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Article

Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex macrocarpa

by
Yuxiao Wang
1,2,
Ning Sun
1,
Wenxi Shi
1,2,
Qiuyue Ma
3,
Liyong Sun
1,
Mingzhuo Hao
1,4,
Changwei Bi
1,2,* and
Shuxian Li
1,2,*
1
Forestry and Grass College, Nanjing Forestry University, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
3
Institute of Leisure Agriculture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
4
Jiangsu Qinghao Landscape Horticulture Co., Ltd., Nanjing 210037, China
*
Authors to whom correspondence should be addressed.
Forests 2023, 14(12), 2372; https://doi.org/10.3390/f14122372
Submission received: 2 November 2023 / Revised: 20 November 2023 / Accepted: 28 November 2023 / Published: 5 December 2023
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
The plant mitochondrial genome (mitogenome) has a complex organization and carries genetic traits of value in exploiting genetic resources. In this study, the Ilex macrocarpa mitogenome was sequenced and assembled to understand the genetic diversity and phylogenetic relationship. The mitogenome has a cyclic molecular structure and is 539,461 bp long with a GC content of 45.53%. It contained 39 protein-coding proteins, 19 tRNA genes, and 3 rRNA genes. The 146 simple sequence repeats and 591 dispersed repeat sequences were identified in the mitogenome. Codon usage analysis revealed a preference for codons ending in A/T. A total of 517 C to U RNA editing sites were predicted, with nad4 and ccmB being edited most frequently (39 times). Phylogenetic analysis via mitochondrial protein-coding genes showed that the Aquifoliales order contains sister plants to Lamiales, Solanales, and Gentianales. In addition, the conflicts between chloroplast and mitochondrial phylogeny were also observed. This study provides a theoretical framework for understanding the evolution, classification, and identification of the Ilex genus, which lays an important foundation for future molecular breeding investigations.

1. Introduction

Mitochondria are key players in plant development, fitness, and reproduction. Their contribution to energy production, metabolism, and cell homeostasis relies on the performance of their genetic system [1,2]. They also regulate cell growth and the cell cycle [3]. The structure of the mitogenome is complex, including circular and linear conformations, as well as branched formations and multiple smaller circular molecules [4,5]. The number of genes in land plant mitogenomes varies widely, typically ranging from 19 to 67 [6,7]. Although the number of genes varies, the encoded products are generally similar, including the rRNAs, tRNAs, and protein subunits required for respiratory chain complexes [8,9]. The mitogenome sizes vary widely among different plant species, ranging from 66 kb (Viscum scurruloideum) [10] to 11.3 Mb (Silene conica) [11]. This difference can be attributed to the repetitive sequences and the DNA from other organisms during evolution [9]. The mitogenome comprises various repetitive sequences, such as simple sequence repeats (SSRs), tandem repeats, and dispersed repeats. These repeats can induce chromosome recombination, altering chromosome structure, and sometimes result in gene loss or duplication [12]. RNA editing is a crucial process that maintains the normal biological functions of chloroplasts and mitochondria in all eukaryotes [13]. Homologous fragment sequences in mitochondria primarily originate from the chloroplast genome and nuclear genome [14,15], suggesting that mitochondria have a propensity to integrate DNA from diverse sources through intracellular horizontal migration [16]. Mitogenome is relatively independent from the nucleus and relatively conserved. Consequently, it is commonly used in evolutionary analyses and interspecific identification studies, particularly for reconstructing ancient phylogenetic relationships and those among closely related species [17,18].
The Ilex genus consists of over 400 species distributed worldwide across tropical and temperate regions, with the highest biodiversity in South America and Asia [19]. Ilex macrocarpa Oliv. is a deciduous tree belonging to the Ilex genus in the Aquifoliaceae family [20]. It is primarily found in southern regions of China [21]. In the past few years, I. macrocarpa has been utilized because of its strong adaptability and high ornamental and medicinal value [22,23]. Additionally, its wood also can be used for construction or furniture. At present, four mitogenomes of Ilex species have been reported, and the other ones have not yet been extensively analyzed [24,25]. This limited exploration hampers our comprehensive understanding of the evolution of mitogenomes in this family.
In this study, we assembled and analyzed the complete I. macrocarpa mitogenome, including the gene content, repetitive sequences, RNA editing sites, synteny sequence analysis, and phylogenetic relationships. The aim of the present study is to enhance the theoretical basis for understanding the evolution, classification, and identification of I. macrocarpa, and facilitate further utilization of I. macrocarpa germplasm resources.

