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Article

Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex rotunda Thunb.

by
Yuanjian Wang
1,2,
Gang Cui
2,
Kaifeng He
2,
Kewang Xu
1,
Wei Liu
3,
Yuxiao Wang
1,
Zefu Wang
4,
Shasha Liu
5,* and
Changwei Bi
1,2,*
1
State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Tree Genetics and Biotechnology of Educational Department of China, Key Laboratory of Tree Genetics and Silvicultural Sciences of Jiangsu Province, Nanjing Forestry University, Nanjing 210037, China
2
College of Information Science and Technology, Nanjing Forestry University, Nanjing 210037, China
3
College of Optical, Mechanical and Electrical Engineering, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
4
Co-Innovation Center for Sustainable Forestry in Southern China, College of Ecology and Environment, Nanjing Forestry University, Nanjing 210037, China
5
School of Intelligent Manufacturing, Nanjing Polytechnic Institute, Nanjing 210044, China
*
Authors to whom correspondence should be addressed.
Forests 2024, 15(7), 1117; https://doi.org/10.3390/f15071117
Submission received: 10 May 2024 / Revised: 9 June 2024 / Accepted: 25 June 2024 / Published: 27 June 2024
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Ilex rotunda Thunb. stands as a representative tree species in subtropical evergreen broad-leaved forests, widely distributed across southeast Asia. This species holds significant value in forestry due to its ecological resilience and adaptability. Although researchers have conducted in-depth research on the plastid genome (plastome) of I. rotunda, the mitochondrial genome (mitogenome) of this species has remained undocumented. In the present study, we successfully sequenced and assembled the I. rotunda mitogenome. The mitogenome has a circular structure and is 567,552 bp in total length, with a GC content of 45.47%. The composition of the mitogenome encompasses 40 protein-coding genes, along with 3 rRNA genes and 19 tRNA genes. Notably, the mitogenome exhibits a universal distribution of repetitive sequences, but the total length of repeats contributes to a relatively small proportion (4%) of the whole mitogenome, suggesting that repeats do not serve as the primary cause of the amplification of the Ilex mitogenomes. Collinear analysis indicates that the I. rotunda mitogenome is very conservative within Aquifoliales species. Additionally, our research identified 51 fragments of plastid genomic DNA, which have migrated from the plastome into the mitogenome, with five genes from the plastome remaining intact. Eventually, the phylogenetic analyses based on the plastomes and mitogenomes of 36 angiosperms determine the Aquifoliales to be the basal group in the campanulids. This study establishes the bedrock for prospective investigations in molecular breeding research.

1. Introduction

As a member of the family Aquifoliaceae, Ilex rotunda Thunb. is widely distributed across the subtropical regions of Asia, including Japan, Korea, Vietnam, and southern China [1]. This widespread distribution indicates the tree’s ecological resilience and adaptability within the diverse environmental conditions found in this pan-Asian biogeographic zone. The study of the I. rotunda mitogenome holds profound significance for breeding research. Comparative analysis of the structure, composition, and evolutionary patterns of Ilex mitogenomes provides essential insights into the genetic diversity and adaptive potential of Ilex species. Such insights are crucial for developing breeding strategies that enhance key traits such as the yield, stress tolerance, and disease resistance [2]. Furthermore, the investigation of the genetic characteristic of I. rotunda holds significant economic value. As a renewable resource, trees like I. rotunda play an indispensable role in maintaining the ecological balance [3,4]. Previous investigations concentrating on the phytochemical analysis of I. rotunda have revealed various biological properties within its crude extract and individual compounds. These activities encompass antimicrobial potential, anti-inflammatory effects, antioxidant properties, and antiplatelet aggregation [5], thus highlighting the multifaceted pharmacological significance of I. rotunda. Although I. rotunda holds ecological and pharmaceutical significance, its comprehensive genetic and molecular basis have not yet been revealed in the mitogenome of I. rotunda.
Mitochondria are characterized as a form of dual membrane organelles that are pivotal to energy production and participate in various cellular processes. These functions span the realms of amino acids, the oxidation of fatty acids, the initiation of cellular apoptosis, and the transmission of cellular signals [6]. Furthermore, mitochondria play a crucial regulatory role in cellular proliferation and the cell cycle [2], thereby underpinning their significance in maintaining cellular homeostasis and overall organismal function. Plant mitogenomes exhibit numerous distinctive evolutionary characteristics and cover an extensive range of sizes, from 66 kb to 12 Mb [7,8]. Remarkable differences exist in the size of the mitogenomes among closely related plant species. For example, the Silene latifolia mitogenome is only 253 kb in size, but the size of Silene conica reaches 11.3 Mb [9]. The presence of substantial repetitive sequences and foreign DNA in the mitogenomes contributes significantly to the observed heterogeneity in their sizes and structures [10]. The mitogenomes of plants demonstrate significant diversity in terms of their structural compositions, genetic contents, rates of nucleotide substitution, and levels of recombination [11,12]. These genomic variations are not only confined to differences between distinct species but also observed within individual species. [13,14]. For instance, notable variations exist in the frequency of mitogenome recombination among distinct Arabidopsis mutants [15]. Considerable distinctions in both the magnitude and the configuration of mitogenomes exist between cultivated and wild lineages of sorghum [16]. Thus, plant mitogenomes are valuable genetic resources for studying plant phylogeny and cellular processes [17].
Plant mitogenomes contain a variety of repetitive sequences, including simple sequence repeats (SSRs), tandemly repeated elements, and dispersed repeats. These repeats have the potential to trigger recombination events within the mitogenome, lead to modifications to its structure and occasionally causing the loss or duplication of mitochondrial genes. The homologous fragments present in mitochondria are predominantly derived from the plastid genome (plastome) and the nuclear genome [18,19], indicating a tendency for mitochondria to assimilate DNA from different sources through a process of intracellular horizontal gene transfer [20]. With the development of new methodologies, more organelle genomes have been assembled and analyzed. The transfer of genes from the plastome to the mitogenome has been increasingly recognized as a characteristic feature of long-term evolution [21,22]. Furthermore, the genetic system of mitogenomes is predominantly maternally inherited, exhibiting a degree of independence from the nuclear genome, and is generally more conserved in nature. The evolutionary pace of plant mitogenome sequences is relatively slow, contrasting with the relatively high recombination pace of genome structures. This characteristic renders mitogenomes more suitable for solving the deep-level and large-scale phylogenetic relationships such as the swift evolution of plastome groups [23] and the early evolution of terrestrial plant systems. For example, researchers have analyzed the evolutionary relationship between Selaginella sinensis and Selaginella sanguinolenta based on their plastomes, leading to a debate over their phylogenetic relationships. By conducting a phylogenetic analysis based on 17 conserved genes from the mitogenomes, the aforementioned controversies can be effectively resolved [23].
In the current study, the first comprehensive assembly and detailed annotation of the mitogenome of I. rotunda are presented. Furthermore, we perform comparative analyses of the gene content, repetitive sequences, fragment migration, collinearity, and phylogeny. These discoveries will provide information of value for the molecular identification, classification, and germplasm conservation research of the Ilex genus. Furthermore, they are instrumental in advancing our understanding of the evolutionary and genetic underpinnings of plant mitogenomes.

