The complete chloroplast genome of Ligusticopsis acaulis (Shan et Sheh) Pimenov (Apiaceae), an endemic species from China

Abstract

Ligusticopsis acaulis, belonging to the family Apiaceae (Umbelliferae), is endemic to China. The complete chloroplast genome sequence of L. acaulis was assembled and annotated for the first time in this study. The results showed that the plastome was 148,509 bp in length and consisted of a pair of inverted repeat regions (IRs: 19,468 bp), a large single-copy region (LSC: 91,902 bp), and a small single-copy region (SSC: 17,671 bp). A total of 114 unique genes were annotated, including 80 protein-coding, 30 tRNA, and four rRNA genes. According to the phylogenetic analysis, L. acaulis belongs to the tribe Selineae, with a close relationship to Ligusticum hispidum (Franch.) Wolff.

Keywords:

• Ligusticopsis acaulis
• complete chloroplast genome
• phylogenetic analyses

Introduction

Ligusticopsis was originally established by Leute in 1969, with the genus containing 14 species from China (Leute 1969). Ligusticopsis acaulis, a species endemic to China, has diverse chemical composition and is used as a substitute for Peucedani Radix in the Yunnan Province (Rao et al. 1995). The species was first described by Shan et al. (1986) and was named Peucedanum acaule Shan et Sheh. Pimenov (2017) classified Ligusticopsis acaulis as a synonym of P. acaule. The genus Peucedanum L. (Apiaceae) contains approximately 120 species widely distributed in Eurasia and Africa (Pimenov and Leonov 1993). There are about 40 species of Peucedanum in China, with 33 species being endemic (Sheh and Watson 2005). However, due to the complex species composition and ambiguous relationship with related taxa, the taxonomy of Peucedanum has long been contentious.

In recent years, the chloroplast (cp) genome was applied to the phylogenetic reconstruction of Apiaceae, whereby a few contentious relationships were successfully resolved. Here, the complete cp genome sequence of L. acaulis was characterized, and its phylogenetic relationships with related taxa were established.

Materials and methods

Sample collection, DNA extraction, and sequencing

The specimen was collected from the Sea Grass Mountain in Huize, Yunnan Province of China (26°23′99″N, 103°24′01″E, and altitude 3570 m), and the voucher was deposited in the School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University (appraiser: Jing Zhou; contact person: Jing Zhou, [email protected]) under voucher number LZ0901 (Figure 1). The modified CTAB method was used to extract total genomic DNA from leaf tissue (Doyle and Doyle 1987).

Figure 1. Morphological characteristics of L. acaulis. Herbs are perennial, 5–10 cm tall, long-cylindric root, 20–24 cm long. The specimen image was taken by the author Junmei Niu in School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University in September 2022, without any copyright issues.

Chloroplast genome assembly and annotation

The genome was sequenced using the Illumina HiSeq 2500 platform (Majorbio, Shanghai, China) with the paired-end (2 × 300 bp) library. The raw reads were filtered using Trimmomatic with default parameters set to remove the adapter and low-quality sequences (Bolger et al. 2014). Clean data were then assembled into circular contigs using GetOrganelle v.1.7.5.3 (Jin et al. 2020). Finally, using Peucedanum praeruptorum Dunn. (GenBank accession number: MN016968) as a reference, the plastome was annotated by PGA (Qu et al. 2019) and manually modified in Geneious (Kearse et al. 2012). The circular genome map was drawn by CPGView (www.1kmpg.cn/cpgview/).

Phylogenetic analyses

A total of 31 published plastome sequences of Apiaceae from NCBI were acquired to investigate the phylogenetic position of L. acaulis. All sequences were aligned using MAFFT (Katoh and Standley 2013) with Bupleurum boissieuanum H. Wolff as the outgroup. The molecular phylogenetic tree was constructed using the maximum-likelihood (ML) analysis in RAxML (Stamatakis 2014) with 1000 rapid bootstrap replicates.

Results and discussion

Chloroplast genome features of L. acaulis

The total length of the cp genome of L. acaulis was 148,509 bp and consisted of a pair of inverted repeat regions (IRs: 19,468 bp), a large single-copy region (LSC: 91,902 bp) and a small single-copy region (SSC: 17,671 bp). The overall GC content of the plastome was 37.4%, with 35.9%, 30.8%, and 43.7% in LSC, IRs, and SSC, respectively. The plastome contained 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Of the 114 unique genes, 18 genes (including 12 protein-coding genes and six tRNA genes) had introns, with 16 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) having one intron each and two genes (ycf3 and clpP) having two introns each (Figure 2, Table 1).

Figure 2. Genomic map of the L. acaulis chloroplast genome generated by CPGView. Genes outside the circle are transcribed in a counterclockwise direction and those inside in a clockwise direction. LSC: large single-copy; SSC: small single-copy; IR: inverted repeat. The inner circle’s dashed region represents the GC content of the chloroplast genome of L. acaulis. Genes belonging to different functional groups are represented using different colors.

Table 1. List of genes found in the Ligusticopsis acaulis chloroplast genome.

