 50%. Notably, the two repeat units of R3 are located on two different circles, possibly acting as an important mediator for recombining the two small circles into a larger circular genome. However, our long-read data show that 93.79% of the long-reads (136 reads) support the independent existence of the two small circles, while only 6.21% of the long-reads (9 reads) support the recombination into a large circle. For the remaining short repeat sequences, we did not observe significant long-reads supporting recombination, but we cannot rule out the possibility of recombination as a lower frequency of recombination events might be difficult to detect. \u003cem\u003eHippophae tibetana\u003c/em\u003e (Xizang Sea Buckthorn) is a single ring structure, which is different from the sea buckthorn in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eNumber of long-reads and recombination rates supporting different configurations mediated by the two pairs of repeat sequences\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"553\" height=\"67\" src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img174543247591.png\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate whether the repetitive sequences can indeed mediate homologous recombination, PCR amplification and Sanger sequencing were conducted to confirm the presence of alternative conformations. The primer design strategy is depicted in Fig. 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of Sequence Transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sea buckthorn mitochondrial genome contains sequences known as mitochondrial plastid sequences (MTPTs). Based on sequence similarity analysis, a total of 30 segments were identified as homologous segments between the mitochondrial genome and the chloroplast genome (Fig. 7), with a cumulative length of 44,950 bp, constituting 9.89% of the total length of mitochondrial genome. Among them, MTPT6 was the longest, spanning 15,994 bp.\u003c/p\u003e\n\u003cp\u003eUpon annotation of these homologous sequences, 28 complete genes were identified within the 30 homologous segments. These genes included 15 protein-coding genes (\u003cem\u003ecem\u003c/em\u003eA\u003cem\u003e, clp\u003c/em\u003eP\u003cem\u003e, ndh\u003c/em\u003eB\u003cem\u003e, ndh\u003c/em\u003eJ\u003cem\u003e, pet\u003c/em\u003eA\u003cem\u003e, pet\u003c/em\u003eB\u003cem\u003e, psb\u003c/em\u003eC\u003cem\u003e, psb\u003c/em\u003eD\u003cem\u003e, psb\u003c/em\u003eH\u003cem\u003e, psb\u003c/em\u003eN\u003cem\u003e, psb\u003c/em\u003eT\u003cem\u003e, rpl\u003c/em\u003e23\u003cem\u003e, rps\u003c/em\u003e4\u003cem\u003e, rps\u003c/em\u003e7\u003cem\u003e, ycf\u003c/em\u003e2) and 13 tRNA genes (\u003cem\u003etrn\u003c/em\u003eD-GUC\u003cem\u003e, trn\u003c/em\u003eF-GAA\u003cem\u003e, trn\u003c/em\u003eH-GUG\u003cem\u003e, trn\u003c/em\u003eI-CAU\u003cem\u003e, trn\u003c/em\u003eL-CAA\u003cem\u003e, trn\u003c/em\u003eL-UAA\u003cem\u003e, trn\u003c/em\u003eM-CAU\u003cem\u003e, trn\u003c/em\u003eN-GUU\u003cem\u003e, trn\u003c/em\u003eP-UGG\u003cem\u003e, trn\u003c/em\u003eS-GGA\u003cem\u003e, trn\u003c/em\u003eT-UGU\u003cem\u003e, trn\u003c/em\u003eV-GAC\u003cem\u003e, trn\u003c/em\u003eW-CCA). The fact that the 13 tRNA sequences are conserved between the chloroplast and mitochondrial genomes suggests that they may still function within the mitochondrial genome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the DNA sequences of 26 conserved mitochondrial Protein Coding Genes (PCGs), a phylogenetic tree was constructed for 33 species from four orders of angiosperms (Fig. 8). The protein-coding genes used for the analysis were \u003cem\u003eatp\u003c/em\u003e1\u003cem\u003e, atp\u003c/em\u003e4\u003cem\u003e, atp\u003c/em\u003e6\u003cem\u003e, atp\u003c/em\u003e8\u003cem\u003e, ccm\u003c/em\u003eB\u003cem\u003e, ccm\u003c/em\u003eC\u003cem\u003e, ccm\u003c/em\u003eFC\u003cem\u003e, ccm\u003c/em\u003eFN\u003cem\u003e, cob, cox\u003c/em\u003e3\u003cem\u003e, mat\u003c/em\u003eR\u003cem\u003e, mtt\u003c/em\u003eB\u003cem\u003e, nad\u003c/em\u003e1\u003cem\u003e, nad\u003c/em\u003e2\u003cem\u003e, nad\u003c/em\u003e3\u003cem\u003e, nad\u003c/em\u003e4\u003cem\u003e, nad\u003c/em\u003e4L\u003cem\u003e, nad\u003c/em\u003e5\u003cem\u003e, nad\u003c/em\u003e6\u003cem\u003e, nad\u003c/em\u003e7\u003cem\u003e, nad\u003c/em\u003e9\u003cem\u003e, rpl\u003c/em\u003e2\u003cem\u003e, rpl\u003c/em\u003e16\u003cem\u003e, rps\u003c/em\u003e3\u003cem\u003e, rps\u003c/em\u003e4\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;rps\u003c/em\u003e19. Two mitochondrial genomes from the Fabales order were designated as outgroups. The topology of the phylogenetic tree based on mitochondrial DNA was consistent with the latest classification by the Angiosperm Phylogeny Group (APG). Sea buckthorn belonged to the order \u003cem\u003eRosales\u003c/em\u003e and the family \u003cem\u003eElaeagnaceae\u003c/em\u003e, and it clustered with \u003cem\u003eHippophae tibetana\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe are annotated \u003cem\u003eHippophae tibetana\u003c/em\u003e and found that its protein coding gene is consistent with the sea buckthorn studied in this study, and the \u003cem\u003erps\u003c/em\u003e19 also has two copies. We also analyzed the RNA editing and repeat sequences of \u003cem\u003eHippophae tibetana\u003c/em\u003e for comparison with \u003cem\u003eHippophae rhamnoides L\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSea Buckthorn (\u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e) RNA Editing Events\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA editing events in the 36 unique PCGs from the sea buckthorn mitochondrial genome were identified. A total of 430 potential RNA editing sites were identified across the 36 mitochondrial PCGs (Fig. 9), all involving the conversion of base C to U. Among the mitochondrial genes, the \u003cem\u003eccm\u003c/em\u003eB gene was found to have 35 RNA editing sites, making it the most extensively edited gene. Following \u003cem\u003eccmB\u003c/em\u003e, the nad5 gene exhibited 31 RNA editing events, indicating the second-highest frequency of editing among all mitochondrial genes.