Complete mitogenome characteristics and phylogenetic analysis of traditional Chinese medicinal plant Tinospora sagittata (Oliv.) Gagnep. from the Menispermaceae family

doi:10.21203/rs.3.rs-5239969/v1

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Complete mitogenome characteristics and phylogenetic analysis of traditional Chinese medicinal plant Tinospora sagittata (Oliv.) Gagnep. from the Menispermaceae family

https://doi.org/10.21203/rs.3.rs-5239969/v1

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Background: Tinospora sagittata, a member belongs to the genus Tinospora of Menispermaceae family. Its tuberous roots have been used as traditional Chinese medicine (TCM) for pharmacological properties and are commonly known name as “Jin Guo Lan”. Although its plastome and nuclear genome had been sequenced, its mitochondrial genome has not been explored, which significantly hampers conservation efforts and further research for this species. In addition, previous efforts based on multiple molecular markers providing profound insights into an intergeneric phylogenetic framework for Burasaieae and sampled species of T. sagittataare placed in a superclades, species delimitation of T. sagittata still need to be comprehensively evaluated.

Results: Flow cytometry revealed that Tinospora sagittata has two cytotypes and a wide range in genome sizes. We further sequenced and assembled the organelle genomes of T. sagittata, including the mitogenome (513,210–513,215 bp) and plastome (163,621–164,006 bp). The plastomes were highly similar in gene content and exhibited a typical quadripartite structure, but a translocation as well as two inversions were detected in mitogenomes. The repeats patterns in both organelles are generally similar, but significant difference in the codon bias of the genes of Tinospora organelle genomes. Interesting, both organelle genomes had shown that inter-gene spacer regions could be used as effective molecular markers for further phylogenetic analyses and species identification. Comprehensive analysis of protein coding genes of organelle genomes showed that significant difference in Ka, Ks, and Ka/Ks values among the organelle genomes. Phylogenetic analysis identified a tree that was basically consistent with the phylogeny of Ranunculales described in the APG IV system.

Conclusions: We provided a high-quality and well-annotated organelle genome for Tinospora sagittata. The study present here advances our understanding of the intricate interplay between plastome and mitogenome. Moreover, our results also laid the foundation for further studying the course, tempo and mode of organelle genome evolution of Menispermaceae.

Organelle

Plastome

Paratinospora

Phylogeny

subspecies

The family Menispermaceae Juss. is not only a characteristic and structural component of modern tropical rainforests [1, 2], but also a well-known plant tribe with medicinal values and all species contain diverse types of alkaloid constituents [3, 4]. Tinospora Miers, a medium genus belonging to the Menispermaceae family, contains about 36 species and is mainly distributed in the tropical and subtropical regions of the Eastern Hemisphere [5]. In China, the genus plants have been used for centuries in traditional Chinese medicine (TCM) for their wide-ranging pharmacological properties [6]. Among the six species distributed in China, Tinospora sinensis (Lour.) Merr. and Tinospora sagittata (Oliv.) Gagnep are the two most famous ones for their long history of pharmaceutical applications [6, 7]. The vine stems of T. sinensis known as ‘Kuan Jin Teng’ are used as the medicinal part in TCM, while for T. sagittate, its tuberous roots as the medicinal part is rich in diterpenoids and benzylisoquinoline alkaloids and are commonly known name as “Jin Guo Lan” in the 2015 edition of the Chinese Pharmacopoeia [7]. Owing to its habitat destruction and over-harvesting of its roots, T. sagittata has become endangered in the wild.

It is worth mentioned that Flora of China recognized that Tinospora sagittata (Oliv.) Gagnep could be divided three varieties: T. sagittata var. sagittate, T. sagittata var. craveniana (S. Y. Hu) H. S. Lo, and T. sagittata var. yunnanensis (S. Y. Hu) H. S. Lo [8], but Forman did not accept those varieties [9]. Wang et al [10] reorganized T. sagittata and T. dentata Diels as a new genus Paratinospora Wei Wang based on six plastid and nuclear sequences, and merged T. sagittata var. craveniana (S. Y. Hu) H. S. Lo and T. sagittata var. yunnanensis in to Paratinospora sagittate (Oliv.) Wei Wang. Interesting, nine samples of T. sagittate from different occurrence always divided into four clades [10]. So that, the interspecies relationships of T. sagittate complex worth further study by integrating different evidence.

