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. 2024 Jul 16;9(7):871–875. doi: 10.1080/23802359.2024.2378996

The complete plastome of Amaranthus roxburghianus (Amaranthaceae)

Liqiang Wang 1,✉, Xiaohan Zhang 1, Hongqin Li 1, Shu Wang 1
PMCID: PMC467102  PMID: 39021392

Abstract

Amaranthus roxburghianus H.W. Kung 1935, belonging to the Amaranthaceae family, is recognized for its significant medicinal properties. However, molecular research on this species has been limited. This study represents the inaugural documentation of the sequencing and assembly of the complete plastome of A. roxburghianus. The genome spans a total length of 149,969 base pairs (bp), exhibiting a conventional quadripartite structure. This structure comprises a large single-copy (LSC) region of 83,917 bp, a small single-copy (SSC) region of 18,124 bp, and two inverted repeat (IR) regions, each extending to 23,964 bp. In its entirety, the A. roxburghianus plastome encompasses 128 genes, of which 107 are unique, encompassing 77 individual protein-coding genes, 26 unique tRNA genes, and four unique rRNA genes. Phylogenetic analysis has shown a close resemblance between A. roxburghianus and A. polygonoides, both part of the subgenus Albersia. Although the genus Amaranthus is roughly divided into three subgenera, additional plastid genomic data are required for a more accurate assignment of A. albus and A. blitoides. The sequencing of this plastome is a significant step forward, likely to expedite the development of molecular markers and significantly contribute to genetic assays involving this distinctive species.

Keywords: Amaranthus roxburghianus, Amaranthaceae, plastome, phylogenetic analysis, Next-generation sequencing technology

Introduction

Amaranthus, a genus within the family Amaranthaceae, includes approximately 70 species of herbaceous plants with a global distribution. These plants are generally annual or perennial, featuring monoecious flowers and leaves of diverse shapes. The Amaranthus genus holds significant economic and cultural value, being utilized in food, medicine, and decoration. Additionally, these plants have been extensively researched for their potential in environmental remediation and biofuel production, among other applications (Kongdang et al. 2021). In classical taxonomy, Amaranthus is roughly categorized into three subgenera: subgenus Albersia, subgenus Amaranthus, and subgenus Acnida (Xu et al. 2020a). The plastome plays a crucial role in the phylogenetic study of the Amaranthus genus. To date, the nuclear genome of five Amaranthus species has been resolved, and the plastomes of 16 Amaranthus species have been sequenced.

Amaranthus roxburghianus H.W. Kung 1935 is an annual herb in the Amaranthaceae family. Traditionally, its roots have been used to treat colic pains. Recent research indicates that the root extract of A. roxburghianus, particularly when combined with piperine, may be effective in treating ulcerative colitis in mice. While further studies are necessary to comprehensively understand its medicinal properties, A. roxburghianus demonstrates potential as a natural remedy for certain health conditions (Nirmal et al. 2013).

Recent research has primarily focused on the medicinal value of Amaranthus roxburghianus, especially its pharmacological activities and the isolation of compounds (Nirmal et al. 2013). However, there has been a scarcity of molecular research on this species. Understanding its genetic diversity is crucial for the conservation of Amaranthus and for elucidating its evolutionary history. To contribute additional genetic data and ascertain the phylogenetic position of A. roxburghianus within the Amaranthus genus, we have sequenced and characterized its complete plastome. This effort aims to support further evolutionary research within the genus.

Materials

The fresh leaves of A. roxburghianus, depicted in Figure 1, utilized in this study were collected from Heze University, located in Heze City, Shandong Province, China (115° 27′ 32.85" E, 35° 16′ 23.65" N). Voucher samples of this species are preserved in the specimen room of Heze University (voucher number: HZ220808; contact: Hongqin Li; email: 463056627@qq.com). The species was identified by Hongqin Li.

Figure 1.

Figure 1.

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Panorama (A) and detail (B) photos of Amaranthus roxburghianus.

