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Comparative analysis of the whole mitochondrial genomes of four species in sect. Chrysantha (Camellia L.), endemic taxa in China

BMC Plant Biology volume 24, Article number: 955 (2024) Cite this article

Abstract

Background

The sect. Chrysantha Chang of plants with yellow flowers of Camellia species as the “Queen of the Tea Family”, most of these species are narrowly distributed endemics of China and are currently listed Grde-II in National Key Protected Wild Plant of China. They are commercially important plants with horticultural medicinal and scientific research value. However, the study of the sect. Chrysantha species genetics are still in its infancy, to date, the mitochondrial genome in sect. Chrysantha has been still unexplored.

Results

In this study, we provide a comprehensive assembly and annotation of the mitochondrial genomes for four species within the sect. Chrysantha. The results showed that the mitochondrial genomes were composed of closed-loop DNA molecules with sizes ranging from 850,836 bp (C. nitidissima) to 1,098,121 bp (C. tianeensis) with GC content of 45.71–45.78% and contained 48–58 genes, including 28–37 protein-coding genes, 17–20 tRNA genes and 2 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the four species. Then, we performed a comprehensive comparison of their basic structures, GC contents, codon preferences, repetitive sequences, RNA editing sites, Ka/Ks ratios, haplotypes, and RNA editing sites. The results showed that these plants differ little in gene type and number. C. nitidissima has the greatest variety of genes, while C. tianeensis has the greatest loss of genes. The Ka/Ks values of the atp6 gene in all four plants were greater than 1, indicating positive selection. And the codons ending in A and T were highly used. In addition, the RNA editing sites differed greatly in number, type, location, and efficiency. Twelve, six, five, and twelve horizontal gene transfer (HGT) fragments were found in C. tianeensis, Camellia huana, Camellia liberofilamenta, and C. nitidissima, respectively. The phylogenetic tree clusters the four species of sect. Chrysantha plants into one group, and C. huana and C. liberofilamenta have closer affinities.

Conclusions

In this study, the mitochondrial genomes of four sect. Chrysantha plants were assembled and annotated, and these results contribute to the development of new genetic markers, DNA barcode databases, genetic improvement and breeding, and provide important references for scientific research, population genetics, and kinship identification of sect. Chrysantha plants.

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Introduction

The sect. Chrysantha Chang plants are a special group of the genus Camellia in the Theaceae family and are known for their golden yellow petals [1]. Li Shizhen’s book “Compendium of Materia Medica” states the following: “Camellia production in the south, late winter flowering, red petals and yellow stamens, there are also yellow ones.” [2]. Camellia petelotii plants are distributed only in China and Vietnam; 42 species and 5 varieties of C. petelotii are known, and there are approximately 20 species in China, of which 18 are endemic. In China, C. petelotii plants are concentrated in southwestern Guangxi, with only a few species distributed in Guizhou and Yunnan [3]. In recent years, as people’s understanding of plants of the sect. Chrysantha has deepened, its multifaceted value has gradually been explored and recognized. Its flowers, fruits, and leaves contain a variety of natural nutrients, such as tea polysaccharides, tea polyphenols, total saponins, total flavonoids, tea pigments, caffeine, and many other microelements and macronutrients that are beneficial to the human body. These components give golden flower tea antioxidant, lipid-regulating, sugar-lowering, anti-allergic, anti-skin photoaging, antibacterial, and other pharmacological activities [4,5,6,7,8]. Its seeds are also used to extract oil and as a raw material for lubricants or other solvents. The wood is hard and dense, making it an excellent material for handicrafts [9]. C. petelotii, an endangered species, has a history spanning hundreds of millions of years. Through the study of C. petelotii, scientists can gain an in-depth understanding of its unique biological properties and genetic mechanisms, providing important references for research in the fields of botany and ecology. Moreover, the identification of the special components in C. petelotii will provide new ideas and methods for drug development. In conclusion, the plants of the sect. Chrysantha are of great significance in terms of ornamental value, medicinal value, economic value, and scientific research value.

The wide distribution of plant species in the sect. Chrysantha, their high morphological variability, and their susceptibility to environmental influences have made the classification of their species more difficult. To solve the problems of species identification and understand interspecific relationships in sect. Chrysantha, most previous studies focused on leaf characteristic, sporology, and karyotype analyses [10,11,12]. However, the morphological characteristics of plants are greatly affected by changes in the environment, and under the same conditions, the floral traits within the sect. Chrysantha plants vary, and natural hybridization can occur [12]. It is difficult to correctly identify species within the sect. Chrysantha based on morphological characteristics. With the development of molecular biotechnology, many scholars have used molecular marker technology and DNA sequencing methods to study the classification of species [13, 14]. However, the different molecular markers have limitations, often lead to different conclusions, and have not been able to resolve long-standing evolutionary controversies. Fang Wei et al. [15] applied DNA fragments to examine the phylogeny and affinities of the genus Camellia, and their results suggested that the sect. Chrysantha may be a complex lineage group. Liufu Yongqing [16] constructed a phylogenetic tree of 33 species of C. petelotii using chloroplast small single copy (SSC) sequences, and his results clarified the phylogenetic positions of the species and possible interspecific hybridization. However, the strong conservation of SSC regions in the chloroplast genome may not provide sufficient information about variation in interspecific or low-level taxonomic unit studies, thus affecting the accuracy of phylogenetic tree construction. Yao Hanya et al. [17] studied 22 species of C. petelotii using DNA barcoding technology. The results showed that rbcL, matK, psbA-trnH, trnL-trnF, and combined sequences could effectively distinguish different species of C. petelotii, and thus, they could be used as barcodes for species identification of C. petelotii. Zhang Xiaoyu [18] conducted a comparative analysis of 23 species of C. petelotii plants by means of chloroplast whole genomes, and the results of this study resolved the taxonomic status of some of the species. However, due to factors such as the fewer number of valid information loci, some of the evolutionary controversies still need to be resolved via further investigation. Inconsistent results between analyses of the chloroplast and nuclear genomes due to hybridization necessitated further exploration of the evolution of sect. Chrysantha species using a wider range of data. Currently, the mitochondrial genomes of sect. Chrysantha plants are rarely reported. Comparative analyses of their whole mitochondrial genomes have not yet been performed.

