Characterization of the complete plastid genome and 45S nrDNA sequences of Murdannia medica (Lour.) D.Y. Hong (Commelinaceae) and its phylogenetic analysis

Abstract

Murdannia medica is a Vietnamese traditional medicinal herb. Here, we report DNA super‑barcodes comprising the complete plastid genome (plastome, 177,455 bp) and 45S nuclear ribosomal DNA (45S nrDNA, 5,741 bp) of M. medica. The plastome has a large single‑copy (LSC, 100,132 bp), a small single‑copy (SSC, 21,125 bp), and a pair of inverted repeats (IRs) of 28,099 bp each. It contains 115 annotated genes and has an overall A/T content of 68.2%. Plastome‑based phylogenetic analyses show that M. medica sister with M. edulis. These new genomic resources provide a valuable foundation for future cultivation, conservation, and systematic investigations of M. medica.

Introduction

Murdannia medica (Lour.) D.Y. Hong, 1974, locally known in Vietnam as “Trường sâm”, is a perennial herbaceous species of Murdannia Royle (Commelinaceae) (Burns et al. 2011, Sengthong et al. 2025). It grows in moist habitats along forest edges, stream banks, and under forest canopies in China, Laos, Cambodia, Thailand, and Vietnam (https://www.gbif.org/species/2764565). A range of Murdannia species have documented ethnomedicinal uses, including as folk remedies for cancer and as treatments for common colds, diabetes mellitus, and various inflammatory conditions (Jiratchariyakul et al. 2006, Panda and Misra 2011, Patwari et al. 2014). M. medica is currently cultivated and conserved ex situ by the Middle Vietnam Research and Manufacturing Medicinal Herb Center for research and pharmaceutical development.

Recent plastome‑based analyses of 42 Commelinaceae accessions supported the topology (Murdannia, (Commelina, (Pollia, Rhopalephora))) (Jung et al. 2021). However, only M. edulis was included, and the plastome of M. medica has not yet been reported, leaving a critical gap in genomic information for this species. To improve understanding of this taxonomically challenging genus, we sequenced, assembled, and characterized the plastome and 45S nrDNA of M. medica. The high‑quality DNA super‑barcodes generated in this study will help resolve species relationships in Murdannia and enable the development of molecular markers and cultivar strategies that advance the effective conservation and medicinal applications of this native germplasm.

Materials and methods

Plant material

The collected M. medica were propagated in Dak Lak Province, Vietnam (12°57’38ʺN 109°18’57ʺE) (Figure 1A–C). Specimens were deposited at the Pharmacognosy and Traditional Pharmacy Department, School of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam (contact: Prof. Tran Thi Van Anh, ttvananh@ump.edu.vn, voucher: UMP26-01) (Figure 1D).

Figure 1.

Photographs of Murdannia medica (A) a subpopulation of M. medica at cultivation site (12°57’38ʺN 109°18’57ʺE). (B) Detail of the whole plant, showing a low-growing, tufted herb with short, light-green, erect stems and a basal rosette of linear-lanceolate leaves. (C) Detail of the inflorescence showing flowers with three brownish-green outer sepals and three distinct reddish-purple inner petals. (D) Herbarium, voucher: UMP26-01. The photo was taken by the author Duong Nguyen Xuan Lam.

DNA extraction and sequencing

The fresh leaf tissues were used to extract total genomic DNA (gDNA) using a cetyltrimethylammonium bromide protocol following Doyle (Doyle 1991), an extra chloroform–isoamyl alcohol (24:1) purification step and RNase A treatment were incorporated to further remove polysaccharides and RNA. gDNA quality was checked on 1% agarose gels, and concentrations were determined using a Thermo Scientific NanoDrop 2000 spectrophotometer. High‑quality gDNA was sequenced as 2 × 150 bp paired‑end reads on an Illumina NovaSeq6000 platform (San Diego, CA, USA) by Macrogen (Seoul, Korea).

Genome assembly and annotation

Raw Illumina reads underwent multiple bioinformatics workflows and data analysis procedures (Table S1, Figure S1). Trimmed reads were remapped to the plastome and 45S nrDNA assemblies using Minimap2, and the resulting alignments were processed with Samtools and inspected in IGV to validate assembly accuracy. The annotated plastome and 45S nrDNA sequences of M. medica have been submitted to GenBank with accession numbers PZ347914.1 and PZ344954.1, respectively.

Phylogenetic analysis

The phylogenetic position of M. medica within Commelinaceae was estimated using plastome sequences from 13 species and Hanguana malayana (Hanguanaceae) as the outgroup (Table S2). The 15 plastomes, including the newly assembled M. medica sequence, were aligned using MAFFT v7 with the L‑INS‑i algorithm (Katoh and Standley 2013), followed by manual inspection in BioEdit (Hall 1999). The final alignment was converted to PHYLIP format and analyzed by maximum likelihood (ML) in RAxML‑NG (Kozlov et al. 2019) under the GTR+G model with 1,000 bootstrap replicates (Table S1). The resulting ML tree was visualized using FigTree v1.4.4 (https://github.com/rambaut/figtree).

Polymorphic sites analysis

The same alignment used for the phylogenetic analysis was employed. Two comparisons were conducted: between the two plastomes of Murdannia and among 14 plastomes of Commelinaceae. Variable sites (defined as one difference per position) were counted in non‑overlapping 1,000 bp windows. For each comparison, the window with the highest count was used to rescale all window counts to percentages, which were then plotted as bar charts in Excel to illustrate the distribution of polymorphic sites across the alignment.

