Complete chloroplast genome of Eleutherococcus divaricatus var. chiisanensis (Araliaceae), an endemic Korean species

Article information

Korean J. Pl. Taxon. 2024;54(4):323-329
Publication date (electronic) : 2024 December 31
doi : https://doi.org/10.11110/kjpt.2024.54.4.323
Division of Forest Biodiversity, Korea National Arboretum, Pocheon 11186, Korea
1Seedvault Center, Korea Arboreta and Gardens Institute, Bonghwa 36209, Korea
2Baekdudaegan National Arboretum, Korea Arboreta and Gardens Institute, Bonghwa 36209, Korea
Corresponding author: Dong Chan SON, E-mail: sdclym@korea.kr
Received 2024 September 6; Revised 2024 November 1; Accepted 2024 November 20.

Abstract

Eleutherococcus Maxim. belongs to the family Araliaceae, with approximately 40 species distributed in Eastern Asia and the Himalayan regions. Among these, Eleutherococcus divaricatus var. chiisanensis is distributed only in the central Korean Peninsula. The complete chloroplast (cp) genome sequence of Eleutherococcus divaricatus var. chiisanensis (Araliaceae) was determined to be 156,765 bp in length, consisting of large (86,592 bp) and small (18,239 bp) single-copy regions and a pair of identical inverted repeats (25,967 bp). The overall GC content of the chloroplast genome was 37.9%, and in the large singlecopy sequence, the small single-copy sequence, and each of inverted repeat sequences, the contents were 36.2%, 32%, and 43%, respectively. The genome consists of 132 genes, made up of 87 protein-coding genes, 37 tRNA genes, and eight rRNA genes. A cp genome phylogenetic analysis of the 12 taxa inferred from the cp genome revealed a close relationship between Eleutherococcus sessiliflorus and E. divaricatus var. chiisanensis. The complete cp genome sequence of E. divaricatus var. chiisanensis provides important information for future phylogenetic and evolutionary studies of the genus Eleutherococcus.

INTRODUCTION

The genus Eleutherococcus Maxim. (Araliaceae) comprises approximately 40 species in Eastern Asia and the Himalayan region distribution (Xiang and Lowry, 2007; Kim, 2017a; 2018). Among these, Eleutherococcus divaricatus var. chiisanensis (Nakai) C. H. Kim & B.-Y. Sun is distributed only in Korea (Kim, 2017a, 2018). In Korea, this species is distributed in Hamgyeongbuk-do, Hamgyeongnam-do, Gangwon-do, Jeollabuk-do, Jeollanam-do, Gyeongsangbuk-do, and Jeju-do (Chung et al., 2017, 2023; Kim, 2017a, 2018). Meanwhile, E. divaricatus var. chiisanensis is distinguished from related taxa by its plant erect, bisexual flower, prickle absent on base of petiole, pubescent and short prickle on abaxial surfaces of leaves, raceme of umbel, pedicel ca. 6 mm, 2-carpeled, and style terete (Kim, 2018).

The chloroplast (cp) is an intracellular organelle that plays an essential role in photosynthesis and other primary and secondary metabolic processes (Shinozaki et al., 1986; Brunkard et al., 2015). The cp genome is a suitable tool for studying plant evolution and phylogeny because of its highly conserved sequence and structure (Maier et al., 1995). The cps have their own genetic replication mechanisms and transcribe their genomes relatively independently (Fu et al., 2016). Most cp genomes range from 120 to 200 kb in length and exhibit a typical quadripartite structure, including a large single-copy sequence (LSC, 80–90 kb), a small single-copy sequence (SSC, 16–27 kb), and two inverted repeat sequences (IRs, 20–28 kb) of subequal length (Palmer, 1985; Wang et al., 2008). The completion of the cp DNA genome provides a large amount of information, including not only related information on protein-coding and non-coding genes but also data to infer gene rearrangement and evolutionary relationships (Golenberg et al., 1993; Reith and Munholland, 1995). Recently, whole cp genomes have been widely used in phylogenetic studies to plants groups with unresolved evolutionary relationships (Gitzendanner et al., 2018).