Plant mitochondria play a crucial role in energy conversion, fatty acid synthesis, amino acid metabolism, and stress response [19]. Together, these effects contribute to the adaptive evolution of plants, enabling them to better adapt to changing environmental conditions. The size of mitochondrial genomes in angiosperms is usually between 100 kb and 10 Mb. Plant mitochondrial genomes are structurally complex, consisting mostly of noncoding DNA sequences containing many short repetitive sequences and homologous sequences, accounting for 2–60% of the total genome length [20]. However, a complete set of tRNA genes sufficient to read all codons is generally lacking in plant mitochondrial genomes, requiring input from the nucleus or cytoplasm. The GC content of the mitochondrial genomes of different plants is closely related to plant evolution and stability. For plants, maintaining the relative stability of the mitochondrial genome is essential for maintaining normal mitochondrial function and adaptation to the environment [21]. Therefore, studying the GC content of plant mitochondrial genomes can provide valuable clues for understanding the evolutionary history and ecological adaptations of plants. The study of plant mitochondrial RNA editing sites has revealed widespread RNA editing in plant mitochondrial genomes. These editing sites are numerous and unevenly distributed across plants and genes, but the vast majority are located in coding regions and are essential for normal protein function [22]. RNA editing regulates gene expression by altering transcript sequences and may play an important role in plant mitochondrial function.

In this study, we assemble the whole mitochondrial genomes of three sect. Chrysantha plants. The mitochondrial genomes of the sect. Chrysantha plants reported in the present study were obtained from the National Center for Biotechnology Information (NCBI) database, and comparative analyses of the basic structure, GC content, codon preference, repetitive sequences, RNA editing sites, Ka/Ks ratio, Haplotypes, and RNA editing sites were carried out for the four Aurelia group plants. Moreover, covariance analyses revealed rearrangements during the evolutionary process of four sect. Chrysantha plants, and high-level homologous sequence transfer between mitochondria and chloroplasts was analyzed. Finally, we constructed a phylogenetic tree of sect. Chrysantha plants based on mitochondrial protein-coding genes. Our study of the mitochondrial genome of plants in the sect. Chrysantha not only enriches the genetic database of this taxon but also provides valuable insights into its speciation, kinship identification, and molecular phylogenetic studies.

Materials and methods

Material collection, DNA extraction and sequencing

The plant samples of Camellia huana T. L. Ming & W. J. Zhang, Camellia liberofilamenta Chang & C. H. Yang, and Camellia nitidissima C. W. Chi collected in this study were from Tianba Village, Luodian County, Luxiong Village, Ceheng County and Ceheng State Forestry Farm, Ceheng County, respectively. The specimens were preserved in the Tree Herbarium of the College of Forestry, Guizhou University (GZAC), under the specimen numbers of LZ-20,221,102, LZ-20,231,102, and LZ-20,230,404, respectively. The botanical specimens were identified by Li Zhi. The selected plants were healthy and disease-free. Young, healthy leaves were collected and stored at -80 °C. The experimental materials were placed in a mortar and pestle, frozen with liquid nitrogen, and then quickly ground to powder form. Genomic DNA was extracted from the leaves using a modified CTAB method [23]. DNA integrity was detected by 1% agarose gel electrophoresis, and DNA purity and concentration were checked by a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA, USA, 2000c UV Vis). Qualified samples were sent to Shanghai Lingen Biotechnology (http://www.biozeron.com/) for Illumina sequencing (Illumina NovaSeq 6000) and Nanopore sequencing (Oxford Nanopore Technologies).

Genome assembly and annotation

First, Illumina sequencing data were assembled using GetOrganelle v1.7.1 [24]. Then, the second-generation assembled sequences were compared to Nanopore third-generation data using BWA v0.7.17 [25] to extract the third-generation data of the target samples, and the extracted third-generation data were mixed and assembled with the second-generation data using SPAdes v3.14.1 software; the sequences with a sufficiently high depth of coverage and a longer assembly length were selected as the candidate sequences, and the sequences were compared to the NT library to confirm the mitochondrial scaffold sequences and link the sequences according to overlap. Clean reads were compared to the mitochondrial genome sequence, and bases were corrected using Pilon v1.23. The final mitochondrial genome sequence was obtained by determining the start position and orientation of the mitochondrial scaffold from the reference genome (Camellia tianeensis; GenBank accession numbers: PP727208).

Mitochondrial genes were annotated using the online GeSeq tool (https://chlorobox.mpimp-golm.mpg.de/geseq) [26] with default parameters to predict protein-coding genes, transfer RNA (tRNA) genes, and ribosomal RNA (rRNA) genes. The location of each coding gene was determined by a BLAST search of the reference mitochondrial gene (C. tianeensis) [27]. Genes with start/end codons and intron/exon boundaries were manually corrected in SnapGene Viewer. Genome mapping was performed using the OGDRAW tool [28].