Results

Plastomes and 45S nrDNA structure analysis

The complete plastome sequence of M. medica was assembled as a 177,455 bp circular molecule with the typical quadripartite organization, comprising a LSC region of 100,132 bp, a SSC region of 21,125 bp, and two IR regions of 28,099 bp each (Figure 2A). The mean read‑mapping depth was 227.58× (Figure S2), and the overall A/T content is 68.2%. A total of 115 genes were annotated, including 80 protein‑coding, 31 tRNA, and 4 rRNA genes (Table S3). The plastome contains 13 cis‑splicing genes (Figure S3) and one trans‑splicing gene, rps12 (Figure S4). Two genes, accD and rpoA, contain multiple internal stop codons, possibly caused by insertions and deletions, and were therefore annotated as pseudogenes (Table S3). The 45S nrDNA unit (Figure 2B) is 5,741 bp in length and comprises the 18S, 5.8S, and 28S rRNA genes (1,806, 165, and 3406 bp, respectively), flanked by ITS1 (148 bp) and ITS2 (216 bp) (Figure S5). Figure S6 summarizes the sequencing depth and coverage across the 45S nrDNA unit, with an average depth of approximately 896.12×.

Figure 2.

DNA super-barcoding of M. medica. (A) Platome of M. medica as a circular diagram. Genes are color-coded according to functional category. Loci on the outer ring are transcribed clockwise, whereas those on the inner ring are transcribed in the opposite direction. The innermost gray bands indicate nucleotide composition, with darker regions marking GC-rich and lighter regions marking AT-rich segments. (B) The 45S rDNA repeat unit consists of the 18S, 5.8S, and 28S rRNA genes, which are flanked by the internal transcribed spacers ITS1 and ITS2.

Phylogenetic analysis

The ML phylogenetic tree was generated to clarify relationships within Commelinaceae (Figure 3). It provides strong support for the monophyly of the Murdannia genus (BS = 100), and within this clade, M. medica is recovered as the sister species to M. edulis (BS = 100).

Figure 3.

ML tree based on the plastome sequences of M. medica and 14 other species. Numbers at nodes indicate bootstrap support values, and the plastome of M. medica is highlighted in red. A scale bar at the bottom indicates a genetic distance of 0.03. GenBank accession numbers and references are as follows: Murdannia medica (PZ347914.1, this study); from study of Jung (Jung et al. 2021): Murdannia edulis (MW617988.1), Commelina communis (MW617984.1), Rhopalephora scaberrima (MW617991.1), Pollia japonica (MW145132.1), Palisota barteri (MW617989.1), Siderasis fuscata (MW617992.1), Dichorisandra thyrsiflora (MW617985.1), Geogenanthus poeppigii (MW617986.1), Cochliostema odoratissimum (MW617983.1), Gibasis geniculata (MW617987.1), Tradescantia virginiana (MW617994.1), Callisia repens (MW617982.1), and Weldenia candida (MW617995.1); and Hanguana malayana (KT312930.1, (Barrett et al. 2016)).

Polymorphic sites analysis

After excluding the IRa region, the alignment was divided into 184 non‑overlapping 1,000 bp segments (Table S4). Among the 14 Commelinaceae plastomes, 37 segments exhibited more than 800 variable sites (Table S4, Figure S7). Of these, six segments also showed more than 500 variable sites (Figure S7) in the pairwise comparison between the plastomes of M. medica and M. edulis, corresponding to the intergenic regions atpH–atpI, trnT–psbD, rpl33–rps18, and rps15–ycf1, and the introns of clpP and rpl16. Segments 86 and 87, which encompass the accD pseudogene, showed low diversity within Murdannia but high divergence among Commelinaceae (Figure S7). Overall, segments in the IR regions displayed lower polymorphism than those in the LSC and SSC regions, consistent with the higher structural conservation of the IRs (Table S4, Figure S7).

Discussion and conclusion

Plastome-based identification in plant systematics has traditionally relied on a few loci, such as matK, rbcL, and trnH‑psbA, which often lack the power to resolve closely related taxa (Chase et al. 2005). Next-generation sequencing enables the de novo assembly of plastomes from low-coverage whole-genome data, offering a cost-effective method to acquire complete plastome sequences from approximately 1–2 Gbp of sequencing output, while concurrently retrieving 45S nrDNA sequences (Kim et al. 2015). The “DNA super‑barcoding” approach, which integrates complete plastome and 45S nrDNA sequences, provides greatly enhanced resolution for distinguishing interspecific divergence and thus extends earlier DNA barcoding strategies (Lee et al. 2026). In this context, the DNA super‑barcodes data can improve the ability to authenticate M. medica and distinguish it from closely related or morphologically similar taxa, which is crucial for preventing misidentification, adulteration in trade and for supporting conservation.

This work provides the first plastome‑level assessment of sequence variation in Murdannia species. The highly polymorphic sites in non‑coding regions may account for the plastome size divergence between M. medica and M. edulis, as well as between Murdannia and other Commelinaceae plastomes (Table S2). The accD pseudogene may serve as a marker for distinguishing the genus Murdannia from close relatives. Nevertheless, broader taxon sampling within Murdannia and Commelinaceae is essential to better characterize plastome diversity and the evolutionary history of this plant family.

Funding Statement

This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh city, Vietnam under contract number 224/2025/HĐ-ĐHYD dated 28/04/2025.

Ethical approval

This study does not involve any endangered or protected species. We confirm that all relevant permissions, licenses, and ethical approvals required for the fieldwork, sample collection, and subsequent analyses in this study were obtained from the appropriate authorities prior to commencement of the research (https://doi.org/10.6084/m9.figshare.32326047).

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The genome sequence data supporting the findings of this study are openly available in GenBank (NCBI) under accession numbers PZ347914.1 (plastome) and PZ344954.1 (45S nrDNA). The corresponding BioProject, BioSample, and SRA identifiers are PRJNA1457851, SAMN57477494, and SRR38282924, respectively.