Abstract
Background
Sabia discolor Dunn is an ethnomedicinally significant plant with underexplored genetic resources. To elucidate its chloroplast genome structure, evolutionary characteristics, and phylogenetic position within the genus Sabia, we sequenced and analyzed its complete chloroplast genome.
Results
Using Illumina HiSeq™ platform for sequencing, followed by assembly with GetOrganelle and annotation with CPGAVAS2, we characterized the genome. The results showed that the chloroplast genome of S. discolor is a 161,581 bp circular DNA molecule with a typical quadripartite structure and a GC content of 39.25%. It harbors 129 genes, including 84 protein-coding genes, 8 rRNAs, and 37 tRNAs. We detected 80 simple sequence repeats (SSRs) and 55 long repeats. Codon usage bias analysis revealed a weak preference, with an effective number of codons (ENC) value of 49.01, primarily shaped by natural selection rather than mutation pressure. Comparative analysis identified a significant contraction in the inverted repeat (IR) regions and six highly variable regions (e.g., trnH-psbA, ndhF), which are proposed as potential molecular markers for species identification. Phylogenetic analysis based on complete chloroplast genomes revealed two major clades within Sabia, with S. discolor placed in Clade I, closely clustered with S. parviflora, S. limoniacea, S. fasciculata, and S. swinhoei, while S. dielsii formed a distinct basal lineage.
Conclusions
This study provides fundamental genetic resources for future research on the conservation, phylogenetics, and molecular identification of S. discolor and its relatives.
Keywords: Sabia discolor, Chloroplast genome, Phylogenomics, Molecular markers
Introduction
The chloroplast is the primary site for photosynthesis in plants and is a semi-autonomous organelle with relatively independent genetic material [1]. Angiosperms possess a highly conserved circular chloroplast genome, with a size generally ranging between 140 and 160 kilobases (kb). It exhibits a circular double-stranded structure composed of four parts, encoding approximately 110 to 130 genes. Typically, it consists of one large single copy (LSC) region, one small single copy (SSC) region, and two inverted repeat (IR) regions [2]. Compared to the nuclear genome, the chloroplast genome has advantages such as being smaller in size, having a higher copy number, and moderate evolutionary rates. It is often used in phylogenetic studies at different taxonomic levels [3]. In addition, features of the chloroplast genome such as simple sequence repeats (SSRs), sequence variability, nucleotide polymorphisms, and codon usage bias provide key information for species identification and evolutionary biology research [4, 5]. In recent years, with the rapid development of high-throughput sequencing technologies, an increasing number of plant chloroplast genomes have been sequenced and widely used in phylogenetic research. Within the family Sabiaceae, the complete chloroplast genome of Meliosma oldhamii was recently characterized, providing additional genomic resources for comparative studies [6]. At the ordinal level, phylogenetic analyses of basal eudicots have confirmed that the order Proteales, to which Sabiaceae belongs, forms a well-supported clade with Sabiales, clarifying the evolutionary position of this lineage [7]. Clearly, chloroplast genome sequences can facilitate phylogenetic studies in higher plants, and for the family Sabiaceae in particular, they provide a powerful tool for resolving long-standing taxonomic controversies.
Sabia is the type genus of the family Sabiaceae and is distributed in southern and southeastern Asia, with about 30 species worldwide. In China, there are approximately 16 species, 5 subspecies, and 2 varieties, primarily distributed in the southwestern and southeastern regions [8]. Several Sabia species have long been used in traditional medicine for their hepatoprotective, anti-inflammatory, and analgesic properties, with roots, stems, and leaves being the primary medicinal parts [9, 10]. Among them, S. discolor Dunn is an important ethnomedicinal plant, particularly valued in the traditional medicine of the Yao people in southwestern China, where it is used to treat rheumatism, traumatic injuries, nephritis, and edema [11].
However, accurate species identification and clear systematic classification relationships are prerequisites for ensuring the quality of medicinal materials and effective resource development. As a reliable genetic marker in plant phylogenetic studies, the chloroplast genome, with its high-resolution characteristic, can effectively resolve taxonomic disputes among closely related species and provide key insights into understanding their evolutionary history. Although the chloroplast genomes of several Sabiaspecies have been characterized in recent years [12]., the complete chloroplast genome of S. discolor has not yet been reported. Currently, studies on S. discolor are largely restricted to its chemical components and pharmacological activities [13], and its evolutionary origin and phylogenetic relationships remain unclear. With the continuous development of high-throughput sequencing technologies, molecular sequencing techniques are also gradually being applied to the identification of plants. Compared with traditional combinations of a few plastid markers, the complete chloroplast genome sequence can provide far more variable loci and phylogenetic information, greatly improving the support rate and resolution of the tree structure [14]. Therefore, this study used the whole chloroplast genome data to more accurately reveal the intrageneric phylogenetic relationships.
