Abstract
The complete chloroplast genome sequence of Kuepferia otophora, a flowering plant occurring in Hengduan Mountains with high altitudes, is determined in this study. The plastome is 139,684 bp in length, with one large single-copy region of 76,787 bp, one small single-copy region of 16,635 bp, and two inverted repeat (IR) regions of 23,131 bp. It contains 128 genes, including 83 protein-coding, 8 ribosomal RNA, and 37 transfer RNA genes. Phylogenetic tree shows that this species is a sister to the clade of genus Gentiana. The first published plastome within Kuepferia provides significant insight for elucidating the phylogenetic relationship of taxa within tribe Gentianeae.
Keywords: Kuepferia otophora, tribe Gentianeae, chloroplast genome, phylogenetic tree
Encompassing two subtribes Gentianinae and Swertiinae, tribe Gentianeae exhibits the highest species diversity of the angiosperm family Gentianaceae (Struwe et al. 2002). One of the genus in tribe Gentianeae, Kuepferia, is a recently described genus including all species of the former Gentiana sect. Otophora, which had been excluded from genus Gentiana in an earlier study (Favre et al. 2014). This genus contains 13 species, distributed in the Himalayas and the south-eastern part of the Qinghai-Tibet Plateau, including China, Nepal, India, Myanmar and Bhutan (Favre et al. 2014; Maity et al. 2016). Kuepferia otophora is the type species within genus Kuepferia, occurring in Hengduan Mountains with a rather wide elevation gradient between 2800 m.a.s.l. and 4200 m.a.s.l. This species has flowers in axillary or terminal cymes, occasionally solitary, and grows in valleys, grassy slopes or Rhododendron scrub. In this study, we reported the complete chloroplast genome of K. otophora, which is the first published plastome sequence within Kuepferia.
Fresh leaves of one K. otophora individual were collected from Yunnan, China (E99°0′30.07′′, N26°35′8.77′′) and dried with silica gels. The voucher specimen was stored in Sichuan University Herbarium. Total DNA was isolated using a modified CTAB method (Doyle and Doyle 1987) and sequenced by the BGISEQ-500 sequencing platform (BIG, Shenzhen, China). A total of 8,326,571 paired-end reads were obtained and de novo assembled using NOVOPlasty v2.7.2 with K-mer = 39 (Dierckxsens et al. 2017). The annotation of the chloroplast genome was performed using GeSeq v1 (Tillich et al. 2017).
The total plastome length of K. otophora (MK937918) is 139,684 bp, with one large single copy (LSC, 76,787 bp), one small single copy (SSC, 16,635 bp), and two inverted repeat regions (IRa and IRb, 23,131 bp for each). The GC content of the whole plastome is 38.0%, while those of LSC, SSC, and IRs are 36.0%, 31.7%, and 43.7%, respectively. The chloroplast genome contains 128 genes, including 83 protein-coding, 8 rRNA, and 37 tRNA genes. A total of 16 genes are duplicated in the inverted repeat regions, seven of which are tRNA genes, four are rRNA genes, and five are protein-coding genes. The complete ycf1 gene was located at the SSC/IRa junction, while one partial ycf1 gene was identified at the IRb/SSC junction as a pseudogene.
In order to further clarify the phylogenetic position of K. otophora, plastome of five representative Gentiana species and two Swertia species were obtained from NCBI to construct the plastome phylogeny, with Catharanthus roseus as an outgroup. All the sequences were aligned using MAFFT v.7.313 (Katoh and Standley 2013) and maximum likelihood phylogenetic analyses were conducted using RAxML v.8.2.11 (Stamatakis 2014). The phylogenetic tree shows that all Gentiana species form a single clade being sister to K. otophora and supports the position of genus Swertia as the sister of clade that contains Kuepferia and Gentiana (Figure 1). This study provides a platform for future molecular phylogenetics of species in tribe Gentianeae.
Figure 1.
A plastome phylogenetic tree for eight species within tribe Gentianeae. Accession numbers: Kuepferia otophora (MK937918); Gentiana crassicaulis (NC_027442); Gentiana macrophylla (NC_035719); Gentiana stipitata (NC_037984); Gentiana caelestis (NC_037979); Gentiana veitchiorum (NC_037985); Swertia mussotii (NC_031155); Swertia verticillifolia (MF795137); Catharanthus roseus (NC_021423).
Disclosure statement
No potential conflict of interest was reported by the authors.
References
- Dierckxsens N, Mardulyn P, Smits G. 2017. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 45:e18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 19:11–15. [Google Scholar]
- Favre A, Matuszak S, Sun H, Liu E, Yuan Y, Muellner-Riehl AN. 2014. Two new genera of Gentianinae (Gentianaceae): Sinogentiana and Kuepferia supported by molecular phylogenetic evidence. Taxon. 63:342–354. [Google Scholar]
- Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 30:772–780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maity D, Dey SK, Favre A. 2016. Kuepferia kanchii sp. nov. (Gentianaceae) from Sikkim Himalaya. Nord J Bot. 34:416–420. [Google Scholar]
- Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 30:1312–1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Struwe L, Kadereit JW, Klackenberg J, Nilsson S, Thiv M, von Hagen KB, Albert VA. 2002. Systematics, character evolution, and biogeography of Gentianaceae, including a new tribal and subtribal classification In: Struwe L, Albert VA, editors. Gentianaceae: systematics and natural history. Cambridge (UK): Cambridge University Press; p. 21–309. [Google Scholar]
- Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S. 2017. GeSeq – versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 45:W6–W11. [DOI] [PMC free article] [PubMed] [Google Scholar]