Mitochondrial genome-shared gene analysis

Mitochondrial genome coding sequence (CDS) sequences were extracted from four plants of the sect. Chrysantha. Homologous sequences were extracted by BLAST software and compared using MAFFT v7.149 software. Phylogenetic trees were constructed for 10 shared genes in mitochondria using MEGA software. Haplotype analysis was performed using DnaSP v5 [29], and mapping was performed with PopART v1.7 [30]. The nonsynonymous/synonymous mutation ratio (Ka/Ks) of the 10 shared genes in the four sect. Chrysantha plants were calculated using Ka/Ks-Calculator v3.0 software [31] and finally plotted using Origin 2022 software.

Preferential use of codons

CDSs larger than 300 bp were selected for analysis according to the selection criteria for codon-preferred sequences [32]. The screened mitochondrial CDSs were analyzed using Codon W software to obtain the effective number of codon (ENC) and relative synonymous codon usage (RSCU) data for each CDS [33]. Parameters such as the GC content of the 1st nucleobase (GC1), 2nd nucleobase (GC2), and 3rd nucleobase (GC3) of the codon, the average GC content of the 1st nucleobase and 2nd nucleobase (GC12), the average GC content of the three nucleobases (GCall), and the contents of A, T, C, and G on the 3rd nucleobase (A3, T3, C3, and G3) were analyzed using CUSP online software [34, 35].

A neutral plot was drawn with mitochondrial GC12 as the vertical coordinate and the GC3 position as the horizontal coordinate. If there is a significant correlation between GC12 and GC3, the slope of the regression curve is close to 1, indicating that codon preference is strongly influenced by base mutations, and vice versa, indicating that it is strongly influenced by natural selection [36]. The ENC plot was drawn with GC3 as the horizontal coordinate and ENC as the vertical coordinate. An ENC of 20 indicates that only one codon per base is used, which is an extreme preference; an ENC of 61 indicates that the codons are biased toward random use, and there is no usage preference. Using G3/(G3 + C3) as the horizontal coordinate and A3/(A3 + T3) as the vertical coordinate, a gene codon bias analysis plot (PR2-plot) was generated. The center point of the PR2 plot represents the codon state when there is no bias in use, i.e., A = U and C = G, and the vector distances of the remaining points from the center point represent the degree and direction of the base bias of the codons of the respective genes [37].

Repetitive sequences and prediction of RNA editing sites

The mitochondrial genome simple repeat sequences (SSRs) of four plants in sect. Chrysantha were identified through the MISA website (https://webblast.ipk-gatersle-ben.de/misa/) [38] with the following parameters: the number of simple repeats was set to 10 for single nucleotides, 5 for dinucleotides, the number of trinucleotide simple repeats was set to 4, and the number of tetranucleotide, pentanucleotide and hexanucleotide simple repeats was set to 3. Forward, reverse, complementary, and palindromic repeats within the genome were identified by the website REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [39] with parameters set to minimum length = 30 bp and Hamming distance = 3. The sequences of protein-coding genes from the mitochondrial genomes of the four sect. Chrysantha plants were predicted for RNA editing sites using Prep-Mt [40]. Statistical analysis and graphing of the data were performed in Excel 2010.

Identification of homologous fragments and covariance analysis

Organellar genome fragments were exchanged using BLASTN software to compare the mitochondrial and chloroplast genomes of the corresponding species with an E value of le-5, setting the minimum comparison length to 50 bp, and mapping the covariance of the chloroplast and mitochondrial genomes using Circos software [41]. The four mitochondrial genomes were compared using MUMmer v3.23 (http://mummer.sourceforge.net/) software, which identified large-scale covariance relationships between the genomes. Interregion comparison was then performed using LASTZ v1.03.54 to confirm the local positional alignment relationships. Mapping was performed using Mauve software.

Phylogenetic tree

To investigate the phylogenetic relationships of mitochondria in four species of the sect. Chrysantha of plants, 24 mitochondrial genome sequences (seven from the genus Camellia) were downloaded from the NCBI database, with Ginkgo biloba serving as the outgroup. Their protein-coding genes were extracted for phylogenetic tree construction, and sequence alignment was performed using MAFFT v7.5 software. Phylosuite software [42] was used to construct the phylogenetic tree by Bayesian inference (BI), and iqtree maximum likelihood (ML) was used to construct phylogenetic trees using MFP as the model. The resulting files were uploaded to the Interactive Tree of Life (iTOL) website [43] to construct the phylogenetic tree.

Results

Characterization of the mitochondrial genomes of the four sect. Chrysantha plants

The mitochondrial DNA compositions of four plants (Camellia tianeensis, Camellia huana, Camellia liberofilamenta and Camellia nitidissima) were statistically analyzed. The results showed that the mitochondrial genomes of the four sect. Chrysantha plants were composed of closed-loop DNA molecules with sizes ranging from 850,836 bp (C. nitidissima) to 1,098,121 bp (C. tianeensis) (Fig. 1). The A, T, C and G contents of the constituent amino acids were 27.1–27.25%, 27.02–27.19%, 22.84–22.91%, and 22.85–22.94%, respectively. The GC content was highly conserved during evolution, with values in the range 45.71–45.78%. CDSs, tRNAs, and rRNAs accounted for 2.49–3.46%, 0.22–0.28% and 0.47–0.71% of the total mitochondrial genome, respectively (Table 1). The number of encoded proteins was consistent and included complex I-complex V, cytochrome c synthase subunits, ribosomal proteins, maturation enzyme proteins, and transit membrane proteins, ranging in number from 48 to 58, while specific gene duplications were observed in different species. Multiple copies were observed to varying degrees in all four plants. A total of nine annotated genes were found to contain introns, of which cox2, rpl2, trnA-UGC, trnI-GAU and ccmFc contained only one intron, and ccmFc appeared to be intronic in all four sect. Chrysantha plants (Table S1). Overall, the mitochondrial genomes of the four plant species varied only slightly in terms of gene type or number. By comparing the PCGs of the mitochondrial genomes of the four individual species, gene loss and gene duplication were found for some genes. For example, rpl1 was present only in C. nitidissima, while it was absent in the other three species of sect. Chrysantha. The rpl2 gene was present only in C. nitidissima and C. huana and was lost in C. liberofilamenta and C. tianeensis. The gene variety was most complete in C. nitidissima, while the gene loss was most severe in C. tianeensis (Table S1).