Photo by Liqiang Wang, plant coordinates 35°16′23.65'' N, 115°27′32.85'' E. Main identifying features: stem erect, light green, 30-65 cm, much branched, glabrous. Petiole 1-2.5 cm, slender; leaf blade ovate-rhombic, obovate, or oblong, 2-5 × 1-2.5 cm, base cuneate, margin undulate, apex notched, mucronate. Flowers few, sparsely clustered in axils. Bracts and bracteoles subulate, ca. 2 mm, abaxially with distinct midvein, apex long pointed. Tepals lanceolate, ca. 2.5 mm, apex acuminate, long pointed. Stamens shorter than perianth; stigmas 3. Utricles ovoid, subequal to perianth, ca. 3 mm, circumscissile. Seeds brownish black, subglobose, ca. 1 mm in diameter. The scale of the scale bar is 10 cm.

Methods

We extracted total DNA from the fresh leaves of A. roxburghianus using a Plant Genomic DNA Kit (Tiangen, China) and sequenced it on the HiSeq2500 platform at Wuhan Benagen Technology Company Limited, Wuhan, China. We filtered the raw reads by eliminating adapters and low-quality bases by Trimmomatic (v0.35) (Bolger et al. 2014). After this filtering process, approximately 13.8 GB of clean reads were assembled using GetOrganelle (v1.7.1) (Jin et al. 2020). After assembly, we verified the accuracy of the assembled plastome using minimap2 (Li 2018) in conjunction with samtools (Li et al. 2009). Subsequently, we annotated the plastome by CPGAVAS2 (Shi et al. 2019).

To establish the phylogenetic relationship of A. roxburghianus, we utilized PhyloSuite (Zhang et al. 2020) to download the plastomes of 32 other Amaranthus species from GenBank. Additionally, plastomes of Celosia cristata (MK470118) and C. argentea (MZ636551) were downloaded to serve as outgroups. We aligned the entire plastome sequences using MAFFT software (https://mafft.cbrc.jp/alignment/software/) with default parameters (Katoh and Standley 2013). Subsequently, a maximum-likelihood (ML) phylogenetic tree was constructed using IQ-TREE (v2.0) (Nguyen et al. 2015), employing the Best-fit model of TVM + F + I + I+R4 and incorporating 1000 bootstrap replicates.

Results

The plastome of A. roxburghianus, as deciphered in this study, is a circular DNA molecule with a total length of 149,969 base pairs (bp). The reliability of the genome assembly was strongly supported by the results of the mapping experiment, with a mean sequencing depth of 1821.4× (Figure S1). The genome exhibits a conventional quadripartite structure, comprising a large single-copy (LSC) region of 83,917 bp, a small single-copy (SSC) region of 18,124 bp, and a pair of inverted repeats (IR) regions, each of 23,964 bp. The overall GC content is 36.52%, which is lower than that of the IR regions (42.68%) but higher than that of the LSC (34.37%) and SSC regions (30.14%). The plastome contains 128 genes, of which 107 are unique, including 77 distinct proteins, 26 distinct tRNAs, and 4 distinct rRNA genes (Figure 2). Seven unique protein-coding genes (atpF, ndhA, ndhB (×2), petB, petD, rpoC1, and rps16) contain one intron, while three unique genes (clpP, rps12 (×2), and ycf3) contain two introns. The cis-splicing genes are rps16, atpF, rpoC1, ycf3, clpP, petB, petD, ndhB (×2), and ndhA, and the trans-splicing gene is rps12 (×2). The structures of the cis- and trans-splicing genes are illustrated in Figure S2. The combined length of the rRNA and tRNA genes constitutes 6.03% and 1.83% of the entire plastome, respectively. Seven tRNA genes (trnK-UUU, trnS-CGA, trnL-UAA, trnE-UUC (×2), and trnA-UGC (×2)) contain one intron.

Figure 2.

Figure 2.