In the present study, we report the complete cp genome of E. divaricatus var. chiisanensis and investigate the phylogenetic relationships of E. divaricatus var. chiisanensis and related species, providing valuable resources for future studies on this species.

MATERIALS AND METHODS

Eleutherococcus divaricatus var. chiisanensis was sampled from Mt. Jirisan, Hwangjeon-ri, Masan-myeon, Gurye-gun, Jeollanamdo, Korea (35.293125°N, 127.525817°E). The collected material was stored in the Herbarium of the Korea National Arboretum (KH) (http://www.nature.go.kr; Dong Chan Son; e-mail: sdclym@korea.kr; voucher number: Jirisan-230831-002).

Fresh leaves were then silica-dried. Total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Seoul, Korea) and verified using 2% agarose gel electrophoresis. The DNA library was constructed using the TruSeq Nano DNA Kit following the sample preparation guide protocol provided by the manufacturer (Macrogen Inc., Seoul, Korea). Genome paired-end sequencing was performed at Macrogen Inc. on an Illumina platform (Illumina Inc., San Diego, CA, USA) based on a 301 bp read size.

The complete cp genome was assembled using Geneious v9.0.5 (Biomatters, Auckland, New Zealand) and annotated using the GeSeq tool (Tillich et al., 2017) and Geneious v9.0.5 (Biomatters). Then, the structure of the cp genome was then plotted using the CPGView web server (Liu et al., 2023).

To infer the phylogenetic relationships among Eleutherococcus species, the complete cp genome sequences of 11 Eleutherococcus species and seven related genera 14 species (three Dendropanax, one Kalopanax, one Fatsia, two Hedera, two Oplopanax, two Panax, and three Aralia species) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Two Pittosporum species were used as the outgroups. Phylogenetic analysis was performed using 87 coding sequences of the Eleutherococcus and related species. Alignments were performed using MAFFT v7.450 (Katoh et al., 2002; Katoh and Standley, 2013). A maximum likelihood (ML) bootstrap analysis with 1,000 replicates was performed, and the best-fit model (Kimura three-parameter model with unequalpurine transitions [K3Pu] + empirical base frequencies [F] + proportion of invariable sites [I]) was determined using the IQ-tree web server (Trifinopoulos et al., 2016). Bayesian inference (BI) analysis was conducted using MrBayes v3.2.6 (Ronquist et al., 2012) in PhyloSuite (Zhang et al., 2020). The best model of molecular evolution for the cp genome dataset (general time reversible [GTR] + empirical base frequencies [F] + proportion of invariable sites [I] + four gammadistributed rates of variation across sites [G4]) was obtained using ModelFider v2.0 (Kalyaanamoorthy et al., 2017).

RESULTS AND DISCUSSION

Eleutherococcus divaricatus var. chiisanensis sequencing produced 9,624,560 reads, 7,417 of which corresponded to the cp genome (depth, 60.8). The complete cp genome sequence of E. divaricatus var. chiisanensis was 156,765 bp (Fig. 1) and it was deposited in GenBank under the accession number PQ153235 and associated BioProject, Sequence Read Archive (SRA), and Bio-Sample numbers of PRJNA1146186, SRR30183904, and SAMN43088289, respectively. The LSC, SSC, and each of IRs of the cp genome sequence were 86,592 bp, 18,239 bp, and 25,967 bp in length, respectively. The overall GC contents of the cp genome was 37.9% and in the LSC, SSC, and IRs were 36.2%, 32%, and 43%, respectively. The cp genome contains 132 unique genes, including 87 coding genes, eight ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes. Eighteen genes (7 coding genes, four tRNA, and seven rRNA) were duplicated in the IRs (Table 1). Among them, a total of 11 unique protein-coding genes (PCGs) (rps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, and ndhA) contained one intron, and two PCG contained two introns (pafI and clpP) (Fig. 2). Additionally, rps12 is a trans-spliced gene as shown in Fig. 3.

Fig. 1.

Gene map of the complete chloroplast (cp) genome of Eleutherococcus divaricatus var. chiisanensis. Genes inside the circle are transcribed in clockwise direction, while genes outside the circle are transcribed in counterclockwise direction. Different gene colors correspond to different gene functions. Inverted repeat (IR), small single-copy (SSC), and large single-copy (LSC) regions are indicated.