In plant, plastid and mitochondria are semiautonomous organelles that originated from symbiotic bacteria within eukaryotic cells [11, 12]. Plastid play a key role in photosynthesis, while the central function of the mitochondria is to energy metabolism, control of stress responses, and serve as a hub for biosynthetic processes [13, 14]. Although the mitogenome of plants is more complex than plastome, animal mitogenome, and fungal mitogenome [15, 16], plant mitogenome as one of the organelles carrying genetic information are an important tool for studying the classification and evolution of plants [17, 18]. As well, plant mitogenome has many unique features, such as a lower substitution rate, variation in size, a large number of repeat sequences, intracellular gene transfer, and abundant RNA editing sites [19, 20, 21, 22, 23]. In addition to the common single loop structure, the plant mitogenome including linear conformations, branched structures, and numerous small circular molecules mediated by repeat sequences [21, 24]. The short reads based on next-generation sequencing which is far from meeting the requirements of mitogenome sequencing, so that the research on plant mitogenome lags behind significantly compared to plastome.

At present, only two mitogenome of Ranunculales had been sequenced (https://www.ncbi.nlm.nih.gov/genome/browse#!/organelles/). With the assembly of the Tinospora sagittate plastome and the first species in Menispermaceae tetraploid T. sagittate chromosomal-scale genome has been performed, it plays an important role in understanding the phylogenetic placement of the Menispermaceae family in flowering plants [25, 26]. Unfortunately, the mitogenome of T. sagittate remains unpublished, and its numerous properties are yet to be uncovered which severely constrained our study of mitogenome evolution in Tinospora species.

During the last decade, phylogeny reconstruction using plastomes or amounts of nuclear genes became routine which enhanced our understanding of evolution and relationships within diverse organism groups [27, 28, 29, 30]. In contrast to the plastome, only a few recent studies have utilized mitochondrial data to infer deep phylogenomic relationships within plant lineages [28, 29, 31, 32]. Interestingly, both mitogenome and plastome were considered to exhibit maternal inheritance patterns [12, 17], but many studies have consistently identified incongruences between mitochondrial and plastid data [28, 29, 33]. Therefore, mitogenome-based phylogenetic analyses are crucial for understanding genetic composition of medicinal plant and fully unleashing the potential of TCM in various applications. It is necessary to adopt third-generation sequencing technology that consider both read length and accuracy, providing an important foundation for the study of plant species evolution and genetic diversity.

With those question in mind, two varieties of Tinospora were as example group based on PacBio HiFi sequencing, Illumina sequencing, and flow cytometry. Our aims were to (1) assemble and annotate the complete organelle genome; (2) characterize the organelle genome structures and sequence features; (3) comprehensively analyze the repeated elements and codon bias; (4) infer species relationships based on mitogenomes to provide essential background information for further understanding the genetics of this plant.

Genome size and sequence data

The results obtained from flow cytometry revealed that Tinospora sagittata has a wide range in genome sizes (Table 1). The genome size ranged from 1.01 Gb in T. sagittata var. craveniana to 6.06 Gb in T. sagittata var. yunnanensis (Table 1). Although flow cytometry results were not supplemented by conventional chromosome counts to confirm the ploidy level, we found a nearly sixfold change in genome size. In addition, Alami et al [26] revealed a monoploid genome size of T. sagittata approximately 553.23 Mb, the assembled tetraploid genome size was 2.33Gb. Therefore, T. sagittata var. yunnanensis genome size based on flow cytometry is six times larger than T. sagittata var. craveniana and is recognized as an hexaploid, but T. sagittata var. craveniana is recognized as diploid.