Table 1 Characterization of the mitochondrial genomes of four species of sect. Chrysantha plants
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Fig. 1
figure 1

Mitochondrial genome mapping of four sect. Chrysantha plants. (A: C. tianeensis; B: C. huana; C: C. liberofilamenta; D: C. nitidissima)

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Comparative analysis of shared genes

To better understand the mitochondrial genomes of the four sect. Chrysantha species, we identified 10 shared genes (atp1, atp6, cob, cox2, ccmFc, nad2, nad5, rpl10, rps1, and sdh3) through analysis of their protein-coding genes and constructed a phylogenetic tree by using these shared genes (Fig. 2). The results indicated that C. huana and C. liberofilamenta are very closely related to each other. Moreover, to gain a deeper understanding of their genetic variation, we analyzed the mutation sites of 10 shared genes in the four plant species and constructed a haplotype network map (Fig. 3). C. nitidissima showed a typical haplotype construction, with C. tianeensis and C. liberofilamenta having the same composition of these 10 shared genes. In addition, C. huana shared these 10 genes with three other plants. The Ka/Ks values were calculated for 10 shared genes from the mitochondrial genomes of the four sect. Chrysantha plants to clarify the evolutionary selection of the mitochondrial genomes under environmental stress (Fig. 4). The Ka/Ks values of the atp6 gene were all greater than 1, indicating positive selection.

Fig. 2
figure 2

A phylogenetic tree was constructed based on 10 shared genes in four sect. Chrysantha plants

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Fig. 3
figure 3

Haplotype network mapping of four sect. Chrysantha plants constructed on the basis of 10 shared genes

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Fig. 4
figure 4

Ka/Ks ratios of 10 shared genes in the mitochondrial genomes of four sect. Chrysantha plants (using C. huana as a reference)

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Codon preference analysis

A total of 26 mitochondrial genome coding sequences (CDSs) were screened for C. tianeensis, 26 for C. liberofilamenta, 27 for C. huana and 27 for C. nitidissima. Among the mitochondrial genome codons, GCall, GC1, GC2, GC3 and GC12 had percentages of 42.22-42.51%, 47.38-48.01%, 42.73-42.87%, 36.33-36.83% and 45.06-45.37%, respectively, and the ENC was 50.22, 49.74, 49.42, and 49.99, respectively, with a weak genomic codon preference (Fig. 5A, Table S2). The frequency distributions of the effective codon ratios and plots of ENC versus GC3 (ENC plot) for the mitochondrial genomes of the four Chrysantha plants showed (Fig. 5B) that the mitochondrial genome ENC mostly deviated from the standard curves, suggesting that their codon preferences were more susceptible to base mutations. Neutral plotting analysis (Fig. 5C) revealed that there was no significant correlation between GC12 and GC3 in any of the four plants, suggesting that the third position of the mitochondrial genome codon in plants of the sect. Chrysantha has a different pattern of base mutation from the first and second bases and that codon preference over base mutation is more strongly influenced by natural selection. The base bias at position 3 of the codons of each gene showed (Fig. 5D) that the genes were unevenly distributed in four different regions, with most being located in the lower and right halves of the plane view. This indicates that the mitochondrial genomic codon of plants in sect. Chrysantha occurs more frequently at position 3 for T bases than for A bases and more frequently for G bases than for C bases. Thus, genomic codon preference is affected not only by mutation but also by natural selection.

Fig. 5
figure 5

Codon preference analysis of the mitochondrial genomes of four sect. Chrysantha plants. (A: Distribution of mean values of different GC contents; B: ENC plot; C: codon neutrality plot; D: PR2 plot; E: relative synonymous codon usage)

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Analysis of the relative usage of synonymous codons (RSCU) of mitochondrial genes showed (Fig. 5E, Table S3) that there were 29 mitochondrial gene high-frequency codons (RSCU greater than 1), including 11 ending in A, 15 ending in T, 2 ending in C, and 1 ending in G. There were 2 codons with RSCU equal to 1: ATG and TGG. There were 31 low-frequency codons (RSCU less than 1) in the gene, 3 for A, 16 for C, and 12 for G. This finding suggests that the most highly used codons in the mitochondrial genomes of the four plant species are those ending in A and T.