\u003c/p\u003e\n\u003cp\u003eThe same RNA editing events were observed in reports of Tibetan sea buckthorn. RNA editing events were also identified in 35 unique PCGs of the mitochondria of Tibetan sea buckthorn, a close relative of sea buckthorn, and a total of 427 potential RNA editing sites were identified on the 35 mitochondrial PCGs (Fig. 10), all of which were base C to U editing, the same as those of sea buckthorn. In the mitochondrial genes, the occurrence times of \u003cem\u003eccm\u003c/em\u003eB, the gene with the highest number of gene edits, and the second \u003cem\u003enad\u003c/em\u003e5 gene were also the same as those of Sea Buckthorn, However, there are differences between \u003cem\u003esdh\u003c/em\u003e3 and \u003cem\u003esdh\u003c/em\u003e4. There is no \u003cem\u003esdh\u003c/em\u003e3 RNA editing site in Tibetan Sea Buckthorn, and\u003cem\u003e\u0026nbsp;sdh\u003c/em\u003e4 RNA editing site is also less than sea buckthorn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-linearity Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-linearity analysis of sea buckthorn and closely related species (Fig. 11). The Chromosome 1 of sea buckthorn is highly coincident with the Tibetan sea buckthorn Chromosome Collinear Block and has a high degree of homology, while the Chromosome 2 of sea buckthorn is heavily inverted compared to the Tibetan sea buckthorn Chromosome. At the same time, homological collinear blocks were also detected in the near-origin species of sea buckthorn and Rosaceae, but the length of these collinear blocks was shorter. In addition, some blank areas were found, sequences that are unique to sea buckthorn and have no homology with the rest of the species. The results showed that the collinear block arrangement order among the mitochondrial genomes of these six species was inconsistent, and the mitochondrial genome of sea buckthorn underwent a large number of genome rearrangements between the mitochondrial genome and its relatives, and the mitochondrial genome was extremely unconserved in structure.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMultiple Circular Configurations of Plant Mitochondrial Genomes\u003c/h2\u003e \u003cp\u003eMitochondria play a crucial role in various metabolic processes related to the synthesis and degradation of intracellular substances, signal transduction, and energy production [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Plant mitochondrial genomes are more complex than animal mitochondrial genomes, exhibiting extensive differences in size, structure, sequence arrangement, and repetitive content [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Previous studies have shown that approximately 10% of plant mitochondrial genomes have a multipartite structure, with most mitochondrial genomes being circular, such as in \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, some are non-circular, and the mitochondrial genome of okra is likely to have multiple conformations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while the oak mitochondrial genome displays various structural variations [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In eukaryotes, not only do the structural shapes differ, but the repetitive evolution of chromosomes also varies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Sea buckthorn, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and \u003cem\u003esagebrush\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] share a similar pattern, cucumber mitochondrial genome has three independent chromosomes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and the mitochondrial genome of \u003cem\u003ePaphiopedilum micranthum\u003c/em\u003e consists of 26 small circular chromosomes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, in-depth studies on plant multipartite mitochondrial genomes will contribute to new discoveries in their genome evolution and molecular functions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHomologous Recombination Mediated by Repetitive Sequences\u003c/h2\u003e \u003cp\u003eDNA sequences in biological cells contain many repetitive elements, which can be broadly categorized into two main types: tandem repeats [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and dispersed repeats [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Among tandem repeats, there is a special type known as Short Sequence Repeat (SSR) or microsatellite repeat sequences. Previous studies have indicated the significance of repetitive sequences in mitochondrial genomes for intermolecular recombination. In plum mitochondrial genomes, three pairs of repetitive sequences mediated genome recombination were discovered, resulting in eight different conformations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In sweet potato, three pairs of direct repeat sequences were found to mediate homologous recombination, forming four independent circular molecules and leading to the formation of seven conformations [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin tandem repeats, Simple Sequence Repeat (SSR), a particular type of microsatellite repeat sequence typically consisting of no more than 6 bp, is often used for molecular marker development due to its dominant genetic characteristics [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The presence of abundant repetitive sequences in the sea buckthorn mitochondrial genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, supplementary data) suggested the frequent occurrence of intermolecular recombination within the mitochondrial genome, resulting in dynamic changes in sequence and conformation during evolution. Repeat-mediated homologous recombination creates a high degree of diversity in plant mitochondrial plant genomes. For example, the mitochondrial genome of \u003cem\u003ePanax notoginseng\u003c/em\u003e has various combinations of repeats that can mediate the recombination of seven subgenome ring molecules to form the main loop [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], high-frequency recombination of two large repeats in the mitochondrial genome of \u003cem\u003eGinkgo\u003c/em\u003e biloba led to the prediction of multipart structure [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The homologous recombination (HR) pathway, on the other hand, relies on the identification of sequences that are highly similar to the region to be repaired, usually other genomic copies or large repeats [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Double-strand break repair (DSBR) involves the recruitment of