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Characterization of the complete mitogenome of Tiarella polyphylla, commonly known as Asian foamflower: insights into the multi-chromosomes structure and DNA transfers

BMC Genomics volume 25, Article number: 883 (2024) Cite this article

Abstract

Background

Tiarella polyphylla D. Don has been traditionally used to cure asthma and skin eruptions. However, the sequence and the structure of the mitogenome of T. polyphylla remained elusive, limiting the genomic and evolution analysis based on the mitogenome.

Results

Using a combination of Illumina and Nanopore sequencing reads, we de novo assembled the complete mitogenome of T. polyphylla. In addition to unveiling the major configuration of the T. polyphylla mitogenome was three circular chromosomes with lengths of 430,435 bp, 126,943 bp, and 55,269 bp, we revealed five (R01-R05) and one (R06) repetitive sequence could mediate the intra- and inter-chromosomal recombination, respectively. Furthermore, we identified 208 short and 25 long tandem segments, seven cp-derived mtDNAs, 106 segments of mtDNAs transferred to the nuclear genome, and 653 predicted RNA editing sites. Based on the sequence of the mitogenomes, we obtained the resolved phylogeny of the seven Saxifragales species.

Conclusions

These results presented the mitogenome features and expanded its potential applications in phylogenetics, species identification, and cytoplasmic male sterility (CMS) in the future.

Peer Review reports

Background

Tiarella polyphylla D. Don (Saxifragaceae) is geographically distributed in Asia from the eastern Himalayas through China to Japan [1]. Traditionally, T. polyphylla has been used to treat asthma, skin eruptions, bruises, and hearing difficulties [2, 3]. Due to its medicinal value, some research focused on the main active components and pharmacological study of T. polyphylla. The compounds isolated from the whole plant of T. polyphylla include some flavonols, triterpenoids, and steroids, such as Tiarellic acid (TA), β-sitosterol, and ergosterol endoperoxide [3]. TA has anti-asthmatic properties [2], and β-sitosterol and ergosterol endoperoxide show anti-complementary activity [3]. Moreover, the 3, 23-dihydroxy-20(29)-Iupen-27-oic acid isolated from T. polyphylla regulated the Type 1-procollagen and MMP-1(matrix metalloproteinases) on the UV-induced damage of cultured old age human dermal fibroblasts [4]. Together, these studies indicated the high medicinal potential of T. polyphylla. Consequently, this underscores the necessity of initiating molecular breeding programs for this species to enhance its therapeutic qualities.

With the increasing number of studies using different sequencing technologies in plants, genomic research on T. polyphylla has also been developed. Plant cells contain three genomic compartments, including the nuclear, mitochondrial (mt), and plastid (pt) genomes, and they interact with each other. Functions of the organellar proteins rely on the import of nuclear-encoded proteins and mutation accumulation in one compartment, causing the nuclear-cytoplasmic co-evolution [5]. The chromosome-level nuclear genome assembly and the complete plastome of T. polyphylla have been constructed [6, 7]. The chromosome-level nuclear genome of T. polyphylla featured seven chromosomes and was 403.10 Mb. Sizes of the seven chromosomes ranged from 44.08 Mb to 79.84 Mb. The size of the complete plastome of T. polyphylla was 154,850 bp. Although previous efforts to assemble the nuclear genome and plastome of T. polyphylla have provided valuable data, the mitogenome has not been reported yet.

Apart from energy production, mitogenomes also hold a crucial role in phylogenetics, species authentication, and cytoplasmic male sterility (CMS). Given the importance of CMS characteristics, the mitochondrial genome becomes crucial for molecular breeding in plants, facilitating the development of varieties with desirable traits. Contrarily to the plastome, whose structure is conserved among most angiosperms, the plant mitogenome is variable in size, structure, and gene order due to recombination through repeated sequences. Recombination mediated by the repeated sequences produced sub-stoichiometric molecules within the same plant cell referred to as “substoichiometric shifting” [8]. Many other features have been reported previously in plant mitogenomes, such as higher RNA editing frequency than the plastome. Moreover, plastid DNA insertions in the mitogenome and mitochondrial DNA insertions in the nuclear genome were commonly found in plants.