2. Materials and Methods

2.1. Plant Materials, DNA Extraction, and Sequencing

Fresh leaves of I. macrocarpa were collected at Nanjing Forestry University (Nanjing, China; 31°24′ N, 119°26′ E). The leaves were cleaned with DEPC water and stored in a freezer at −80 °C. The DNA of I. macrocarpa was extracted by using a Plant Genomic DNA kit (Tiangen Co., Ltd., Nanjing, China). The assessment of the purity and quality of DNA was conducted using 1.0% agarose gel and a NanoDrop spectrophotometer 2000 (NanoDrop, Wilmington, DE, USA). The high-integrity genomic DNA was then used for constructing sequencing libraries using an SMRTbell Express Template Prep Kit 2.0 (PacBio Biosciences, Menlo Park, CA, USA). The HiFi sequencing data were generated from the PacBio Revio platform.

2.2. Assembly and Annotation of Mitogenomes

The HiFi sequencing data of I. macrocarpa were input into PMAT v1.3.1 (https://github.com/bichangwei/PMAT, accessed on 4 July 2023) to assemble the mitogenome with the ‘autoMito’ mode. The parameters used for assembly were ‘-st HiFi -g 730M -ml 40 -mi 90’. The genome size was estimated using the I. polyneura genome as reference [26]. The I. macrocarpa mitogenome exhibited a typical circular structure and was composed of a single contig (contig00556).
Protein-coding genes (PCGs), tRNA genes, and rRNA genes were predicted using the GeSeq [27] with default parameters. The position of each coding gene was determined by performing a BLASTn search against the I. pubescens [24] and I. metabaptista mitogenomes [25]. The final annotation results were obtained after careful review and manual correction of the initial results. The mitogenome was then visualized using the drawing software OGDRAW v1.3.1 [28].

2.3. Analysis of Repeated Sequences and Codon Usage

The online website MISA (https://webblast.ipk-gatersle-ben.de/misa/, accessed on 5 July 2023) was utilized to identify SSRs, with minimum repetition numbers of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively [29]. Dispersed repeats were identified using REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer, accessed on 7 July 2023), with a hamming distance of 3, a maximum computed repeat of 5000, and a minimum repeat size of 30 [30].
Tandem Repeats Finder (https://tandem.bu.edu/trf/home, accessed on 10 July 2023) was utilized to identify tandem repeats. The parameters used for this analysis were a minimum alignment score to repeat of 60, a maximum period size of 500, and maximum TR array size (bp, millions) of 2 [31]. In addition, CodonW v1.4.4 software was used to analyze codon preference in the mitogenome PCGs and calculate RSCU values.

2.4. Analyses of Chloroplast to Mitochondrion DNA Transformation and RNA Editing

The chloroplast genome of I. macrocarpa (NC_069020) was downloaded from NCBI. The BLASTn v2.14.0 software on NCBI was employed to identify the fragments transferred from chloroplasts to mitochondria [32]. Screening criteria were established based on the following parameters: a minimum matching rate of 70% and a sequence length requirement of at least 30 bp. The parameters used for BLASTn were ‘-evalue 1e-5 -word_size 9 -gapextend 2 -reward 2 -penalty -3 -gapopen 5’.
RNA editing sites in the PCGs of I. macrocarpa were predicted using the Deepred-mt [33]. To obtain more accurate predictions, we selected results with probability values greater than 0.9. The HiFi sequencing data were then mapped to the mitochondrial PCG sequence using Minimap2 with the parameter “-x map-hifi” to identify potential single-nucleotide polymorphism (SNP) sites. The mpileup module of samtools was set to “-Ou -f” and the bcftools call was set to “-mv -Ov -o”. Finally, the custom script was used to calculate the coverage and base composition of each locus. The coverage was set to 50× and SNP loci with quality values greater than 50 retained. The sites excluded from the SNPs were high-quality RNA editing sites in the I. macrocarpa mitogenome PCGs.