the second strand of the DSB and the formation of two Holliday nodes (HJ). HJs can be resolved by dissolution, intermigration of branches, and topoisomerase dechaining, or by analytic enzymatic cleavage, resulting in crossover or no crossing, depending on how they are resolved. The Break-Induced Replication (BIR) pathway uses homologous DNA to resynthesize DNA that has passed through DSB. BIR can lead to significant genomic rearrangements when the homologous sequence is not an allele, such as repetitive sequences [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In plant mitochondria, HR is the main DNA repair pathway, is also responsible for the rapid evolution of genomic organization, and plays an important role in the replication and isolation of mtDNA [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mitochondrial genome of plants typically has very low mutation rates [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. On the other hand, they exhibit excessively high rates of genomic rearrangements, resulting in poor homology conservation, even within the same species [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the absence of stress, ectopic recombination mediated by medium-sized repeats is rare, and the smaller the repeat size, the lower the frequency and irreversibility of recombination [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and seems to depend on the repeat length and its location in the genome [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and whether they are in two different subgenomic molecules (intermolecular recombination) or in a single DNA molecule (intramolecular recombination).\u003c/p\u003e \u003cp\u003eThe distinction between single-ring and double-ring mitochondrial genome structures arises from extensive sequence rearrangements and recombination events evident in long-read sequencing data. Motivated by the hypothesis that additional substructures might exist beyond the canonical single-ring configuration, we performed hybrid assembly using Unicycler with default parameters. The algorithm successfully aligned long reads to repetitive elements, and subsequent analysis revealed reads spanning entire repeat regions, leading to the conclusion that the dimeric conformation represents the most plausible mitochondrial genome architecture.\u003c/p\u003e \u003cp\u003eExperimental validation demonstrated that repeat element R1 (coting4) mediates dual structural conformations in Hippophae rhamnoides mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We propose that repetitive sequence-mediated homologous recombination, potentially through double-strand break repair (DSBR) and break-induced replication (BIR) mechanisms, drives this structural polymorphism. Notably, the R3 repeat exhibited limited capacity to generate large circular conformations, likely attributable to its shorter length (287 bp vs. R1's 1,024 bp).\u003c/p\u003e \u003cp\u003eComparative analysis revealed significant structural divergence between Tibetan and reference sea buckthorn mitochondrial genomes, with Chromosome 2 demonstrating near-complete inversion (93% collinearity loss). While these findings provide mechanistic insights, definitive validation of repeat-mediated recombination in sea buckthorn mitochondria requires further experimental investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Relationship of Homologous Species\u003c/h2\u003e \u003cp\u003eThe rapid evolution of plant mitochondria has led to extensive gene recombination, genome heterogeneity, and gene fusion among mitochondria of different species [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. While the size and structure of plant mitochondrial genomes vary significantly, functional genes remain conserved [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Here, we employed IQ-TREE to construct a sequence-based phylogenetic tree to explore the evolutionary relationships between sea buckthorn and representative taxa of angiosperms (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This tree clearly reflected distinct classification relationships among these groups, and the results also suggested a closer phylogenetic relationship between \u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e and \u003cem\u003eHippophae tibetana\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA Editing Site Prediction\u003c/h2\u003e \u003cp\u003eRNA editing is widely present in higher plant mitochondria, and it is an essential step in gene expression in plant mitochondrial genomes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. RNA editing falls under the category of post-transcriptional modification, with the most common type being C-to-U editing, which tends to occur at the second codon position [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and the majority of these edits are complete. Predicting potential RNA editing sites can facilitate to understand the expression of genes in plant mitochondria.\u003c/p\u003e \u003cp\u003eLiu J et al. reported 28 RNA editing sites in \u003cem\u003ecannabis.\u003c/em\u003e Among these, 14 were in intergenic regions, and 14 were in the protein-coding regions of genes, involving both C-to-U and G-to-A editing events [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Liu Q et al. identified 269 RNA editing sites in \u003cem\u003eOat mitochondria\u003c/em\u003e, all of which were C-to-U edits [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In addition, RNA editing may contribute to the stability and quantity of proteins [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Although the average coverage is not high due to low expression levels or limited RNA sequencing data, it still provides a wealth of information to further understand the potential functional role of RNA editing in the mitogenome[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In this study, we predicted a total of 430 potential editing sites in 36 genes, all of which involved C-to-U editing. The prediction and characterization of potential RNA editing sites provide important clues for predicting gene functions using the new codons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEndosymbiotic Sequence Transfer Events in the Sea Buckthorn Mitochondrial Genome\u003c/h2\u003e \u003cp\u003eEndosymbiotic or intracellular gene transfer (EGT/IGT) occurs within plant cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. For example, Stupar et al. discovered a 630 kb-long mitochondrial sequence on chromosome 2 of Arabidopsis thaliana, with sequence homology of over 99% [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In this study, 30 homologous segments that may be MTPT sequences were found, among which 15 protein-coding genes and 13 complete tRNA genes migrated from the chloroplast genome to the mitochondrial genome (see supplementary table). These sequences have a total length of 44,950 bp, accounting for 9.89% of the size of the mitochondrial genome. In this study, only three homologous fragments of mitochondria and chloroplasts were analyzed, which proved that two plastids had sequence migration, and found that 28 of them were complete, and 15 of them were protein-coding genes, and two genes were detected as incomplete. This phenomenon was also observed in other plant mitochondria [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Similar occurrences have been reported in species like \u003cem\u003eCatharanthus roseus\u003c/em\u003e and \u003cem\u003eBupleurum chinense\u003c/em\u003e [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the sea buckthorn mitochondrial genome was assembled successfully using a hybrid strategy involving Illumina and Nanopore sequencing. The sea buckthorn mitochondrial genome consists of two circular chromosomes with a total length of 454,544 bp. A total of 64 genes were annotated within the sea buckthorn mitochondrial genome, including 36 Protein Coding Genes (PCGs), 25 tRNA genes, and 3 rRNA genes. One repetitive sequence with a length of 410.2 bp that mediated homologous recombination and led to the formation of two possible conformations, was identified in the sea buckthorn mitochondrial genome. This research provides valuable insights for a better understanding of the genetics of sea buckthorn and other higher plants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Materials\u003c/h2\u003e \u003cp\u003eThe experimental samples were collected from a 405 years old sea buckthorn orchard located in Liupingdi Village, Changsheng Town, Aohan Banner, Chifeng City, and fresh young leaves were collected. The material used in the experiment is the high yield variety \"Yulu 1\" recognized by the provincial level of sea buckthorn. The collected leaves were rapidly frozen using liquid nitrogen and stored in a -80\u0026deg;C freezer. Later, DNA was extracted from the leaf samples through liquid nitrogen grinding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Determination Indicators\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial Genome Assembly\u003c/h2\u003e \u003cp\u003eThe sea buckthorn mitochondrial genome was assembled using long-read sequencing data. The Flye software [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] was employed with default parameters to perform direct assembly of the long-read sequencing data, resulting in a graphical assembly in GFA format. For all contigs assembled in fasta format, a database was constructed using the make BLASTb tool. Subsequently, the BLASTn program was used with the conserved mitochondrial genes from Arabidopsis thaliana as query sequences to identify contig fragments containing mitochondrial genome sequences. The parameters used for BLASTn were \"-evalue le-5 -outfmt 6 -max_hsps 10-word_size 7-task blastn-short.\" The Bandage software (version 0.8.1) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] was used to visualize the GFA file, and contigs containing the sea buckthorn mitochondrial genome were selected based on the BLASTn results, resulting in a draft mitochondrial genome map for sea buckthorn. The sequencing strategy of this study is Illumina (second generation)\u0026thinsp;+\u0026thinsp;Nanopore (third generation). We used DNBSEQ and Nanopore GridION (Oxford Nanopore Technology, Oxford Science Park) sequencing platforms for sequencing and library construction to obtain raw sequence data. Low quality sequences are then removed using Trimmomatic. removing sequences with a mass value of Q\u0026thinsp;\u0026lt;\u0026thinsp;19.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eValidation and Correction of Mitochondrial Genome\u003c/h2\u003e \u003cp\u003eThe long-reads and short-reads data were aligned to the mitochondrial contigs using the BWA software (version 0.7.17) [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Reads that aligned to the mitochondrial contigs were filtered and exported for further use in the assembly. These filtered reads were saved for subsequent hybrid assembly.\u003c/p\u003e \u003cp\u003eA hybrid assembly strategy was employed to assemble the sea buckthorn mitochondrial genome, utilizing both the short-reads and long-reads data. The Unicycler software (version 60) [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] was used with default parameters to perform the hybrid assembly, resulting in the final assembly of the sea buckthorn mitochondrial genome. The Bandage software (version 0.8.1) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] was used for the visualization of this mitochondrial genome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Genome Annotation\u003c/h2\u003e \u003cp\u003eThe protein-coding genes of the mitochondrial genome were annotated using Geseq software (version 2.03) [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], with Arabidopsis thaliana (NC_037304) and Liriodendron tulipifera (NC_021152.1) as reference genomes. The tRNA genes within the mitochondrial genome were annotated using tRNA scan-SE software (version 2.0.11) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], while the rRNA genes were annotated using BLASTN software (version 2.13.0) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Any annotation errors in the mitochondrial genome were manually corrected using Apollo software (version 1.11.8) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Genome Codon Usage Bias Analysis\u003c/h2\u003e \u003cp\u003eThe protein-coding sequences of the mitochondrial genome were extracted using Phylosuite software (version 1.1.16) [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Codon usage bias analysis of the protein-coding genes in the mitochondrial genome was performed using Mega software (version 7.0) [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], and the Relative Synonymous Codon Usage (RSCU) values were calculated.