| Home | E-Submission | Sitemap | Editorial Office |  
top_img
Korean J. Pl. Taxon > Volume 53(3); 2023 > Article
SON and CHOI: The complete chloroplast genome of Scrophularia kakudensis and a comparative analysis of S. kakudensis and S. cephalantha

Abstract

The genus Scrophularia L. (Scrophulariaceae) comprises 200–270 species worldwide and is a taxonomically challenging lineage, displaying morphological diversity and hybridization. S. kakudensis is morphologically similar to the closely related taxa S. kakudensis var. microphylla, S. pilosa, and S. cephalantha. Therefore, the purpose of this study was to sequence the chloroplast (cp) genome of S. kakudensis using next-generation sequencing and compare it to those of related taxa. The complete cp genome sequence of Scrophularia kakudensis was found to be 152,355 bp long, consisting of a pair of inverted repeats of 25,485 bp that separate a large single-copy (LSC) of 83,479 bp from small single-copy regions of 17,909 bp. The cp genome contained 78 protein-coding genes, 30 tRNAs, and four rRNAs. A phylogenetic analysis based on 78 protein-coding genes from six Scrophularia species showed S. kakudensis and S. cephalantha formed with 100% boot-strap values. We compared the complete cp genomes of S. kakudensis and S. cephalantha and identified seven sequence divergence regions: matK/rps16, rps16/trnQ, trnS/trnG, rpoB/trnC, trnS/trnG, rpl32/trnL, and ndhD/psaC. These regions may be useful for determining the phylogenetic relationships among S. kakudensis-related species.

INTRODUCTION

Scrophularia L. belongs to the family Scrophulariaceae, which comprises approximately 200–270 species (Ortega Olivencia, 2009; Jang et al., 2021). This genus is distributed throughout the Northern Hemisphere and has square stems (sometimes winged stems) and generally opposite leaves. Stiefelhagen (1910) divided this genus into two sections, sect. Anastomosantes Stiefelhagen (sect. Scrophularia) and sect. Scorodoniae (Benth.) Stiefelhagen (sect. Canina) which are characterized by perennial subshrubs, petal length, corolla tube shape, and life form. The two series in sect. Scrophularia are Grayanaea T. Yamaz and Kakudenses. T. Yamaz recognized (Yamazaki, 1949).
Seven species of Scrophularia (S. buergeriana, S. koraiensis, S. kakudensis, S. takesimensis, S. cephalantha, S. kakudensis var. microphylla, and S. alata) are distributed throughout Korea (Jang and Oh, 2013). All Korean Scrophularia species are included in sect. Scrophularia. Two species (S. alata and S. takesimensis) belong to the series Grayanae and five species (S. buergeriana, S. kakudensis, S. kakudensis var. microphylla, S. koraiensis, and S. cephalantha) belong to series Kakudenses. However, identifying S. kakudensis is difficult due to similar morphological characteristics among S. kakudensis var. microphylla, S. cephalantha, and S. pilosa. Jang et al. (2011) suggested that S. pilosa should be treated as a synonym for S. kakudensis. Scrophularia cephalantha and S. kakudensis var. microphylla are distinct from S. kakudensis during the flowering season, with fewer nodes numbers on the stem and the sizes of the leaves, respectively. Recently, the phylogenetic relationships and evolution of several Scrophularia species have been studied using chloroplast (cp) genomes (Xu et al., 2018; Jang et al., 2021; Wang et al., 2022). In this study, we sequenced and analyzed the cp genome of S. kakudensis and performed a comparative analysis of S. kakudensis and S. cephalantha.

MATERIALS AND METHODS

We collected young leaves of S. kakudensis from Mt. Bohyeonsan (36.16625°N, 128.98097°E). The voucher specimens were deposited at the Daegu National Science Museum. Total DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA, USA). Genomic DNA was sequenced using the Illumina HiSeq X platform (San Diego, CA, USA). We obtained 37,322,320 reads from the 150 bp paired-end sequences. The chloroplast genomes were assembled using GetOrganelle (Jin et al., 2020) and Geseq (Tillich et al., 2017) and Geneious Prime v.2022.1.1 (http://www.geneious.com) were used to annotate the S. kakudensis cp genome. Chloroplast genome mapping was performed using OGDRAW v. 1.3.1 (Greiner et al., 2019).
The chloroplast genome sequences of six taxa (Table 1), including one outgroup (Verbascum phoenieum, MN983301), were included in the phylogenetic analyses. The 78 protein-coding genes shared across taxa were extracted from each chloroplast genome and concatenated. The sequences were aligned using MAFFT (Katoh et al., 2022), and ML analysis was conducted using RAxML (Stamatakis, 2014) with the GTR + GAMMA + I model (rapid bootstrap of 1,000 replications). The complete cp genomes of S. kakudensis and S. cephalantha were compared. The nucleotide diversity (Pi) was determined using DnaSP (Rozas et al., 2017). The step size was set to 200 bp, and the window length was set to 600 bp.