Here, we assembled the complete mitogenome of T. polyphylla, combining Illumina and Nanopore sequencing data. We characterized the main structural feature of the mitogenome of T. polyphylla and documented the repeat-mediated recombination supported by the Nanopore reads. Also, we identified the transfer of plastomic DNA to the mitogenome and mitochondrial DNA segments to the nuclear genome. Finally, based on the mitogenome sequences, we performed a phylogenetic analysis of the seven Saxifragales species. The complete mitogenome sequence of T. polyphylla was the first sequenced mitogenome of the family Saxifragaceae, provided new sequences that can be used to infer evolutionary relationships, and offered a valuable genetic resource for comparative genomics within the order Saxifragales.

Methods

Plant materials and total DNA extraction

The fresh leaves were collected from the whole plants of T. polyphylla in Chongdugou scenic spot, Henan, China (111◦39′41.64″E, 33◦56′23.87″N). Dr. Luxian Liu (School of Life Sciences, Henan University, China) formally identified the plant material. T. polyphylla is neither classified as an endangered species nor subject to protection regulations, so no special permissions were required for its collection. A voucher specimen (LLX20160814) is deposited at the Herbarium of Henan University (HHU). The fresh leaves were used to extract total DNA using a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.). The purity and quantity of DNA were assessed using the Nanodrop spectrophotometer 2000 (Thermo Fisher Scientific, America).

DNA sequencing and mitogenome assembly

PromethION libraries were constructed from 1 µg of intact genomic DNA using the LSK108 ligation kit (Oxford Nanopore Technologies) following the manufacturer’s protocol. Sequencing was performed on a PromethION Flow Cell (Oxford Nanopore Technologies, UK).

Next-generation sequencing (NGS) sequencing libraries were prepared using Illumina’s Nextera DNA Flex library. Libraries were sequenced on Illumina’s NovaSeq 6000 System, obtaining 2 × 150 paired-end reads using the NovaSeq 6000 S2 Reagent Kit. All sequencing data utilized in this study were derived from our previous whole-genome sequencing research [6].

The mitogenome of T. polyphylla was assembled using a hybrid assembly method. We used Unicycler (v0.4.9) [9] to assemble the NGS and Nanopore reads. In brief, the hybrid assembly method for the T. polyphylla mitogenome combines the accuracy of short reads with the structural resolving power of long reads to create a complete mitogenome assembly. Initially, a primary assembly graph was constructed using NGS reads with SPAdes software, and Unicycler simplified this graph using NGS and Nanopore reads, resolving ambiguities and spanning repetitive regions. The NGS and Nanopore reads were mapped back to the assembled sequences using BWA (v0.7.12-r1039) [10] to validate the assembly results of the Unicycler. The number of reads mapped to the mtDNA was counted using the samtools (v1.3.1) [11].

Mitogenome annotation

We annotated the T. polyphylla mitogenome using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) with manual checking using Apollo [12]. We used IPMGA (http://www.1kmpg.cn/ipmga/), which provides origin information from 423 tRNA genes across 12 species, to determine the origin of each tRNA. A map of the T. polyphylla mitogenome was constructed using OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html). The sequences and the annotation of the T. polyphylla mitogenome were deposited in GenBank under the accession number OR866907 for chromosome 1, OR866908 for chromosome 2, and OR866909 for chromosome 3 respectively.

Identification of the repeat-mediated recombination

To identify the recombination events mediated by repetitive sequences in the T. polyphylla mitogenome, we detected the intra-chromosomal repetitive sequences using ROUSfinder v2 [13] and inter-chromosomal repetitive sequences using BLASTN with the e-value < 1e-5. Then, the 1000 bp-long non-repeat regions flanking the repetitive sequences and the repetitive sequences were extracted and generated the four configurations corresponding to both 5’-and 3-ends of each non-repeat contig encompassing one single recombination event. Based on the inferred alternative genome configurations, we calculated the stoichiometry of each configuration using Nanopore long reads. The configurations with the more supporting reads were the major configurations (Mac1, 2), and those with the less supporting reads were the minor configurations (Mic1, 2). To calculate the recombination efficiency, we mapped the Nanopore long reads to the four configurations of each recombination event. We extracted all mapped reads and calculated the read numbers for each configuration. The mapped reads of each configuration for the long reads supporting the recombination event were visualized using IGV (v 2.15.1) [14].