List of genes annotated in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

Fig. 2.

Schematic map of the cis-spliced genes in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

Fig. 3.

Schematic map of the trans-spliced gene rps12 in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

We used sequences from 28 species (twelve Eleutherococcus species, three Dendropanax species, one Kalopanax species, one Fatsia species, two Hedera species, two Oplopanax species, two Panax species, three Aralia species, and two Pittosporum species as outgroups) to infer the ML-based and BI-based phylogenies, both of which showed the same topology and high support for each branch (Fig. 4). The phylogenetic analysis revealed the monophyly of the genus Eleutherococcus. In addition, E. divaricatus var. chiisanensis, E. sessiliflorus, and E. henry belonging to the genus Eleutherococcus formed a group and was well supported (bootstrap value = 100; posterior probability = 1) and branched first within the genus Eleutherococcus to form a basal group.

Fig. 4.

Phylogenetic tree of Eleutherococcus divaricatus var. chiisanensis and related taxa based on 87 protein-coding gene sequences from the chloroplast genome sequences using maximum likelihood (ML) and Bayesian inference (BI) techniques across different datasets. Numbers on each node indicate bootstrap values/posterior probability values. Pittosporum illicioides and P. tobira were set as the outgroups.

We performed a comparative of the cp genome of E. sessiliflorus, which was identified as the most closely related to E. divaricatus var. chiisanensis in phylogenetic analysis (Fig. 4). The cp genome of E. divaricatus var. chiisanensis was 14 bp longer than that of E. sessiliflorus (156,751 bp; ON422332). However, E. divaricatus var. chiisanensis and E. sessiliflorus cp genomes pairwise identity was 99.98%.

Nine single-nucleotide polymorphisms and 29 insertions/deletions (indels) were identified from the pairwise alignment of Eleutherococcus divaricatus var. chiisanensis and E. sessiliflorus cp genomes (Tables 2, 3). Of the 29 indels, five were found in four protein-coding regions (rps16, rpoC1, pafI, and rpl16) and 24 in the non-coding region between the two plastomes (Table 3).

Single-nucleotide polymorphisms in Eleutherococcus divaricatus var. chiisanensis relative to those in the E. sessiliflorus chloroplast genome.

Indels in Eleutherococcus divaricatus var. chiisanensis relative to those in the E. sessiliflorus chloroplast genome.

Eleutherococcus divaricatus var. chiisanensis can be morphologically distinguished from E. sessiliflorus by the leaf abaxial surface of leaflets densely to sparsely villose, setose and prickles, flowers distinctly pedicellate, arranged in loose umbels, and pedicels ca. 6 mm long (Kim, 2017a; Kim, 2018). Although morphologically similar, the results of the present study support that E. divaricatus var. chiisanensis is closely related but distinct from E. sessiliflorus, as evidenced by the genome differences and phylogenetic analysis. In addition, E. sessiliflorus is distributed in northeast China and Korea, whereas E. divaricatus var. chiisanensis is distributed only in South Korea. Meanwhile, although not mentioned in this study, a more detailed comparative analysis of the original species, Eleutherococcus divaricatus, is necessary. The results of this study will provide important basic information for future phylogenetic and evolutionary studies of the genus Eleutherococcus and Araliaceae, as they provide a comprehensively characterized cp genome of a novel species belonging to this genus by adding one species to them. Additionally, the comparative analyses between the two species of the genus Eleutherococcus will provide useful information for developing species identification markers and conducting genetic diversity analyses.

Acknowledgements

This study was supported by the grant “Silvics of Korea” [KNA-1-1-18, 15-3] financed by the Korea National Arboretum.

Notes

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

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Article information Continued

Fig. 1.

Gene map of the complete chloroplast (cp) genome of Eleutherococcus divaricatus var. chiisanensis. Genes inside the circle are transcribed in clockwise direction, while genes outside the circle are transcribed in counterclockwise direction. Different gene colors correspond to different gene functions. Inverted repeat (IR), small single-copy (SSC), and large single-copy (LSC) regions are indicated.