2. Materials and Methods

2.1. Plant Material and DNA Sequencing

Samples of I. rotunda were obtained from the Nanjing Forestry University campus, located at coordinates 32°04′41″ N, 118°48′23″ E, and were promptly frozen at −80 °C for subsequent DNA extraction. The extraction of the total genomic DNA was carried out utilizing the Hi-DNA Secure Plant Kit (TiangenDP350), with the DNA purity and quality assessed by running a 1.0% agarose gel and using a NanoDrop 2000 Spectrophotometer (Thermo Fisher, Nanjing, China). Following this, the sequencing libraries were constructed based on the high-integrity genomic DNA by employing the SMRTbell Express Template Prep Kit 2.0 from PacBio Biosciences, located in Menlo Park, CA, USA. Ultimately, the PacBio Revio platform was employed to produce the HiFi sequencing data, and the quality of the reads was assessed using SeqKit software v2.2.0 [24].

2.2. Assembly and Annotation of the Mitochondrial Genomes

The I. rotunda mitogenome was assembled using two different types of software. First, we used PMAT v1.31 (https://github.com/bichangwei/PMAT, accessed on 20 July 2023) in ‘autoMito’ mode to perform the assembly [25,26]. The assembly parameters specified were ‘-st HiFi-g 804M-ml 40-mi 90’. The genome size of I. rotunda was approximated by referencing the genome of Ilex asprella [27]. Following the elimination of complete plastid contigs and tip contigs, the primary assembly graph of I. rotunda was composed of two contigs (contig1133: 566,766 bp; contig23348: 786 bp). Subsequently, we used Hifiasm v0.16.1 to confirm the assembly of PMAT with default parameters [28]. Finally, we found that the circular contig shared by PMAT and Hifiasm was the mitogenome of I. rotunda.
The mitogenomes of four Ilex species (I. rotunda NC_084321.1, I. macrocarpa NC_082235.1, I. metabaptista NC_081509.1, and I. pubescens NC_045078.1) were first annotated by the online program IPMGA (www.1kmpg.cn/mgavas, accessed on 5 August 2023). Subsequent verification of the tRNAs and rRNAs was conducted by employing the tRNAscan-SE v2.0 software [29] and BLASTn [30], respectively. After that, we used deepred-mt v3 software [31] to predict the C–to–U RNA editing sites. The intron contents of the I. rotunda mitogenome were detected using BLASTn. A meticulous manual inspection was performed of all the PCGs, tRNAs and rRNAs, as well as the intron contents, utilizing the MacVector v18.5 software. The mitogenome map of I. rotunda was drawn using the web-based tool OrganellarGenomeDRAW v1.3.1 (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 5 April 2024) [32].