Analysis of the SSRs and long tandem repeats

Simple sequence repeats (SSRs) in the T. polyphylla mitogenome were searched by MISA [15]. The thresholds applied were 10, 5, 4, 3, 3, and 3 base pairs for all mono-, di-, trinucleotide, tetra-, penta-, and hexanucleotide repeat units, respectively. The tandem repeats in the T. polyphylla mitogenome were identified by the Tandem Repeats Finder (v 4.09) [16]. The parameters applied were two base pairs for matches, seven base pairs for mismatches and indels, and 50 and 500 base pairs for the minimum alignment score and maximum period size, respectively.

Identification of mitochondrial plastid and nuclear DNA sequences

We assembled the plastome of the T. polyphylla using GetOrganelle with the parameters: “-R 15 -k 21,45,65,85,105 -F embplant_pt”. To find the mitochondrial DNA sequences that are derived from inserted plastid DNA (MTPTs), we searched the sequence of T. polyphylla plastome against a custom database constructed by the sequence of the mitogenome, using BLASTN with the e-value < 1e-5 and removing hits < 100 bp with < 80% identity [17]. We extracted the 1000 bp-long regions flanking the MTPTs, and the sequences of MTPTs were extracted. We mapped the Nanopore long reads to these sequences and visualized the mapping results using IGV. We annotated these sequences using CPGAVAS2 (http://www.1kmpg.cn/cpgavas2) to check the genes located in the MTPTs.

To identify the mitochondrial DNA insertions in the nucleus (NUMTs), we searched the sequence of the T. polyphylla mitogenome against a custom database constructed by the sequence of the nuclear genome of T. polyphylla. The nuclear genome was downloaded from the CNCB-NGDC database (https://ngdc.cncb.ac.cn/gwh/Assembly/26102/show) under the accession number GWHBKAH00000000. The NUMTs were identified using the same procedure as MTPTs. Circos implemented in TBtools [18] were used to plot the MTPTs and NUMTs located in the assembled mitochondrial chromosomes of T. polyphylla.

Prediction C-to-U RNA editing sites

To predict the cytidines (C) for uridines (U) RNA editing sites in the protein-coding regions of the T. polyphylla mitogenome, we applied Deepred-mt [19] to analyze the coding sequences. The RNA editing sites predicted in the protein-coding regions with the > 0.9 probability were included in the current study.

Phylogenetic analysis of the seven Saxifragales species based on mitogenomes

For phylogenetic reconstructions based on the mitogenomes, the sequence of the complete mitogenomes of T. polyphylla was obtained in this study, and fourteen other species of Saxifragales were downloaded from the GenBank database (Table 1). The coding sequences of twelve shared genes among these fifteen species were extracted using PhyloSuite (v1.2.1) [20] and were aligned with MAFFT (v7.450) [21]. We selected the best-fit evolutionary model and partitioning scheme using PartitionFinder2 [22]. The twelve shared genes were atp1, atp4, atp6, atp8, ccmC, ccmFc, cox3, matR, nad4, nad4L, nad9, and rpl5. The phylogenetic tree was reconstructed using the Maximum Likelihood (ML) method in RAxML (v8.2.4) [23] with the model, and the partitioning scheme was obtained from PartitionFinder2 and 1000 bootstrap replicates. We also performed the Bayesian (BI) phylogenetic analysis using MrBayes (v3.2.7) [24] with the model and the partitioning scheme obtained from PartitionFinder2. Phylogenetic trees were displayed using the Interactive Tree of Life (iTOL v4) web server (https://itol.embl.de).

