Euchresta tubulosa Dunn, commonly referred to as Hu Dou Lian, is a climbing shrub species within the Fabaceae family and the genus Euchresta. This medicinal plant is predominantly found in southern China’s provinces of Guangxi, Guangdong and Guizhou, where it typically grows in humid environments at altitudes between 650 and 900 meters. Lin et al. 2013 reported that the folk anticancer medicine known as “Hu Dou Lian” primarily refers to E. tubulosa and its closely related species (Lin et al., 2013; Wang and Li, 1983). The dried roots of this plant contain a variety of alkaloids and flavonoids, which are widely used to alleviate sore throats, gum pain and oral ulcers. Among the Tujia ethnic group inhabiting the Wuling Mountains (including Hunan, Hubei, Sichuan, Chongqing and Guizhou), E. tubulosa is traditionally used to treat esophageal and throat cancers (Li et al., 2014; Lo et al., 2002; Toda and Shirataki, 2006). There is even a saying among the Tujia people: “Having Hu Dou Lian at home ensures a good year.”
       
Due to its limited reproductive strategies, specific habitat requirements and low pollination efficiency, the wild population of E. tubulosa has declined significantly (Li et al., 2014, Yao et al., 2017, Han et al., 2013). It is now listed as a second-class national key protected wild plant and categorized as an endangered species in the “China Species Red List.” Current studies on E. tubulosa mainly focus on its cultivation, domestication and the extraction of active medicinal compounds. However, despite its medicinal significance, no prior research has addressed the complete chloroplast genome of Euchresta species, including E. tubulosa.
       
Chloroplast genomes play a central role in photosynthesis, amino acid and secondary metabolite synthesis and are maternally inherited, making them a powerful tool for plant taxonomy, population genetics and phylogenetic analysis. Although the chloroplast genomes of many angiosperms have been sequenced, genomic resources for the genus Euchresta remain scarce. Recent studies have demonstrated the value of chloroplast genomes in resolving taxonomic ambiguities in medicinal plants (Cui et al. 2012, Wang et al. 2020). For example, Zhao et al. 2022 sequenced the complete chloroplast genome of Asarum sieboldii and reconstructed phylogenetic relationships within the Aristolochiaceae family. Zhou et al. 2018 established super DNA barcodes in Rheum, while analyzed codon usage patterns in Alpinia species.
       
Within the Fabaceae family, a growing number of chloroplast genome studies have shed light on genome structure, codon usage bias and evolutionary dynamics of key genera. Notably, the complete chloroplast genome of Medicago sativa cv. Qingda no.1 has been sequenced and analyzed, revealing a genome length of 125,637 bp, a relatively high GC content of 38.33% and the absence of a typical quadripartite structure. The study also demonstrated strong A/T-ending codon usage bias and identified 62 SSR loci, highlighting important structural and evolutionary features of alfalfa chloroplast genomes (Ren et al. 2023). Other genera, including Cajanus (Kaila et al. 2016), Glycine (Asaf et al. 2017) and Desmodium (Yen and Park 2022), have also been studied for their plastome characteristics, such as SSR distribution and GC content variation, which offer insights into legume phylogeny and taxonomy. These studies collectively reinforce the importance of plastome data in clarifying taxonomic relationships and evolutionary origins in Fabacea.
       
To address this gap, this study aims to sequence, annotate and analyze the chloroplast genome of E. tubulosa using high-throughput sequencing technology (Tai and Tanksley 1990, Zhang et al. 2012, Yang et al. 2022, Mu et al. 2022). The results will provide insights into its genome structure, codon usage and phylogenetic placement, laying a foundation for future research on species identification, genetic diversity, conservation and molecular systematics of this endangered medicinal plant.

Materials
 
Leaves of Euchresta tubulosa were collected from Fanjingshan in Jiangkou County, Guizhou Province (27.9112^oN, 108.7033^oE) and taxonomically identified by Professor Shuwen Zhao of Inner Mongolia Agricultural University. Healthy leaf samples were collected into sterile bags, rinsed with tap water followed by 3-4 washes with sterile water, air-dried and subsequently preserved at -80/^oC in an ultra-low temperature freezer.
 