2.3. Identification of Repetitive Sequences

The mitogenomes of four Ilex species (I. rotunda NC_084321.1, I. macrocarpa NC_082235.1, I. metabaptista NC_081509.1, and I. pubescens NC_045078.1) were obtained from the NCBI nucleotide database. The presence of SSRs in the mitogenomes of the four Ilex species was discerned by employing the MISA web service (https://webblast.ipk-gatersleben.de/misa, accessed on 7 April 2024) [33]. The parameter for the motif sizes was set to 1 to 6 nucleotides and the thresholds were set to 10, 5, 4, 3, 3 and 3. The parameter for the maximum distance between any two SSRs was established at a threshold of 100 bp. Subsequently, the tandem repeats of the assembled mitogenomes were recognized by employing Tandem Repeats Finder (TRF) v4.09 [34]. The parameters for alignment were designated as 2 for matches, 7 for mismatches, and 7 for indels. The minimum threshold for the alignment scores was set to 60, while the maximum period size was capped at 500. Additionally, we used the online tool REPuter service (https://bibiserv.uni-bielefeld.de/reputer, accessed on 7 April 2024) [35] to identify the dispersed repeats in the mitogenomes of the four Ilex species. The parameters for analysis were specifically set with a hamming distance of 3, a minimum repeat size of 30, and a maximum of 5000 computed repeats. The visualization of the dispersed repeats within the I. rotunda mitogenome was achieved using the ‘Advanced Circos’ module in TBtools v1.132 [36].

2.4. Identification of Mitochondrial Plastid Sequences (MTPTs)

The plastomes and mitogenomes of four Ilex species (I. rotunda, I. macrocarpa, I. metabaptista, and I. pubescens) were sourced from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore, accessed on 10 March 2024). We employed BLASTn version 2.15.0 [30] for the purpose of detecting the homologous fragment sequences between the plastome and the mitogenome of I. rotunda. The specific parameters used for the BLASTn software were ‘-evalue 1 × 10−5-word_size 9-gapopen 5-gapextend 2-reward 2-penalty-3’. The MTPTs with matching rates ≥ 80% and lengths ≥ 40 bp were selected for further analysis. All the results were manually annotated to verify the presence of genes within the MTPTs. Finally, the visualization of the MTPTs was achieved by employing the ‘Advanced Circos’ module in TBtools v1.132 [36].

2.5. Collinearity Analysis

In order to investigate the collinearity among I. rotunda and other mitogenomes within the Ilex genus, we retrieved four additional mitogenomes from the NCBI nucleotide database (I. macrocarpa NC_082235.1, I. metabaptista, NC_081509.1, I. pubescens, NC_045078.1, Lactuca sativa NC_042756.1). The sequence alignment between I. rotunda and the aforementioned species was conducted using the BLASTn v2.15.0 software [30]. To facilitate the collinearity analysis, the BLASTn results with the minimum alignment length of 200 bp, the minimum identity of 80% and the minimum e-value of 1 × 10−5 were selected. The collinearity was finally visualized using the NGenomeSyn tool (https://github.com/hewm2008/NGenomeSyn, accessed on 10 February 2024) [37].

2.6. Phylogenetic Analysis

To ascertain the phylogenetic position of I. rotunda, phylogenetic trees based on the plastomes and mitogenomes were constructed separately. A total of 35 additional plant species were obtained from the NCBI nucleotide database (Table S1). A total of 22 conserved PCGs from mitogenomes and 31 conserved PCGs from plastomes were selected for the individual multiple sequence alignments (Table S7). The shared PCGs across the 36 species representing various families were aligned by employing MAFFT software (v7.505, —auto mode) [38] and then trimmed by employing trimAl (v1.2) [39]. Subsequently, the conserved regions were extracted from the alignment by employing the Gblocks (v0.91b) software [40]. After that, maximum likelihood phylogenetic analyses were conducted with IQ-TREE v2.2.0 [41] with the ‘Auto’ option and supported by 1000 ultrafast bootstraps [42]. Oryza sativa and Allium cepa were designated as the outgroup. The visualization of the resultant phylogenetic tree was ultimately achieved and further edited by employing the online iTOL platform (https://itol.embl.de, accessed on 10 April 2024) [43].