Table 1 Summary of the sequences used in phylogenetic analysis
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For phylogenetic reconstructions based on the plastid genomes, the sequences of the complete mitogenomes of thirteen species of Saxifragales were downloaded from the GenBank database (Table 1). These thirteen species’ whole plastid genome sequences were aligned with MAFFT (v7.450). The phylogenetic tree was reconstructed using the Maximum Likelihood (ML) method in RAxML (v8.2.4) with the GTRGAMMA substitution model and 1000 bootstrap replicates. We also performed the BI phylogenetic analysis using MrBayes with the model parameters obtained from jModelTest (v2.1.0) [25]. Phylogenetic trees were displayed using the iTOL.

Results

General features of the T. polyphylla mitogenome

We obtained the assembled mitogenomes of T. polyphylla, three circular chromosomes with lengths of 430,435 bp, 126,943 bp, and 55,269 bp (Fig. 1). They were the dominant configurations of T. polyphylla mitogenome. The average Illumina sequencing depth of three mitochondrial chromosomes was estimated at 326×, 404×, and 313×, respectively (Figure S1A-C). The average Nanopore sequencing depth of three mitochondrial chromosomes was estimated at 32×, 41×, and 22×, respectively (Fig S1D-F).

Fig. 1
figure 1

Genome maps of the three circular chromosomes of T. polyphylla mitogenome. Genes shown on the inside were on the negative strand, whereas those on the outside were on the positive strand. Genes with introns were highlighted using “*”. The gray circle represents the GC contents. The circle inside the GC content graph marks the 50% threshold. The colors indicate different functional categories shown in the legend

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T. polyphylla mitogenome encoded the conserved set of genes consisting of 36 protein-coding genes (three subunits of cytochrome oxidase, four cytochrome c biogenesis genes, nine subunits of NADH dehydrogenase, five subunits of ATP synthase, one cytochrome b gene, one transport membrane protein gene, and one maturases gene), three rRNA genes, and 16 tRNA genes (Table 2, Table S1). 27 protein-coding genes were intron-less genes, and nine protein-coding genes were intron-containing gene. In the T. polyphylla mitogenome, the coding sequence of rps3 overlaps with that of rpl16, with an overlap of 29 bp. This overlap is also observed in other plant mitogenomes [26, 27]. Chromosome 1 of the T. polyphylla mitogenome encoded 26 protein-coding genes and 15 tRNA genes. Chromosome 2 of the T. polyphylla mitogenome encoded nine protein-coding genes, one tRNA gene, and three rRNA genes. Chromosome 3 of the T. polyphylla mitogenome encoded four protein-coding genes and one tRNA gene (Fig. 1).

Table 2 Summary of genes annotated in T. polyphylla mitogenome
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The plant mitochondrial tRNAs have been reshaped through a series of gene transfer events over time [28]. We identified the origins of the 17 tRNAs in the T. polyphylla mitogenome. Among the 17 tRNA genes identified, 12 were native mt-tRNA genes (Table S1). Four tRNAs (trnN-GUU, trnD-GUC, trnW-CCA, and trnM-CAU) originated from plastids, signifying the presence of intracellular gene transfers. Among them, only trnD-GUC was identified as the MTPT (mitochondrial plastid DNA) transferred from the plastid genome of T. polyphylla. This finding supports previous conclusions showing that plastid-derived tRNA genes have been retained for hundreds of millions of years [29, 30]. Additionally, trnP-UGG was identified as an unknown source gene, suggesting it may have originated from bacteria.

Repeat-mediated recombination in the T. polyphylla mitogenome

Plant mitogenome showed a high repeat-mediated recombinational activity, producing structurally dynamic genome configurations [31]. Homologous sequence analysis and Nanopore sequencing read showed that the T. polyphylla mitogenome contains six recombinationally active repetitive sequences (Table S2, Figure S2). The lengths of the longest and shortest repetitive sequences of these six repetitive sequences were 9,703 bp and 350 bp. Four repetitive sequences mediated recombination within chromosome 1 (R01-R04), and the other two mediated recombination between chromosomes 1 and 2 (R05-R06). Five repetitive sequences were direct repeats (R01, R02, R04, R05, and R06), and one repetitive sequence was an inverted repeat (R03).

