The Whole Mitochondrial Genome Sequence of Dendrobium loddigesii Rolfe, an Endangered Orchid Species in China, Reveals a Complex Multi-Chromosome Structure

Dendrobium loddigesii is a precious traditional Chinese medicine with high medicinal and ornamental value. However, the characterization of its mitochondrial genome is still pending. Here, we assembled the complete mitochondrial genome of D. loddigesii and discovered that its genome possessed a complex multi-chromosome structure. The mitogenome of D. loddigesii consisted of 17 circular subgenomes, ranging in size from 16,323 bp to 56,781 bp. The total length of the mitogenome was 513,356 bp, with a GC content of 43.41%. The mitogenome contained 70 genes, comprising 36 protein-coding genes (PCGs), 31 tRNA genes, and 3 rRNA genes. Furthermore, we detected 403 repeat sequences as well as identified 482 RNA-editing sites and 8154 codons across all PCGs. Following the sequence similarity analysis, 27 fragments exhibiting homology to both the mitogenome and chloroplast genome were discovered, accounting for 9.86% mitogenome of D. loddigesii. Synteny analysis revealed numerous sequence rearrangements in D. loddigesii and the mitogenomes of related species. Phylogenetic analysis strongly supported that D. loddigesii and D. Amplum formed a single clade with 100% bootstrap support. The outcomes will significantly augment the orchid mitochondrial genome database, offering profound insights into Dendrobium’s intricate mitochondrial genome architecture.


Introduction
The genus Dendrobium, belonging to the Orchidaceae family, comprises approximately 1500 species worldwide, with about 80 species native to China [1].The stems of several plants within this genus have been utilized for centuries in traditional Chinese medicine [2].D. loddigesii is a perennial epiphytic herb in the Orchidaceae family.It is primarily distributed in provinces such as Guizhou, Guangxi, Yunnan, and Guangdong in China [3].The stems of D. loddigesii are a commonly used and precious Chinese medicinal herb, which has antiinflammatory, antimicrobial, antioxidant, antitumor, and immunomodulatory effects [3][4][5].It mainly contains polysaccharides, alkaloids, and other chemical components [6].Due to the low germination rate of D. loddigesii seeds in natural environments, as well as excessive harvesting and utilization, the germplasm resources and wild populations of D. loddigesii have rapidly decreased [7].Therefore, it is necessary to analyze the genetic relationship through mitochondrial sequencing to provide a theoretical basis for the sustainable utilization and protection of D. loddigesii germplasm resources.
The plant mitochondrial genome exhibits semi-autonomous genetic characteristics and serves as a crucial cellular organelle for respiratory metabolism and the supply of energy for life activities [8,9].Compared with other organelle genomes, the mitochondrial genome is relatively large and has frequent structural changes, such as genome rearrangement, repeat sequence recombination, transfer, deletion, and repetition of foreign DNA [10].
This complexity makes it more challenging to assemble the mitochondrial genome [11].While most plant mitogenomes are typically circular, the actual physical architecture of these genomes shows a diverse array of structures, including circles, linear molecules, and intricate branching patterns [12,13].Furthermore, investigations into the structure of plant mitogenomes are predominantly focused on model species, including Arabidopsis thaliana [14], tobacco (Y Sugiyama, 2005) [15], and maize [16], which limits the application of generalized inferences about the plant mitochondrial genome evolution and function.Currently, studies have found that Dendrobium mitochondria exhibit a multiple chromosome phenomenon.However, only four mitochondrial genomes of Dendrobium have been published, including D. wilsonii, D. Henanense, D. wilsonii, and D. Henanense [17].Hence, more mitogenomes should be explored to enhance our understanding of mitochondrial structures of Dendrobium.In this study, we aimed to sequence, assemble, and annotate the complete mitogenome of D. loddigesii.A comprehensive analysis of repeat sequences, synonymous codon usage, and RNA editing was performed, and synteny and phylogenetic relationships were compared with several previously reported mitochondria genomes.Our findings aim to demonstrate that the plant mitochondrial genome comprises various sequence elements characterized by complex structures.