3. Results

3.1. Genome and Gene Characteristics of the I. rotunda Mitogenome

Using the Revio sequencing platform, we successfully obtained 611,832 HiFi sequencing reads. These reads exhibited a maximum length of 46,899 bp and an average length of 15,841 bp. In terms of the sequence quality, the reads achieved a peak Phred score of 40, with an average score of 27.29. With the highly accurate long-read sequencing data, the I. rotunda mitogenome was assembled into a typical circular structure (Figure S1) of 567,552 bp in size (accession number: NC_084321.1) (Figure 1). Furthermore, we annotated the mitogenomes of three additional Ilex species in the same way, thereby completing the missing PCGs in the GenBank entries (Table 1) (Table S2) and removing tRNAs with low scores. The mitogenome of I. rotunda was found to contain a total of 62 genes, comprising 40 PCGs, 3 rRNAs, and 19 tRNAs. The GC content of the I. rotunda mitogenome was determined to be 45.47%, aligning with the GC content observed in other Ilex species (Table 1). The total length of these 40 PCGs was 33,189 bp, representing 5.85% of the I. rotunda genome. The collective lengths of the rRNA and tRNA genes constituted 0.92% (5247 bp) and 0.25% (1428 bp), respectively. Notably, the intergenic regions accounted for nearly 93% of the mitogenome’s total length. All the tRNA genes were present in single copies, whereas two of the PCGs (atp9 and rps19) were found in two copies each.
In the mitogenome of I. rotunda, most of the PCGs started with the standard start codon ATG. Nevertheless, a deviation was observed in the genes cox1, nad4L, and rps4, which used ACG as their start codons. Among the 40 PCGs, 3 distinct stop codons were identified, including TAA (23 genes), TAG (5 genes), and TGA (12 genes). The two atp9 genes possessed different stop codons (TAA and TGA). Furthermore, the analysis of the I. rotunda mitogenome indicated the presence of 18 cis-spliced introns in 10 PCGs and 6 trans-spliced introns distributed across the NADH dehydrogenase genes.

3.2. Repetitive Sequence Analysis of the I. rotunda Mitogenome

Repetitive sequences are prevalent in plant mitogenomes, which can be categorized into three types: SSRs, tandem repeats, and dispersed repeats (Figure 2A). The I. rotunda mitogenome was found to contain 178 SSRs (Table S3) through the online tool MISA, including 45 (25.28%) monomers, 32 (17.98%) dimers, 14 (7.87%) trimers, 71 (39.89%) tetramers, 15 (8.43%) pentamers and 1 (0.56%) hexamer, which is similar to the other three types of Ilex mitogenomes (Figure 2B). The I. rotunda mitogenome possessed the most A/T repeats, with 43 occurrences representing 95.6% of all the mononucleotide repeats. In addition, the trimeric repeat units AAG/CTT and the tetrameric repeat units AAAG/CTTT were found to be more prevalent compared to other units within their respective classes. Tetramers exhibited the highest frequency among the various repeat types of SSRs identified within the genus Ilex (Figure 2B).
Using another online tool, Tandem Repeats Finder, we identified 58 tandem repeats within the mitogenome of I. rotunda. The largest tandem repeat span 137 bp in length, while most tandem repeats are less than 100 bp (Table S4). The dispersed repeats are evenly scattered across the mitogenome, contrasting with the clustered nature of the tandem repeats, and can enhance or suppress gene expression at the sites where they are inserted. The mitogenome of I. rotunda contains 528 dispersed repeats totaling 22,704 bp, which constitutes 4.09% of the whole mitogenome. Most of the repeats (450 repeats, 85.23%) are shorter than 50 bp, and only 6 (1.14%) repeats are larger than 200 bp. The short repeats (<100 bp) of I. pubescens are significantly more than those of other three Ilex mitogenomes (Figure 2C).

3.3. Analysis of Mitochondrial Plastid Sequences (MTPTs)

In the present study, we identified multiple instances of plastid-derived sequence transfers within the mitogenomes of I. rotunda (Figure 3) and three other Ilex species. Through our analysis, we identified a total of 51 MTPTs in I. rotunda, with a total length of 11,458 bp, which constitutes 2.02% of the whole mitogenome. The results of similar comparative analyses are presented in Table S5, respectively. Among the MTPTs in the I. rotunda mitogenome, 44 are larger than 50 bp, with the longest being 1375 bp. By annotating these plastid-like fragments in the I. rotunda mitogenome, 21 plastid genes were found to be located at MTPTs, including 5 protein-coding gene fragments (ycf2, ndhB, rpl14, rpl2, and ycf15), 3 rRNA gene fragments (rrn16 and rrn5), and 10 tRNA gene fragments (trnV-GAC, trnP-UGG, trnW-CCA, trnD-GUC, trnN-GUU, trnH-GUG, trnM-CAU, trnI-CAU, trnA-UGC, and trnN-GUU). Among them, five plastid-derived tRNA genes (trnN-GUU, trnM-CAU, trnV-GAC, trnD-GUC, and trnH-GUG) remained intact after transfer to the mitogenome.

3.4. Collinearity Analysis

In the current study, an analysis of the collinearity was conducted for the mitogenome of I. rotunda alongside another three reported Ilex mitogenomes (I. macrocarpa, I. pubescens, and I. metabaptista) and one Asterales mitogenome (L. sativa) (Figure 4 and Table S6). The homologous collinear regions detected between the mitogenomes of I. rotunda and I. metabaptista were in the aggregate 43. The most extensive region spanned 54,589 bp, accounting for 76.06% (431,644 bp) of the whole I. rotunda mitogenome. The homologous colinear regions detected between the mitogenomes of I. rotunda and I. macrocarpa were in the aggregate 72. The most extensive region spanned 90,029 bp, accounting for 73.77% (418,660 bp) of the entire mitogenome of I. macrocarpa. The homologous colinear regions detected between the mitogenomes of I. metabaptista and I. pubescens were in the aggregate 41, with the most extensive region spanning 42,348 bp. These regions accounted for 81.78% (433,082 bp) of the whole mitogenome of I. metabaptista (529,560 bp) [44]. The homologous colinear regions detected between the mitogenomes of I. macrocarpa and L. sativa were in the aggregate 107, with the largest region spanning only 3974 bp, accounting for only 23.66% (127,621 bp) of the whole I. macrocarpa mitogenome (539,461 bp) [45]. This reveals that there are numerous homologous collinear regions between the mitogenomes of I. rotunda and other Aquifoliales, which have longer lengths and achieve higher scores than the collinear regions between the mitogenomes of Aquifoliales and Asterales (L. sativa), with variations in the arrangement order among different mitogenomes.