In this context, this study conducted chloroplast genome sequencing, assembly, and annotation of S. discolor under the genus Sabia for the first time, and analyzed its structural characteristics and codon usage bias. In addition, a phylogenetic tree was constructed by comparing it with the chloroplast genomes of other reported Sabia species. The aims of this study are as follows: (1) to analyze the characteristics of the chloroplast genome of S. discolor; (2) to explore the characteristics and influencing factors of its codon usage bias; (3) to screen effective candidate molecular marker sequences and optimal codons; and (4) determine the phylogenetic placement of S. discolor within the genus Sabia. The results will provide fundamental genomic resources and valuable insights for the conservation, molecular identification, and evolutionary studies of S. discolor and its related species.
Materials and methods
Materials
Sixty fresh leaves of S. discolor Dunn under the same growth conditions were picked from Changdong Township, Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region, China (latitude 24.003036°N, longitude 110.091274°E; altitude 500 m) and stored in a desiccant. The voucher specimen is stored in Room 404, Laboratory of the School of Biology and Food Engineering, Guangxi Science & Technology Normal University, with the specimen number DSGF2025JX (Fig. 1). The plant material was formally identified by Prof. Song Guo (Guangxi Science and Technology Normal University, China).
Fig. 1.
Photo of S. discolor Dunn, taken on May 11, 2024. A: The plant leaves are elliptical, with a broad - ovate shape, 4–6 cm long and 2.2–4.4 cm wide, papery, entire, revolute, grayish - white beneath, light black above when dry, with a rounded, acute or sometimes slightly acuminate apex, and a rounded base, glabrous on both sides; the petiole is 7–10 mm long. B: The plant is a climbing woody vine or grows prostrate on the ground
Sequencing, assembly, annotation, and analysis
Total genomic DNA was extracted from approximately 100 mg of silica-dried leaves using a modified cetyltrimethylammonium bromide (CTAB) method [15]. DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and a Qubit 3.0 fluorometer (Invitrogen, USA). DNA integrity was verified by 1% agarose gel electrophoresis. High-quality DNA was used to construct a paired-end library with an insert size of 350 bp, which was then sequenced on the Illumina HiSeq™ platform (2 × 150 bp) [16]. After quality control and assessment of the sequencing data using FastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), the complete chloroplast genome was assembled using GetOrganelle v1.7.7.0 [17]. The assembly graph was visualized and inspected using Bandage v0.8.1 [18], and manual corrections were performed using Apollo [19] with reference to the closely related species Sabia yunnanensis (GenBank accession NC_029431.1).
Genome annotation was performed using the CPGAVAS2 online tool (http://www.herbalgenomics.org/cpgavas2), followed by manual validation and correction using Geneious v2023.2 (https://www.geneious.com). The annotated chloroplast genome was submitted to the NCBI GenBank database under the accession number PP733984. The circular chloroplast genome map was drawn using OGDRAW v1.3.1 [20].
Analysis of repetitive sequences
Simple sequence repeats were detected using the MISA online software [21] with the following parameters: mononucleotide repeats were set to a minimum of 10 repeats, dinucleotide repeats to 5, trinucleotide repeats to 4, and tetranucleotide, pentanucleotide, and hexanucleotide repeats to 3. Long repeat sequences were detected using the REPuter online software [22] with a minimum repeat length of 30 base pairs (bp), a Hamming distance of 3 and a Minimal Repeat Size of 8.
Codon usage bias analysis
The repetitive and protein-coding sequences shorter than 300 bp were removed, and the CodonW 1.4.2 software [23] was used to calculate the effective number of codons (ENC) and the relative synonymous codon usage (RSCU) for 48 protein-coding sequences. The RSCU value represents the ratio of the actual frequency of codon use to the expected frequency and is used to measure codon usage bias [24]. An RSCU value greater than 1 indicates a strong bias, with the codon being classified as a high-frequency codon; an RSCU value of 1 indicates no bias; and an RSCU value less than 1 indicates a weaker bias, with the codon being classified as a low-frequency codon. The ENC value reflects the degree of codon usage bias, with a theoretical range of 20 to 61—a smaller ENC value indicates a stronger bias [25]. The GC content of the first, second, and third codon positions (GC1/GC2/GC3) was calculated using the cusp program from the EMBOSS website [26] GC12 represents the average of GC1 and GC2. The neutrality plot (GC3-GC12), ENC plot, and analysis of optimal codons were analyzed using the methods described by Liu [4] and others.