Plant Materials and DNA Sequencing
Fresh leaves of D. loddigesii were rapidly collected and preserved at −80 • C. Genomic DNA was extracted using the modified CTAB method [18].The qualified DNA samples were then sequenced on the Illumina NovaSeq 6000 platform (Genepioneer Biotechnologies Co., Ltd.; Nanjing, China).

Assembly and Annotation
For acquiring a high-quality D. loddigesii mitochondrial genome, the second-generation data were used to extract high-quality reads, while the original third-generational data were employed for correction.Then, the genome assembly was conducted utilizing NextPolish v1.3.1 (https://github.com/Nextomics/NextPolish,accessed on 18 August 2023).The annotation process for the draft mitochondrial genome of D. loddigesii followed established procedures [19].Encoded proteins and rRNAs were annotated through Blastn searches of published plant mitochondrial sequences at the National Center for Biotechnology Information (NCBI).Transfer RNA genes (tRNAs) were annotated using tRNA scan-SE [20].An ORF finder was utilized to analyze open reading frames (ORFs), and an Organellar Genome DRAW was employed for the construction of the mitochondrial genome [21].

Synonymous Codon Usage Analysis
To assess the synonymous codon usage patterns within the mitochondrial genome, we employed the relative synonymous codon usage (RSCU) using the CodonW1.4.4 (http: //codonw.sourceforge.net/,accessed on 16 August 2023) [24].Subsequently, the R package (3.5.1) ggplot2 was utilized to generate visualizations of the RSCU data, providing a clear and informative representation of the codon usage patterns.

RNA Editing Analyses and Chloroplast to Mitochondrion DNA Transfer
To identify RNA-editing sites within the mitochondrial genes of D. loddigesii, the plant mitochondrial gene-encoding proteins were used as the reference proteins.The plant predictive RNA editor (PREP) suite (http://prep.unl.edu/,accessed on 16 August 2023) was employed to analyze the editing sites [25].The chloroplast genome sequence of D. loddigesii (accession number: NC_036355.1)was retrieved from the NCBI Organelle Genome Resources Database.The homologous fragments were identified utilizing BLAST v2.10.1 software.

Synteny and Phylogenetic Analysis
A dot plot comparing pairwise sequences was generated to visualize conservative colinear blocks.Furthermore, a multiple synteny plot was created to depict the mitogenome of D. loddigesii in comparison with related species.For phylogenetic tree analysis, we utilized the conserved PCGs extracted from the mitochondrial genome of D. loddigesii along with those from 15 other taxa.The 15 mitochondrial genomes were obtained from NCBI Organelle Genome Resources database, and the conserved PCGs were extracted using the Tbtools2.07software.The gene sequences were then aligned using the Muscle v5 software, and a Neighbor-joining (NJ) tree was constructed using the Mega 11.0 software [26].

Repeat Sequence Analysis
A total of 403 repeats were found in the D. loddigesii mitochondrial genome, including 221 dispersed sequences, 146 SSRs, and 36 tandem sequences (Figure 2, Table S4).The dispersed sequences included 90 forward repeat sequences and 131 palindromic repeat sequences (Table S5).The largest forward repeat sequence was 631 bp in length, while the palindromic repeat sequence had a size of 467 bp.The total length of the dispersed repeat sequences was 24,272 bp, accounting for 4.73% of the total length of the mitochondrial genome.Moreover, among 146 SSRs, 48 mononucleotide, 34 dinucleotide, 18 trinucleotide, 35 tetranucleotide, 8 pentanucleotide, and 3 hexanucleotide repeat types were explored.Among the mononucleotide repeat types, the repeats of A/T were the most ordinary, and only one type was C/C repeats (Table S6).A total of 36 tandem repeats were detected, ranging from 6 to 34 bp in length, with a matching degree surpassing 78% (Table S7).Chromosomes 3, 5, and 7 exhibited the highest occurrence of tandem repeats, whereas chromosomes 9, 10, 11, 16, and 17 displayed an absence of such repeats.This observation indicated an uneven distribution of tandem repeats across the mitochondrial genome of D. loddigesii.

