Chloroplast genome of plantago major, a medicinal plant in China

Abstract

Plantago major (Plantaginaceae) is a medicinal plant in Chinese folk culture, known for its famous medicinal components such as plantagomain. In this study, we conducted genome sequencing of P. major using Illumina sequencing technology. The assembled complete chloroplast genome had a length of 165,044 bp, comprising a large single-copy regions (82,963 bp), a small single-copy regions (4,633 bp), and a pair of inverted repeat regions (38,724 bp). A total of 140 genes were detected, including 94 CDS, 38 tRNA, and 8 rRNA. Phylogenetic analysis revealed a close genetic relationship between P. major and P. rigida. These findings provide valuable data for a comprehensive understanding of the biological characteristics of P. major.

Keywords:

• Plantago
• Plantago major
• medicinal plant
• phylogenetic analysis
• China

Introduction

Plantago major (Linaeus 1753) is an accepted species in the genus Plantago (family Plantaginaceae) distributed in the temperate regions of the Eurasian continent, with the type specimen collected from Europe. However, it has now become naturalized in most areas of the world (Iwanycki Ahlstrand et al. 2022). Due to the existence of a large number of transitional morphologies, the deep evolutionary relationships among different species still need further clarification, which requires more samples and DNA data to be supplemented (Bagheri et al. 2022; Iwanycki Ahlstrand et al. 2022). Combining the released chloroplast genomes of other plants in this genus, we will be able to more clearly reveal the complex genetic relationships of this genus.

In China, P. major is renowned as a medicinal plant with abundant nutritional components (Lukova et al. 2020; Zhang et al. 2021). Its young shoots and tender stems are edible and can be prepared in various ways, including blanching in boiling water, pickling, stir-frying, and stewing. Moreover, this plant contains bioactive constituents like plantamajoside, demonstrating significant pharmacological activities (Samuelsen 2000; Li et al. 2014; Wu et al. 2016; Liu et al. 2019; Zeng et al. 2022), which have sparked great interest among researchers. The effects of its defatted methanol extract are similar to the commercially-used drug silymarin, indicating its potential as an affordable dietary supplement or nutritional medicinal formulation, offering liver support to patients with hepatic dysfunction (Eldesoky et al. 2018). Additionally, seed extracts can be utilized for meat preservation (Noshad et al. 2021). Currently, the lack of data on the chloroplast genome of this species hinders in-depth research in genetic field. In this study, the whole chloroplast genome of P. major was characterized and assembled for the first time to reveal the genetic characteristics of this species at the molecular level and to deepen our understanding of the evolutionary relationships of P. major within the Plantago genus.

Materials and methods

In this study, we utilized Illumina sequencing technology to assemble and analyze the complete chloroplast genome of P. major plant, which was grown in the organic environments in Mashan county, Nanning, China (22°51′34″N, 108°22′10″E; Figure 1; identified by Yingying Liu with contact information: [email protected]). Fresh leaves were collected and voucher specimen (No. LYY20230515002) was deposited in the herbarium of Guangxi Botanical Garden of Medicinal Plants (abbr. GBGMPP).

Figure 1. The morphological characteristics of P. major in our research. It is a perennial herb with basal leaves. Photoed by Yingying Liu in Guangxi Botanical Garden of Medicinal Plants, Nanning city, China.

Genome sequencing was performed using the Illumina platform (HiseqPE150). The clean data was assembled into the chloroplast genome using NOVOPlasty (Dierckxsens et al. 2017). The assembled sequence was annotated using PGA-master (Qu et al. 2019), with reference to the chloroplast genome annotation of P. media (NC_028520), and manually corrected using Genious 10.2 (Kearse et al. 2012).

Twenty-four chloroplast genomes of the genus Plantago were obtained from the National Center for Biotechnology Information (NCBI), using the chloroplast genomes of Crepidiastrum sonchifolium (NC_046513) and Saxifraga perpusilla (NC_070499) as outgroup for phylogenetic analysis, for these two species are representative taxa of closely related clades. A total of 79 shared CDS were selected and aligned using MAFFT (Katoh and Standley 2013). ModelFinder v2.2.0 (Kalyaanamoorthy et al. 2017) was used to select the best-fit model using bayesian information criterion (BIC TVM + F + I + I+R2) criterion. A maximum likelihood (ML) tree was constructed using IQtree (Nguyen et al. 2015) with 10,000 ultrafast bootstraps.