RESULTS AND DISCUSSION

The complete cp genome of S. kakudensis (NCBI accession number: OR004236) comprised 152,355 bp with a quadripartite structure and two inverted repeat regions (IRs, 25,485 bp) separated by large single-copy (LSC, 83,476 bp) and small single-copy (SSC, 17,909 bp) regions (Fig. 1). The average GC content was 38.0%. It contained 112 genes, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. Six protein-coding genes (rpl2, rpl23, ycf2, ndhB, rps7, and rps12), seven tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC) and four rRNA genes (4.5S, 5S, 16S, and 23 rRNA) were duplicated in two IR regions. Fifteen genes had one intron, and three genes (rps12, clpP, and ycf3) had two introns.
Comparative genomic analyses of Scrophularia revealed that the six cp genomes were highly conserved (Table 1). The total length of the Scrophularia species ranged from 152,355 bp (S. kakudensis) to 153,175 bp (S. ningpoensis). The GC content ranged from 38% to 38.1% (S. takesimensis), and the seven Scrophularia species contained the same genes (CDS, tRNA, and rRNA).
A phylogenetic analysis was conducted using 78 protein-coding genes from seven Scrophularia species and one outgroup (Verbascum phoeniceum). The genus Scrophularia is a monophyletic group. S. kakudensis was closely related to S. cephalantha with a 100% bootstrap value, and this clade was a sister to the S. buergeriana + S. ningpoensis clade (Fig. 2). This result supports the hypothesis that S. kakudensis and S. cephalantha are included in series Kakudenses (Yamazaki, 1949; Jang and Oh, 2013). Scrophularia cephalantha is an endemic species (Chung et al., 2023), and this study confirmed that it shows a close relationship between S. kakudensis and S. cephalatha.
Internal transcribed spacer (ITS) and cpDNA non-coding regions have been widely used to investigate molecular phylogeny at the interspecific level (Taberlet et al., 1991; Baldwin, 1992). In Scrophularia, Scheunert and Heubl (2010) tested the nrDNA (ITS) and chloroplast DNA intergenic spacers (psbA/trnH and trnQ/rps16). However, the relationship among Scrophularia species is not well supported. Recently, cp genome sequences have been used as genetic markers for DNA barcoding and phylogenetic relationships, and several studies on the genus Scrophularia have analyzed the chloroplast genome (Choi and Park, 2016;Yi and Kim, 2016; Jang et al., 2021; Wang et al., 2022; Guo et al., 2023).
The nucleotide diversities between S. kakudensis and S. cephalantha were compared. Nucleotide diversity (Pi) ranged from 0–0.01167. Most of the variable regions were located between the ndhD/psaC regions (Pi = 0.01167). This result suggests that seven regions (matK/rps16, rps16/trnQ, trnS/trnG, rpoB/trnC, trnS/trnG, rpl32/trnL, and ndhD/psaC) were highly informative markers to identify species (Fig. 3).
Xu et al. (2018) suggested that nine markers (trnH/psbA, rps15, rps18/rpl20, rpl32/trnL, trnS/trnG, ycf15/trnL, rps4/trnT, ndhF/rpl32, and rps16/trnQ) could be used as DNA barcodes to distinguish Scrophularia. The results from this study indicate that, compared to the nine markers presented in the previous study, the rpl32/trnL and ndhD/psaC regions are more useful for studying S. kakudensis and its relatives. This study reports the complete chloroplast genome of S. kakudensis and provides useful information on the phylogenetic relationships within Scrophularia species.

ACKNOWLEDGMENTS

This research was funded by Collect and Research Native plants on the Korean Peninsula for the Natural History exhibition of the Daegu National Science Museum (DNSM).

NOTES

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.

Fig. 1.
A circular map and annotation of the chloroplast genome of Scrophularia kakudensis. The genes are transcribed clockwise on the inside and counterclockwise on the outside. The darker gray in the inner circle corresponds to the GC content.
kjpt-53-3-237f1.jpg
Fig. 2.
A maximum-likelihood tree of Scrophularia kakudensis and five other Scrophularia species based on 78 chloroplast proteincoding genes.
kjpt-53-3-237f2.jpg
Fig. 3.
Comparison of the nucleotide variability (Pi) values between Scrophularia kakudensis and S. cephalantha. LSC, large single-copy; IR, inverted repeat; SSC, small single-copy.
kjpt-53-3-237f3.jpg
Table 1.
Characteristics of the cp genomes in six species of Scrophularia.
Length (bp) GC contents (%) Genes Reference


Total LSC SSC IR CDS tRNA rRNA
S. buergeriana 153,631 84,454 17,929 25,624 38 78 30 4 Yi and Kim (2016)
S. takesimensis 152,436 83,542 17,938 25,478 38.1 78 30 4 Choi and Park (2016)
S. henryi 152,868 84,020 17,941 25,454 38 78 30 4 Wang et al. (2022)
S. ningpoensis 153,175 84,257 17,938 25,490 38 78 30 4 Guo et al. (2023)
S. cephalantha 153,016 84,124 17,922 25,485 38 78 30 4 Jang et al. (2021)
S. kakudensis 152,355 83,479 17,909 25,485 38 78 30 4 This study

cp, chloroplast; LSC, large single-copy; SSC, small single-copy; IR, inverted repeat; CDS, coding sequence.