DNA extraction and sequencing
 
Leaves were rapidly frozen in liquid nitrogen and finely ground into a powder using a mortar and pestle. Genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method as described by (Doyle 1987), followed by purification using a TIANquick Midi Purification Kit (Tiangen Biotech, Beijing, China). The quality and quantity of DNA were assessed using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1.0% agarose gel electrophoresis.
       
The DNA was fragmented by ultrasonication using a Covaris S220 system (Covaris, Woburn, MA, USA). End repair, A-tailing and adapter ligation were performed following the protocol of (Meyer and Kircher 2010), with minor modifications. The ligated products were purified and enriched via PCR amplification using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) to construct the sequencing library. The final library was assessed for size distribution using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using a Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) before sequencing on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA).
 
Chloroplast genome assembly
 
Following the removal of adapter sequences and low-quality reads, high-quality clean reads were acquired. The chloroplast genome of Euchresta tubulosa was then assembled using NOVOPlasty (Perl script, default settings), with the chloroplast genome of Euchresta japonica serving as the reference seed sequence (Sun et al., 2020).
 
Genome annotation and comparison
 
The Geneious Prime Find Repeats plugin was used to detect the inverted repeat (IR) sequences in the chloroplast genome and to define the boundaries of the large single-copy (LSC) and small single-copy (SSC) regions (Kearse et al. 2012). Genome annotation was performed using the Dual Organellar GenoMe Annotator (DOGMA, [http://dogma.ccbb.utexas.edu/] (http://dogma.ccbb.utexas.edu/)) and manually adjusted. tRNA genes were predicted using tRNAscan-SE ([http://lowelab.ucsc.edu/tRNAscanSE/] (http://lowelab.ucsc.edu/tRNAsc anSE/)) and ARAGORN tools. Finally, the Organellar Genome DRAW ([https://chlorobox. mpimp-golm.mpg.de /OGDraw.html] (https://chlorobox. mpimp-golm.mpg.de/OGDraw.html)) was used to generate the circular map with the “Tidy up annotation” option selected and other parameters set to default.
 
Simple sequence repeat (SSR) analysis
 
SSR loci were analyzed using the Perl script provided by MISA (MIcroSAtellite Identification Tool) (Beier et al. 2017). The script was run in the Windows command prompt, setting the minimum number of repeats for mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides to 10, 5, 4, 4, 4 and 4, respectively. Nucleotide diversity was assessed using DnaSP version 5.0, applying a sliding window approach with a window length of 600 bp and a step interval of 200 bp (Rozas 2009).
 
Codon usage bias analysis
 
The relative synonymous codon usage (RSCU) values for genes within the chloroplast genome of Euchresta tubulosa were analyzed. Codon with an RSCU greater than 1 was considered a high-frequency codon. Highly expressed genes have lower effective number of codons (ENC) values, while lowly expressed genes have higher ENC values. Genes were ranked based on their ENC values and the top and bottom 5% were selected as the high and low expression gene sets, respectively. The difference in RSCU (DRSCU) between the high and low expression gene sets was calculated, with a DRSCU greater than 0.08 considered an optimal codon (Duret and Mouchiroud 1999, Liu et al. 2020).
 
Phylogenetic analysis
 
Complete chloroplast genome sequences from 14 species were retrieved from the NCBI database, including three species of Lespedeza (L. buergeri, L. maritima and L. bicolor), one of Kummerowia (K. striata), three of Campylotropis (C. trigonoclada, C. polyantha and C. wilsonii), one of Christia (C. vespertilionis), one of Urariopsis (U. brevissima), one of Pseudarthria (P. viscida) and two species from the genus Saxifraga (Bergenia crassifolia and Saxifraga stolonifera) as outgroups (Katoh et al. 2002).
       
Sequences were aligned using MAFFT version 7.388. The best substitution model was determined by jModelTest and a maximum likelihood (ML) tree was constructed using PhyML with 1,000 bootstrap replicates.