The relative abundance of alternative genome configurations generated by the six repetitive sequences ranged from 4.35 to 50%, quantified using the Nanopore sequencing reads (Table S2). Two longer repetitive sequences (R01 and R06) with lengths of 9,446 bp and 9,703 bp showed recombination frequencies of 21.43% and 45.45%, respectively. The shortest repetitive sequence (R05) had a lower recombination frequency of 4.35%. The repetitive sequence R02 exhibited the highest recombination frequency at 50.00%. A partial sequence of the cox2 and nad6 genes, located within the direct repetitive sequences R01 and R05, mediated genome recombination with frequencies of 21.43% and 4.35%, respectively (Table S2). This resulted in the duplication of cox2 and nad6 gene fragments within two copies of R01 and R05. Notably, the entire sequence of the rps7 gene, found within the direct repetitive sequence R06, mediated genome recombination with a frequency of 45.45%, leading to the duplication of the rps7 gene within two copies of R06 (Table S2).

Starting with a dominant configuration of three circular chromosomes and assuming each repeat pair recombines only once, we can predict the six alternative configurations (Fig. 2). Each alternative configuration maintained the same genomic content but varied in the number of chromosomes, ranging from an increase to four circular chromosomes to a decrease to two, diverging from the original three-chromosome structure.

Fig. 2
figure 2

Predicted alternative configurations (ACs) generated through recombination of six pairs of repetitive sequences in the T. polyphylla mitogenome. The dominant configuration of three circular chromosomes was shown in the middle (Mito_chr1/2/3: mitogenome chromosome 1/2/3). This figure assumes that each pair of repeats recombines only once, as the black arrows indicate. Red arrows on genome maps show six pairs of repetitive sequences, with the direction of each arrow indicating the orientation of these sequences. Single-copy genomic regions are represented in gradients of color, with consistent coloring across all configurations to represent each specific single-copy region

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SSRs and long tandem repeats in the T. polyphylla mitogenome

Simple sequence repeats (SSRs) with a tandem repeat motif of 1–6 nucleotides and long tandem repeats with a tandem repeat motif of > 7 nucleotides have been used for genetic fingerprinting, phylogenetic analysis, and population genetic studies [32, 33]. Mitochondrial SSRs (mt-SSRs) have specific advantages over nuclear SSRs due to their uniparental inheritance, providing clearer insights into phylogenetic relationships between individuals [34]. In addition, mt-SSRs are particularly valuable when used as mitotype-specific markers to differentiate mitotypes or cytoplasms, such as distinguishing normal CMS lines from the lines with floral deformities in plant breeding [35]. A total of 208 SSR loci were mined in three chromosome sequences of the T. polyphylla mitogenome (Tables S3), with an average frequency of 0.34 SSR per kb. The total SSRs were 138, 36, and 34, identified in chromosomes 1, 2, and 3, respectively (Fig. 3). The mononucleotide repeat was the most abundant repeat type in the T. polyphylla mitogenome, comprising 30.29%, followed by tetra-, di-, tri-, penta, and hexa-nucleotide repeat that comprised 28.85%, 21.15%, 12.98%, 4.81%, and 1.92% respectively. The A/T motif was the most abundant mononucleotide repeat in the T. polyphylla mitogenome. Several genes with associated SSRs are featured (Table S4). Notably, the rps3 gene exhibits the highest count of mt-SSRs, including a monomeric repeat of 11 thymine bases (T)11 and a tetranucleotide repeat (AATG)3. Additionally, the nad1 gene possesses a monomeric repeat of 10 thymine bases (T)10. The matR gene is associated with a pentanucleotide microsatellite repeat (CTAGT)3, while the rps1 gene contains a trinucleotide repeat (GAA)4.