Of the two generated Illumina data, we generated PE clean reads (average read length 150 bp) ranging from 115.88 Mbp reads for Tinospora sagittata var. craveniana to 135.06 Mbp reads for T. sagittata var. yunnanensis (Table 1). The next generation sequencing total reads were 17.35 Gb and 20.20 Gb (Table 1). For hexaploid plant T. sagittata var. yunnanensis, Pacbio HiFi generated a total base of 25.00 Gb (average read length 15,745 bp), while the diploid T. sagittata var. craveniana with 13.40 Gb base (average read length 14,584 bp) (Table 1). The average coverage of T. sagittata var. craveniana with 17.52× short reads and 13.53× long reads, and 3.33× short read and 4.13× long reads for T. sagittata var. yunnanensis.

Characteristics of organelle genomes

The complete mitogenomes of Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis were obtained by combing Illumina and PacBio reads and visualized using Bandage (Fig. S1a, b). The assembly circle mitogenome with a total length of 513,210–513,215 bp and a GC content of 48% (Fig. 1a, b). A total of 70 genes were annotated, including 38 protein-coding genes (PCGs), 6 rRNAs, and 26 tRNAs, and identified in the mitogenome of T. sagittata var. craveniana and T. sagittata var. yunnanensis (Fig. 1a, b; Table 2). It was found that only the rps19 protein-coding gene and had two copies (Fig. 1a, b; Table 2). Furthermore, three rRNA (rrn5, rrn18, rrn26) and four tRNA (trnF-GAA, trnfM-CAU, trnS-GCU, trnW-CCA) genes also has two copies (Fig. 1a, b; Table 2). Eight PCGs and one tRNA contained at least one intron (Fig. 1a, b; Table 2).

The chloroplast genomes displayed the typical quadripartite structure, consisting of a pair of Inverted Repeats (termed IRA and IRB), separated by the Large Single Copy regions (LSC) and Small Single Copy regions (SSC) (Fig. 1c). Besides, the plastomes of the different Tinospora species showed little variation in size (163,621–164,006 bp) and GC content (38%) (Fig. 1c; Table S1). We observed only marginal variation in the LSC (92,488–93,027 bp), SSC (20,203–20,909 bp), and IR (25,112–25,388bp) (Table S1). As well, the GC content in the LSC, SSC, and IR regions were 35.50–35.70%, 32.00–32.40%, and 43.40–43.50% (Table 3), respectively. The plastomes of all two accessions, encoded an identical set of 130 genes with 16 being duplicated in the IR regions. Among the 130 genes, there were 85 PCGs, 37 tRNA genes, and eight rRNA genes (Fig. 1c). It is worth mentioning that the 5-end exon of the rps12 gene was located in the LSC region, and 3-end exon of the gene were situated in the IR region. Besides, six tRNA genes (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) and nine protein-coding genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, and rps16) contained a single intron, and three genes including rps12, clpP, and ycf3 contained two introns (Fig. 1c).

Collinearity and repeat sequences

To explore the rearrangements and conserved sequence blocks within the mitogenomes, we employed the Mauve to analyze the mitogenome collinear regions (Fig. S1c). When comparing the two mitogenomes (Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis), we identified five locally collinear blocks (LCBs) (Fig. S1c). We further found a translocation ca.11kb (including nad1 and cemB genes), as well as two inversions from trnW-CCA to mttB (ca. 73 kb) and from trnH-GUG to rrn5 (ca. 82 kb), respectively (Figs. 1a, b, S1c).

