Complete chloroplast genome sequence of Campanula punctata Lam. (Campanulaceae).

The complete chloroplast genome of Campanula punctata Lam. was determined in this study. The total genome size was 169 341 bp in length, containing a pair of inverted repeats (IRs) of 29 637 bp, which were separated by large single copy (LSC) and small single copy (SSC) of 102 323 bp and 7744 bp, respectively. The overall GC contents of the plastid genome were 38.8%. One hundred thirteen unique genes were annotated, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In these genes, 15 genes contained one intron and 2 genes had two introns. A phylogenetic tree showed that Campanula punctata was closely related to Campanula takesimana.

Campanulaceae are known for having highly rearranged chloroplast genomes, based on earlier gene mapping and restriction site, and whole chloroplast genome sequencing studies (Cosner 1993; Cosner et al. 1997; Haberle et al. 2008). Therefore, the structure and sequences of chloroplast genome of Campanulaceae are useful data to clarify their phylogeny and evolutionary tendency. Haberle et al. (2008) first analysed the whole chloroplast genome sequence of Trachelium caeruleum in Campanulaceae. Recently, Cheon and Yoo (2014), Cheon et al. (2014) and Kim et al. (2015) analysed whole chloroplast genome sequences of Hanabusaya asiatica, Campanula takesimana and Adenophora remotiflora, respectively. However, chloroplast genome data are not enough to clarify their phylogenetic relationships of inter- and intrageneric level of Campanulaceae.

In this study, we reported the complete chloroplast genome of C. punctata to gain information for further studies on the phylogenomics of Campanulaceae. The sampled C. punctata was collected from Mt Jeombong, Gangwon-do Province in Korea. The specimen (voucher number KWNU77388) was deposited at the Kangwon National University Herbarium (KWNU). The genomic DNA was sequenced using the Illumina Hiseq2000 (Illumina, San Diego, CA) platform. Using these sequence data, we assembled the complete chloroplast genome with Geneious 7.1 (Biomatters Ltd, Auckland, New Zealand). Annotation was performed with Dual Organellar GenoMe Annotator (DOGMA) using default parameters to predict protein-coding genes, transfer RNA (tRNA) genes and ribosome RNA (rRNA) genes (Wyman et al. 2004). BLASTX was used to further identify the positions of genes with intron by searching against published plastid genome database. The complete chloropalst genome sequence of C. punctata was submitted to GenBank under accession number KU198434.

The plastid genome of C. punctata is 169 341 bp in length and composed of LSC region of 102 323 bp, two IR copies of 29 637 bp and SSC region of 7744 bp. The whole plastid genome of C. punctata was 210 bp shorter than that of closely related C. takesimana (GenBank accession KP006497). Also, the gene content and order of plastid genome were almost identical to C. takesimana. The GC content was 38.8%, and in the LSC, SSC and IR regions were 37.8%, 32.6% and 41.4%, respectively. The chloroplast genome of C. punctata includes 113 unique genes, composed of 79 protein coding genes, 30 tRNA genes and 4 rRNA genes. The trnI-CAU was duplicated once in the LSC regions. Among the 79 protein-coding genes, 4 genes (rpl23, ycf15, infA and clpP) were identified as pseudogenes. The pseudogene of ndhE, which had various lengths were located at the IRb/SSC boundary, and parts of psbB, ycf3 and rrn23 are duplicated in the IRs. For the rps12 gene, 3’ exon was located in the LSC region and 5’ exon was duplicated in the IRs. Seventeen genes (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC, atpF, clpP, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12 and ycf3) contain one or two introns.

To investigate the phylogenetic relationships of C. punctata and related taxa we conducted the phylogenetic analysis. The cpDNA sequence except intergenic spacer region (IGS) was used to their relationships among the Asterales due to many rearrangements. A total of 74 protein-coding gene sequences from 19 Asterales species and 1 outgroup (Vitaceae) were aligned using MAFFT (Katoh et al. 2002). Phylogenetic analysis was carried out in PAUP* v4.0b10 (Swofford 2003) using maximum parsimony and 1000 bootstrap replicates. The maximum parsimony (MP) tree showed that C. punctata was closely related to C. takesimana with a high bootstrap value (BP = 100) within the monophyletic Campanulaceae clade (Figure 1).

Figure 1.

Phylogenetic relationship based on chloroplast genome sequences from 19 Asterales and one Vitaceae species. Accession numbers: Adenophora remotiflora KP889213, Hanabusaya asiatica KJ477692, Campanula punctata KU198434, Campanula takesimana KP006497, Trachelium caeruleum NC010442, Ageratina adenophora JF826503, Plaxelis clematidea KF922320, Guizotia abyssinica EU549769, Helianthus annuus NC007977, Artemisia montana KF887960, Leontopodium leiolepis NC027835, Aster spathulifolius KF279514, Jacobaea sativa HQ234669, Lactuca sativa DQ383816, Centaurea diffusa KJ690264, Cynara baetica KP842706, Cynara cadunculus var. scolymus KP842712, Cynara humilis NC027113, Silybum marianum KT267161, Vitis vinifera NC007957.