Sequence variation analysis and IR boundary analysis
Sequence variation analysis was conducted using the mVISTA online software [27], under the Shuffle-LAGAN model. Nucleotide polymorphism (Pi) was calculated using DnaSP 6.0 software [28], with the running parameters set to a window length of 600 bp and a step size of 200 bp [29]. Analysis of the IR region boundary contraction and expansion was performed using the IRscope online software [30].
Phylogenetic analysis
The complete chloroplast genome sequences of 10 Sabia species were downloaded from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) (Table 1). Whole-genome multiple sequence alignment was performed using MAFFT [31] with the ‘--auto’ strategy. The resulting alignment was manually inspected and adjusted where necessary. Maximum likelihood (ML) analysis was conducted using IQ-TREE v2.2.0 [32]. The best-fit nucleotide substitution model was selected using ModelFinder [33] according to the Bayesian Information Criterion (BIC). Nodal support was assessed using 1,000 ultrafast bootstrap replicates [34].
Table 1.
Species used for phylogenetic analysis
| Species name | GenBank | Family |
|---|---|---|
| Sabia fasciculata | NC_069947.1 | Sabia |
| Sabia swinhoei | NC_069951.1 | Sabia |
| Sabia limoniacea | NC_069949.1 | Sabia |
| Sabia parviflora | NC_059863.1 | Sabia |
| Sabia japonica | NC_069948.1 | Sabia |
| Sabia dielsii | NC_069946.1 | Sabia |
| Sabia schumanniana | NC_069950.1 | Sabia |
| Sabia yunnanensis | NC_029431.1 | Sabia |
| Sabia campanulata subsp. ritchieae | OP310790.1 | Sabia |
Phylogenetic trees were visualized and annotated using the online tool iTOL (Interactive Tree Of Life, https://itol.embl.de/). The resulting tree was midpoint-rooted (no outgroups were included).
Results
Features of the chloroplast of S. discolor Dunn
The chloroplast genome of S. discolor Dunn is 161,581 bp in length and exhibits a circular, double-stranded structure composed of four parts: two inverted repeat regions (IRa and IRb), a large single copy region (LSC), and a small single copy region (SSC) (Fig. 2). The GC and AT contents are 39.25% and 60.75%, respectively, with the GC contents of the IR, LSC, and SSC regions being 39.04%, 38.82%, and 38.01%, respectively (Table 2). A total of 129 genes have been annotated, including 84 protein-coding genes, 8 ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes (Table 3).
Fig. 2.
Gene map of the S. discolor Dunn chloroplast genome. The quadripartite structure includes two inverted repeat (IRa and IRb) regions separating the large single-copy (LSC) and small single-copy (SSC) regions. Genes drawn inside the circle are transcribed clockwise, whereas those outside are transcribed counter-clockwise. The inner gray plot represents the GC content, with the dark gray area marking the 50% threshold. Colored bars indicate known protein-coding genes, transfer RNA (tRNA) genes, and ribosomal RNA (rRNA) genes
Table 2.
Summary of the chloroplast genome features of Sabia discolor Dunn
| Parameter | Value | Parameter | Value |
|---|---|---|---|
| Genome size (bp) | 161,581 | Total GC content(%) | 39.25 |
| Length of LSC region (bp) | 89,933 | GC content of LSC region(%) | 38.82 |
| Length of IR region (bp) | 26,361 | GC content of IR region(%) | 39.04 |
| Length of SSC region (bp) | 18,926 | GC content of SSC region(%) | 38.01 |
| Base composition (%) | |||
| A | 30.33 | T | 31.06 |
| C | 19.66 | G | 18.96 |
Table 3.