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial Genome Repeat Sequence Analysis\u003c/h2\u003e \u003cp\u003eWe identified dispersed repetitive sequences in the mitochondrial genome using Rousfinder with default parameters, with a minimum repeat unit length of 50 bp. Repeat sequences, including microsatellite sequence repeats, tandem repeats, and dispersed repeats, were identified using the following tools:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMISA (v2.1): Microsatellite sequences were identified using the MISA software, accessible at \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=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTRF (v4.09): Tandem repeats were identified using the Tandem Repeats Finder (TRF) software, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tandem.bu.edu/trf/trf.unix.help.html\u003c/span\u003e\u003cspan address=\"https://tandem.bu.edu/trf/trf.unix.help.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eRousfinder web server: Dispersed repeats were identified using the REPuter web server, which can be found at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bibiserv.cebitec.uni-bielefeld.de/reputer/\u003c/span\u003e\u003cspan address=\"https://bibiserv.cebitec.uni-bielefeld.de/reputer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe results were visualized using Excel (2021) software and the Circos package (v0.69-9) [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Genome Structure Analysis and PCR Amplification Validation\u003c/h2\u003e \u003cp\u003eThe mixed assembly was performed using default parameters with Unicycler [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Based on the sea buckthorn mitochondrial genome data, sequences of 500 bp upstream and downstream of the junction sites were extracted and recombined to form two additional configurations.Primers were designed using Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and synthesized by Genewiz (Shanghai) Co., Ltd.\u003c/p\u003e \u003cp\u003eThe PCR reaction system had a total volume of 50 \u0026micro;L, consisting of 1 \u0026micro;L template DNA, 2 \u0026micro;L forward primer, 2 \u0026micro;L reverse primer, 25 \u0026micro;L 2\u0026times; Phanta Max Master Mix, and 20 \u0026micro;L ddH2O. The PCR program included an initial denaturation step at 95\u0026deg;C for 3 min, followed by 35 cycles of denaturation at 95\u0026deg;C for 15 s, annealing at 55\u0026deg;C for 15 s, and extension at 72\u0026deg;C for 30 s. The final extension was performed at 72\u0026deg;C for 15 min.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e \u003cp\u003eBased on phylogenetic relationships, 32 closely related species were selected, and their mitochondrial genomes were downloaded. Vigna radiata (mitochondrial: NC_015121.1) and Glycine max (mitochondrial: NC_020455.1) were chosen as outgroups. Common genes were extracted using PhyloSuite software [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], followed by multiple sequence alignment analysis using MAFFT software (v7.505) [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Systematic phylogenetic analysis was conducted using IQ-TREE software (v1.6.12) [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The results of the phylogenetic analysis were visualized using ITOL software (v6) [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eRNA Editing Event Analysis\u003c/h2\u003e \u003cp\u003eThe sequences of all Protein Coding Genes (PCGs) encoded by the mitochondrial genome of this species were used as input files for the prediction of C to U RNA editing sites in mitochondrial PCGs using Deepred-mt [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. This tool is based on a Convolutional Neural Network (CNN) model for prediction and offers higher accuracy compared to previous prediction tools. All results with a probability value greater than 0.9 were retained. PhyloSuite software was used to extract shared genes, multiple sequence alignment analysis was performed using MAFFT software (v7.505), then phylogenetic analysis was performed using IQ-TREE software (v1.6.12), and the results of phylogenetic analysis were visualized using ITOL software (v6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eCo-linearity Analysis\u003c/h2\u003e \u003cp\u003eBLAST program was used to obtain pairwise comparisons of mitochondrial genomes for all species, and sequences with a length exceeding 500 bp were retained as conserved syntenic blocks for generating a Multiple Synteny Plot. Based on sequence similarity, we utilized the source code of MCscanX [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e] to create a Multiple Synteny Plot comparing the mitochondrial genome of the sea buckthorn with its closely related species.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the demonstration and popularization of rapid propagation and intensive management technology of seabuckthorn\u0026nbsp;（2022ZY0094）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.D. and Y.N.T. contributed equally to this work. X.J. M. and X.Y. L. designed the study. L.D., Y.N. T.,T.L. and X.J L. performed the work, analyzed data,and wrote the manuscript. J.M.Z. and X.S.W. provided advice on interpretation of data, and editing of the manuscript. All authors contributed to and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all researchers who unselfishly.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYou C, Cui T, Zhang C, et al. Assembly of the Complete Mitochondrial Genome of Gelsemium elegans Revealed the Existence of Homologous Conformations Generated by a Repeat Mediated Recombination[J]. Int J Mol Sci. 2022;24(1):527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpinelli JB, Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism[J]. Nat Cell Biol. 2018;20(7):745\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence[C]//Seminars in cell \u0026amp; developmental biology. Acad Press. 2020;98:139\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMar\u0026eacute;chal A, Brisson N. Recombination and the maintenance of plant organelle genome stability[J]. New Phytol. 2010;186(2):299\u0026ndash;317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao M, Zhu W, Han H, et al. Mitochondrial proteomic analysis reveals the regulation of energy metabolism and reactive oxygen species production in Clematis terniflora DC. leaves under high-level UV-B radiation followed by dark treatment[J]. J Proteom. 