Fig. 2.

Schematic map of the cis-spliced genes in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

Fig. 3.

Schematic map of the trans-spliced gene rps12 in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

Fig. 4.

Phylogenetic tree of Eleutherococcus divaricatus var. chiisanensis and related taxa based on 87 protein-coding gene sequences from the chloroplast genome sequences using maximum likelihood (ML) and Bayesian inference (BI) techniques across different datasets. Numbers on each node indicate bootstrap values/posterior probability values. Pittosporum illicioides and P. tobira were set as the outgroups.

Table 1.

List of genes annotated in the chloroplast genome of Eleutherococcus divaricatus var. chiisanensis.

Category for genes Group of genes Name of genes
Self-replication Large subunit ribosomal proteins rpl2(×2)*, rpl14, rpl16*, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
DNA-dependent RNA polymerase rpoA, rpoB, rpoC1*, rpoC2
Small subunit ribosomal proteins rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2)**T, rps14, rps15, rps16*, rps18, rps19
Ribosomal RNAs rrn4.5S(×2), rrn5S(×2), rrn16S(×2), rrn23S(×2)
Transfer RNAs trnA-UGC(×2)*, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC*, trnH-GUG, trnI-CAU(×2), trnI-GAU(×2)*, trnK-UUU*, trnL-CAA(×2), trnL-UAA*, trnLUAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnRACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV-UAC*, trnW-CCA, trnY-GUA
Photosynthesis Subunits of ATP synthase atpA, atpB, atpE, atpF*, atpH, atpI
Subunits of NADH-dehydrogenase ndhA*, ndhB(×2)*, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complex petA, petB*, petD*, petG, petL, petN
Subunits of photosystem I psaA, psaB, psaC, psaI, psaJ
Subunits of photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
Subunit of rubisco rbcL
Photosystem assembly factors pafI**, pafII
Other genes Subunit of acetyl-CoA-carboxylase accD
C-type cytochrome synthesis gene ccsA
Envelop membrane protein cemA
ATP-dependent protease subunit P clpP**
Translational initiation factor ψinfA
Maturase matK
Unknown function Conserved open reading frames ycf1, ycf2(×2), ycf15(×2)
*

, genes containing one intron;

**

, genes containing two introns;

T

, trans-spliced genes; (×2), genes have two copies;

ψ

, pseudogene.

Table 2.

Single-nucleotide polymorphisms in Eleutherococcus divaricatus var. chiisanensis relative to those in the E. sessiliflorus chloroplast genome.

No. E. divaricatus var. chiisanensis E. sessiliflorus Site Region Gene Position
1 T A 2,206 LSC matK Gene
2 C A 15,867 LSC atpH-atpL IGS
3 G C 20,491 LSC rpoC2 Gene
4 A C 23,937 LSC rpoC1 intron Intron
5 A T 29,489 LSC rpoB-trnC-GCA IGS
6 T G 67,835 LSC psbE-petL IGS
7 T G 74,370 LSC clpP-psbB IGS
8 G T 86,491 LSC rps19 Gene
9 A C 123,383 SSC ndhA intron Intron

LSC, large single-copy; IGS, intergenic spacer; SSC, small single-copy.

Table 3.

Indels in Eleutherococcus divaricatus var. chiisanensis relative to those in the E. sessiliflorus chloroplast genome.

No. E. divaricatus var. chiisanensis E. sessiliflorus Start End Length Region Position
1 - C 5,466 5,466 1 LSC rps16 intron
2 A - 6,570 6,570 1 LSC rps16-trnQ-UUG
3 T - 7,237 7,237 1 LSC rps16-trnQ-UUG
4 - C 23,927 23,927 1 LSC rpoC1 intron
5 - TA 46,452 46,453 2 LSC pafI intron
6 - TTATATAAT 76,867 76,875 9 LSC psbN-psbH
7 T - 84,965 84,965 1 LSC rpl16 intron
8 CTTATAGATCTAA - 112,594 112,606 13 SSC trnN-GUU-ndhF

LSC, large single-copy; SSC, small single-copy.