We have identified amount of SSRs in the plastomes and mitogenomes (Fig. 2a, TableS2). Within the plastome, mononucleotide repeats (163 out of 287 in Tinospora sagittata var. craveniana;169 out of 272 in T. sagittata var. yunnanensis) and dinucleotide repeats (85 out of 287 in T. sagittata var. craveniana; 90 out of 272 in T. sagittata var. yunnanensis) being more abundant, other repeats being less abundant (Fig. 2a, TableS2). The majority of the mitogenome SSR of T. sagittata var. yunnanensis were mononucleotide repeats (140 out of 442) and dinucleotide repeats (199 out of 442), followed by tetranucleotide repeats (69), and trinucleotide repeats (28). However, the majority of the mitogenome SSR of T. sagittata var. craveniana was dinucleotide repeats (202 out of 330), followed by tetranucleotide repeats (67), trinucleotide repeats (30), and mononucleotide repeats (24) (Fig. 2a, TableS2). It was obviously that both hexanucleotide repeats and pentanucleotide repeats was the least in plastomes and mitogenomes (Fig. 2a, TableS2). REPuter identified 50 dispersed repeats in each organelle genomes (Fig. 2b, TableS3). When dispersed repeats in the plastomes are further divided into four types, including 26–29 forward repeats, 8–12 reverse repeats, 10–12 palindromic repeats, and 1–2 complement repeats (Fig. 2b, TableS3). Additionally, no reverse (R) or complement (C) repeats were detected in the mitogenome (Fig. 2b, TableS3). Among the dispersed repeats, there are 20–222 forward repeats and 28–30 palindromic repeats in the mitogenome (Fig. 2b, TableS3). The A/T type mononucleotide was the most abundant SSR in the plastomes, while AG/AT type was the most abundant SSR in the mitogenomes (Fig. 2c, TableS4).

Protein coding gene codon usage and comparative analysis of genomic variation

There was significant difference in the codon bias of the genes of Tinospora organelle genomes (Fig. 3a, TableS5). In general, the two organelles genomes share the same most frequent codon for each amino acid (Fig. 3a, TableS5). It was found that there were 28 codons (UCC, UUU, AAA, UCA, GUU, GUA, CCA, UGU, UUG, CCU, CGU, GGU, UUA, UAA, CGA, AUU, GAA, ACU, AAU, CUU, UCU, AGA, GAU, GGA, CAA, CAU, UAU, GCU) with RSCU > 1 in both plastomes and mitogenomes, of which 15 end with U and 11 end with A (Fig. 3a, TableS5). Two codons RSCU value of 1 in both plastomes and mitogenomes, which were AUG and UGG encoding methionine (Met) and tryptophan (Trp) (Fig. 3a, TableS5). It worth mentioned that two codons (ACC and UGA) RSCU > 1 in mitogenomes, but the two codons RSCU < 1 in plastomes (Fig. 3a, TableS5).

Comparative analysis of nucleotide polymorphisms of mitogenomes and plastomes, the variability in plastomes were higher than that mitogenomes (Fig. 3b, c). The Pi values ranged from 0 to 0.40 in plastome and 0 to 0.02 in mitogenomes (Fig. 3b, c). Among mitogenomes, there were five hypervariable regions in plastomes with Pi > 0.016 were trnS–rpl10, rps3_exon2–rps3_exon1, trnP–nad5_exon5, ccmFN–rps2, nad4_exon3–nad4_exon4 (Fig. 3b). Compare with mitogenomes, the plastome generally with lower Pi values in IR regions, and four (trnV_exon2–ndhC, trnL_exon1–trnT, trnG–psbZ, trnK_exon2–psbA) were common high variation hotspots in the LSC and SSC (Fig. 3c).

Selective pressure analysis of organelle genome CDS

The evolutionary pressure among the protein coding sequences of two organelle genomes were analyzed, the Ka/Ks value of protein coding sequences (CDS) were calculated (Fig. 4). The results showed that the Ka values of both plastome and mitogenomes were almost all less than 0.3 (Fig. 4a). The Ks values of both organelle genomes and Ka/Ks of plastome were ranged from 0 to 1.0 (Fig. 4b, c). There was no significant difference in Ka values between the two different organelle genomes (Fig. 4a), but there was significant difference in Ka values and Ka/Ks values between two different organelle genomes (Fig. 4b, c). In addition, there is no significant difference in Ka values, Ks values, and Ka/Ks values among the same organelle genomes of different species (Fig. 4).