2022;254:104410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi F, Xiang R, Liu Y et al. Approaches and challenges in identifying, quantifying, and manipulating dynamic mitochondrial genome variations[J]. Cell Signal, 2024: 111123.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShtolz N, Mishmar D. The mitochondrial genome\u0026ndash;on selective constraints and signatures at the organism, cell, and single mitochondrion levels. Front Ecol Evol. 2019;7:342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGras DE, Mansilla N, Rodr\u0026iacute;guez C, Welchen E, Gonzalez DH. Arabidopsis thaliana SURFEIT1-like genes link mitochondrial function to early plant development and hormonal growth responses. Plant J Cell Mol Biol. 2020;103:690\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c[J]. Cell. 1996;86(1):147\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang J, Luo Z, Zhang J, et al. Multi-Chromosomal mitochondrial genome of medicinal plant Acorus tatarinowii (Acoraceae): Firstly reported from Acorales Order[J]. Gene. 2024;892:147847\u0026ndash;.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoller IM, Rasmusson AG, Van Aken O. Plant mitochondria\u0026ndash;past, present and future[J]. Plant J. 2021;108(4):912\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaluška F, Lyons S. Archaeal origins of eukaryotic cell and nucleus[J]. BioSystems. 2021;203:104375.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakenaka M. Quantification of mitochondrial RNA editing efficiency using sanger sequencing data[J]. Plant Mitochondria: Methods Protocols, 2022: 263\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetersen G, Anderson B, Braun HP, et al. Mitochondria in parasitic plants[J]. Mitochondrion. 2020;52:173\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlverson AJ, Wei XX, Rice DW, et al. Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)[J]. Volume 27. Molecular biology and evolution; 2010. pp. 1436\u0026ndash;48. 6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi C, Lu N, Xu Y, et al. Characterization and analysis of the mitochondrial genome of common bean (Phaseolus vulgaris) by comparative genomic approaches[J]. Int J Mol Sci. 2020;21(11):3778.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi Y, Odahara M, Sekine Y, et al. Holliday junction resolvase MOC1 maintains plastid and mitochondrial genome integrity in algae and bryophytes[J]. Plant Physiol. 2020;184(4):1870\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng L, Wang W, Yao Y, et al. Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis[J]. PLoS Biol. 2021;19(8):e3001357.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOdahara M, Nakamura K, Sekine Y, et al. Ultra-deep sequencing reveals dramatic alteration of organellar genomes in Physcomitrella patens due to biased asymmetric recombination[J]. Commun Biology. 2021;4(1):633.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Li D, Yao X, et al. Evolution and diversification of kiwifruit mitogenomes through extensive whole-genome rearrangement and mosaic loss of intergenic sequences in a highly variable region[J]. Genome Biol Evol. 2019;11(4):1192\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChustecki JM, Johnston IG. Collective mitochondrial dynamics resolve conflicting cellular tensions: From plants to general principles[C]//Seminars in Cell \u0026amp; Developmental Biology. Volume 156. Academic; 2024. pp. 253\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026acirc;tlan AM, Gutt G. Sea buckthorn in plant based diets. An analytical approach of sea buckthorn fruits composition: Nutritional value, applications, and health benefits[J]. Int J Environ Res Public Health. 2021;18(17):8986.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Z, Zhang Z, Tso N, et al. Complete mitochondrial genome of Hippophae tibetana: insights into adaptation to high-altitude environments[J]. Front Plant Sci. 2024;15:1449606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFajardo D, Schlautman B, Steffan S, et al. The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants[J]. Gene. 2014;536(2):336\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Yu S, L\u0026uuml; P, et al. De novo assembly and characterization of the complete mitochondrial genome of Phellodendron amurense reveals three repeat-mediated recombination[J]. Gene. 2025;935:149031.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Zhang Q, Jiang J, et al. Pullout properties of \u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e roots in the loess area[J]. Arch Agron Soil Sci. 2023;69(11):2170\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Liu Y, Tang Z, et al. Total flavonoids of \u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e improves type 2 diabetes symptoms in rats through down-regulating of the DAG/PRKCA/MAPK10/p65/TNF-α signalling pathway[J]. J Ethnopharmacol. 2024;318:116962.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Y, Dong L, Fu X, et al. HrTCP20 dramatically enhance drought tolerance of sea buckthorn (\u003cem\u003eHippophae rhamnoides L\u003c/em\u003e). by mediating the JA signaling pathway[J]. Plant Physiol Biochem. 2022;174:51\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe ZS, Zhu A, Yang JB, et al. Organelle genomes and transcriptomes of Nymphaea reveal the interplay between intron splicing and RNA editing[J]. Int J Mol Sci. 2021;22(18):9842.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Aken O. Mitochondrial redox systems as central hubs in plant metabolism and signaling[J]. Plant Physiol. 2021;186(1):36\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozik A, Rowan BA, Lavelle D, Berke L, Schranz ME, Michelmore RW, Christensen AC. The alternative reality of plant mitochondrial DNA: One ring does not rule them all. PLoS Genet. 2019, 15, e1008373.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Chen H, Ni Y, et al. De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species[J]. Int J Mol Sci. 2022;23(22):14267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Li J, Ma Y, et al. The complete mitochondrial genome of okra (Abelmoschus esculentus): Using nanopore long reads to investigate gene transfer from chloroplast genomes and rearrangements of mitochondrial DNA molecules[J]. BMC Genomics. 2022;23(1):481.