2.5. Analysis of Phylogeny and Synteny

The chloroplast and mitochondrial phylogenetic trees were constructed using 19 other chloroplasts and mitogenomes downloaded from NCBI, with Oryza sativa and Phoenix dactylifera set as outgroups. The common mitochondrial genes were filtered with BLASTn [32] and extracted, then concatenated with PhyloSuite. A total of 22 conserved PCGs were selected for multiple sequence alignment using MAFFT v7.407 [34]. Next, these aligned sequences were concatenated to construct the phylogenetic trees using the maximum likelihood (ML) method implemented in IQ-TREE v2.0.3. The alignment results were calculated using MRBAYES [35], with the best evolutionary model chosen as GTR+F+I+G4 according to the Bayesian Information Criterion (BIC) scores. The bootstrap analysis was performed with 1000 replicates. Finally, the maximum-likelihood tree was visualized using iTOL (https://itol.embl.de/, accessed on 11 July 2023) [36].
In addition, MUMmer v3.23 [37] was used to visualize the multiple synthetic dot plots of I. macrocarpa and three other Ilex species (I. metabaptista, I. pubescens, and I. aquifolium). The delta-filter program of MUMmer was then employed to filter the alignment results with a minimum identity > 80% and a minimum alignment length > 100 bp. An in-house R script was used to visualize the co-linear results.

3. Results

3.1. Features of the I. macrocarpa Mitogenome

The I. macrocarpa mitogenome was 539,461 bp in size and had a circular DNA structure (Figure 1). The coding regions only accounted for 7.31% of the whole mitogenome, including the PCGs (6.07%), tRNA genes (0.26%), and rRNA (0.97%), respectively (Table 1). The GC content varies among different gene types in the I. macrocarpa mitogenome. The highest GC content was shown in rRNA, which was 51.85%, followed by tRNA genes (50.63%) and PCGs (43.21%). A total of 61 functional genes were found in the I. macrocarpa mitogenome, including 39 PCGs, 19 tRNA genes, and 3 rRNA genes (Table S1). Among these genes, atp9, trnE-TTC, trnM-CAT, and trnP-TGG are multi-copy genes. Furthermore, no introns were found in most of the PCGs, except for ccmFc, cox2, nad1, nad2, nad4, nad5, nad7, and rps3 (one or more introns).

3.2. Codon Usage Analysis of PCGs

Codon usage was analyzed in the 39 PCGs of the I. macrocarpa mitogenome (Table S2). A total of 10,900 codons were identified and most of them had ATG as the start codon, while ACG was found to be the start codon in nad4L and rps4, and ATA was the start codon in matR, which were possibly due to RNA editing alterations. Three stop codons (TAA, TGA, TAG) were identified in the PCGs, waith utilization rates of 56.41%, 30.77%, and 12.82%, respectively. This indicated a preference for A/T bases among mitogenomes.
In the I. macrocarpa mitogenome, we also assessed the relative synonymous codon usage (RSCU) of 39 PCGs. Codons (RSCU > 1) were considered to be used preferentially by amino acids [38]. The 28 codons were found with RSCU > 1, indicating a higher frequency of these codons compared to other synonymous codons (Figure 2). In the I. macrocarpa mitogenome, the GCU codon for Ala had the highest RSCU, with a value of 1.60. The codon usage analysis indicated that all RSCU values of NNT and NNA codons, excluding (Ala) GCA, Ile (ATA), Leu (CTA), and Thr (ACA), were higher than 1.0 (Table S3). The results indicated a strong A or T bias at the third codon position in the I. macrocarpa mitogenome.

3.3. Prediction of RNA Editing Sites in PCGs

In our study, a total of 569 C to U RNA editing sites were predicted in the 39 PCGs of the I. macrocarpa mitogenome (Figure 3). nad4 had the highest number of edits, with 39 RNA editing sites, followed by ccmB and mttB, with 38 and 36 editing sites, respectively. Interestingly, we found that one protein-coding gene (rps14) did not have any RNA editing sites, while rpl2, rpl10, and rps7 were predicted to have only two RNA editing sites, which was the lowest among all PCGs except for rps14. In addition, RNA editing in the mitogenome of I. macrocarpa primarily occurred at the first and second codon positions, with 32.34% and 63.27% predicted for those positions, respectively, and only 4.39% for the third position (Table S4).