Phylogenetic analysis of the mitogenome

In our phylogenetic inference, 108 species representing 18 order of Angiosperm were included (Table S2; Fig. 5). After carefully alignment check and avoid long branch attraction influence, the combined dataset was 32,927 nucleotides in length and ModelFinder identified GTR + F + I + G4 as the best model of evolution for both Maximum-Likelihood (ML) and Bayesian Inference (BI) analyses (Table S3). ML and BI analyses produced trees sharing the same general topology, including 18 main clades each supported by high statistical values (Fig. 5). At the order level, the overall phylogenetic tree closely aligns with the APG IV system (Fig. 5). Amborellales as the first divergence lineage, the remaining order formed two super clades (Fig. 5). On of the super clade was consisted of Alismatales, Pandanales, Asparagales, Arecales, and Poales (Fig. 5). Another super clade was consisted of Asterales, Solanales, Lamiales, Ericales, Fabales, Fagales, Rosales, Brassicales, Myrtales, Malpighiales, Proteales, and Ranunculales. In addition, the two species of Ranunculaceae formed a well-supported monophyletic clade and sister to the newly sequenced species of Menispermaceae with full support (ML-BS = 100, BI-PP = 1.0; Fig. 5).

In this study, the organelle genomes of two individuals from the genus Tinospora were newly sequenced, annotated and compared (Figs. 1, S1). The results showed that two plastomes of the Tinospora had a typical tetrad structure, and the genome sizes were similar, about 163 kb (163,621–164,006 bp), and the GC contents of the plastomes were 38%, which were similar to the sequence length and composition of the previously obtained Tinospora plastomes [34]. Compared with plastomes, the final assembly of the mitogenomes have complex circular structures (Fig. S1). We also found that abundant repeats existed in the mitogenomes, which had been demonstrated that can promote the formation of large/small subrings and isomers in plants [35]. Benefiting from the emergence of PacBio HiFi sequencing, the mitogenomes of Tinospora are structured as a circular DNA molecule with a GC content of 48% and a size of 513,210–513,215 bp (Fig. 1a, b). The two newly sequenced mitogenomes are larger in size to the majority of seed plants [36, 37], as is the fully assembled Ranunculales mitogenome from Aconitum kusnezoffii, at 440 kb, but smaller than the mitogenome of Pulsatilla dahurica (ca. 824 kb; Ranunculales) [38].

The collinear analysis of homologous fragments showed that the mitogenome of Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis had experienced rearrangement events (Fig. S1c). Based on a review of previous studies [39, 40], arrangement of repetitive fragments occurs between shorter repeats and play an important role in promoting species evolution. As seen in Fig. 2, amounts of SSRs and dispersed repeats were detected in the mitogenomes of T. sagittata var. craveniana and T. sagittata var. yunnanensis. Those repetitive fragments constitute the complex circular structures of the mitogenome of genus Tinospora. The codon usage pattern of the organelle genomes plays an important role in exploring its evolutionary process [36, 41, 42]. RSCU can directly reflect the preference of codon usage [43]. We found that the third base of most codons ends with A or U (Fig. 3a, TableS5), this result is consistent with the results of studies such as Crataegus [44], Engelhardia [45], Pisum [46], and Miscanthus [47]. Besides, comparative analyses have also indicated that mitogenomes and plastomes were some similarities in in the codon usage bias (Fig. 3a) [41, 42]. However, the PCGs in mitogenomes show a more balanced usage of synonymous codons (Fig. 3a). DNA barcode is a novel species identification technique that uses short gene regions to quickly, and accurately identify species [48]. In the present study, five significantly different variable regions of T. sagittata var. craveniana and T. sagittata var. yunnanensis were identified among the mitogenomes and plastomes (Fig. 3b, c), respectively. These regions exhibited high Pi values, indicating their potential for developing molecular markers to facilitate accurate identification and rational utilization of this genus Tinospora. Due to the effect of purification selection, the substitution rate of non-synonymous nucleotides is lower than that of synonymous nucleotides, so the ratio of Ka/Ks is less than 1 in most cases [e.g., 45]. In order to gain a clear understanding of the adaptive evolution of the Tinospora organelle genomes. Both mitogenomes and plastomes were calculated the Ka/Ks ratios of protein-coding genes, and results showed that the Ka/Ks ratio of many PCGs of mitogenomes was greater than 1 were subject to positive selection (Fig. 4). In addition, the Ka/Ks ratios of most of the PCGs of plastomes were less than 1, indicating a strong purification selection pressure (Fig. 4).

