The complete plastome of Amaranthus roxburghianus (Amaranthaceae)

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.


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 0 32.85"E, 35 � 16 0 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.

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).
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.
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, Bay� on categorized monoecious species into three subgenera (Bay� on 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 Gal� apagos 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.

Figure 2 .
Figure2.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.