Characterization of the chloroplast genome of Adenophora koreana (Campanulaceae), a species endemic to Korea

Plant material

The plant material of A. koreana was sampled from Mt. Woraksan National Park in Chungcheongbuk-do of South Korea, and a specimen (voucher no. SJUH001459) was deposited at the Sangji University Herbarium (SJUH).

DNA extraction and chloroplast genome determination

Total DNA was extracted from approximately 100 mg of fresh leaves using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). The genomic DNA library was constructed with the TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) following the sample preparation guide protocol provided by the manufacturer (Macrogen Inc., Seoul, Korea). Paired-end sequencing of the chloroplast genome of A. koreana was performed using the MiSeq platform (Illumina). Approximately 9,553,764 raw reads of 301 bp in length were obtained. The chloroplast genome assembly was performed by de novo assembly using Geneious Prime v.2025.0.3 software package (Biomatters Ltd., Auckland, New Zealand) and NOVOPlasty v4.3.1 pipeline (Dierckxsens et al., 2016). The assembled chloroplast genome was annotated using Geneious Prime v.2025.0.3 based on the previously published seven Adenophora cp genomes (A. divaricata, A. erecta S. T. Lee, J. K. Lee & S. T. Kim, A. kayasanensis Kitam., A. racemosa S. T. Lee & J. K. Lee, A. remotiflora (Siebold & Zucc.) Miq., A. stricta Miq. and A. triphylla (Thunb.) A. DC.), and annotated transfer RNAs (tRNAs) were confirmed using tRNAscan-SE (Chan and Lowe, 2019). Additionally, OGDRAW (Greiner et al., 2019) and CPGview (Liu et al., 2023) were used for genome structure visualization and manually corrected for start and stop codons as well as for intron/exon boundaries to match gene predictions. Finally, short reads were mapped to the assembled cp genome, and read mapping depth at all positions of the assembled genome was statistically obtained.

The structure and repeat sequences of the complete chloroplast genome were compared with those of seven previously analyzed Adenophora. The genome structure was referenced from two previous studies (Cheon et al., 2017; Kim and Cheon, 2021). Additionally, simple sequence repeats were identified using MISA software (Beier et al., 2017), with minimum repeat numbers set to 10 for mononucleotides, 5 for dinucleotides, 4 for trinucleotides, and 3 each for tetra-, penta-, and hexanucleotides.

Phylogenetic analysis

To construct the phylogenetic tree, the complete cp genomes of 15 species were selected within Campanulaceae s. str. as the ingroup and Lobelia chinensis Lour. was selected as the outgroup. A total of 76 protein-coding genes were aligned separately using the MAFFT v7.490 (Katoh and Standley, 2013) plugin of the Geneious Prime v.2025.0.3 software (Dotmatics, Boston, MA, USA). A maximum likelihood (ML) tree analysis was conducted using raxmlGUI v.2.0.14 with 1,000 bootstrap replicates, and the optimal nucleotide substitution model was GTR+I+Gamma. Bayesian inference (BI) analysis was performed using MrBayes v3.2.7a (Huelsenbeck and Ronquist, 2001), and the analysis was run for 1,000,000 generations. The resulting ML and BI trees were visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Complete chloroplast genome of A. koreana and comparison to congeneric species

As a result of assembly, the cp genome of A. koreana exhibits the typical quadripartite structure characteristic of most angiosperms, consisting of a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeat (IR) regions (Jansen and Ruhlman, 2012; Park and Cheon, 2021; Quang and Huynh, 2024). The complete chloroplast genome is 175,721bp in length with 37.4% GC content, composed of the LSC region of 129,189bp, the SSC region of 29,294bp, and two IR regions of 8,619bp each (Fig. 2).

Fig. 2

Map of the chloroplast genome of Adenophora koreana. Genes inside the circle are transcribed clockwise, and genes outside are transcribed counterclockwise. The dark gray inner circle corresponds to the GC contents, and the light gray circle corresponds to the AT content.

The cp genome contains a total of 110 unique genes consisting of 76 protein-coding genes, 30 tRNA genes, and four ribosomal RNA (rRNA) genes. Among the 110 genes, seventeen (atpF, clpP, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, ycf3, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnLUAA, and trnV-UAC) contained one or two introns (Fig. 3, Table 1). It was deposited in GenBank under the accession number PV339537 and associated BioProject, Sequence Read Archive (SRA), and Bio-Sample numbers of PRJNA1236940, SRR32731664, and SAMN47409543, respectively.

Fig. 3

Schematic map of the cis-splicing genes (A) and trans-splicing rps12 gene (B), in the chloroplast genome of Adenophora koreana. The exons are shown as black and the introns as white. The arrow indicates the direction of the gene.

Table 1

The list of genes annotated in the chloroplast genome of Adenophora koreana.

Compared to the chloroplast genomes of seven published Adenophora species, the total length of the chloroplast genome of A. koreana was the third longest after A. triphylla (239,431 bp) and A. divaricata (176,331 bp), and the GC content was the lowest at 37.4% (Table 2). It is known that Adenophora species have very different chloroplast genome structures among them due to various rearrangements such as inversions in the LSC region and expansions or contractions of the IR or SSC regions (Cheon et al., 2017; Kim and Cheon, 2021). In this study, it was confirmed that A. koreana has the same structure as A. racemosa and A. kayasanensis. When compared with the above-mentioned taxa having the same cp genome structure with A. koreana the LSC region was longest in A. koreana, but the SSC region was longest in A. racemosa, while the IR regions were found to have identical lengths in all three taxa.

Table 2

Comparison of chloroplast genome features of A. koreana and seven other Adenophora species included in this study.

Phylogenetic analysis of Campanulaceae

In the results of the phylogenetic tree reconstruction, the genus Adenophora formed a monophyletic group, and sect. Remotiflorae being the earliest diverging lineage within the genus and clustering independently, while sect. Platyphyllae formed a polyphyletic group (Fig. 4). These results are similar to previous studies (Kim and Cheon, 2021; Kim et al., 2021), and it is thought that studies including more diverse species need to be conducted to more clearly define the boundaries between sections within the genus. Meanwhile, A. koreana showed a close phylogenetic relationship with A. racemosa and A. kayasanensis than with A. divaricata and A. triphylla in the sect. Platyphyllae.

Fig. 4

The maximum likelihood tree from 15 Campanulaceae species and one outgroup, Lobelia chinensis. The 100% bootstrap value and 1.00 posterior probability value are marked with *. The newly analyzed Adenophora koreana is represented by red color.