Repeat sequence analysis and RNA editing sites

Simple repetitive sequences have the characteristics of high reproducibility, codominant inheritance, uniparental inheritance, and strong relative conservatism, which make them highly efficient molecular markers and most suitable for species identification and evaluation of genetic variation at the population and individual levels. Comparative SSR analyses of the mitochondrial genomes of the four sect. Chrysantha plants sequenced in this study revealed a total of 1099 SSR loci. These included 123 mononucleotides, 314 dinucleotides, 146 trinucleotides, 434 tetranucleotides, 72 pentanucleotides, and 10 hexanucleotides. The number of SSRs in different species ranged from 233 to 306, with the highest number of simple repetitive sequences in C. tianeensis and the lowest number of simple repetitive sequences in C. huana (Fig. 6A). Among all the types, tetranucleotide repeats were the most abundant, ranging in number from 91 to 124 and accounting for 39% of all SSRs. This was followed by dinucleotides, with an average of 78 per species for a total of 29%, trinucleotides (Tri-) at 13%, mononucleotides (Mono-) at 11%, pentanucleotides at 7%, and hexanucleotides at 1% (Fig. 6B). There are two types of single nucleotide repeats, of which A/T was the most numerous. There are three types of dinucleotide repeats. There are seven types of trinucleotide repeats. Among the 25 types of tetranucleotide repeats, the ACAT/ATGT and ACGG/CCGT types were unique to C. huana, the ACAG/CTGT type was unique to C. nitidissima, and the ATCC/ATGG type was absent from C. liberofilamenta. Among the 15 types of pentanucleotide repeats, the AAGGT/ACCTT and ACTAT/AGTAT types were unique to C. huana, and the AAAGG/CCTTTT, AAATT/AATTT, AACTT/AAGTT, and AAGGC/CCTTG types were unique to C. nitidissima. There were five types of hexanucleotide repeats, all of which were present in C. huana (Fig. 6C).

Fig. 6
figure 6

Repeat sequence analysis of the mitochondrial genomes of the four sect. Chrysantha plants. (A: Number of six SSR types; B: proportion of six SSR types; C: number of SSR repetitive sequence types; D: number of four dispersed repetitive sequence types; E: length of four long repetitive sequence types)

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Long repetitive sequences can be classified according to length into 9 types (30–34, 35–39, 40–44, 45–49, 50–54, 55–59, 60–64, 65–69, and ≥ 70 bp) and according other features into four types (forward repetitive sequence (F), reverse repetitive sequence (R), complementary repetitive sequence (C), and palindromic sequence (P)). Analysis of the long repetitive sequences of the complete mitochondrial genomes of the four species revealed numbers ranging from 1000 to 2010 for each species, with F and P sequences exhibiting the highest percentage, followed by R and C. The long repetitive sequences of the complete mitochondrial genomes of the four species were analyzed in the same way. The fewest types of duplicates were found in C. huana, and no type C duplicates were found in C. tianeensis or C. huana (Fig. 6D). In all four species, 30–34 bp repeats occurred most frequently, followed by 35–39 bp repeats, and 60–64 bp repeats occurred least frequently. Interestingly, repeat sequences greater than 70 bp in length were present in all four species. Analysis of the sequence lengths of the repetitive elements of different repeat types revealed an inverse relationship between 30 and 69 bp repeats among all four types of repeats detected, accompanied by a gradual decrease in the number of repetitive elements as the sequence length increased. The sequence lengths of the repeats in most species repeats were between 30 and 34 bp (Fig. 6E).

RNA editing sites were predicted in the four plants by Prep-Mt. A total of 24 genes were found to undergo RNA editing, with a total of 1,587 editing sites, all of which were C-to-U transitions. Among them, no editing occurred in the rpl2 genes of C. tianeensis and C. liberofilamenta, and no editing occurred in the rps7 gene of C. huana (Fig. 7A). A total of 549 (35%) site edits occurred at the first base of the codon, 1,038 (65%) site edits occurred at the second base of the codon, and none occurred at the third base of the codon (Fig. 7B, C). Amino acid changes occurred at all sites, with the main patterns of change being A(Ala)→V(Val), H(His)→Y(Tyr), L(Leu)→F(Phe), P(Pro)→F, P→L, P→S(Ser), Q(Gln)→*(Stop codon), R(Arg)→*, R→C(Cys), R→W(Trp), S→F, S→L, T(Thr)→I(Ile), and T→M(Met). Of these, the highest frequency of changes occurred at P→L, followed by S→L, and the least at Q→* (Fig. 7D). Before RNA editing, of the 1,587 edited sites, 61% were hydrophilic amino acids, whereas after editing, 13% were hydrophilic amino acids; the percentage of hydrophobic amino acids increased from 39 to 87% (Fig. 7E, F). This resulted in a shift from hydrophilic to hydrophobic amino acids, which led to an increase in the hydrophobicity of the protein. Many of the amino acid changes triggered by RNA editing introduced more hydrophobic amino acids into the protein structure, thereby altering the hydrophilic nature of the protein, and may play a key role in maintaining the regulation of mitochondrial gene expression.

Fig. 7
figure 7

Prediction of RNA editing sites in four plant species based on protein-coding genes. (A: number of RNA editing sites in each gene; B: ratio of codons in different positions; C: number of codons in different positions; D: type of RNA editing site; E: ratio of hydrophilicity to hydrophobicity of amino acids before RNA editing; F: ratio of hydrophilicity to hydrophobicity of amino acids after RNA editing)

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Horizontal gene transfer and covariance in the organellar genome

The mitochondrial genomes of the four sect. Chrysantha plants in this study were approximately 4.6–7.0 times longer than their chloroplast genomes (Table S4). However, the protein-coding gene sequences in their mitochondrial genomes accounted for only 2.5–3.5% of their full length, most of which consisted of noncoding regions, and the function of this portion remains unknown. Twelve, six, five, and twelve gene exchange fragments were found in C. tianeensis, C. huana, C. liberofilamenta, and C. nitidissima, respectively, and their total lengths were 42,875 bp, 6,469 bp, 10,377 bp, and 11,269 bp, respectively (Fig. 8D, Table S4). In C. tianeensis, all 12 migrating fragments exceeded 100 bp, 6 fragments were in the range of 100–500 bp, 4 fragments were in the range of 500–1000 bp, and 2 fragments were more than 1 kb in length. There were 9 integrative genes on these fragments, which were CDSs (5), tRNAs (3), and rRNAs (1). There were three fragment sequences of 100–500 bp in C. huana, and the remaining three were above 1 kb in size. There were six genes, comprising three each of CDSs and rRNAs (Fig. 8A). The C. liberofilamenta fragments were all more than 1 kb in length and were integrated into the genes rpl2, trnA-UGC, and 23 S (Fig. 8B). There were 7 transfer fragments of 100–500 bp in C. nitidissima, and the remaining 5 were more than 1 kb in size and were integrated into 11 genes, i.e., 8 and 3 each of CDSs and rRNAs, respectively (Fig. 8C). Numerous chloroplast protein-coding genes migrated from chloroplasts to mitochondria, most of which lost their integrity during evolution, and partial sequences of these genes, e.g., nad1, ccmC, and rrn18, are found in the mitochondrial genome. Transferred tRNA genes are much more conserved, suggesting that they have indispensable roles in mitochondria.