Functional classification statistics of genes of the S. discolor Dunn chloroplast genome
| Category of gene | Group of gene | Name of genes |
|---|---|---|
| photosynthesis | photosystem I | psaA, psaB, psaC, psaI, psaJ |
| photosystem II |
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbM, psbN, psbT, psbZ, |
|
| cytochrome b/f complex | petA, petB*, petD*, petG, petL, petN | |
| ATP synthase | atpA, atpB, atpE, atpF*, atpH, atpI | |
| NADH-dehydrogenase |
ndhA*, ndhB* (×2), ndhC,ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK |
|
| Rubisco large subunit | rbcL | |
| Self-replication | RNA polymerase | rpoA, rpoB, rpoC1*, rpoC2 |
| Small subunit of ribosome (SSU) |
rps11, rps12**(×2), rps14, rps15, rps16*, rps18, rps19, rps2, rps3, rps4, rps7(×2), rps8 |
|
| Large subunit of ribosome (LSU) |
rpl14, rpl16*, rpl2* (×2), rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36 |
|
| Transfer RNAs |
trnA-UGC* (×2), trnC-GCA, trnD-GUC, trnE-UUC* (×2), trnE-UUC, trnF-GAA, trnG-GCC, trnH-GUG, trnK-UUU* trnL-CAA(×2), trnL-UAA*, trnL-UAG, trnM-CAU(×4), trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnW-CCA, trnY-GUA |
|
| Ribosomal RNAs |
rrn16S(×2), rrn23S(×2), rrn4.5 S(×2) ,rrn5S(×2), |
|
| Other genes | Subunit of Acetyl-CoA-carboxylase | accD |
| C-type cytochrom synthesis gene | ccsA* | |
| Envelop membrane protein | cemA | |
| Protease | clpP** | |
| Translational initiation factor | infA | |
| Maturase | matK | |
| Hypothetical chloroplast reading frames (ycf) | ycf1, ycf2(×2), ycf3**ycf4 |
* and ** indicates the gene contains 1 and 2 introns; (×2) and (×4) indicates the gene has 2 and 4 copies
Repetitive sequence analysis
A total of 80 SSR loci were detected in the chloroplast genome of S. discolor Dunn, with most of them located in the LSC region. The most numerous were single nucleotide repeats (54), with 98.1% of these being A/T types, followed by dinucleotide and tetranucleotide repeats (Table 4). We found that the repeat units of A/T, AT/AT, AAT/ATT, and AAAT/ATTT accounted for 82.5%, indicating a bias towards the use of A/T bases in SSRs. A total of 55 long repeat sequences were detected, including 26 forward repeats and 29 palindromic repeats. The length of long repeat sequences mainly ranged between 30 and 39 bp, with forward and palindromic repeats being the most abundant types (26 and 29, respectively).
Table 4.
Type and number of SSRs of the S. discolor Dunn
| SSR type | Repeat unit | Number | Ratio(%) |
|---|---|---|---|
| Mononucleotide | A/T | 53 | 98.1 |
| C/G | 1 | 1.9 | |
| Dinucleotide | AT/AT | 10 | 76.9 |
| AG/CT | 2 | 15.4 | |
| AC/GT | 1 | 7.7 | |
| Trinucleotide | AAG/CTT | 3 | 60 |
| AAT/ATT | 2 | 40 | |
| Tetranucleotide | AGAT/ATCT | 2 | 25 |
| AATG/ATTC | 2 | 25 | |
| AATC/ATTG | 2 | 25 | |
| AAAT/ATTT | 1 | 12.5 | |
| AAAG/CTTT | 1 | 12.5 |
Codon usage bias characteristics and influencing factor analysis
The ENC value of S. discolor Dunn is 49.01, indicating a weak codon usage bias. A total of 32 high-frequency codons (excluding stop codons) were detected in the chloroplast genome of S. discolor Dunn, with 15 ending in A, 13 ending in U, 2 ending in G, and 2 ending in C. There were also 30 low-frequency codons, with 14 ending in C, 12 ending in G, 3 ending in U, and 1 ending in A (Fig. 3). This suggests that the high-frequency codons in S. discolor Dunn tend to end in A/U, while the low-frequency codons tend to end in G/C.
Fig. 3.
Analysis of relative synonymous codon usage of the S. discolor Dunn chloroplast. Genome, The blocks below represent all codons encoding each amino acid, while the height of the bars above indicates the total RSCU values for all codons
Codon usage bias is influenced by various factors, with natural selection and mutation pressure being the main influencing factors. The ENC-plot analysis found that the majority of genes were located below the standard curve, indicating a significant difference between the actual and expected ENC values, which suggests that natural selection plays a primary role in shaping codon usage bias (Fig. 4: A). Furthermore, the analysis of the ENC ratio frequency distribution revealed that 36 genes (accounting for 75%) were within the range of -0.05 to 0.05, indicating that the codon usage bias of these genes was influenced by internal mutation pressure (Table 5). The remaining 12 genes (accounting for 25%) were outside this range, indicating that their codon usage bias was primarily influenced by natural selection. In the neutrality plot analysis, we found that the GC12 values ranged from 0.343 to 0.561, and the GC3 values ranged from 0.224 to 0.370, with all genes located above the diagonal line (Fig. 4: B). The comprehensive analysis results revealed that the base composition at the first and third codon positions had a small difference and similar evolutionary patterns, mainly influenced by base mutations; while the second base significantly differed from the third base, mainly influenced by natural selection. Additionally, the slope of the regression curve was 0.0854, indicating that the influence of mutation pressure on codon bias reached 8.54%, with natural selection accounting for 91.46%. The R² value of 0.0035 suggests a weak correlation between GC12 and GC3 values, further confirming that codon usage bias is primarily influenced by natural selection. In summary, both natural selection and internal mutation pressure affect the codon usage bias of the chloroplast genome in S. discolor Dunn, with natural selection playing a primary role.