Results

As evidence of correct genome assembly, the reads obtained from sequencing exhibit a high coverage depth (Figure S1). The results of the study revealed that the chloroplast genome of P. major (GenBank accession OR187835) is organized into four regions, with a total length of 165,044 bp (Figure 2). Number of cis-splicing genes and trans-splicing genes are shown in Supplemental Figure S2. The chloroplast genome consists of a large single-copy regions (LSC; 82,963 bp), a small single-copy regions (SSC; 4,633 bp), and a pair of inverted repeats (IRs, 38,724 bp). A total of 140 genes were detected, including 94 coding sequence (CDS), 38 transfer RNA (tRNA), and 8 ribosomal RNA (rRNA). The overall GC content of this chloroplast genome was found to be 38.1%. The phylogenetic analysis revealed that P. major was taxonomically classified within the genus Plantago. Furthermore, it was observed that P. major formed a monophyletic clade together with P. rigida, while P. asiatica, P. fengouensis, P. depressa, P. rugelii, P. australis were found to be their sister taxon (Figure 3).

Figure 2. Circular map of the complete chloroplast genome of P. major generated by CPGview. The map contains six tracks in default. From the center outward, the first track shows the dispersed repeats. The dispersed repeats consist of direct (D) and palindromic (P) repeats, connected with red and green arcs. The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors. The colors, the type of repeat they represent, and the description of the repeat types are as follows. Black: c (complex repeat); green: p1 (repeat unit size = 1); yellow: p2 (repeat unit size = 2); purple: p3 (repeat unit size = 3); blue: p4 (repeat unit size = 4); orange: p5 (repeat unit size = 5); red: p6 (repeat unit size = 6). The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. The genes are shown on the sixth track. The optional codon usage bias is displayed in the parenthesis after the gene name. Genes are color-coded by their functional classification. The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively. The functional classification of the genes is shown in the bottom left corner.

Figure 3. Maximum-likelihood phylogenetic tree of 25 plantago species was constructed based on 76 CDS sequences from chloroplast genomes of P. major and its closely related specie, with Crepidiastrum sonchifolium and Saxifraga perpusilla as outgroups. The number on each branch indicates the boot support value. The following sequences were used: P. asiatica NC 060521 (Si et al. 2022), P. fengdouensis NC 047190 (Wang et al. 2020), P. depressa NC 041161 (Kwon et al. 2019), P. rugelii MW 877573, P. australis MW 877569, P. rigida MW 877572, P. elongata MW 877571, P. tenuiflora MW 877574, P. coronopus MW 877566, P. macrorhiza MW 877568, P. crassifolia MW 877567, P. alpina MW 877565, P. atrata MW77580, P. amplexicaulis MW 877576, P. indica MW 877577, P. afra MW 877575, P. brasiliensis MW 877579, P. nubicola MW 877564, P. sericea MW 877584 (Mower et al. 2021), P. media NC 028520, P. maritima NC 028519 (Zhu et al. 2016), P. lagopus NC 041420, P. ovata NC 041421 (Sun et al. 2019), P. lanceolata NC 068049 (Zhao et al. 2023), C. sonchifolium NC 046513 (Cho et al. 2020), and S. perpusilla NC 070499 (Yuan et al. 2023).

Discussion and conclusion

In this study, we first utilized Illumina sequencing technology to assemble and analyze the complete chloroplast genome of P. major and found that P. major formed a monophyletic clade together with P. rigida. It is interesting to note that P. rigida is primarily distributed in Ecuador and exhibits significant morphological differences from P. major, such as greatly reduced plant size and leaves. Whereas, the close proximity of the two species on the phylogenetic tree indicates a close genetic relationship between P. major and P. rigida. Chloroplasts play a crucial role in the biological characteristics and distribution of plants. The unveiling of the chloroplast genome sequence of P. major will provide meaningful information for the phylogeny and plant molecular research of the genus Plantago.

Ethical approval

P. major L. is not a threatened or endangered species in the Red list of the International Union for Conservation of Nature (IUCN). This Chloroplast Genome research on P. major L., including the collection of plant and DNA material (cultivated) and field work, were permitted and granted by GBGMP. No ethical items were involved in the research.

Authors’ contributions

L. Jiang & L. Chen designed the research and gave financial support. D. Hu, W. Zeng, Z. Yan, Y. Liu & L. Gui. collected plant and DNA materials. Y. Liu & L. Gui conducted the research and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data of this study are openly accessible in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession number OR187835. The associated BioProject, SRA, and BioSample numbers are PRJNA987869, SRR25033257, and SAMN35994512, respectively.

Additional information

Funding

This study was supported by the Nanning Science & Technology Programme [No. 20212131] and the Research Project from Guangxi Administration of Traditional Chinese Medicine[(No. GZZC2020003].