Codon Usage Analysis
The relative synonymous codon usage (RSCU) is generally thought to reflect the result of biological natural selection, with RSCU values exceeding 1 indicating a preference for specific amino acid codons [27].Codon usage analysis was conducted on 36 PCGs within D. lodigesii mitochondria.The results revealed that all genes were encoded by 8154 codons, encoding 20 amino acids (Figure 3, Table S8).Notably, the RSCU values of 4338 codons were greater than 1, indicating that they were used more frequently.Furthermore, in addition to the high frequency of use of the three-stop codons, UAU, UGA, and UAG, a general preference for specific codons was observed in mitochondrial PCGs.For example, GCA, GCC, GCG, and GCU were the most commonly used codons in D. lodigesii.

Codon Usage Analysis
The relative synonymous codon usage (RSCU) is generally thought to reflect the result of biological natural selection, with RSCU values exceeding 1 indicating a preference for specific amino acid codons [27].Codon usage analysis was conducted on 36 PCGs within D. lodigesii mitochondria.The results revealed that all genes were encoded by 8154 codons, encoding 20 amino acids (Figure 3, Table S8).Notably, the RSCU values of 4338 codons were greater than 1, indicating that they were used more frequently.Furthermore, in addition to the high frequency of use of the three-stop codons, UAU, UGA, and UAG, a general preference for specific codons was observed in mitochondrial PCGs.For example, GCA, GCC, GCG, and GCU were the most commonly used codons in D. lodigesii.

RNA Editing Site Analysis
In higher plants, RNA editing is a post-transcriptional process necessary for mitochondrial gene expression [28].In the D. loddigesii mitochondria, 538 RNA-editing sites were predicted in PCG genes (Figure 4).Among those PCG genes, the most RNA-editing

RNA Editing Site Analysis
In higher plants, RNA editing is a post-transcriptional process necessary for mitochondrial gene expression [28].In the D. loddigesii mitochondria, 538 RNA-editing sites were predicted in PCG genes (Figure 4).Among those PCG genes, the most RNA-editing sites were the nad4 gene (56 sites, 10.41%) followed by the ccmFn gene with 40 RNA editing sites.The minimum number of RNA-editing sites were the rps14 and rps7 genes, with only two and three editing sites, respectively.In addition, further analysis showed that 262 RNA-editing sites (48.70%) of the amino acids changed from hydrophilic to hydrophobic, 156 sites (29.00%) from hydrophobic to hydrophobic, 70 sites (13.01%) from hydrophilic to hydrophilic, 48 sites (8.92%) from hydrophobic to hydrophilic, and only 2 editing sites (0.37%) of amino acids became stop codons (X) (Table 3).The studies also found that 230 editing sides (42.74%) of the amino acids were changed to leucine (L), showing a leucine tendency.with only two and three editing sites, respectively.In addition, further analysis showed that 262 RNA-editing sites (48.70%) of the amino acids changed from hydrophilic to hydrophobic, 156 sites (29.00%) from hydrophobic to hydrophobic, 70 sites (13.01%) from hydrophilic to hydrophilic, 48 sites (8.92%) from hydrophobic to hydrophilic, and only 2 editing sites (0.37%) of amino acids became stop codons (X) (Table 3).The studies also found that 230 editing sides (42.74%) of the amino acids were changed to leucine (L), showing a leucine tendency.