The inter-species relationship of Tinospora has long been overlooked, and different authors have recognized different numbers of species in Tinospora [8, 9, 10]. Wang et al [10] recovered that Tinospora sagittata s.l. was divided into three or four clades based on concatenated datasets (rbcL + atpB + matK + ndhF + trnL-F + ITS). In our phylogenetic analysis, T. sagittata s.l. formed a well-supported clade, sister to Aconitum kusnezoffii + Pulsatilla dahurica (Fig. 5). Alami et al [26] had assembled a tetraploid genome of Tinospora sagittata (2n = 4X = 52). However, flow cytometry analysis revealed that of Tinospora sagittata s.l. accessions here has two cytotypes (diploid and hexaploid) and a wide range in genome sizes (553 Mb–2.33Gb; Table 1). In addition, we found the organelle genome sequenced here were varied (Figs. 1, S1). Therefore, we advocate that integrate more evidence to study the boundaries between species. The research presented here contributes valuable insights into Menispermaceae evolution. Besides, expanding our understanding of plant mitochondrial genomes, we can better elucidate the factors driving species evolution and better utilize traditional Chinese medicine (TCM).

Plant materials, DNA extraction, and Sequencing

The samples (fresh and health leaf tissues) of Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis were sampled from the green house in Yunnan Agricultural University, Yunnan Province, China, and immediately stored in liquid nitrogen. All samples were sent to Beijing Novogene Technology Co., Ltd. (Tianjin, China) for genomic sequencing. Total genomic DNA was extracted using TIANGEN plant genomic DNA extraction kit (TIANGEN Biotech., Beijing, China) following the manufactures’ protocols, and evaluated using a NanoDrop One spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). For the Illumina short reads, the DNA libraries with 350 bp insert sizes were constructed and sequenced using an Illumina X Plus platform. For PacBio HiFi library with 20 kb insertions construction, more than 50 µg of sheared DNA was subjected to size-selection on a BluePippin instrument (Massachusetts, USA). PacBio HiFi sequencing was performed on the PacBio Sequel II platform (Pacific Biosciences, CA, USA) according to the manufacturer’s instructions.

Estimation of DNA ploidy and genome size

Ploidy levels and absolute genome sizes of Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis were estimated using flow cytometry. Camellia sinensis (L.) Kuntze ‘Yunkang No. 10’ (Genome Size = ca. 3 Gb) was used as internal standard. About 30 mg of fresh leaves was chopped with a razor blade in a plastic Petri dish containing 0.8 mL of ice-cold MGb buffer (45 mM MgCl₂·6H₂O, 20 mM MOPS, 30 mM C₆H₅Na₃O₇, 1% (w/v) PVP 40, 0.2% (v/v) Triton X-100, 10 mM Na₂EDTA, 20 µL/mL β-mercaptoethanol, pH 7.5). The crude suspension was filtered through a 40 µm nylon mesh to remove tissue debris, RNAase was added and the nuclei were then stained with propidium iodide. Samples were stained for 30 min on ice. After incubation, each sample was run on a flow cytometer. DNA quantities were measured using a BD FACScalibur laser flow cytometer with Modifit v3.0. DNA ploidy and absolute genome sizes were determined on the basis of the sample/standard ratio.