Chloroplast genome structure, gene content and codon usage bias of E. tubulosa
 
The chloroplast genome of Euchresta tubulosa exhibits a typical quadripartite structure, comprising a large single-copy (LSC) region, a small single-copy (SSC) region and two inverted repeats (IRs) (Fig 1). The total length is 153,960 bp and the overall GC content is 36.3%. Specifically, the LSC spans 84,107 bp (42.63% GC), SSC 18,053 bp (33.77% GC) and each IR 51,800 bp (29.80% GC), consistent with typical angiosperm chloroplast genome features.


       
Genome annotation identified 127 functional genes, including 44 photosynthesis-related genes, 72 self-replication-related genes, 5 genes with other known functions and 6 genes of unknown function. Among the photosynthesis-related genes, ndhB (encoding NADH dehydrogenase subunit) occurs in two copies and contains one intron. The petB (cytochrome b/f complex subunit) and atpF (ATP synthase subunit) genes each contain one intron (Table 1). For self-replication-related genes, duplicated genes include ribosomal proteins (rpl2, rpl23, rps12, rps7), ribosomal RNAs (rrn16S, rrn23S, rrn4.5S, rrn5S) and several tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC). Genes with one intron include rpl16, rpl2, rpoC1, trnA-UGC, trnG-UCC, trnI-GAU, trnL-UAA and trnV-UAC, while clpP contains two introns. Additionally, the pseudogenes ycf1 and ycf2 are both duplicated and ycf3 has two introns.


       
Compared with other legumes, the E. tubulosa chloroplast genome is relatively large and gene-rich. For instance, Medicago sativa cv. Qingda No.1 possesses a smaller genome (125,637 bp) with a distinct structure showing IR region contraction and gene loss such as ndh genes (Ren et al. 2023). This suggests significant evolutionary divergence within Fabaceae. Codon usage analysis of E. tubulosa indicates a strong preference for codons ending in A or U, with 29 out of 31 high-frequency codons terminating in either A or U and only two ending in G, consistent with findings from M. sativa and other angiosperm chloroplast genomes (Jiang et al. 2020). 
       
This AT-rich codon usage bias likely reflects underlying mutational pressure and natural selection, as reported in related Fabaceae species (Zhao et al., 2022). Codon usage bias analysis plays an important role in exploring gene expression efficiency, genome evolution and species adaptation, providing insights into phylogenetic relationships (Chen et al., 2010).
 
Codon usage bias in the chloroplast genome of E. tubulosa
 
To investigate codon usage bias, we analyzed all chloroplast coding sequences (CDSs) longer than 200 bp. The results showed that leucine (Leu) was the most frequently encoded amino acid, with 2,644 codons representing 10.52% of the total codon count (Table 2). Relative synonymous codon usage (RSCU) values greater than 1 were found for 31 codons, indicating that these codons are used more frequently than expected. Among these, only two codons ended in G, while the remaining 29 codons ended in either A or U (Fig 2).




       
This pronounced preference for A/U-ending codons suggests an AT-rich bias in the chloroplast genome of E. tubulosa. Similar A/U-ending codon usage patterns have also been reported in the chloroplast genomes of Medicago sativa, Glycine max and other legume species, where the vast majority of preferred codons terminate in A or U (Jiang et al. 2020). These consistent findings among Fabaceae species indicate that codon usage bias in their chloroplast genomes is likely driven by mutational pressures and nucleotide composition constraints.
       
Such codon bias plays an important role in understanding gene expression regulation, organelle genome evolution and species adaptation. It also aids in optimizing gene expression for transgenic research and synthetic biology applications (Zhou et al., 2018).
 