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Guo H, Zhu J, et al. Complex physical structure of complete mitochondrial genome of Quercus acutissima (Fagaceae): A significant energy plant[J]. Genes. 2022;13(8):1321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu ZQ, Liao XZ, Zhang XN, et al. Genomic architectural variation of plant mitochondria\u0026mdash;A review of multichromosomal structuring[J]. J Syst Evol. 2022;60(1):160\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Chen H, Ni Y, et al. Mitochondrial Genome Sequence of Salvia officinalis (Lamiales: Lamiaceae) Suggests Diverse Genome Structures in Cogeneric Species and Finds the Stop Gain of Genes through RNA Editing Events[J]. Int J Mol Sci. 2023;24(6):5372.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlverson AJ, Rice DW, Dickinson S, et al. Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber[J]. Plant Cell. 2011;23(7):2499\u0026ndash;513.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang JX, Dierckxsens N, Bai MZ, et al. Multichromosomal mitochondrial genome of Paphiopedilum micranthum: Compact and fragmented genome, and rampant intracellular gene transfer[J]. Int J Mol Sci. 2023;24(4):3976.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebrauwere H, Gendrel CG, Lechat S, et al. Differences and similarities between various tandem repeat sequences: minisatellites and microsatellites[J]. Biochimie. 1997;79(9\u0026ndash;10):577\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehrotra S, Goyal V. Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function[J]. Genomics Proteom Bioinf. 2014;12(4):164\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang B, Li J, Zhao Q et al. Assembly of the complete mitochondrial genome of Chinese plum (Prunus salicina): Characterization of genome recombination and RNA editing sites[J]. Genes, 2021, 12(12): 1970.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Ni Y, Lin Z, et al. De novo assembly of the complete mitochondrial genome of sweet potato (Ipomoea batatas [L.] Lam) revealed the existence of homologous conformations generated by the repeat-mediated recombination[J]. BMC Plant Biol. 2022;22(1):285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi YC, Korol AB, Fahima T, et al. Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review[J]. Mol Ecol. 2002;11(12):2453\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo W, Grewe F, Fan W, et al. Ginkgo and Welwitschia mitogenomes reveal extreme contrasts in gymnosperm mitochondrial evolution[J]. Mol Biol Evol. 2016;33(6):1448\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu X, Ma Z, Liu S, et al. Analysis of the Rhodomyrtus tomentosa mitochondrial genome: Insights into repeat-mediated recombination and intra-cellular DNA transfer[J]. Gene. 2024;909:148288.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMileshina D, Ibrahim N, Boesch P, et al. Mitochondrial transfection for studying organellar DNA repair, genome maintenance and). aging[J]. Mech Ageing Dev. 2011;132(8\u0026ndash;9):412\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChevigny N, Schatz-Daas D, Lotfi F, Gualberto JM. DNA Repair and the Stability of the Plant Mitochondrial Genome. Int J Mol Sci. 2020;21(1):328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms21010328\u003c/span\u003e\u003cspan address=\"10.3390/ijms21010328\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 31947741; PMCID: PMC6981420.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe KH, Li WH, Sharp PM. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc. Natl. Acad. Sci. USA. 1987; 84:9054\u0026ndash;9058. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.84.24.9054\u003c/span\u003e\u003cspan address=\"10.1073/pnas.84.24.9054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmer JD, Herbon LA. Plant mitochondrial DNA evolved rapidly in structure, but slowly in sequence[J]. J Mol Evol. 1988;28:87\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLovett ST, Hurley RL, Sutera VA Jr., Aubuchon RH, Lebedeva MA. Crossing over between regions of limited). homology in E. coli. RecA-dependent and RecA-independent pathways. Genetics. 2002;160:851\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLovett ST, Hurley RL, Sutera VA Jr., Aubuchon RH, Lebedeva MA. Crossing over between regions of limited). homology in E. coli. RecA-dependent and RecA-independent pathways. Genetics. 2002;160:851\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavila JI, Arrieta-Montiel MP, Wamboldt Y, et al. Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis[J]. BMC Biol. 2011;9:1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller-Messmer M, K\u0026uuml;hn K, Bichara M, et al. RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis mitochondrial genome[J]. Plant Physiol. 2012;159(1):211\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun M, Zhang M, Chen X, et al. Rearrangement and domestication as drivers of Rosaceae mitogenome plasticity[J]. BMC Biol. 2022;20(1):181.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynn EL, Christensen AC. Repeats of unusual size in plant mitochondrial genomes: identification, incidence and evolution. G3 (Bethesda). 2019;9:549\u0026ndash;J559.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong Z, Liao X, Ye Y, et al. A complete mitochondrial genome for fragrant Chinese rosewood (Dalbergia odorifera, Fabaceae) with comparative analyses of genome structure and intergenomic sequence transfers[J]. BMC Genomics. 2021;22(1):1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Zhao N, Li S, et al. Plant mitochondrial genome evolution and cytoplasmic male sterility[J]. CRC Crit Rev Plant Sci. 2017;36(1):55\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo W, Grewe F, Mower JP. Variable frequency of plastid RNA editing among ferns and repeated loss of uridine-to-cytidine editing from vascular plants[J]. PLoS ONE. 2015;10(1):e0117075.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Liu H, Ge L, et al. Identification and analysis of RNA editing sites in the chloroplast transcripts of Aegilops tauschii L[J]. Genes. 2016;8(1):13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Ni Y, Liu C. Polymeric structure of the Cannabis sativa L. mitochondrial genome identified with an assembly graph model[J]. Gene. 2023;853:147081.