Organelle genome assembly and annotation

Quality assessment of raw short reads and raw HiFi reads were performed using Fastp v0.23.1 [49] and PacBio SMRT-Analysis v2.3.0 (https://www.pacb.com) and remove low-quality and adapter-contaminated reads. A hybrid assembly approach was conducted to assemble complete organelle genome. For plastome assembly, the remaining high-quality short reads were assembled into contigs using GetOrganelle v.1.7.5 [50]. Following the instruction manual, the complete plastome of Paratinospora sagittata (MN646885) were employed as the reference and other parameter were kept as default settings. Then, the assembled ‘fastg’ file was converted to “gfa” file though Bandage v0.8.1 [51] to visualize and learn the plastome structure. Subsequently, both gfa file and/or the filtered HiFi reads were chosen as input files to perform de novo assembly of plastome long reads using GSAT v1.1.2 [52] and PMAT v1.5.3 [53], respectively. For the assembly of the mitochondrial genome through a similar hybrid assembly strategy, GetOrganelle was initially used to assemble mitochondrial reads from the short reads, employing parameters “-R 50 -k 21,45,65,85,105 -P 1000000 -F embplant_mt” and the complete mitogenome of Pulsatilla dahurica (NC071219) as reference. Bandage was then used to visualize and disentangle the raw assembly graph of mitogenome. The complete plastome was initially annotated using PGA [54], GeSeq [55], and CPGAVAS2 [56], respectively. Online program IPMGA (http://www.1kmpg.cn/ipmga/) was used to annotate the mitogenome. The start and stop codons of all organelle genes were then manually checked in Geneious Prime 2019.2.1. For any uncertain protein-coding genes, a Blastn search was performed with default parameter settings. All tRNAs were verified using the tRNAscan-SE v2.0 web server [57]. The circular organelle genome maps were drawn by the OmicsSuite v1.3.9 [58] and followed by manual modification.

Collinearity analysis and identification of repeat sequences

The collinearity among the complete mitogenomes was assessed by comparison with linear genome maps generated with Geneious Prime based on the result of Locally Colinear Blocks (LCBs) via the progressive Mauve algorithm in Mauve v2.3.1 [59]. We analyzed two different types of repeat sequences in both organelles. Simple sequence repeats (SSRs), also known as microsatellites, were identified using MISA v2.1 [60], with parameter settings of 8, 4, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. Four types of dispersed repeats, namely forward (F), palindromic (P), reverse (R), and complement (C) repeats, were searched by the online version of REPuter with the parameters minimal repeats and hamming Distance set to 30 and 3 bp, respectively [61]. The results were plotted in OriginPro v2022b (OriginLab Corporation, Northampton, MA, USA).

Analysis of nucleotide polymorphism and relative synonymous codon usage

We analyzed nucleotide polymorphisms to show the nucleotide diversity based on the Pi values of plastome, and mitogenome, respectively. Both organelle genomes were adjusted collinearity based on the result of LCBs and aligned using Mafft v7.450 [62]. The Pi of protein-coding genes, noncoding genes, and the intergenic regions extracted from the organelle genomes were calculated using DnaSP v6.0 [63] (set window length = 600 bp, step size = 200 bp). The protein-coding genes (PCGs) sequence of Tinospora sagittata var. craveniana and T. sagittata var. yunnanensis mitogenomes and plastomes were extracted using PhyloSuite v1.1.2 [64]. MEGA-X [65] was applied to analyze the relative synonymous codon usage (RSCU) of PCGs and calculate the RSCU value. RSCU = 1 indicated that there was no preference for the use of this codon, and RSCU > 1 indicates that the codon was used preferentially by amino acids, while if RSCU < 1, the codon usage is contrary.