Identification and distribution of ssrs in the chloroplast genome of e. tubulosa
 
A total of 109 simple sequence repeats (SSRs) were identified in the E. tubulosa chloroplast genome, including 85 mononucleotide repeats, 10 dinucleotide repeats, 1 trinucleotide repeat and 13 multinucleotide repeats (Table 3). No tetranucleotide or other complex SSR types were detected. Most SSRs (n = 80, 73.40%) were distributed in intergenic spacer (IGS) regions. Additionally, 16 SSRs were located in coding regions of genes such as rps18, ycf4, psbC, rpoC2, rpoB, atpB, matK, ndhF and ycf1-2, with ycf1-2 containing four SSR loci. Thirteen SSRs were found in intronic regions, including those of rpl16, petD, petB, clpP, ycf3, atpF, trnV-UAC, rpl2 and ndhA. Regionally, 74 SSRs were located in the LSC region, 21 in the SSC and 14 in the IRs.


       
The predominance of SSRs in IGS regions is consistent with previous findings in other legumes such as Glycine max and Cajanus cajan, where SSRs are frequently localized to non-coding areas (Zhao et al. 2022). The observed pattern-primarily short polyA or polyT mononucleotide repeats-matches earlier studies indicating a strong AT-rich bias in chloroplast SSR motifs (Kuang et al. 2011). These SSRs, due to their high polymorphism, maternal inheritance and genomic abundance, are highly valuable molecular markers for species identification, genetic diversity analysis and phylogenetic reconstruction (Du et al. 2012).
       
Therefore, the SSR loci identified in this study offer potential for future molecular breeding, population genetics and evolutionary biology research in Euchresta and related genera.
 
Phylogenetic analysis of e. tubulosa based on chloroplast genome
 
To clarify the phylogenetic position of Euchresta tubulosa, a phylogenetic tree was constructed using complete chloroplast genome sequences. Species from Lespedeza (3), Kummerowia (1), Campylotropis (3), Christia (1), Urariopsis (1), Pseudarthria (1) and Saxifraga (2) were selected as outgroups. The resulting tree showed robust topology with a high bootstrap support of 100%, indicating strong confidence in the inferred relationships.
       
Within the phylogenetic tree, species of the same genus clustered together, forming two major clades (Clade 1 and Clade 2). Clade 1 was further subdivided into a Lespedeza subclade and a Desmodium subclade. The Lespedeza subclade included Lespedeza buergeri, L. maritima, L. bicolor, Kummerowia striata, Campylotropis trigonoclada, C. polyantha and C. wilsonii. The Desmodium subclade included Urariopsis brevissima, Christia vespertilionis, Uraria lagopodoides, Desmodium heterocarpon and D. styracifolium. Notably, E. tubulosa formed an independent clade (Clade 2), distinct from the Desmodieae tribe, confirming its unique phylogenetic position (Fig 3).


       
The phylogenetic placement of E. tubulosa supports the proposal by Hiroyoshi Ohashi that Euchresta may belong to a distinct monogeneric tribe, although it is closely related to the tribe Sophoreae (Zhang et al., 2012). These results demonstrate that complete chloroplast genomes are highly effective in resolving taxonomic ambiguities among closely related legume genera (Jiang et al., 2020). Moreover, the strong bootstrap values observed in this study further affirm the utility of chloroplast genome data in molecular systematics.
       
Our findings not only clarify the systematic position of Euchresta tubulosa within Fabaceae but also provide foundational data for future research in phylogeny, evolution and taxonomic classification of the genus Euchresta.

In this study, we sequenced and analyzed the complete chloroplast genome of Euchresta tubulosa. The genome exhibits a typical quadripartite structure with a total length of 153,960 bp and 127 annotated genes. Codon usage analysis revealed a strong preference for A/U-ending codons, reflecting an AT-rich bias similar to that observed in other Fabaceae species. A total of 109 SSRs were identified, most of which were located in intergenic spacer regions, providing valuable molecular markers for future genetic studies. Phylogenetic analysis based on the complete chloroplast genome clearly distinguished E. tubulosa from related genera within the Desmodieae tribe, forming an independent clade with strong bootstrap support. This study provides important genomic resources and phylogenetic evidence that enhance our understanding of the evolutionary relationships and systematic position of Euchresta, laying a foundation for its conservation, molecular taxonomy and future breeding research.

This work was supported by the National Natural Science Foundation of China (32160334).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.