Fig. 3
figure 3

Number of short tandem repeats of 1–6 base for SSRs in the three circular chromosomes of T. polyphylla mitogenome

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A total of 25 long tandem repeats were identified in three chromosome sequences of the T. polyphylla mitogenome (Tables S5). 12, 7, and 3 long tandem repeats were identified in chromosomes 1, 2, and 3, respectively. Moreover, the length of all the tandem repeats in T. polyphylla mitogenome was < 100 bp.

Mitochondrial plastid and nuclear DNA sequences in the T. polyphylla mitogenome

DNA transfers of MTPTs and mtDNAs to the nuclear genome (NUMTs) were documented and suggested to be signatures of the evolutionary processes [36, 37]. In the T. polyphylla mitogenome, we identified seven MTPTs listed in Table S6 and shown in Fig. 4A. The length of these transferred organelle genome copies ranged from 133 bp (MTPT03) to 2,023 bp (MTPT01). These transferred copies were 80.46% (MTPT04) to 100% (MTPT01), identical to their ptDNA counterparts. Most of these MTPTs were part of sequences of some protein-coding genes, indicating that they are non-functional. However, MTPT02 and MTPT03 contained the complete coding sequences of the ndhK and one cp-derived tRNA (trnD-GUC). Moreover, Nanopore long reads confirmed the presence of these seven MTPTs in the T. polyphylla mitogenome (Figure S3).

Fig. 4
figure 4

Circos plot shows the seven MTPTs in the three circular chromosomes of T. polyphylla mitogenome (A) and the NUMTs in the seven chromosomes of T. polyphylla nuclear genome (B)

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As depicted in Table S7 and Fig. 4B, we identified 1,012 NUMTs in the seven chromosomes of the T. polyphylla nuclear genome involving all three chromosomes of the T. polyphylla mitogenome. 646, 262, and 104 NUMTs transferred from chromosomes 1, 2, and 3 of the T. polyphylla mitogenome, respectively. The lengths of the longest and shortest sequences of these NUMTs were 24,772 bp and 34 bp. After removing the duplicate statistics, these NUMTs have a total length of 234,840 bp, accounting for 38.33% of the mitogenome. The divergence of the largest NUMTs in the seven nuclear chromosomes of T. polyphylla was relatively low (2.45-8.53%), indicating their recent origin. However, none of these largest NUMTs exhibited the lowest divergence among the NUMTs in each chromosome (Table S7, Figure S4).

RNA editing sites predicted in the T. polyphylla mitogenome

In plant organelles, post-transcriptionally RNA editing (numerous C-to-U and some U-to-C sites) is highly prevalent [38], resulting in changes in the coding sequences of transcripts and creating AUG start sites or eliminating premature stop codons or changes in the RNA structure [39]. The complete mitogenome of T. polyphylla contained 37 protein-coding genes, and 653 edited sites were predicted for all these genes (Table S8, Fig. 5A). The frequency of editing across these genes ranged from 0.15% (1 site in 303 bp) for rps14 to 7.35% (48 sites in 1,488 bp) for nad4 (Table S8). RNA editing produced new start codons for cox1, nad1, and nad4L and new stop codons for atp6, atp9, ccmFc, and rps10.

Fig. 5
figure 5

Bar plots show the numbers of RNA editing sites of protein-coding genes (A) and the frequency of the top ten amino changes generated by the RNA editing events (B)

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The most frequent amino acid changes are Pro to Leu, occurring at 151 sites (23.12% of the changes), followed by Ser to Leu at 137 sites (20.98%), and Ser to Phe at 92 sites (14.09%) (Fig. 5B). In the T. polyphylla mitogenome, the proportion of RNA editing at silent and nonsilent editing sites was biased. 33 of 653 editing sites were silent (5.05%), and 620 were nonsilent sites (94.95%).