Fig. 4.
(A) ENC-plot analysis; (B) Neutrality plot analysis. Analysis of codon usage bias factors of the S. discolor Dunn chloroplast genome. A: Effective number of codons ENC-plot showing relationship between ENC and GC3. B: Neutrality plots (GC12 vs. GC3). GC12 stands for the average value of GC content in the first and second position of the codons (GC1 and GC2). While GC3 refers to the GC content in the third position. The solid line is the linear regression of GC12 against GC3, R2 = 0.0035, p < 0.01
Table 5.
Frequency distribution of ENC ratio
| Class boundry | Class middle value | Number | Class frequency (%) |
|---|---|---|---|
| -0.15~-0.05 | -0.1 | 9 | 18.75 |
| -0.05 ~ 0.05 | 0 | 36 | 75.00 |
| 0.05 ~ 0.15 | 0.1 | 3 | 6.25 |
Optimal codon determination
In the chloroplast genome of S. discolor Dunn, the optimal codons were determined by satisfying the criteria of having a RSCU greater than 1 (indicating high-frequency codons) and a ΔRSCU (the difference between RSCU of high-expression genes and RSCU of low-expression genes) of 0.08 or higher. A total of 15 optimal codons were identified: GCA, GCU, UGU, GAA, AUU, AAA, UUA, UUG, CCC, CCU, CAA, CGA, UCC, ACU, and GUA (Table 6). Of these, 13 (accounting for 87%) have A or U at the third position, indicating a preference for codons ending with A and U in the chloroplast genome of S. discolor Dunn.
Table 6.
Optimal codon analysis of the S. discolor Dunn chloroplast genome
| Amino acid | Codon | RSCU | Amino acid | Codon | RSCU | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Genome | High expression gene | Low expression gene | △RSCU | Genome | High expression gene | Low expression gene | △RSCU | ||||
| Ala | GCA | 1.11 | 1.28 | 0.71 | 0.58 | Asn | AAC | 0.60 | 0.69 | 0.34 | 0.35 |
| GCC | 0.97 | 0.36 | 0.86 | -0.5 | AAU | 1.40 | 1.31 | 1.66 | -0.35 | ||
| GCG | 0.63 | 0.46 | 0.63 | -0.17 | Pro | CCA | 1.19 | 1.17 | 1.33 | -0.17 | |
| GCU | 1.29 | 1.9 | 1.8 | 0.09 | CCC | 1.02 | 1.33 | 0.76 | 0.57 | ||
| Cys | UGC | 0.78 | 0 | 1.25 | -1.25 | CCG | 0.64 | 0.25 | 0.76 | -0.51 | |
| UGU | 1.22 | 2 | 0.75 | 1.25 | CCU | 1.15 | 1.25 | 1.14 | 0.11 | ||
| Asp | GAC | 0.58 | 0.4 | 0.26 | 0.14 | Gln | CAA | 1.40 | 1.6 | 1.52 | 0.08 |
| GAU | 1.42 | 1.6 | 1.74 | -0.14 | CAG | 0.60 | 0.4 | 0.48 | -0.08 | ||
| Glu | GAA | 1.33 | 1.77 | 1.56 | 0.21 | Arg | AGA | 1.89 | 1.34 | 1.53 | -0.19 |
| GAG | 0.67 | 0.23 | 0.44 | -0.21 | AGG | 1.14 | 0.27 | 0.47 | -0.2 | ||
| Phe | UUC | 0.82 | 1.22 | 0.83 | 0.39 | CGA | 1.05 | 1.7 | 1.18 | 0.53 | |
| UUU | 1.18 | 0.78 | 1.17 | -0.39 | CGC | 0.44 | 0.45 | 0.59 | -0.14 | ||
| Gly | GGA | 1.48 | 1.16 | 1.9 | -0.74 | CGG | 0.75 | 0.18 | 0.82 | -0.64 | |
| GGC | 0.61 | 0.35 | 0.52 | -0.17 | CGU | 0.73 | 2.06 | 1.41 | 0.65 | ||
| GGG | 0.95 | 0.46 | 0.52 | -0.07 | Ser | AGC | 0.57 | 0.44 | 0.55 | -0.1 | |
| GGU | 0.96 | 2.03 | 1.05 | 0.98 | AGU | 0.85 | 1.76 | 1 | 0.76 | ||
| His | CAC | 0.58 | 1 | 0.64 | 0.36 | UCA | 1.24 | 0.79 | 1 | -0.21 | |