Chloroplast to Mitochondrion DNA Transfer
Sequence similarity analysis showed that there were 27 homologous fragments shared between mitochondrial and chloroplast genomes, with a total length of 50,632 bp, accounting for 9.86% of the total length of the D. lodigesii mitogenome (Figure 5, Table 4).Among these, six fragments exceeded 1000 bp, with fragments 1 and 2 being the longest at 8595 bp, while the smallest fragment was 23 for 29 bp.Through annotation of these homologous sequences, 18 integrated chloroplast-derived genes were identified, specifically including trnL-CAA, trnR-ACG, trnN-GUU, trnV-GAC, trnA-UGC, trnL-UAG, trnS-GGA, trnT-UGU, trnG-GCC, trnM-CAU, trnT-GGU, trnE-UUC, trnY-GUA, trnW-CCA, trnP-UGG, trnF-GAA, trnQ-UUG, and trnS-GCU, and two incomplete rRNA genes (rrn18 and rrn26) were also discovered.Notably, all transferred genes were tRNA genes and partial rRNA genes, and no PCGs were found, which indicated that tRNA genes were more conserved in the D. lodigesii plastid genome.To determine the phylogenetic location of D. lodigesii, the mitochondrial genome sequences of 15 angiosperms were retrieved from GenBank (Table S10).The sequences were based on 25 conserved PCGs, which were to establish a phylogenetic tree, using Aegilops speltoides as the outgroup (Figure 8).The phylogenetic tree provided robust evidence, with 100% bootstrap support, for a close phylogenetic affinity between D. lodigesii and D. amplum.Both species are native to south-central and south-eastern China, and phylogenetic analysis further confirms this geographical proximity.

Characterization of the D. lodigesii Mitogenome
Mitochondria play an important role as organelles within eukaryotic cells [29].Unlike animal cells, plant mitochondrial DNA exhibits amazing structural diversity, with

Characterization of the D. lodigesii Mitogenome
Mitochondria play an important role as organelles within eukaryotic cells [29].Unlike animal cells, plant mitochondrial DNA exhibits amazing structural diversity, with its structure being able to rapidly switch between linear, circular, and branched forms within the organism [30].Generally, plant mitochondria are usually assembled into a large circle structure, but their true morphology may consist of smaller circles combined with branching DNA molecules [31].In this study, we assembled the mitochondria of D. lodigesii, which contained 17 chromosomes and exhibited a multi-chromosome structure.The Dendrobium genus is generally polychromosome, with the mitochondrial genome sequences of D. wilsonii and D. henenense possessing 22 and 24 independent chromosome structures, respectively [32].The rapid acquisition or loss of chromosomes has been postulated as a pivotal evolutionary process that explains these observed disparities [16].The D. loddigesii mitogenome was annotated with 70 genes, which was similar to the number in D. wilsonii (77) and D. henanense (83) [32].However, the most significant differences among the three mitogenomes were primarily observed in the tRNA genes.Specifically, the mitogenome of D. loddigesii contained 31 tRNA genes, while the mitogenomes of D. Wilsonii and D. henanense had 33 and 40 tRNA genes, respectively.These findings indicated that Dendrobium species exhibited the greatest variation in the number of tRNA genes within their mitochondrial genomes.

The Repeat Sequences in the D. lodigesii Mitogenome
The repeat sequences are potentially important markers for population and evolutionary studies [33].In this study, a total of 403 repeat sequences were identified in the D. loddigesii mitochondrial genome, including 146 SSRs.SSRs have the advantages of codominance, high reproducibility, and the requirement of a small amount of DNA template [34,35].These features enable their application in various scenarios, including DNA fingerprinting, gene mapping, and marker-assisted breeding [36][37][38].