The phylogeny analysis based on the mitogenomes and plastid genomes of Saxifragales species

The mitogenome has been one of the informative loci of molecular evolutionary biology [40]. Phylogenetic analysis based on coding sequences of twelve protein-coding genes of the fourteen Saxifragales species was conducted in this study using the ML and BI methods. The results suggested robust phylogenetic inferences, with many nodes having ML bootstrap support values > 80 and BI posterior probabilities = 1 (Fig. 6). We also performed the phylogenetic analysis based on the complete plastid genomes of the same species except for one species that didn’t have the available plastid genome in Genbank. We found the incongruence between mitochondrion and plastid datasets. Paeoniaceae was sister to Hamamelidaceae in the plastid tree (Fig. 6B), the same as the APG IV system. However, it was sister to Cynomoriaceae in the mitochondrion tree (Fig. 6A). And we also found the congruence between mitochondrion and plastid datasets. Saxifragaceae was sister to Grossulariaceae in both mitochondrion and plastid tree.

Fig. 6
figure 6

Phylogenetic trees reconstructed using the coding sequences of 26 conserved genes of the seven species of Saxifragales. The ML bootstrap support values and BI posterior probabilities were labeled. Two species of superasterids, including Chrysanthemum boreale (NC_039757.1) and Synotis nagensium (NC_082299.1), were used as outgroups

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Discussions

Multi-chromosome organizations of T. polyphylla mitogenome

Although often assembled as one master circle, the plant mitogenome structure physically existed as a variety of circles, linear molecules, and complex branching structures [41]. Moreover, a few cases in the genus Silene L., Cucumis sativus L., and sugarcane identified the mitogenome consisting of complex multichromosomal structures instead of finding a single master circle [31, 42]. Our study showed that three circular-mapping chromosomes were also found to be the dominant configuration of the T. polyphylla mitogenome, and our sequencing data did not support the presence of a master circle.

In the mitogenome of Silene species and Cucumis sativus, no repetitive sequences that could mediate recombination were found between at least two circular chromosomes. Similar to our study, the Nanopore long reads only supported two repetitive sequences (R05 and R06) that mediated the inter-chromosomal recombination in chromosomes 1 and 2.

Imbalanced stoichiometry in recombination products of repetitive sequences in T. polyphylla mitogenome

Frequent homologous recombination involving repetitive sequences could generate recombination products as the alternative genome configurations in plant mitogenome [43]. These recombination products showed balanced and imbalanced stoichiometry, indicating reciprocal and non-reciprocal recombination events [44]. In the T. polyphylla mitogenome, one of the six repetitive sequences (R02) that mediate homologous recombination displayed precisely balanced stoichiometry, with an equal number of reads supporting both the principal and alternative configurations, resulting in a 50% recombination frequency. The occurrence of imbalanced stoichiometry in recombination products with more reads supporting the major configuration was observed in most of the repetitive sequences (R01, R03, 04, 05, and 06), with recombination frequencies varying between 4.35% and 47.06%. These non-reciprocal recombination events of repeat pairs were also found in the mitogenomes of Oenothera species and Mimulus guttatus [17, 45].

The mechanism underlying these reciprocal and non-reciprocal homologous recombination events was complex. Specifically, non-reciprocal recombination events may be induced by the break-induced replication pathway within the homologous recombination process [46]. Therefore, we postulated that non-reciprocal homologous recombination events in T. polyphylla mitogenome were associated with the break-induced replication of homologous recombination. Moreover, nuclear genes involving the DNA repair pathways by homologous recombination and mismatch repair modulated these processes [43]. For example, it has been reported in Arabidopsis that OSB1 (for Organellar Single-stranded DNA Binding protein 1) played a role in controlling the stoichiometry of alternative mitogenome configurations generated by homologous recombination [47]. Further empirical research is essential to elucidate these processes fully in the T. polyphylla mitogenome in the future.

DNA transfers of cp-derived mtDNAs (MTPTs) and mtDNAs to nuclear genome (NUMTs)

MTPTs have been shown to transfer randomly from any position in the ptDNAs, and some of the tRNA transferred from the ptDNA was still functional in mtDNA [29]. In agreement with these conclusions, the MTPTs in the T. polyphylla mitogenome were randomly distributed across the plastome, and the pt-derived tRNA sequence (trnD-GUC) was intact, suggesting its function. However, in this study, the coding sequence of the ndhK protein-coding gene was intact, which appears to differ from the results, indicating that all the protein-coding MTPTs degenerate [29].