3.4. Analysis of Repeat Sequences in the I. macrocarpa Mitogenome

In this study, 591 long repeats (≥30 bp) were identified in the I. macrocarpa mitogenome. These repeats were classified into three categories: forward repeats (331), palindromic repeats (254), and reverse repeats (6). Collectively, these long repeat sequences accounted for 18.24% of the whole mitogenome (26,608 bp) (Figure 4B and Table S5). The longest forward repeat sequence was 331 bp, while the longest palindromic repeat sequence was 410 bp. Most of the repeat sequences (477 repeats, 80.71%) were between 30 and 50 bp in length, with only 20 repeats longer than 100 bp. Furthermore, we identified nine tandem repeats in the mitogenomes of I. macrocarpa (Table S6). These tandem repeats had a matching degree exceeding 81% and varied in length from 18–39 bp.
SSR markers are reliable molecular markers for DNA fragments consisting of short sequence repeat units, which can range from 1 to 6 bp in length [9]. In the current study, we also analyzed SSR loci in the I. macrocarpa mitogenome. A total of 170 SSRs were identified (Table 2). Among the identified SSRs, tetranucleotide repeats were the most common, accounting for 36.47% of the total SSRs, followed by mononucleotide repeats of 23.53%. The lowest value was observed for the hexanucleotide category. Specifically, mononucleotide repeats containing A/T bases represented 92.50% of the monomeric SSRs, while dimeric SSRs composed of AT/AT bases accounted for 41.03% of the total dimeric SSRs (Table S7).

3.5. Chloroplast to Mitochondrion DNA Transfers

In this study, the 66 fragments were identified and transferred from chloroplast genomes to mitogenomes of I. macrocarpa (Figure 5, Table S8). These fragments had a combined length of 14,882 bp, accounting for 2.76% of the whole I. macrocarpa mitogenome. Two of these fragments were more than 1000 bp in length, and the longest one was 1565 bp. Some of the PCGs, rRNAs, and tRNAs were found among the fragments. Interestingly, the trnW-CCA, trnP-UGG, trnD-GUC, trnN-GUU, trnM-CAU, trnT-GGU, and trnI-CAU genes were all transferred in our study.

3.6. Synteny Sequence Analysis

The synteny of the I. macrocarpa mitogenome with the other three reported mitogenomes (I. metabaptista, I. pubescens, and I. aquifolium) was analyzed in this study (Figure 6, Table S9). A total of 63 homologous co-linear blocks were identified between the I. macrocarpa and I. aquifolium mitogenomes, with the largest block being 80,581 bp in length. These blocks accounted for 72.99% (393,761 bp) of the whole I. macrocarpa mitochondrial genome, showing 98.52% identity. A total of 54 homologous co-linear blocks were identified between the I. macrocarpa and I. metabaptista mitogenomes, with the largest block being 48,915 bp in length. These blocks accounted for 70.37% (379,624 bp) of the whole I. macrocarpa mitochondrial genome. Similarly, a total of 53 homologous co-linear blocks were identified between the I. macrocarpa and I. pubescens mitogenomes, with the largest block being 49,660 bp in length. These blocks accounted for 74.03% (399,381 bp) of the whole I. macrocarpa mitochondrial genome, showing 99.25% identity. The dot plot also revealed that I. macrocarpa and I. pubescens shared longer synteny sequences and higher similarity (Figure 6).

3.7. Phylogenetic Analysis

In this study, a phylogenetic analysis was conducted with a set of 22 conserved single-copy orthologous genes present in the I. macrocarpa mitogenome and 19 other plant species. As shown in Figure 7, the phylogenetic tree strongly supported (bootstrap was 100%) the close phylogenetic relationship between I. macrocarpa and two other Ilex species. The Ilex species clustered on the same branch in phylogenetics and mitogenomes, and I. pubescens showed a closer relationship to I. metabaptista (Figure 7). Simultaneously, the Aquifoliales were sister plants to Lamiales, Solanales, and Gentianales in the mitochondrial phylogeny. To further determine the evolutionary position of I. macrocarpa, the chloroplast phylogenetic tree was also constructed (Figure S1). The results showed that I. macrocarpa and I. metabaptista were clustered together, while I. pubescens was located on a separate branch within the chloroplast genome. In addition, the phylogenetic topology of the chloroplast genome supported the most recent classification of the Angiosperm Phylogenetic Group (APG IV) [39]. However, based on the analysis of the chloroplast phylogeny, it appeared that the Aquifoliales order had a closer genetic relationship with the Asterales and Apiales orders.