RNA Editing in the D. lodigesii Mitogenome
In plants, RNA editing is a pivotal process that significantly contributes to mitochondrial gene expression and functionality [39].Numerous RNA editing events have the potential to introduce changes in RNA sequences, ultimately resulting in variations in the amino acid sequences of the translated protein products [40,41].A total of 538 RNA-editing sites were detected in D. loddigesii mitochondria.Consistent with observations in other plant species [42][43][44], a majority of RNA-editing sites in this mitochondrion occurred at the first or second positions within the RNA sequence.RNA editing events at two specific sites lead to the creation of stop codons in D. loddigesii mitochondria, which is frequently linked to the production of proteins that exhibit high conservation to those identified in other species [40].This mechanism facilitates efficient gene expression within mitochondria.

MTPTs in the D. lodigesii Mitogenome
Horizontal gene transfer (HGT) occurs between the genomes of organelles (such as plastids and mitochondria) and the nuclear genomes of plant cells, and it is a general phenomenon that significantly impacts plant evolution [45].One of the more intriguing phenomena in plant genetics is the transformation of DNA fragments from plastids into mitochondrial genomes, referred to as plastid-to-mitochondrial transfers (MTPTs) [46].These transfers involve the movement of genetic material, typically short DNA sequences, from the plastid genome into the mitochondrial genome [47].In this study, eighteen complete tRNA genes, including trnL-CAA, trnR-ACG, trnN-GUU, trnV-GAC, trnA-UGC, trnL-UAG, trnS-GGA, trnT-UGU, trnG-GCC, trnM-CAU, trnT-GGU, trnE-UUC, trnY-GUA, trnW-CCA, trnP-UGG, trnF-GAA, trnQ-UUG, and trnS-GCU, were found to migrate from the chloroplast to the mitogenome in D. lodigesii.Interestingly, the mitogenome of D. lodigesii harbored only 13 native tRNA genes, suggesting that over half of its tRNA genes had undergone HGT from the chloroplast.During the entire evolutionary process, the horizontal transfer of tRNA genes from the chloroplast to the mitochondrion in D. lodigesii had resulted in the acquisition of functionally conserved tRNAs, which were prevalent across the angiosperms [48].Among the tRNA genes' horizontally transferred events, trnW-CCA frequently appeared in the mitochondrial genomes of diverse angiosperms [49,50].Prior research had demonstrated that trnM-CAU possessed a potential functional role in plant mitochondria genomes, suggesting that it underwent transfer during an initial phase of evolutionary development [51].Both tRNA genes, trnW-CCA, and trnM-CAU, were also discovered as part of the level gene transfer involving the organelles of D. lodigesii.

Synteny and Phylogenetic Analyses in the D. lodigesii Mitogenome
We conducted homologous collinear alignments to delve into the rearrangement and conservative sequence patterns within the mitochondrial genome.The findings indicated that D. lodigesii and several other genera (Phoenix, Allium, Cocos, Asparagus, and Chlorophytum) displayed low collinearity, whereas D. amplum exhibited high collinearity (71.42%).This suggests that closely related species tended to have longer collinear regions, while distantly related genomes showed poorer collinearity [52].Moreover, analysis of collinear alignments revealed inconsistent alignment of collinear blocks in the mitogenomes of D. lodigesii and D. Amplum, which may suggest that the mitogenome had undergone substantial genomic rearrangements, resulting in a highly variable and unconserved structure.This outcome aligned with earlier findings from mitochondrial collinearity analysis of D. Wilsonii and D. Henanense, demonstrating a substantial presence of mitochondrial rearrangements in both Dendrobium species [32].
Here, we built the phylogeny of Dendrobium using conserved mitochondrial PCG sequences from 15 angiosperm species retrieved from GenBank.Unlike chloroplast and nuclear genomes, mitogenomes are rarely used for phylogenetic analysis in higher plants, primarily due to their slow mutation frequency, high rate of genome recombination, and integration of exogenous DNA [53,54].However, in this study, the D. lodigesii clade was sister to the D. Amplum clade with strong support (100%) in the present study, and the issue of weakly supported nodes in mitochondrial gene trees has been well addressed.These results suggest that PCG genes in plant mitochondrial genomes can be used for phylogenetic analysis.