It has been recognized that some large mtDNA segments are transferred into nuclear genomes through evolution [48]. For example, a 620-kb long NUMT was found in Arabidopsis thaliana [49]. The present study found a large mtDNA segment, 24.7 kb in length, in the T. polyphylla nuclear genome.

RNA editing events were conserved

RNA editing in plant organelle plays a critical role in correct mtDNA expression [50]. In our study, new start codons for cox1, nad1, and nad4L and new stop codons for atp6, atp9, ccmFc, and rps10 were generated by RNA editing. These start and stop gain RNA editing events were described previously [51,52,53]. It has been shown that the most frequent amino acid changes are Pro to Leu, Ser to Leu, and Ser to Phe [54, 55], similar to this study’s findings.

Conclusions

The multichromosomal structure of the T. polyphylla mitogenome described here offers unique insights into both variable structures and the recombination events mediated by the repetitive sequences, achieving a better understanding of the mitogenome of Saxifragaceae species. Using sequences of our T. polyphylla mitogenome and six other Saxifragales species, we constructed the resolved phylogenetic tree consistent with the APG IV system. The investigation of the T. polyphylla mitogenome expanded our genomic resources to investigate the structure and evolution of the plant mitogenome.

Data availability

The raw sequencing reads for T. polyphylla genome assembly have been deposited in the NCBI database with BioProject ID PRJNA870970. The Nanopore and Illumina raw data are available under the accession nos. SRR21133002 and SRR21133003. The T. polyphylla mitogenome assembly has been deposited in the NCBI database with the accession nos. OR866907, OR866908, and OR866909.

Abbreviations

CMS:

Cytoplasmic Male Sterility

NGS:

Next-Generation Sequencing

MTPTs:

Mitochondrial Plastid DNAs

NUMTs:

Nuclear Mitochondrial DNAs

tRNA:

Transfer RNA

rRNA:

Ribosomal RNA

C:

TCytidines

U:

Uridines

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Acknowledgements

We sincerely thank Yonghua Zhang and Xinjie Jin from Wenzhou University for the help with mitochondrial genome assembly and annotation.

Funding

This research was supported by Xinyang Academy of Ecological Research Open Foundation (Grant No. 2023DBS09), the National Natural Science Foundation of China (Grant No. 31900188).

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Author notes

  1. Bo Liu and Qian Long contributed equally to this work.

Authors and Affiliations

  1. School of Agriculture, Henan Institute of Science and Technology, Xinxiang, 453003, China

    Bo Liu

  2. State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, 475000, China

    Bo Liu, Qian Long, Weiwei LV, Yu Shi & Luxian Liu

  3. Henan Dabieshan National Field Observation and Research Station of Forest Ecosystem, Zhengzhou, 450000, China

    Yu Shi & Luxian Liu

  4. Xinyang Academy of Ecological Research, Xinyang, 464000, China

    Yu Shi & Luxian Liu

  5. Systematic & Evolutionary Botany and Biodiversity Group, MOE Key Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

    Pan Li

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  4. Yu Shi
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  5. Pan Li
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  6. Luxian Liu
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Contributions

PL and LXL designed the study. BL and QL conducted the field sampling. BL, QL and WWL produced and analyzed the data. BL and QL wrote the manuscript. YS, PL and LXL revised the manuscript. All authors approved the final manuscript.

Corresponding authors

Correspondence to Pan Li or Luxian Liu.

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All plant materials were collected following national and international standards and local laws and regulations. No specific permission is required to collect all samples described in this study.

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The authors declare no competing interests.

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Liu, B., Long, Q., LV, W. et al. Characterization of the complete mitogenome of Tiarella polyphylla, commonly known as Asian foamflower: insights into the multi-chromosomes structure and DNA transfers. BMC Genomics 25, 883 (2024). https://doi.org/10.1186/s12864-024-10790-5

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Keywords

BMC Genomics

ISSN: 1471-2164

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