4. Discussion

4.1. Characterization of the I. macrocarpa Mitogenome

Mitochondria play crucial roles in supplying plant cells with the necessary energy for vital processes. Plant mitochondria contain various types of repetitive sequences and conserved coding sequences [40,41,42]. In higher plants, mitogenomes exhibit intricate composite structures, including circular, linear, and even numerous small circular structures with different sizes [43,44]. Circular structures appeared to be the common form among mitogenomes assembled in Ilex species. The mitogenome of I. macrocarpa is a circular DNA molecule spanning 539,461 bp in size, as we found in this study. The GC content, which is an important indicator for species evaluation, is typically stable at 43%–46% in higher plants [45,46]. In this study, the GC content of I. macrocarpa was 45.53%, which is similar to other angiosperm mitogenomes [47,48]. The result supports the conclusion that GC content is highly conserved in higher plants.
In general, the coding region is more conserved compared to the non-coding region, and the latter is the primary contributor to differences in mitogenomes [49]. The coding region makes up only 6.07% of the whole genome, while the non-coding region accounts for 92.69% of the I. macrocarpa mitogenome. The present study showed the presence of 39 PCGs, 35 tRNA genes, and 4 rRNA genes in the mitogenome. Some of these genes contain one or more introns, which have a significant impact on the regulation of gene expression [25].

4.2. Repeated Sequences and RNA Editing

Repetitive sequences, such as SSRs, tandem repeats, short repeats, and large repeats, are commonly found in plastid genomes [5]. Repetitive sequence analysis is crucial for studying the sequence duplications and intermolecular recombination in mitochondrial genomes [50]. The results revealed that a significant number of repetitive sequences was found in the I. macrocarpa mitogenome. The longest repeat was 410 bp and the A/T proportion was higher in many SSR repeats.
RNA editing is enriched in mitochondrial genomes, which plays an important role in protein folding. It primarily affects post-transcriptional gene expression and modifies genetic information in mRNA [51]. The number of RNA editing sites varies greatly among plant species mitogenomes. For example, Arabidopsis thaliana [52] has 441 RNA editing sites in 36 PCGs, Suaeda glauca [18] has 216 RNA editing sites in 26 PCGs, and Oryza sativa [53] has 491 RNA editing sites in 34 PCGs. In this study, we identified 517 C to U RNA editing sites in 39 PCGs within the I. macrocarpa mitogenome. RNA editing primarily targets the first and second positions of codons, resulting in alterations to the encoded amino acids. However, the largest numbers of RNA editing sites were associated with cytochrome c biogenesis and NADH dehydrogenase genes, a trend similar to that in Acer truncatum and Clematis acerifolia [54,55].

4.3. DNA Fragment Transfer Events

The length and sequence similarity of the migrated fragments have gradually changed during mitochondrial evolution. Consequently, the homologous sequences of higher plant mitogenomes have been transferred to chloroplast DNA, which has aided the movement of genetic material throughout organisms [56]. The DNA fragment transfer events have been found in many mitogenomes, such as A. truncatum [54] and Bupleurum chinense [50], and they have similar percentages of homologous fragments (2.36% and 2.56%, respectively) to I. macrocarpa (2.76%). However, this proportion is lower than the transfer of fragments between mitochondrial and chloroplast genomes in Vitis vinifera (8.8%) [57] and Oryza sativa (6.3%) [53]. This indicates significant variation in the migration of mitogenome sequences from chloroplasts across different plant species. This study identified 66 fragments of the mitogenome that were homologous to the chloroplast genome, which accounts for 2.76% (14,882 bp) of the total length of the mitogenome. These fragments may have important implications for evolution, which could facilitate normal transport functions. Due to numerous sequence variations and the absence of RNA editing, the transferred protein-coding sequences are often nonfunctional [58]. It has been proposed that tRNA genes in the mitogenome are highly conserved compared to PCGs, a characteristic that may be unique to the mitochondria of higher plants during their evolutionary development [59]. Based on the results of this research, the enlargement of the mitogenome of I. macrocarpa is primarily due to the increase in repetitive and homologous sequences between genomes.