Fig. 8
figure 8

Analysis of homologous fragments in different organelles of four sect. Chrysantha plants. The green arcs represent chloroplast DNA, and the yellow arcs represent mitochondrial DNA. (A: C. huana; B: C. liberofilamenta; C: C. nitidissima; D: number of horizontal gene transfers in different species)

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The results of the rearrangement and collinearity of the mitochondrial genomes of the four sect. Chrysantha plants showed that the four plants split into numerous localized colinear blocks. The size of these local colinear blocks and their relative positions varied greatly (Fig. 9). Their genes were arranged in an inconsistent order, with poor covariance and internal structural inversions or gene rearrangements.

Fig. 9
figure 9

Mitochondrial genome covariance mapping in four species of sect. Chrysantha plants

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Phylogenetic tree

In this study, the mitochondrial genome sequence was used for the first time for the evolutionary analysis of plants in the sect. Chrysantha, and a phylogenetic tree was constructed by Bayesian inference and maximum likelihood methods by combining 28 species from 12 families. The results show (Fig. 10) clear clustering of plants from each of the different families, with the four species of the sect. Chrysantha clustered on the same branch and the 12 species of Theaceae clustered into one group. The four species of sect. Chrysantha plants are more closely related, with C. liberofilamenta and C. huana closer together and then clustered with C. tianeensis and C. nitidissima, suggesting that they have closer affinities. In this study, the whole mitochondrial genome sequence was applied to the evolutionary analyses of sect. Chrysantha plants, which will provide new ideas for future genetic relationship studies of sect. Chrysantha plants.

Fig. 10
figure 10

A phylogenetic tree based on protein-coding genes constructed from the mitochondrial genomes of 28 plant species

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Discussion

This study is the first to comparatively analyze the mitochondrial genomes of four plants from the sect. Chrysantha. The mitochondrial genomes of all four plants were ring shaped, which is consistent with the fact that most of the mitochondrial genomes were reported to be circular in structure [44]. The mitochondrial genome size ranged from 850,836 bp to 1,098,121 bp. The GC content was evolutionarily conserved, with values ranging from 45.71 to 45.78%, and was higher than that of Sunflower (45.22%), Angelica dahurica (45.06%), and Prunella vulgaris (43.92%); these are high levels among higher plants [45,46,47]. The protein-coding region occupies only approximately 2.5–3.5% of the full length, and the noncoding region occupies approximately 90%. The relatively similar functional classification of protein-coding genes in the mitochondrial genomes suggested that the number of mitochondrial genomes of closely related species is essentially the same and that the protein-coding gene sequences are highly conserved [48, 49]. The coding regions of the genome are more conserved than the noncoding regions, and the noncoding regions are also a major source of mitochondrial genomic variation [50]. The mitochondrial genome intergenic region is composed mainly of repetitive sequences, chloroplast genome homologous sequences, and nuclear genome homologous sequences. Repetitive sequences containing tandem, short and long repeats, are widely present in the mitochondrial genome [51] and are essential for intermolecular recombination of the mitochondrial genome. They are usually considered the main cause of mitochondrial genome differences in plants [52]. Most protein-coding genes begin with a typical ATN codon [53]. Some genes contain one or more introns, and these introns may play an important role in the regulation of gene expression [54]. At the same time, we annotated two identical rRNA genes (rrnL and rrnS) in the mitochondrial genomes of the four species, as is the case for most land plants [44]. The occurrence of partial deletions of ribosomal protein genes in the four species is consistent with the tendency of these genes to be readily lost during angiosperm evolution [55]. Therefore, the results of this study deepen the understanding of the genome structure of sect. Chrysantha plants and provide a reference for the discovery of their new functional genes and genetic improvement.

The Ka/Ks ratio is important for evaluating the effects of plants on environmental stresses during evolution and can reveal the effects of genetic changes on the phenotypes of different individual seed plants in terms of the evolutionary selection of genes [31]. We screened atp6 as a positively selected gene from 10 shared protein-coding sequences of four mitochondrial sect. Chrysantha plants. Their high ratios suggest that they play an important role in species adaptation to selection. From the distribution of haplotypes and the haplotype network structure, it was found that haplotype H1 was distributed in all 10 shared genes of C. tianeensis and occupied a dominant position, which indicated that H was an obvious shared haplotype and that it was presumed to be an ancient ancestral haplotype. This analysis is of great scientific value for understanding the molecular evolutionary pathways, the conservation and innovation of gene functions, and the genetic basis of adaptive differences between species in plants of the sect. Chrysantha.