3.5. Phylogenetic Analysis

A phylogenetic analysis was undertaken in order to elucidate the evolutionary position of the mitogenome of I. rotunda, incorporating both the mitogenomes and the plastomes of I. rotunda and 35 other plant species, including 34 eudicots and 2 monocots (which served as the outgroup). The phylogenetic tree (Figure 5A) based on the mitogenomes revealed that the Aquifoliales showed high bootstrap support (100%) at the base of the campanulids, while the Asterales, Apiales, and Dipsacales clustered together with relatively high bootstrap support. The genus Ilex was observed to be clustered on the same branch, with I. rotunda exhibiting a particularly close evolutionary relationship to I. macrocarpa. The topological branching conflict outside of the Aquifoliales between the mitochondrial and plastid phylogenetic trees lies in the branching relationships of the Apiales, Asterales, and Dipsacales (Figure 5B). In the Ilex, plastid phylogeny indicates that I. rotunda is located at the base of the Ilex, which requires further research. The phylogenetic trees with the branch lengths are presented in Figures S2 and S3.

4. Discussion

Plant mitochondria play a crucial role in evolutionary history investigations, attributed to their relatively low rate of sequence mutation and high frequency of genomic recombination [46,47]. The structural characteristics of plant mitogenomes are not only crucial for understanding plant development and stress tolerance mechanisms [2] but also hold important value in forestry, given their implications for enhancing tree species’ resilience and productivity. Moreover, this research contributes to the economic value of agriculture by suggesting strategies for developing crops with improved stress resistance and yield, thereby supporting sustainable agricultural practices. However, sequencing and analyzing plant mitogenomes pose significant challenges owing to the complex situation of the mitogenome, marked by an abundance of repeats, the integration of plastid DNA, and a high degree of recombination. These complexities present hurdles to the mitogenome assembly of plant species [11,48]. Fortunately, the recent and rapid progress in high-throughput sequencing technologies and assembly methods has led to more plant mitogenome projects and the production of high-quality assemblies [49]. In higher plants, mitogenomes often exhibit complex structures, which can include circular, linear, and even a variety of smaller circular forms of different sizes [21,50,51,52]. In the present research, we successfully assembled the I. rotunda mitogenome based on PacBio HiFi sequencing data. In order to ensure the clear identification and assembly of repeat structures, the following specific parameters were set: a minimum overlap length (-ml) of 40 bp and a minimum overlap identity (-mi) of 90%. With these parameters, repeat structures longer than 40 bp were identified and assembled [24]. Consequently, the mitogenome of I. rotunda was successfully assembled into a typical single-circular structure. The mitogenome size (567,552 bp) and GC content (45.47%) of I. rotunda are similar to those of other Ilex mitogenomes (Table 1). The numbers of PCGs, tRNAs, and rRNAs in four Ilex mitogenomes are basically the same. The variance in the gene number primarily stems from gene duplication events (Table S2). In our analysis of PCGs, we observed that the cox1 and nad4L genes exhibit C–to–U RNA-editing events at their start codons. However, the start codon of gene rps4 is probably ACG. This is a phenomenon that has been observed in other plants, such as Nicotiana tabacum [53] and Ricinus communis [54]. Moreover, we identified two gene-loss events: the rpl16 gene, which appears to be absent in the I. pubescens mitogenome, and the rps10 gene, which is uniquely conserved in I. metabaptista. The loss of these genes may be attributed to their transfer to the nuclear genome, which is frequently observed in the long-term evolution of angiosperms [55]. Further research is needed to investigate the implications of these genetic variations for gene expression and evolutionary relationships. These results indicate that the genetic structure of I. rotunda is highly conserved in the genus Ilex and circular structures seem to be the prevailing configuration of the Ilex mitogenomes [45].
Repetitive sequences are typically prevalent in plant mitogenomes but relatively less abundant in plastomes [56]. Analyzing these sequences is vital for gaining insights into the duplication of sequences and the recombination between different molecules within mitogenomes [57]. The variation in the size of Ilex mitogenomes is relatively limited (Table 1), spanning from 517 kb (I. pubescens) to 567 kb (I. rotunda). The comparative analysis of repeats within the four Ilex mitogenomes demonstrated that the I. pubescens mitogenome exhibited a larger size and a higher abundance of repeats (33,648 bp). This underscores the significance of repeats in the amplification of mitogenomes in plants. Repeats > 50 bp account for 12.17% of the whole mitogenome of I. rotunda, which is similar to that of other Ilex species (11.96%–16.99%) (Table 1). The largest repeats of the four Ilex species all come from dispersed repeats that are 410 bp in length. Meanwhile, we found that these repeats among the four species were completely identical, indicating that the size and structure of these repeats are highly conserved in the Ilex mitogenomes. Repeat-mediated rearrangement can also lead to gene loss and multiple copies [27,50,58]. However, the tRNA genes in the I. rotunda mitogenome are all single-copy genes, while multiple copies are evident solely in the PCGs. The absence of repeats larger than 500 bp in the I. rotunda mitogenome may contribute to maintaining the conservation of its genomic structure and gene content. Additionally, a higher proportion of A/T proportion was observed in many of the SSRs, aligning with previous observations. This phenomenon indicates that mitochondrial SSRs are primarily made up of short repeats dominated by polyadenine (polyA) or polythymidine (polyT) motifs [59].