4.4. Analysis of Phylogeny and Synteny

The functional genes are conserved in terms of number, type, and sequence in plant mitochondrial genomes. However, the location and order of these genes vary greatly among different species [60]. In this study, we compared the I. macrocarpa mitogenome with those of three other Ilex species. The results showed that synteny sequences between them exceeded 70%, indicating a high degree of conservation among different Ilex mitogenomes.
The maternal inheritance of chloroplast and mitochondrial genomes in many plants has simplified genetic research, making it a popular choice for phylogenetic inference in taxonomy. This genetic process has gained increasing utilization in recent years [61]. Establishing a scientifically reliable phylogenetic tree is essential for understanding the mechanisms of origin, diffusion, trait evolution, and species formation in biological groups [2,61]. Many phylogenetic inconsistencies between nuclear and organelle gene trees have been identified in plants, including Quercus acutissima [59] and Eucalyptus [62]. These inconsistencies might be caused by the occurrence of Horizontal Gene Transfer (HGT), hybridization, and Incomplete Lineage Sorting (ILS) in plant mitochondria [60,62,63,64,65]. Research on Dendrobium officinale has shown mitonuclear discordance, suggesting the possibility of hybridization [63]. Similarly, the phylogenetic relationships of Oreochromis exhibited incongruence between the mitochondrial and nuclear phylogenies, indicating potential ILS [64]. Previous studies have proposed that some plants might have acquired mitochondrial genes from other plants, leading to inconsistencies in phylogenetic relationships [65,66]. The conflicts between chloroplast and mitochondrial phylogeny were also observed in this study. According to the mitochondrial phylogeny, Aquifoliales are sister plants to Lamiales, Solanales, and Gentianales. However, the chloroplast phylogeny results indicated that the Aquifoliales order was more closely related to the Asterales and Apiales orders. Some researchers speculated that this inconsistency may arise from the relatively independent genetic system of the mitochondria, which could potentially conflict with nuclear or chloroplast genomes in terms of evolution [67,68]. Meanwhile, it was also hypothesized that ancient and recent hybridization affect the inconsistent evolution results between mitochondrial and chloroplast phylogenetic trees [69,70]. Moreover, employing more comprehensive sampling strategies, including sufficient samples for each species, and utilizing advanced analysis methods may yield a more robust conclusion on the evolutionary patterns within the genus.

5. Conclusions

In this study, we sequenced, assembled, and analyzed the features of the I. macrocarpa mitogenome. The genome has a circular molecular structure with a length of 539,461 bp and a GC content of 45.53%. A total of 61 genes were identified, including 39 PCGs, 19 tRNA genes, and 3 rRNA genes. In addition, the repetitive sequences, codon preferences, RNA editing, and fragment transfer events were also analyzed. To confirm the evolutionary status, a phylogenetic analysis of the I. macrocarpa organelle genomes and 19 other plants was conducted. Our study provides comprehensive information on the features of the I. macrocarpa mitogenome and contributes to our understanding of evolutionary relationships between Aquifoliaceae and Asterids.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14122372/s1, Figure S1: Maximum-likelihood phylogenetic tree based on chloroplast genomes; Table S1: Gene contents of the I. macrocarpa mitogenome; Table S2: Gene profile and organization of the I. macrocarpa mitogenome; Table S3: The relative percentage of amino acid residue in I. macrocarpa mitochondrial proteins; Table S4: The distribution of RNA editing sites in the PCGs of the I. macrocarpa mitogenome; Table S5: The repeat sequences distributions in the I. macrocarpa mitogenome genome; Table S6: Distribution of tandem repeats in the I. macrocarpa mitogenome; Table S7: The SSR types detected in the I. macrocarpa mitogenome; Table S8: Fragments transferred from chloroplasts to mitochondria in the I. macrocarpa genome; Table S9: The synteny sequences analysis of three Ilex mitogenomes.

Author Contributions

Y.W. and C.B. conceived the study. C.B. assembled and annotated the genome. N.S., W.S. and Q.M. carried out the repeat analyses and phylogenetic analysis. L.S. and M.H. analyzed codon usage and synteny sequences. Q.M., S.L. and C.B. revised and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Natural Science Foundation of Jiangsu Province (BK20220414), the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (22KJB220003), Jiangsu Students’ Innovation and Entrepreneurship Training Program (202210298119Y), Jiangsu Provincial Key Research & Development Programme—Modern Agriculture (BE2021307), and the Natural Science Foundation of China (32001357).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy restrictions.

Acknowledgments

We acknowledge Mingwei Zhu, Xu Wang, and Jinyan Mao from Nanjing Forestry University for their help and suggestions on the manuscript.