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The schematic map of the Amaranthus roxburghianus plastome. The species name is shown at the top left of the map. The map displays six tracks representing various genetic elements. Track one illustrates dispersed repeats, with direct and palindromic repeats connected by red and green arcs. Long tandem repeats in track two are shown as short blue bars. Track three depicts short tandem repeats/microsatellite sequences, differentiated by colored bars: black for complex repeats and green, yellow, purple, blue, orange, and red for repeat units of sizes 1 to 6, respectively. The fourth track indicates single-copy and inverted repeat regions, while the fifth track presents the genome’s GC content. The sixth track details genes, color-coded by functional classification and potentially including codon usage bias, with gene transcription direction indicated (inner genes clockwise, outer genes anticlockwise). The functional classification of genes is provided in the bottom left corner.

In the phylogenetic analysis, the maximum-likelihood (ML) phylogenetic tree comprises 33 nodes (excluding the root), among which 27 nodes have bootstrap values of not less than 97. This phylogenetic tree provides a dependable topological structure that indicates the evolutionary relationships among different Amaranthus species. According to the tree, A. roxburghianus and A. polygonoides form a monophyletic group with a bootstrap value of 100 (Figure 3), indicating their placement within the Subgenus Albersia.

Figure 3.

Figure 3.

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The maximum likelihood phylogeny of Amaranthus roxburghianus and its close relatives using whole plastome sequences. The bootstrap values based on 1000 replicates were shown on each node in the cladogram tree (a). The corresponding phylogram tree is shown in panel B without outgroups. The tree was constructed with 35 species, they were A. albus (MT526785) (Xu et al. 2022), A. arenicola (MN091969) (Xu et al. 2022), A. arenicola (MZ152791) (Xu et al. 2022), A. blitoides (MT526786) (Xu et al. 2022), A. blitum (MT526777) (Xu et al. 2021), A. blitum (MW255966), A. capensis (MT526779) (Xu et al. 2021), A. caudatus (MG836508), A. cruentus (MG836506), A. cruentus (MG836507), A. deflexus (MT526776) (Xu et al. 2021), A. dubius (MN091971) (Xu et al. 2022), A. dubius (MZ397802) (Xu et al. 2021), A. hybridus (MT993471) (Bai et al. 2021), A. hypochondriacus (KX279888), A. hypochondriacus (MG836505) (Xu et al. 2022), A. palmeri (MT312257) (Xu et al. 2022), A. polygonoides (MT472619) (Xu et al. 2022), A. retroflexus (MN091972) (Xu et al. 2022), A. retroflexus (MW646089) (Lou and Fan 2021), A. roxburghianus (OQ354384, this study), A. spinosus (MT526783) (Xu et al. 2022), A. spinosus (MT526784) (Xu et al. 2022), A. spinosus (OP718299) (Xu et al. 2022), A. crispus (MT526778) (Xu et al. 2021), A. standleyanus (MT526780) (Xu et al. 2021), A. standleyanus (MT526781) (Xu et al. 2021), A. standleyanus (MT526782) (Xu et al. 2021), A. tricolor (KX094399) (Xu et al. 2022), A. tricolor (OM419215) (Xu et al. 2022), A. tuberculatus (MN091967) (Xu et al. 2022), A. tuberculatus (MN091968) (Xu et al. 2022), A. viridis (MW679034) (Ding et al. 2021), Celosia cristata (MK470118, outgroup), and C. argentea (MZ636551, outgroup). Bootstrap supports were calculated from 1000 replicates. The A. roxburghianus was labeled in bold font in the phylogenetic tree. The blue, red and green box represents subgenus Albersia, subgenus Acnida and subgenus Amaranthus in the Amaranthus.