KaKs analysis and phylogenetic inference

The non-synonymous (Ka) and synonymous (Ks) substitution (Ka/Ks) was analyzed for shared PCGs between Tinospora sagittata var. craveniana, T. sagittata var. yunnanensis and two other species of Ranunculales (Pulsatilla dahurica [plastome: MK860685; mitogenome: NC071219]; Aconitum kusnezoffii [plastome: MK782808; mitogenome: NC053920]) through Ka/Ks calculator v2.0 [66]. A total of 106 mitogenomes of plant were download from the GenBank database to construct a phylogenetic tree. To generate a well-supported mitogenome phylogeny backbone, 41 PCGs were extracted by using a python script (get_annotated_regions_from_gb.py; https://github.com/Kinggerm/PersonalUtilities/) and aligned using Mafft. Ambiguously aligned regions were excluded and concatenated by using plugins trimAI v1.3 (set -automated1) [67]. In addition, TreeShrink v1.3.7 [68] was selected to reduce the long branch attraction (LBA) influence. The input gene trees for TreeShrink were generated using IQ-tree v2.1.3 [69] with 1,000 rapid bootstrap replicates [70]. The retained sequences were used to construct the phylogenetic trees. The nucleotide substitution rate variation model of Maximum Likelihood (ML) and Bayesian Inference (BI) was fitted using the ModelFinder tool under the corrected Akaike Information Criterion (AICc) in IQ-tree. The ML analyses were performed with 5,000 ultrafast bootstraps replicates in IQ-tree. The BI analyses were performed using MrBayes v.3.2.2 [71] with a random tree and sampling one tree every 100 generations for a total 2,000,000 generations. Finally, the concatenated trees were generated and visualized with their Maximum-Likelihood bootstrap support values (ML-BS) and Bayesian Inference posterior probability (BI-PP) in Figtree v1.4.3 [72].

Author contributions

Y.Z., J.G.W., and Y.Y.Z. contributed to the study conception and design. Material preparation was performed by P.C.H. and L.H.W. Experiments and data analyses were performed by J.Z., Z.H. C. and L.W.C. Data collection and interpretation were performed by J.Z., Z.H.C. and X.Z.M. The first draft of the manuscript was written by J.Z., Z.H.C.

Funding

This study was supported by grants from the “Yunnan Province Academician and Science and Technology Leading Talents Special Project-Academician Free Exploration Project” (Grant No. 202405AA350011), Postdoctoral Research Funds of Yunnan University (Grant No. W8163003).

Data availability

The new sequenced chloroplast genome of this study has been submitted to GenBank under the accession number of *** to ***, respectively. Multiple sequence alignments and phylogenetic trees are available on the Figshare repository: ************, and other data and materials provided in this manuscript are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical Trial Number

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Tables 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

Table1.xlsx
Table 1. Summary of the flow cytometer and sequence results.
Table2.xlsx
Table2. Gene composition of the mitogenome
FigureS1.tif
Figure S1. Map of mitochondrial genome assembly and annotation of Tinosporasagittata var. cravenianaand T. sagittata var. yunnanensis. (a) The map of the preliminary assembly of the mitogenome of T. sagittata var. yunnanensis. (b) The map of the preliminary assembly of the mitogenome Tinospora sagittata var. craveniana. (c) Whole mitogenome collinearity analysis.
TableS1.xlsx
TableS1. Complete features of two newly assembled Tinospora plastomes.
TableS2.xlsx
TableS2. The frequency of identified SSRs.
TableS3.xlsx
TableS3. Statics of dispersed repeat sequences.
TableS4.xlsx
TableS4.Type and quantity of SSR motifs.
TableS5.xlsx
TableS5. Codon usage bias analysis of Tinospora organelle genomes.
TableS6.xlsx
TableS6. The list of the mitogenomes used to construct phylogenetic trees.
TableS7.xlsx
TableS7. Best-fitting models and characteristics of the datasets in this study.

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Complete mitogenome characteristics and phylogenetic analysis of traditional Chinese medicinal plant Tinospora sagittata (Oliv.) Gagnep. from the Menispermaceae family

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Abstract

Figures

Introduction

Results

Genome size and sequence data

Characteristics of organelle genomes

Collinearity and repeat sequences

Protein coding gene codon usage and comparative analysis of genomic variation

Selective pressure analysis of organelle genome CDS

Phylogenetic analysis of the mitogenome

Discussion

Materials and methods

Plant materials, DNA extraction, and Sequencing

Estimation of DNA ploidy and genome size

Organelle genome assembly and annotation

Collinearity analysis and identification of repeat sequences

Analysis of nucleotide polymorphism and relative synonymous codon usage

KaKs analysis and phylogenetic inference

Declarations

Author contributions

Funding

Data availability

Ethics approval and consent to participate

Consent for publication

Competing interests

Clinical Trial Number

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1