Conflicts of Interest

The authors declare that they have no competing interests. Author M.H. is employed by Jiangsu Qinghao Landscape Horticulture Co., Ltd. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Forests 14 02372 g001
Figure 1. Circular map of the I. macrocarpa mitogenome. Genes located in the interior of the circle undergo transcription in a clockwise direction, while those on the exterior undergo transcription in a counterclockwise direction. The various colors on the circle indicate distinct functional genes. The dark gray shading on the inner circle denotes the GC content, and genes that possess introns are marked with an asterisk.
Figure 1. Circular map of the I. macrocarpa mitogenome. Genes located in the interior of the circle undergo transcription in a clockwise direction, while those on the exterior undergo transcription in a counterclockwise direction. The various colors on the circle indicate distinct functional genes. The dark gray shading on the inner circle denotes the GC content, and genes that possess introns are marked with an asterisk.
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Forests 14 02372 g002
Figure 2. Analysis of relative synonymous codon usage (RSCU) in the I. macrocarpa mitogenome. The X-axis displays various codon families, while RSCU values reveal the frequency of specific codons compared to their anticipated appearance with uniform synonymous codon usage.
Figure 2. Analysis of relative synonymous codon usage (RSCU) in the I. macrocarpa mitogenome. The X-axis displays various codon families, while RSCU values reveal the frequency of specific codons compared to their anticipated appearance with uniform synonymous codon usage.
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Forests 14 02372 g003
Figure 3. The number of RNA editing sites identified in the PCGs of the I. macrocarpa mitogenome.
Figure 3. The number of RNA editing sites identified in the PCGs of the I. macrocarpa mitogenome.
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Figure 4. Detected repeats in the I. macrocarpa mitogenome. (A) Type and proportion of detected repeats. (B) Frequency distribution of repeat lengths.
Figure 4. Detected repeats in the I. macrocarpa mitogenome. (A) Type and proportion of detected repeats. (B) Frequency distribution of repeat lengths.
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Figure 5. Fragments transferred from chloroplasts to mitochondria in the I. macrocarpa genome.
Figure 5. Fragments transferred from chloroplasts to mitochondria in the I. macrocarpa genome.
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Figure 6. The synteny sequences analysis of three Ilex mitogenomes, with the I. macrocarpa mitogenome as the reference.
Figure 6. The synteny sequences analysis of three Ilex mitogenomes, with the I. macrocarpa mitogenome as the reference.
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Figure 7. Maximum-likelihood phylogenetic tree based on mitogenomes. Oryza sativa and Phoenix dactylifera as outgroups. Numbers at nodes are bootstrap support values. The position of I. macrocarpa is indicated in bold.
Figure 7. Maximum-likelihood phylogenetic tree based on mitogenomes. Oryza sativa and Phoenix dactylifera as outgroups. Numbers at nodes are bootstrap support values. The position of I. macrocarpa is indicated in bold.
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Table 1. Genomic features of the I. macrocarpa mitogenome.
Table 1. Genomic features of the I. macrocarpa mitogenome.
FeatureA %C %G %T %GC %Size (bp)Proportion in Genome (%)
Whole genome27.2022.5422.9927.2745.53539,461100
Protein-coding genes26.4021.4021.8130.3843.2132,7676.07
tRNA genes a26.1927.4523.1823.1850.6314280.26
rRNA genes a26.1422.6629.1922.0151.8552480.97
Non-coding regions27.2722.6023.0027.1345.60500,01892.69
a Protein-coding genes, cis-spliced introns, tRNAs, and rRNAs belong to coding regions.
Table 2. Frequency of identified SSR motifs in the I. macrocarpa mitogenome.
Table 2. Frequency of identified SSR motifs in the I. macrocarpa mitogenome.
Motif TypeNumber of RepeatsTotalProportion (%)
34567891011121315161820
Monomer 2166312 14023.53
Dimer 2782 2 3922.94
Trimer 112 137.65
Tetramer592 1 6236.47
Pentamer1111 2 158.82
Hexamer1 10.59
Total7114308212216631221170100
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Wang, Y.; Sun, N.; Shi, W.; Ma, Q.; Sun, L.; Hao, M.; Bi, C.; Li, S. Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex macrocarpa. Forests 2023, 14, 2372. https://doi.org/10.3390/f14122372

AMA Style

Wang Y, Sun N, Shi W, Ma Q, Sun L, Hao M, Bi C, Li S. Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex macrocarpa. Forests. 2023; 14(12):2372. https://doi.org/10.3390/f14122372

Chicago/Turabian Style

Wang, Yuxiao, Ning Sun, Wenxi Shi, Qiuyue Ma, Liyong Sun, Mingzhuo Hao, Changwei Bi, and Shuxian Li. 2023. "Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex macrocarpa" Forests 14, no. 12: 2372. https://doi.org/10.3390/f14122372

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