Conclusions
In this study, we have successfully assembled and annotated the mitogenome of an orchid plant, D. loddigesii, revealing a complex multi-chromosome structure.The total length of D. loddigesii mitogenome was 513,356 bp, which consisted of 17 circular chromosomes.The genome was annotated with 70 genes, including 36 PCGs, 31 tRNA genes, and 3 rRNA genes.The repeat sequences, codon preference, and RNA-editing sites were also characterized.In addition, we also identified MTPTs and performed synteny and phylogenetic analyses to gain a deeper insight into the evolutionary trajectory of the mitogenome in Dendrobium.The results of this research further validate the intricate structure of mitogenomes in the orchid family.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes15070834/s1,Table S1: Statistics of Illumina sequencing data.Table S2: Statistics of Nanopore sequencing data.Table S3: The annotation of the D. loddigesii mitogenome.Table S4: The repeat sequences in the D. loddigesii mitogenome.Table S5: the dispersed sequences in the D. loddigesii mitogenome.Table S6: SSRs in the D. loddigesii mitogenome.Table S7: The tandem sequences in the D. loddigesii mitogenome.Table S8.Codon usage analysis in the D. loddigesii mitogenome.Table S9: Species list used for synteny analysis.Table S10: Species list used for phylogenetic analysis.

Figure 2 .
Figure 2. Repeated sequence distribution in the D. loddigesii mitogenome.The outermost circle was the SSRs, followed by tandem repeat sequences, and the innermost was the dispersed repeat sequences.

Figure 2 . 17 Figure 3 .
Figure 2. Repeated sequence distribution in the D. loddigesii mitogenome.The outermost circle was the SSRs, followed by tandem repeat sequences, and the innermost was the dispersed repeat sequences.Genes 2024, 15, x FOR PEER REVIEW 7 of 17

Figure 3 .
Figure 3. RSCU in the D. loddigesii mitogenome.The x-axis represents the different kinds of amino acids.The y-axis represents the value of RSCU.

Figure 4 .
Figure 4. Distribution of RNA-editing sites in PCGs of the D. loddigesii mitogenome.Figure 4. Distribution of RNA-editing sites in PCGs of the D. loddigesii mitogenome.

Figure 4 .
Figure 4. Distribution of RNA-editing sites in PCGs of the D. loddigesii mitogenome.Figure 4. Distribution of RNA-editing sites in PCGs of the D. loddigesii mitogenome.

Figure 5 .
Figure 5. Homologous fragments were distributed between mitochondria and chloroplast in D. loddigesii.

Figure 5 .
Figure 5. Homologous fragments were distributed between mitochondria and chloroplast in D. loddigesii.

Figure 6 .
Figure 6.Dot plot graphs show similar sequences between mitogenomes primarily in D. loddigesii and related species.The red line in the box is a forward comparison, while the blue line is a reverse complementary comparison.

Figure 6 .
Figure 6.Dot plot graphs show similar sequences between mitogenomes primarily in D. loddigesii and related species.The red line in the box is a forward comparison, while the blue line is a reverse complementary comparison.

Figure 7 .
Figure 7. Collinearity plots of the mitogenomes of D. loddigesii and related species.The boxes in each row represent the mitogenomes, and the lines in the middle represent homologous regions.

Figure 7 . 17 Figure 8 .
Figure 7. Collinearity plots of the mitogenomes of D. loddigesii and related species.The boxes in each row represent the mitogenomes, and the lines in the middle represent homologous regions.Genes 2024, 15, x FOR PEER REVIEW 13 of 17

Figure 8 .
Figure 8.The phylogenetic relationships of D. loddigesii and other 15 species based on conserved mitochondrial genes.

Table 3 .
Prediction of RNA-editing sites in the D. loddigesii mitogenome.

Table 4 .
Fragments transferred from chloroplast to mitochondria in D. loddigesii.