The evolution of the mitogenomes is associated with numerous structural rearrangements and sequence transfer events [59]. One of the most important features of the evolutionary process is the exchange of sequences between mitogenomes and plastomes [49,60,61]. Consequently, monitoring the transfer of sequences between these two types of organelle genomes is crucial for elucidating the evolutionary processes within the mitogenomes of plants [62,63]. Throughout the evolutionary history of mitochondria in higher plants, the lengths and sequence similarities of the fragments that have been transferred have shown considerable variation [64]. Sequence transfers between plastomes and mitogenomes occur frequently over extended periods of evolution, with plastid-derived sequences typically occupying 0.1 to 10.3% of the mitogenome [65]. From an evolutionary perspective, the transfer of plastid sequences to mitogenomes occurred in the earlier common ancestor of certain relative species as a single event [64]. In the current research, the percentage of sequence fragments transferred between the mitogenome and the plastome in I. rotunda (2.02%) was closely similar to previously documented rates for I. metabaptista (2.02%), I. pubescens (1.91%) and I. macrocarpa (1.28%). Nevertheless, the transfer rate is notably lower than what has been observed in Vitis vinifera (8.8%) [66] and O. sativa (6.3%) [67]. The similarity rate of MTPT among the four Ilex mitogenomes may suggest that they have undergone comparable genomic dynamic changes during their evolutionary processes. It is worth noting that in angiosperms, the transfer of tRNA genes from plastomes to mitogenomes is a prevalent phenomenon [44]. Our research has revealed that the transfer of tRNA genes from plastomes to mitogenomes is a common occurrence within I. rotunda. This finding aligns with the patterns observed in both I. metabaptista [44] and I. macrocarpa [45], highlighting a consistent trend in the genetic mobility of tRNA genes across these related plant species. The frequent transfer confirms the universality of tRNA gene transfer in the mitogenomes of angiosperms.
The collinear sequences between the mitogenomes of Oryza species accounted for less than 60% of the whole O. granulate mitogenome. In addition, numerous short homologous collinear regions were detectable among the Oryza species. However, the arrangement of these collinear regions within the mitogenomes of Oryza species appears to be unstable, indicating that Oryza species lack collinearity. During the early stages of its evolutionary history, S. bicolor experienced a substantial loss of genes within its homologous fragments, which led to the low conservation of the species. Different from the Poales, plants in Populus exhibit a high degree of conservation among themselves [26]. The collinear sequences among the mitogenomes of Populus has a high proportion (89%–99%) [26], indicating that the internal species of Populus are closely related and that their mitogenome is conserved. In the current research, we conducted a comparative analysis of the I. rotunda mitogenome alongside those of three additional Ilex species and one Asterales species, revealing high collinearity (>70%) within the genus Ilex (Figure 4). The collinear sequences between the mitogenomes of Ilex and L. sativa originating from different genera of close species accounted for only 23.66% of the whole I. macrocarpa mitogenome. The collinearity analysis we conducted shows that the Ilex mitogenomes have likely undergone fewer genomic rearrangements throughout their evolutionary history and are highly conserved in size.
Mitogenomes serve as a testament to the unique evolutionary trajectory of angiosperms [68]. The content, structure, and genetic arrangement within plastomes are of great importance in elucidating plants’ evolutionary relationships [69]. The content of plastid genes in terrestrial plants seems to have remained relatively stable, with minimal occurrences of gene loss throughout the evolutionary history of these organisms [70]. Compared to plastomes, the mitogenomes are noted for their reduced nucleotide substitution rates and decreased homoplasy. This characteristic renders mitogenomes particularly suitable for the investigation of more ancient and deeper phylogenetic relationships within the tree of life [71]. Plastomes and mitogenomes have distinct evolutionary trajectories within certain groups of plants. Instances of paternal leakage have been observed in natural plant populations. This phenomenon can lead to discrepancies between the phylogenies inferred from organellar genomes [72,73]. Numerous cases have been documented where the phylogenetic trees derived from nuclear and organelle DNA do not align, comprising Eucalyptus [74] and Dendrobium [75]. These observed incongruencies can be ascribed to various evolutionary processes, such as the horizontal gene transfer (HGT) between different species, the interbreeding of different plants in hybridization events, and the phenomenon of incomplete lineage sorting (ILS), which occur throughout the course of plants’ evolution [74,75]. This study analyzed the phylogenetic position of I. rotunda and other 35 plants based on mitogenomes and plastomes. The results revealed incongruent topologies between the phylogenetic trees constructed from plastomes and mitogenomes. The mitochondrial and plastid phylogenetic trees both corroborate the positioning of the Aquifoliales as the basal group within the campanulids, a classification that aligns with the APG IV taxonomic tree [76]. However, phylogenetic inconsistency is found within the campanulids. The phylogenetic tree based on mitogenomes supports a close branching relationship between the Asterales and Apiales groups, with the Dipsacales group located near the basal branch of the campanulids. In the genus Ilex, I. rotunda is a sister to I. macrocarpa, with strong support in mitochondrial phylogenetic tree. However, the plastid phylogenetic tree indicates that the Asterales group is closely related to the basal branches of the campanulids, whereas I. rotunda is located at the base of the Ilex. Additionally, the high degree of support of leaf nodes in the mitochondrial and plastid phylogenetic trees demonstrates the accuracy of the topological structures. This inconsistency may be due to the relatively independent genetic system of mitochondria, or it may be caused by species hybridization and ILS. More accurate speculation requires a more comprehensive sampling strategy and more detailed evolutionary analysis.
The I. rotunda mitogenome offers profound insights into the tree’s evolutionary trajectory and its role in the forestry ecosystem of subtropical regions. Comparative analyses across Ilex mitogenomes can reveal the molecular basis of its speciation and adaptation. Moreover, research on plant mitogenomes is foundational for advancing plant breeding, facilitating the creation of plants that are more resilient to shifting environmental conditions. The findings of this study are not only academically significant but also have practical implications for forestry. The detailed characterization of the I. rotunda mitogenome can inform conservation efforts aimed at preserving the genetic integrity of this species.