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Yuan H, Xu J, et al. The mitochondrial genome of the diploid oat Avena longiglumis[J]. BMC Plant Biol. 2023;23(1):218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang H, Lu Q, Qiu S et al. Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice[J]. Proceedings of the National Academy of Sciences, 2022, 119(34): e2208759119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai C, Wang J, Kan S, et al. Comparative analysis of mitochondrial genomes of Broussonetia spp.(Moraceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing[J]. Front Plant Sci. 2022;13:1052151.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimmis JN, Ayliffe MA, Huang CY, et al. Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[J]. Nat Rev Genet. 2004;5(2):123\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStupar RM, Lilly JW, Town CD et al. Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: implication of potential sequencing errors caused by large-unit repeats[J]. Proceedings of the National Academy of Sciences, 2001, 98(9): 5099\u0026ndash;5103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao P, Huang Y, Zong M, et al. De Novo Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Chaenomeles speciosa (Sweet) Nakai Revealed the Existence of Two Structural Isomers[J]. Genes. 2023;14(2):526.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi C, Paterson AH, Wang X et al. Analysis of the complete mitochondrial genome sequence of the diploid cotton Gossypium raimondii by comparative genomics approaches[J]. BioMed Research International, 2016, 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Y, He X, Priyadarshani S, et al. Assembly and comparative analysis of the complete mitochondrial genome of Suaeda glauca[J]. BMC Genomics. 2021;22(1):1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019;37(5):540\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWick RR, Schultz MB, Zobel J, Holt KE. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics. 2015;31(20):3350\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichael T, Pascal L, Tommaso P, Ulbricht-Jones ES, Axel F, Ralph B, Stephan G. GeSeq \u0026ndash;versatile and accurate annotation of organelle genomes. Nucleic Acids Res 2017(W1):W1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25(5):955\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Ye W, Zhang Y, Xu Y. High speed BLASTN: an accelerated MegaBLAST search tool. Nucleic Acids Res. 2015;43(16):7762\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis SE, Searle S, Harris N, Gibson M, Iyer V, Richter J, Wiel C, Bayraktaroglu L, Birney E, Crosby M. Apollo: a sequence annotation editor. Genome Biol. 2002;3(12):1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT. PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Resour. 2020;20(1):348\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol biology Evol. 2016;33(7):1870\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeier S, Thiel T, M\u0026uuml;nch T, Scholz U, Mascher M. MISA-web: a web server for microsatellite prediction. Bioinformatics. 2017;33(16):2583\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999;27(2):573\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynn EL, Christensen AC. Repeats of unusual size in plant mitochondrial genomes: identification, incidence and evolution[J]. Volume G3. Genes, Genomes, Genetics; 2019. pp. 549\u0026ndash;59. 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Meltzer P, Davis S. RCircos: an R package for Circos 2D track plots. BMC Bioinformatics. 2013;14(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Bork P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47(W1):W256\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdera AA, Small I, Milone DH, Sanchez-Puerta MV. Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria. Comput Biol Med. 2021;136:104682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, Lee T-h, Jin H, Marler B, Guo H. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sea buckthorn, Mitochondrial genome, Homologous recombination, Phylogenetic analysis, RNA editing events","lastPublishedDoi":"10.21203/rs.3.rs-5607252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5607252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSea buckthorn (\u003cem\u003eHippophae rhamnoides L.\u003c/em\u003e) is a plant of significant economic, culinary, medicinal, ecological, and societal value. In order to gain a deeper understanding of the sea buckthorn mitochondrial genome, sequencing and assembly of the sea buckthorn mitochondrial genome was performed, and its substructures were explored in this study.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe mitochondrial genome of seabuckthorn consists of two circular chromosomes, with lengths of 297,507 bp and 167,037 bp, respectively. It encompasses 36 annotated protein-coding genes, 3 rRNA genes, and 25 tRNA genes. In addition, 212 pairs of repeats were detected, including a pair that mediated homologous recombination of seabuckthorn mitochondrial chromosomes to form two conformations. The existence of these conformations was confirmed through PCR amplification and Sanger sequencing. A total of 162 SSR loci were identified in the sea buckthorn mitochondrial genome. There are 30 homologous fragments in chloroplast and mitochondrial genomes, with a total length of 44,950 bp, accounting for 9.89% of the total length of mitochondrial genomes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, this study conducted the assembly and annotation of the sea buckthorn mitochondrial genome, providing a comprehensive understanding of the genome of this plant. This knowledge is of great significance for effective utilization and genetic improvement of seabuckthorn, especially in breeding and evolutionary analysis of cytoplasmic male sterility.\u003c/p\u003e","manuscriptTitle":"Structural Features and Synteny Analysis of the Sea Buckthorn Mitochondrial Genome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 18:38:44","doi":"10.21203/rs.3.rs-5607252/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e93c32c-0ead-449a-8f02-7cc469a6621e","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T08:26:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-23 18:38:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5607252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5607252","identity":"rs-5607252","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}