Conclusion and discussion

This study presents the primary characterization of the plastome of A. roxburghianus for the first time, which exhibits a typical annular tetrad structure with a size of 149,969 base pairs (bp) and contains 128 predicted genes. Compared with other reported plastomes of the Amaranthus genus (Lou and Fan 2021; Chaney et al. 2016; Xu et al. 2022; Xu et al. 2020b; Ding et al. 2021; Xu et al. 2021; Bai et al. 2021), the A. roxburghianus plastome shows a high degree of similarity in terms of plastome length and gene content.

The classification of the Amaranthus genus presents considerable challenges due to interspecific hybridization and gene introgression, leading to numerous intricate taxa that are challenging to delineate (Xu et al. 2022). Various authors have explored the taxonomy and evolution of this genus. In 2015, Bayón categorized monoecious species into three subgenera (Bayón 2015), while in 1955, Sauer (1955) classified dioecious species into three subgenera, drawing on both morphological and molecular biology data. Despite these efforts, some controversies still persist within the classification (Xu et al. 2022).

In this study, we reconstructed a phylogenetic tree based on an expanded set of plastome sequences. Our findings revealed that not only did the subgenus Albersia and the subgenus Acnida form monophyletic taxa, but the subgenus Amaranthus also exhibited a similar pattern. In Xu et al.'s report (2022), while the subgenus Amaranthus and subgenus Acnida were described as forming monophyletic taxa, the subgenus Albersia was not considered a monophyletic taxon. Contrary to Xu et al.'s reports (2022), our analysis suggests that A. albus and A. blitoides, which belong to the Galápagos Clade, do not fit within the subgenus Albersia. Instead, they should be tentatively classified under the subgenus Amaranthus. A significant finding of our study is that the genus Amaranthus can tentatively be divided into three subgenera. However, more plastid genomic data is required to determine the classification of A. albus and A. blitoides. Ultimately, these results lay a foundation for taxonomic revision, the understanding of phylogenetic evolution, the study of weed biology, and the development of genetic resources within the Amaranthus species.

Supplementary Material

Supplemental Material
TMDN_A_2378996_SM1105.docx (4MB, docx)

Funding Statement

This work was supported by the Doctoral Fund Project of Heze University [XY20BS09] and the Shandong Provincial Natural Science Foundation [ZR2021MC136]. The funders were not involved in the study design, data collection and analysis, publication decision, or manuscript preparation.

Author contributions

The manuscript reflects the contributions of all authors. Liqiang Wang conceptualized and designed the study. Xiaohan Zhang collected the sample and extracted the total DNA of the species. Hongqin Li was responsible for species identification. Liqiang Wang carried out the assembly and annotation of the plastome. Hongqin Li and Shu Wang analyzed the structure of the plastome and drafted the manuscript. All authors have approved the version to be published and agree to be accountable for all aspects of the work.

Ethics statement

The A. roxburghianus specimen is not designated as an endangered species. It requires no specific permissions or licenses. In this study, the collection of A. roxburghianus leaves was conducted following the guidelines provided by Heze University.

Disclosure statement

The authors declare no competing interests in the preparation or execution of this study. Dr. Liqiang Wang is the author of this manuscript and also serves as the Associate-Editor of Mitochondrional DNA Part B journal. Dr. Liqiang Wang declares no conflicts of interest regarding the research findings in this study.

Data availability statement

The plastome sequence has been deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with the accession number of OQ354384 (https://www.ncbi.nlm.nih.gov/nuccore/OQ354384). The associated BioProject, Bio-Sample and SRA numbers are PRJNA928567, SAMN36852648 and SRR23251385. (https://www.ncbi.nlm.nih.gov/sra/?term=SRR12620715).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
TMDN_A_2378996_SM1105.docx (4MB, docx)

Data Availability Statement

The plastome sequence has been deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with the accession number of OQ354384 (https://www.ncbi.nlm.nih.gov/nuccore/OQ354384). The associated BioProject, Bio-Sample and SRA numbers are PRJNA928567, SAMN36852648 and SRR23251385. (https://www.ncbi.nlm.nih.gov/sra/?term=SRR12620715).

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