5. Conclusions

This investigation successfully assembled and annotated the I. rotunda mitogenome using PacBio HiFi sequencing technology. The circular mitogenome of I. rotunda is 567,552 bp in total length and 45.47% of the GC content, which is consistent with other Ilex mitogenomes. Moreover, we performed comprehensive analyses of the gene content, repetitive sequences, mitochondrial plastid sequences, and collinear sequences within the mitogenome. To ascertain the evolutionary position, a comprehensive phylogenetic analysis was performed, incorporating the organelle genomes of I. rotunda along with 35 other plant species. This research not only offers detailed insights into the characteristics of the I. rotunda mitogenome but also enhances our comprehension of the evolutionary relationships within the Aquifoliales and the campanulids. The availability of the mitogenome of I. rotunda, a member of the Aquifoliales family, facilitates future studies on the genetic evolution, phylogenies, and conservation strategies. Therefore, unraveling the mitochondrial molecular background has implications for ecological benefits, economic efforts, and molecular breeding strategies for enhancing stress tolerance, disease resistance and pharmaceutical use of Ilex species in subtropical regions.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f15071117/s1, Table S1: Species included in the phylogenetic analyses with their accession numbers; Table S2: Gene profile and organization of PCGs in four Ilex mitogenomes; Table S3: Frequency of identified SSR motifs within the I. rotunda mitogenome; Table S4: Tandem repeats in the I. rotunda mitogenome; Table S5: Fragments transferred from the plastome to the mitogenome in four Ilex species; Table S6: An analysis of the sequence collinearity between the mitogenome of I. rotunda and those of related species; Table S7: Conserved genes selected in the phylogenetic trees; Figure S1: The circular structure of the mitogenome of I. rotunda; Figure S2: A visualization of the phylogenetic tree’s branch lengths derived from the mitogenomes; Figure S3: A visualization of the phylogenetic tree’s branch lengths derived from the plastomes.

Author Contributions

C.B., Z.W. and W.L. planned and designed the research. Y.W. (Yuanjian Wang), G.C. and K.H. analyzed the data and prepared the figures. K.X. provided materials. C.B. and S.L. conducted the experiments. Y.W. (Yuanjian Wang), G.C. and K.H. wrote the initial version of the manuscript. Y.W. (Yuxiao Wang) polished the initial version of the manuscript. C.B. and S.L. revised this and provided comments. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20220414 and BK20210612), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (22KJB220003 and 23KJB480005), and the Beidou Scientific Research Program of Nanjing Polytechnic Institute (NJPI-RC-2023-13).