The numbers of codons in the mitochondrial genomes of the four species of plants in sect. Chrysantha were 42.22–42.51%, GCall, GC3, and 36.33–36.83%, with most values below 50%, indicating that base 3 of the mitochondrial gene codons of the four species was predominantly A/T, which is consistent with the mitochondrial codon preferences of Ganoderma and Setaria italica [56, 57]. This shows that the plant organelle genome evolutionary trends were roughly the same. The RSCU is an important indicator for evaluating the codon usage pattern of biological organelle genomes. In this study, there were 29 high-frequency codons, 26 of which ended in A/T; this finding is consistent with the codon usage pattern of the mitochondrial genome of Oenanthe javanica [58]. The results of neutral mapping analysis, ENC plot analysis, and PR2 plot analysis all showed that mitochondrial genome codons are strongly influenced by natural selection, which is consistent with the findings for Hemerocallis citrina codons [59], unlike those for Masteraria chamomilla codons, which are influenced mainly by base mutations [60], suggesting that diverse factors influence species codon preferences. This convergence phenomenon may have originated from the adaptive selection of the four sect. Chrysantha plant species by similar environmental factors during long-term natural evolution. This preliminary study revealed that the main reason for the formation of codon preferences in the mitochondrial genomes of the four species is natural selection, which will be highly important for exploring the molecular properties and genetic diversity of sect. Chrysantha, determining the evolutionary pressure of genes, and performing molecular breeding.

From an evolutionary point of view, differences in repetitive sequences between species are the result of natural selection [61], and the more concentrated distribution of plants in the sect. Chrysantha and the study of the characteristics of the repetitive sequences within their genomes are of great significance to the evolution of these species. In this study, it was found that the AAGGT/ACCTTT and ACTAT/AGTAT types were unique to C. huana, and the AAAGG/CCTTTT, AAATT/AATTT, AACTT/AAGTT, and AAGGC/CCTTG types were unique to C. nitidissima, which is helpful for tracing the roots of interspecific crosses in the sect. Chrysantha. Mitochondrial genomic SSRs are short (1–6 bp) repetitive unit sequences in the mitochondrial genome that are shorter in length and more limited in number and distribution than SSRs in the nuclear genome [62, 63]. The types of repetitive SSR units in the mitochondrial genome of the four sect. Chrysantha plants were more consistent, indicating that the mitochondrial genome sequences of the sect. Chrysantha of plants are relatively conserved, which is similar to the results of a previous study [64]. In this study, we chose to analyze the differences among the four plant species of the sect. Chrysantha under the same setup conditions, and the results can reflect the differences between the species more realistically; moreover, how these simple repeat sequences actually affect the evolution of the species still needs to be further investigated.

The phenomenon of RNA editing is prevalent in the organelle genomes of higher plants and plays an important role in organelle development, morphogenesis, and stress response in higher plants [65]. In this study, we found that RNA editing sites in the mitochondrial genomes of four plant species were prone to causing changes in the bases at the first and second positions in the corresponding amino acids, with a greater frequency of editing occurring at the second position, which is consistent with other plant mitochondrial RNA editing features [44]. A total of 24 genes were detected with editing at locus 1038, and all were typical C-to-U transitions, similar to previous studies [66]. During RNA editing, 14 types of amino acid changes can occur: A→V, H→Y, L→F, P→F, P→L, P→S, Q→*, R→*, R→C, R→W, S→F, S→L, T→I, and T→M. Most of the amino acids are converted to hydrophobic amino acids, and the hydrophobicity of the edited proteins increases [67]. Hydrophilic amino acids are distributed on the surface of protein molecules, while hydrophobic amino acids are predominantly distributed in the interior, and the correlation of hydrophilic and hydrophobic amino acids can often be used to determine the general trend of protein folding [68]. An increase in hydrophobic amino acids during RNA editing may increase protein stability [65]. The identification of these RNA editing sites provides essential clues for future studies on the evolution and neo-codon prediction of gene function and can help us better understand gene expression in the mitochondrial genome of plants.

The genomes of plant mitochondria vary greatly in size and structure, which is mainly due to the large number of repetitive sequences contained therein on the one hand and, on the other hand, due to frequent horizontal migration [44]. The plant mitochondrial genome often exchanges genetic material with the nucleus and the chloroplast genome, and some genes in chloroplasts are often integrated into the mitochondrial genome. DNA migration is common in plants, and the mitochondrial genome contains many sequences that have migrated from the chloroplast genome [69]. It varies from species to species during autophagy, gametogenesis, and fertilization [70] and plays an important role in the evolution of plant mitochondrial genomes. DNA from chloroplasts can be found in virtually every plant whose mitochondrial genome has been sequenced to date. Typically, this portion of the migrating integrated sequence accounts for 1–12% of the total length of the mitochondrial genome [71]. In the mitochondrial genomes of the four sect. Chrysantha plants, the full length of the sequences from chloroplasts ranged from 6,469 bp–11,269 bp, which accounted for 2.5–3.5% of the total length. Except tRNAs, the vast majority of sequences in the mitochondrial genome that have migrated from the chloroplast genome are now considered “dead on arrival” [72]. The mitochondrial genome has been shown to express chloroplast-derived tRNA genes [73], and the expression of these genes is associated with the nuclear genome-encoded rpoTm polymerase [74]. This feature is also specific to the mitochondrial genomes of higher plants, as no similar phenomenon has been found to exist in some of the lower plants for which organelle genomes have been determined. It is unlikely that sequences found within the chloroplast genome that come from the mitochondrial genome exist in higher plants [75]. However, evidence of the migration of the mitochondrial genome to the chloroplast genome has been analyzed in studies of the mitochondrial genome in Vitis vinifera and Daucus carota by researchers [76, 77]. In contrast to the migration of chloroplast sequences to the mitochondrial genome, the migration of mitochondrial sequences to the chloroplast genome is not present in all species, and the lengths of the migrating sequences found thus far are very limited. Our results did not reveal sequence migration of mitochondria towards the chloroplast genome.