| CAU | 1.42 | 1 | 1.36 | -0.36 | UCC | 1.18 | 1.15 | 1 | 0.15 | ||
| Ile | AUA | 1.04 | 0.81 | 1.04 | -0.23 | UCG | 0.73 | 0.35 | 0.55 | -0.19 | |
| AUC | 0.74 | 0.7 | 0.74 | -0.04 | UCU | 1.43 | 1.5 | 1.91 | -0.41 | ||
| AUU | 1.22 | 1.48 | 1.21 | 0.27 | Thr | ACA | 1.18 | 1.1 | 1.4 | -0.3 | |
| Lys | AAA | 1.34 | 1.6 | 1.35 | 0.25 | ACC | 0.94 | 1.25 | 0.6 | 0.65 | |
| AAG | 0.66 | 0.4 | 0.65 | -0.25 | ACG | 0.69 | 0.16 | 0.7 | -0.54 | ||
| Leu | CUA | 0.84 | 1.12 | 0.8 | 0.33 | ACU | 1.19 | 1.49 | 1.3 | 0.19 | |
| CUC | 0.85 | 0.2 | 0.61 | -0.41 | Val | GUA | 1.18 | 2.25 | 1.58 | 0.67 | |
| CUG | 0.58 | 0.2 | 0.73 | -0.54 | GUC | 0.71 | 0.31 | 0.68 | -0.37 | ||
| CUU | 1.30 | 1.05 | 1.35 | -0.29 | GUG | 0.71 | 0.31 | 0.6 | -0.29 | ||
| UUA | 1.19 | 1.91 | 1.47 | 0.44 | GUU | 1.39 | 1.13 | 1.13 | -0.01 | ||
| UUG | 1.24 | 1.52 | 1.04 | 0.48 | Trp | UGG | 1.00 | 1 | 1 | 0 | |
| Met | AUG | 3.00 | 1 | 1 | 0 | Tyr | UAC | 0.65 | 0.56 | 0.34 | 0.22 |
| UAU | 1.35 | 1.44 | 1.66 | -0.22 | |||||||
Sequence variability analysis
Using the mVISTA chloroplast genome alignment tool, with the chloroplast genome of Sabia japonica as a reference, the chloroplast genomes of nine species within the genus Sabia were analyzed for sequence variability. The results indicated that the structures and gene orders of these chloroplast genomes were largely conserved. The variability in non-coding regions was greater than that in coding regions, and the variability in the inverted repeat (IR) regions was significantly less than that in the large single copy (LSC) and small single copy (SSC) regions (Fig. 5).
Fig. 5.
Complete chloroplast genome alignment of Sabia species with mVISTA. The vertical scale indicates the average percent identity, ranging from 50% to 100%. The horizontal scale indicates the coordinates within the chloroplast genome. Gray arrows above the alignment indicate genes with their orientation
Analysis of nucleotide polymorphism revealed a total of 795 polymorphic sites, with the nucleotide diversity (Pi) ranging from 0 to 0.02537, with an average value of 0.00399. Six regions with high variability (Pi > 0.015) were identified: trnH-GUG-psbA, rps16, petN, ndhC, ndhF, and ndhA (Fig. 6).
Fig. 6.
Nucleotide polymorphism analysis of the nine Subgen. Sabia species chloroplast. Genomes, The X-axis represents the position of the chloroplast genome, and the Y-axis represents the nucleotide diversity of each window
In summary, our comparative analysis not only reaffirmed the conservation and variation patterns of coding and non-coding regions in the chloroplast genome. More importantly, we successfully screened six highly variable regions (trnH-psbA, rps16, petN, ndhC, ndhF, and ndhA). Due to their high content of informative sites, these regions are considered excellent candidate DNA barcodes and molecular markers for species identification and population genetics research in the genus Sabia.