LITERATURE CITED

Baldwin, B.G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae. Molecular Phylogenetics and Evolution 1: 3-16.
crossref pmid
Choi, K.S and Park, S. 2016. The complete chloroplast genome sequence of the Korean endemic plant Scrophularia takesimensis . Mitochondrial DNA Part A 27: 2058-2059.
crossref pmid
Chung, G. Y. Jang, H.-D. Chang, K. S. Choi, H. J. Kim, Y.-S. Kim, H.-J and Son, D. C. 2023. A checklist of endemic plants on the Korean Peninsula II. Korean Journal of Plant Taxonomy 53: 79-101.
crossref pdf
Greiner, S. Lehwark, P and Bock, R. 2019. OrganellarGenome-DRAW (OGDRAW) version 1.3.1: Expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Research 47: W59-W64.
crossref pmid pmc
Guo, L. Wang, X. Wang, R and Li, P. 2023. Characterization and comparative analysis of chloroplast genomes of medicinal herb Scrophularia ningpoensis and its common adulterants (Scrophulariaceae). International Journal of Molecular Science 24: 10034.
crossref pmid pmc
Jang, H. D. Kim, T. H and Oh, B. U. 2011. A taxonomic review of Scrophularia kakudensis Franch. and its relatives. Korean Journal of Plant Taxonomy 41: 345-352 (in Korean).
crossref pdf
Jang, H.-D. Nam, G.-H. Park, M.-S and Jun, J. 2021. Characterization of the complete chloroplast genome of Scrophularia cephalantha endemic to Korea. Mitochondrial DNA Part B Resources 6: 3179-3180.
crossref pmid pmc
Jang, H.-D and Oh, B.-U. 2013. A taxonomic study of Korean Scrophularia L. (Scrophulariaceae) based on morphological characters. Korean Journal of Plant Resources 26: 271-283 (in Korean).
crossref
Jin, J.-J. Wu, W.-B. Yang, J.-B. Song, Y. dePamphilis, C. W. Yi, T.-S and Li, D.-Z. 2020. GetOrganelle: A fast and versatile tool-kit for accurate de novo assembly of organelle genomes. Genomic Biology 21: 241.
crossref pmid pmc pdf
Katoh, K. Misawa, K. Kuma, K.-I and Miyata, T. 2002. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059-3066.
crossref pmid pmc
Ortega Olivencia, A. 2009. Scrophularia. Flora Iberica, Plantaginaceae to Scrophulariaceae, Vol 13. Benedί, C. Rico, E. Güemes, J. Herrero, A (eds.), Real Jardίn Botánico, CSIC, Madrid. 97-134.

Rozas, J. Ferrer-Mata, A. Sánchez-DelBarrio, J.C. Guirao-Rico, S. Librado, P. Ramos-Onsins, S. E and Sánchez-Gracia, A. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution 34: 3299-3302.
crossref pmid
Scheunert, A and Heubl, G. 2010. Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae): New insights inferred from DNA sequence data. Plant Systematics and Evolution 291: 69-89.
crossref pdf
Stamatakis, A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.
crossref pmid pmc pdf
Stiefelhagen, H. 1910. Systematiche und Pflanzengeographische Studien zur Kenntnis der Gattung Scrophularia . Botanische Jahrbücher für Systematic, Pflanzen-geschichte und Pflanzengeographie 44: 406-496.

Taberlet, P. Gielly, L. Pautou, G and Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109.
crossref pmid pdf
Tillich, M. Lehwark, P. Pellizzer, T. Ulbricht-Jones, E. S. Fischer, A. Bock, R and Greiner, S. 2017. GeSeq: Versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45: W6-W11.
crossref pmid pmc
Yamazaki, T. 1949. Scrophularia Asiae orientalis (1). The Journal of Japanese Botany 23: 79-88.

Yi, D.-K and Kim, K.-J. 2016. The two complete plastomes from Scrophularia (Scrophulariaceae): Scrophularia buergeriana and S. takesimensis . Mitochondrial DNA Part B Resources 1: 710-712.
crossref pmid pmc pdf
Wang, R. Gao, J. Feng, J. Yang, Z. Qi, Z. Li, P and Fu, C. 2022. Comparative and phylogenetic analyses of complete chloroplast genomes of Scrophularia incisa complex (Scrophulariaceae). Genes 13: 1691.
crossref pmid pmc
Xu, W.-Q. Losh, J. Chen, C. Li, P. Wang, R.-H. Zhao, Y.-P. Qiu, Y.-X and Fu, C.-X. 2018. Comparative genomics of figworts (Scrophularia, Scrophulariaceae), with implications for the evolution of Scrophularia and Lamiales. Journal of Systematics and Evolution 57: 55-65.
crossref pdf
Editorial Office
Korean Journal of Plant Taxonomy
Department of Biology, Daejeon University, Daejeon 34520, Korea
TEL: +82-42-280-2434   E-mail: kjpt1968@gmail.com
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Copyright © Korean Society of Plant Taxonomists.                 Developed in M2PI