Data Availability Statement

The mitochondrial genome supporting this study is available in GenBank under accession number NC_084321.1. The HiFi sequencing data of I. rotunda is deposited in the SRA repository under SRR28365293.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Circular structure of the I. rotunda mitogenome. The genes encoded within the inner circle are transcribed in a clockwise orientation, and those situated on the outer circle are transcribed in a counterclockwise orientation. Different colors on the map serve to differentiate genes based on their specific functions.
Figure 1. Circular structure of the I. rotunda mitogenome. The genes encoded within the inner circle are transcribed in a clockwise orientation, and those situated on the outer circle are transcribed in a counterclockwise orientation. Different colors on the map serve to differentiate genes based on their specific functions.
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Figure 2. The circle on the left (A) illustrates the distribution of repeats within the mitogenome of I. rotunda. Different concentric circles represent different types of repeats, with the SSRs located in the outermost circle, followed by the tandem repeats, and concluding with the dispersed repeats in the innermost circle. The darker color of the lines represents for the longer fragments of the repeats. The two bar charts on the right show repeats of the genus Ilex. (B) Comparison of different types of SSRs in four Ilex mitogenomes. (C) The count of dispersed repeats of varying lengths within four Ilex mitogenomes.
Figure 2. The circle on the left (A) illustrates the distribution of repeats within the mitogenome of I. rotunda. Different concentric circles represent different types of repeats, with the SSRs located in the outermost circle, followed by the tandem repeats, and concluding with the dispersed repeats in the innermost circle. The darker color of the lines represents for the longer fragments of the repeats. The two bar charts on the right show repeats of the genus Ilex. (B) Comparison of different types of SSRs in four Ilex mitogenomes. (C) The count of dispersed repeats of varying lengths within four Ilex mitogenomes.
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Figure 3. The shared transfer sequences of the mitogenome and plastome of I. rotunda. The folded line indicates the GC skew across the mitogenome and plastome. The lines between the arcs correspond to the genomic homologous fragments. The darker shade of the connecting lines signifies longer fragments (>1000 bp) of the MTPTs.
Figure 3. The shared transfer sequences of the mitogenome and plastome of I. rotunda. The folded line indicates the GC skew across the mitogenome and plastome. The lines between the arcs correspond to the genomic homologous fragments. The darker shade of the connecting lines signifies longer fragments (>1000 bp) of the MTPTs.
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Figure 4. Collinearity plots of I. rotunda’s and four other campanulids’ mitogenomes. Each row comprises boxes representing the mitogenomes. The homologous regions between these genomes are highlighted by connecting lines situated in the center of the plots.
Figure 4. Collinearity plots of I. rotunda’s and four other campanulids’ mitogenomes. Each row comprises boxes representing the mitogenomes. The homologous regions between these genomes are highlighted by connecting lines situated in the center of the plots.
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Figure 5. The phylogenetic trees of I. rotunda and 35 other plants. Oryza sativa and Allium cepa served as the outgroup. Each node in the phylogenetic trees is annotated with its corresponding bootstrap value. Figure (A) illustrates the phylogenetic tree constructed based on the mitogenomes, while Figure (B) depicts the phylogenetic tree inferred from the plastomes.
Figure 5. The phylogenetic trees of I. rotunda and 35 other plants. Oryza sativa and Allium cepa served as the outgroup. Each node in the phylogenetic trees is annotated with its corresponding bootstrap value. Figure (A) illustrates the phylogenetic tree constructed based on the mitogenomes, while Figure (B) depicts the phylogenetic tree inferred from the plastomes.
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Table 1. Genomic characteristics of four Ilex mitogenomes.
Table 1. Genomic characteristics of four Ilex mitogenomes.
FeatureI. rotundaI. macrocarpaI. metabaptistaI. pubescens
Accession numberNC_084321.1NC_082235.1NC_081509.1NC_045078.1
Size of genome (bp)567,552539,461529,560517,520
GC content (%)45.4745.5345.6145.55
Length of protein coding region (bp)33,189 (5.85%)32,817 (6.08%)33,123 (6.25%)32,385 (6.26%)
Length of rRNAs (bp)5247 (0.92%)5248 (0.97%)5250 (0.99%)5243 (1.01%)
Length of tRNAs (bp)1428 (0.25%)1354 (0.25%)1438 (0.27%)1303 (0.25%)
Number of PCGs40394239
Number of rRNAs3333
Number of tRNAs19181917
Total genes62606459
Number of >50 (bp) repeats88 (12.17%)81 (11.96%)92 (14.02%)168 (16.99%)
Longest repeat (bp)410410410410
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MDPI and ACS Style

Wang, Y.; Cui, G.; He, K.; Xu, K.; Liu, W.; Wang, Y.; Wang, Z.; Liu, S.; Bi, C. Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex rotunda Thunb. Forests 2024, 15, 1117. https://doi.org/10.3390/f15071117

AMA Style

Wang Y, Cui G, He K, Xu K, Liu W, Wang Y, Wang Z, Liu S, Bi C. Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex rotunda Thunb. Forests. 2024; 15(7):1117. https://doi.org/10.3390/f15071117

Chicago/Turabian Style

Wang, Yuanjian, Gang Cui, Kaifeng He, Kewang Xu, Wei Liu, Yuxiao Wang, Zefu Wang, Shasha Liu, and Changwei Bi. 2024. "Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex rotunda Thunb." Forests 15, no. 7: 1117. https://doi.org/10.3390/f15071117

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