IR region boundary analysis
The length of the inverted repeat (IR) regions in the chloroplast genomes of the nine Sabia species ranged from 26,360 to 26,604, with relatively minor differences at the IR region boundaries (Fig. 7). In all species, the boundary between the LSC region and IRb (JLB) was located within the rps19 gene. Similarly, the boundary between the LSC region and IRa (JSA) was located within the ycf1 gene in all species. Except for S. discolor Dunn, the ycf1 gene was distributed in the IRb region in the other species, with a variable distance of 1 to 3 base pairs from the SSC region. Except for S. discolor Dunn, the rps19 gene was also distributed in the IRb region in the other species, all at a distance of 1 bp from the LSC region. This indicates that there are significant differences in the length of the IR region and the boundary genes of the chloroplast genome in S. discolor Dunn compared to other Sabia plants, and that the IR boundaries show a clear contraction phenomenon when compared to other species.
Fig. 7.
Comparison of small single-copy region (SSC), large single-copy (LSC), and inverted repeat regions (IRa and IRb) boundaries in 9 chloroplast genomes of Sabiaceae. LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC denote the junction sites between each corresponding two regions of the genomes
Phylogenetic analysis
To avoid the potential artifact of long-branch attraction caused by distantly related outgroups, we reconstructed a maximum likelihood (ML) tree using the complete chloroplast genome sequences of 10 Sabia species only. The tree was midpoint-rooted and is presented in Fig. 8. The ML analysis yielded a well-resolved topology with high bootstrap support (BS) for most internal nodes. The tree recovered S. dielsii as the earliest-diverging lineage. The remaining nine species formed a strongly supported clade, which further split into two distinct subclades. Clade I comprised five species: S. parviflora, S. limoniacea, S. discolor, S. fasciculata, and S. swinhoei. Clade II consisted of four species: S. japonica, S. schumanniana, S. campanulata subsp. ritchieae, and S. yunnanensis. The best-fit substitution model selected by ModelFinder was TVM + F+R7.
Fig. 8.
Maximum likelihood (ML) phylogeny of Sabia species based on complete chloroplast genome sequences. Numbers above branches indicate ultrafast bootstrap support values (≥ 78% shown). Branch lengths are proportional to the number of nucleotide substitutions per site (scale bar = 0.001). The star (★) marks the focal species S. discolor. Two major clades (I and II) are strongly supported
These results place S. discolor firmly within Clade I, closely allied with S. limoniacea, S. parviflora, S. fasciculata, and S. swinhoei, while S. dielsii stands as a distinct basal lineage.
Discussion and conclusion
Through comparative analysis, it was found that the chloroplast genome of Sabia discolor is similar to previously reported Sabia species in terms of length, structure, and gene content. contractions were observed in the boundaries of the inverted repeat (IR) regions, indicating the relative instability of the S. discolor chloroplast genome. Additionally, compared to the large single copy (LSC) and small single copy (SSC) regions, the IR regions have the highest GC content and the least sequence variability. This may be related to the presence of RNA genes with high GC content (55.1%) within the IR regions. Studies by Wolf et al. [35] have shown that the length of angiosperm chloroplast genomes is typically between 120 and 180 kb, with IR regions being 20 to 30 kb. In this study, the full length of the S. discolor chloroplast genome is 161,581 bp, with the IR region being 26,361 bp, which further indicates the conservation of the chloroplast genome. Similar phenomena have also been observed in other plants [36, 37], suggesting that the IR regions play a crucial role in maintaining the structural stability of the chloroplast genome.
Codons are crucial for the accurate expression of genetic information, and the bias in codon usage is specific to different species and even different genes within the same species. This specificity arises from the combined effects of selection, mutation, and drift during the long-term evolutionary process of genes and species. Species that are more closely related or share similar living environments are more likely to adopt similar codon selection strategies [38, 39]. By analyzing and comparing differences in codon usage bias, it is possible to infer the degree of translational selection acting on genes, which is important for exploring the evolutionary patterns of Sabia species. Previous studies have shown that neutral mutations occur at the third codon position, with G/C and A/T being equally present in the codons within the chloroplast genome [40]. In this study, among the 32 high-frequency codons (excluding stop codons) with RSCU values greater than 1, 28 codons end with A/U; among the 30 low-frequency codons with RSCU values less than 1, 26 codons end with C/G. This indicates that synonymous codons ending with G/C are more involved in the protein-coding gene process of the S. discolor Dunn chloroplast genome. Comprehensive analysis reveals that the base composition at the first and third positions of codons has a small difference and similar evolutionary patterns, mainly influenced by base mutations; while the second base position shows a significant difference from the third, primarily influenced by natural selection. In the ENC-plot analysis, it was found that most genes are located below the standard curve, indicating a significant difference between the actual and expected ENC values, further suggesting that natural selection plays a major role [41]. In this study, we found that natural selection and internal mutational pressure together influence the codon usage bias of the S. discolor Dunn chloroplast genome, with natural selection being the primary factor [42].