Category for genes	Group of genes	Name of genes
Photosynthesis	Rubisco	rbcL
	Subunit of photosystem I	psaA, psaB, psaC, psaI, psaJ
	Subunit of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunit of ATP synthase	atpA, atpB, atpE, atpF^a, atpH, atpI
	Subunit of cytochrome b/f complex	petA, petB^a, petD^a, petG, petL, petN
	Subunit of NADPH dehydrogenase	ndhA^a, ndhB^a(×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Self-replication	Large subunit of ribosome	rpl2^a, rpl14, rpl16^a, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36
	DNA dependent RNA polymerase	rpoA, rpoB, rpoC1^a, rpoC2
	Small subunit of ribosome	rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12^a(×2), rps14, rps15, rps16^a, rps18, rps19
	tRNA genes	trnA-UGC^a(×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC^a, trnH-GUG, trnI-GAU^a(×2), trnI-CAU(×2), trnK-UUU^a, trnL-CAA(×2), trnL-UAA^a, trnL-UAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV^a-UAC, trnW-CCA, trnY-GUA
	rRNA genes	rrn5(×2), rrn4.5(×2), rrn16(×2), rrn23(×2)
Other genes	Subunit of acetyl-CoA-carboxylase	accD
	Maturase	matK
	Translational initiation factor	infA
	Protease	clpPb
	c-type cytochrome synthesis gene	ccsA
	Envelope membrane protein	cemA
Unknown function	Conserved open reading frames	ycf1, ycf2, ycf3^b, ycf4, ycf15(×2)

^a Genes with one intron.

^b Genes with two introns.

Phylogenetic analysis

To confirm the phylogenetic position of L. acaulis, an ML phylogenetic tree was constructed using 31 cp genome sequences from NCBI. Using MAFFT, sequences were aligned. B. boissieuanum served as the outgroup. Our phylogeny showed that L. acaulis was closely related to Ligusticum hispidum and belonged to the tribe Selineae (Figure 3). Meanwhile, the phylogenetic inference provided an opportunity to delimit the species of the genus Ligusticopsis and understand their evolutionary relationships. The complete cp genome of L. acaulis provided important information for the phylogenetic analysis of the genus Ligusticopsis and laid the foundation for further reconstruction of the Apiaceae phylogeny.

Figure 3. Phylogenetic tree of 31 species in the family Apiaceae based on the complete chloroplast sequences (bootstrap support values are shown above each node). The tree was constructed by ML analysis using RAxML and the GTR + G+I nucleotide model, with 1000 rapid bootstrap replicates. The following sequences were used: G. littoralis NC_034645 (Lee et al. 2019), A. nitida MF594405 (Deng et al. 2017), O. grosseserratum NC_028618 (Choi et al. 2016), S. montanum NC_027451 (Samigullin et al. 2016), S. divaricata MN539269 (Bao et al. 2019), L. hispidum MT409614 (Ren et al. 2020), L. sinense NK9214541 (Wu et al. 2020), C. officinale NC_039760 (Park et al. 2018), L. tenuissimum NC_029394 (www.ncbi.nlm.nih.gov/), C. sativum NC_029850 (www.ncbi.nlm.nih.gov/), A. sinensis MH430891 (Tian et al. 2019), H. candicans NC_045184 (Kang et al. 2019), S. gyirongensis NC_042912 (Xiao et al. 2019), A. graveolens NC_029470 (Peery 2015), F. vulgare NC_029469 (www.ncbi.nlm.nih.gov/), P. crispum NC_015821 (Downie and Jansen 2015), A. graveolens NC_041087 (www.ncbi.nlm.nih.gov/), P. fedtschenkoi KY652265 (Mustafina et al. 2019), C. carvi NC_029889 (www.ncbi.nlm.nih.gov/), C. maritimum NC_015804 (Downie and Jansen 2015), D. carota NC_008325 (Ruhlman et al. 2006), C. cyminum MN901636 (www.ncbi.nlm.nih.gov/), A. cerefolium NC_015113 (Downie and Jansen 2015), T. filiformis subsp. greenmannii HM596071 (Downie and Jansen 2015), C. virosa NC_037711 (www.ncbi.nlm.nih.gov/), H. forbesii NC_035054 (www.ncbi.nlm.nih.gov/), C. violaceum KU921430 (www.ncbi.nlm.nih.gov/), P. camtschaticum NC_033343 (www.ncbi.nlm.nih.gov/), C. delavayi MN119367 (Guo et al. 2020), and B. boissieuanum NC_036017 (Wu et al. 2018).

Author contributions

Zhenwen Liu and Jing Zhou conceived and designed the study. Junmei Niu, Xinyue Wang, Jiarui Yue, and Shilin Zhou analyzed the experimental results and interpreted the data. Junmei Niu drafted the manuscript. Zhenwen Liu and Jing Zhou revised the manuscript. All authors have read and approved the final manuscript.

Ethical approval

Investigations of the authors with human participants or animals are not included in this article. Hence, no ethical approval or permissions were required.

Disclosure statement

The authors declare no conflicts of interest.

Data availability statement

Genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under accession no. ON359911. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA849278, SRR19693381, and SAMN28870233, respectively.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant No. [31960048], the Hundred-Talent Program of Kunming Medical University under Grant No. [60118260127], and the Department of Science and Technology of Yunnan Province under Grant No. [202201AT070118].