Plant mitochondrial genomes are characterized by structural rearrangements, large numbers of genes lost or gained, the transfer of mitochondrial genes to nuclear genes, and very low nucleic acid mutation rates [78], providing unique information for phylogenetic analyses [79] and analyses of homology among plant mitochondrial genes that can reveal the relationships and evolutionary history among different species [80]. Evolutionary analyses and comparisons revealed that the mitochondrial genomes of the four sect. Chrysantha species underwent frequent genetic recombination events during the evolutionary process. These colinear regions and inverted structures not only reveal the conserved patterns of the mitochondrial genomes but also reflect the evolutionary relationships among species and the evolutionary history of sect. Chrysantha plants, as well as revealing the affinities and evolutionary histories of the species, which provides a new perspective for exploring their genetic bases and evolutionary histories.

A large amount of data demonstrates that the chloroplast genome sequence, with its moderate rate of base variation and other characteristics, serves as an ideal material for constructing phylogenetic trees [81]. However, there are relatively few phylogenetic studies on mitochondrial genome construction. In this first application of whole mitochondrial genome sequences to the evolutionary analyses of sect. Chrysantha plants, the mitochondrial genomes of four sect. Chrysantha plants that have been sequenced and the published mitochondrial genome sequences of seven other families were selected for phylogenetic relationship analysis. The results showed that the classification of each family was still clear, and the four species of sect. Chrysantha were the closest relatives, indicating that the mitochondrial genome can be used to analyze the phylogenetic relationships of sect. Chrysantha plants. Our present study on the mitochondrial genome represents only the tip of the iceberg, and the mitochondrial genomes of the sect. Chrysantha of plants need to be further investigated before accurate conclusions can be drawn.

Conclusion

In this study, we successfully assembled three high-quality mitochondrial genomes from plants of the sect. Chrysantha and, by using the published C. tianeensis sequence, a comprehensive comparative analysis of the mitochondrial genomes of the four species was carried out in terms of their compositional structure, genotypes, haplotypes, codon preferences, and RNA editing sites, thus revealing the structure of the mitochondrial genomes of sect. Chrysantha and their compositions. A comparison of PCGs of the mitochondrial genomes of four individual species revealed the presence of gene loss and gene duplication in some genes. In addition, RNA editing site and phylogenetic tree analyses revealed a shift from hydrophilic to hydrophobic amino acids, leading to an increase in protein hydrophobicity. Twelve species of Theaceae were clustered into one group. C. huana and C. liberofilamenta clustered together, and then C. tianeensis and C. nitidissima clustered together as a group, indicating that they have a closer relationship. This study provides more comprehensive genetic information for the genomes of C. tianeensis, C. huana, C. liberofilamenta, and C. nitidissima, which is important for revealing the function of the mitochondrial genome and for studying the genetic characteristics, origin evolution, conservation, and utilization, as well as the taxonomic status, of plants in the sect. Chrysantha.

Data availability

The mitochondrial genomes of Camellia nitidissima, Camellia liberofilamenta and Camellia huana in this study have been uploaded to NCBI. Their Gene Bank accession numbers are PP975885-PP975887.

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Acknowledgements

We thank the editor and the anonymous reviewers for their careful comments and suggestions on the manuscript. We also thank the Jiangxi Provincial Key Laboratory of Improved Variety Breeding and Efficient Utilization of Native Tree Species (2024SSY04093) for providing the platform conditions and financial support.

Funding

This study was supported by the Guizhou Provincial Basic Research Program (Natural Science) 2022 (072) and the National Natural Science Foundation of China (31960043, 32400179).

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Authors and Affiliations

  1. College of Forestry, Guizhou University, Guiyang, 550025, China

    Zhi Li, Zhaohui Ran, Xu Xiao, Chao Yan & Mingtai An

  2. Jiangxi Provincial Key Laboratory of Improved Variety Breeding and Efficient Utilization of Native Tree Species, Jiangxi Agricultural University, Nanchang, 330045, China

    Ming Tang

  3. Jiangxi Provincial Key Laboratory of Conservation Biology, Jiangxi Agricultural University, Nanchang, 330045, China

    Ming Tang

  4. Guizhou Botanical Garden, Guiyang, 550000, China

    Jian Xu

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  1. Zhi Li
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Contributions

Z.L. conceived the study. Z.L. and Z.H.R. were responsible for analyzing and writing this manuscript; X.X. and M.T.A. were responsible for collecting and identifying the material; C.Y. and J.X. helped with the analysis of the data; and M.T. and Z.L. revised the manuscript. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Ming Tang or Mingtai An.

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Ethics approval and consent to participate

All materials used in this study comply with international and national legal standards. The collected species material does not pose a threat to other species, and the collection of the species is recognized by the relevant authorities.

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The authors declare no competing interests.

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Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1: Table S1 Classification of mitochondrial genes in four sect. Chrysantha plants.

12870_2024_5673_MOESM2_ESM.xlsx

Supplementary Material 2: Table S2 GC content and effective codon number of mitochondrial genome codons in four sect. Chrysantha plants.

12870_2024_5673_MOESM3_ESM.xls

Supplementary Material 3: Table S3 RSCU values of mitochondrial genome codons in four species of sect. Chrysantha plants.

Supplementary Material 4: Table S4 Mitochondrial genome horizontal gene transfer in four sect. Chrysantha plants.

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Li, Z., Ran, Z., Xiao, X. et al. Comparative analysis of the whole mitochondrial genomes of four species in sect. Chrysantha (Camellia L.), endemic taxa in China. BMC Plant Biol 24, 955 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-024-05673-6

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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