Repeated sequences and simple sequence repeats (SSRs) are widely present in the chloroplast genomes of plants and are characterized by a rich polymorphism, high variability, and a high content of genetic information. They are often used in studies of genetic diversity, species identification, and molecular marker-assisted breeding [43]. In this study, the LSC region had the highest number of long repeated sequences, and the nucleotide polymorphism in the LSC and SSC regions was higher than that in the IR region, indicating a higher conservation of the IR region in S. discolor Dunn, which is consistent with the findings in other angiosperms. Six highly variable regions with Pi > 0.015 were detected, including trnH-GUG-psbA, rps16, petN, ndhC, ndhF, and ndhA, which can be used as molecular markers for genetic studies of S. discolor Dunn. In the S. discolor Dunn chloroplast genome, 80 SSR loci were detected, most of which were located in the LSC region, with the majority being mononucleotide repeats (54). Of these, 98.1% were A/T types, which may be related to the higher content of A and T bases (60.75%) in its chloroplast genome.
In the phylogenetic analysis, we reconstructed a maximum likelihood (ML) tree based on the complete chloroplast genome sequences of ten Sabia species, with no outgroups included to avoid long-branch attraction. The tree was midpoint-rooted and is presented in Fig. 8. The analysis recovered S. dielsii as the earliest-diverging lineage. The remaining nine species formed a strongly supported clade, which further split into two distinct clades. Clade I comprised five species: S. parviflora, S. limoniacea, S. discolor, S. fasciculata, and S. swinhoei. Clade II consisted of four species: S. japonica, S. schumanniana, S. campanulata subsp. ritchieae, and S. yunnanensis. These results place S. discolor firmly within Clade I, closely allied with S. limoniacea, S. parviflora, S. fasciculata, and S. swinhoei, while S. dielsii stands as a distinct basal lineage. Our results do not support a close relationship between S. discolor and S. japonica, highlighting the value of whole chloroplast genome data for resolving phylogenetic relationships within morphologically similar groups. To further elucidate the evolutionary history of Sabiaceae, future efforts should focus on sequencing chloroplast genomes from additional Sabia species and from other genera within the family. In summary, this study reports for the first time the chloroplast genome sequence of S. discolor Dunn and analyzes its structural features and phylogenetic relationships within the genus. The results indicate that the chloroplast genome of S. discolor Dunn, in terms of size, structure, and IR region boundaries, is relatively conservative. The codon usage bias is weak, primarily influenced by natural selection. Additionally, the high variable regions and repetitive sequences identified can serve as potential molecular markers for the identification of S. discolor Dunn and other species within the genus Sabia. The phylogenetic analysis reveals for the first time the phylogenetic relationships within the genus Sabia and the systematic position of S. discolor Dunn. The data and results presented provide an important reference for further studies on the evolutionary characteristics and phylogenetic relationships of plants in the genus Sabia.
Acknowledgements
We thank the Editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript.
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Authors’ contributions
Q.L. and S.G. jointly designed the study; S.G. and X.-L.L. collected the samples; Q.L. and X.-L.L. performed the experiments; P.Z. and S.G. conducted the data analysis; Q.L. wrote the initial draft of the manuscript; X.-L.L., P.Z., and S.G. revised the manuscript. All authors have read and approved the final version of the manuscript.
Funding
The author(s) declare financial support was received for the research and/or publication of this article. This work was supported by Guangxi Special Project for Science and Technology Bases and Talents (Project No. GuikeAD22080058); The Scientific Research and Technology Development Plan of Guangxi Laibin, China (Laibin Sci - Tech Tackling 240102); Youth Scientific Research and Innovation Team for Smart Fisheries Guangxi Science &Technology Normal University (GXKS2025QNTD17).
Data availability
The dataset generated and analyzed during the current study is available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) , accessed on 4 May 2024) and the accession number is PP733984.
Declarations
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.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
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Associated Data
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Data Availability Statement
The dataset generated and analyzed during the current study is available in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) , accessed on 4 May 2024) and the accession number is PP733984.