The complete chloroplast genome sequence of Viola verecunda (Violaceae)

Abstract Viola verecunda is a perennial plant native to the mountainous areas of Northeast Asia. Here the complete chloroplast genome of V. verecunda and its phylogenetic relationships to other species within the genus Viola are reported. The complete chloroplast genome of V. verecunda is 157,843 bp in length and circular in structure with four regions: a large single-copy region (86,345 bp), a small single-copy region (17,292 bp), and a pair of inverted repeat regions (27,103 bp each). The chloroplast genome contains 111 unique genes comprising 77 protein-coding, 30 unique tRNA, and 4 unique rRNA genes. Based on the protein-coding gene sequences from eight Viola chloroplast genomes, with Balanops balansae designated as the outgroup, maximum likelihood tree analysis indicates that V. verecunda is more closely related to V. raddeana than to other Viola species. The complete chloroplast genome of V. verecunda contributes to a better understanding of the phylogenetic relationships among Viola species.

The infrageneric phylogenetic relationships in Viola remain poorly understood. Since polyploidization and interspecific hybridization occur very frequently during Viola speciation, simple phylogenetic studies using limited numbers of molecular markers fail to provide sufficient resolution to elucidate the complex phylogenetic relationships among Viola species (Liang and Xing 2010;Yoo and Jang 2010;Marcussen et al. 2015). Determining the whole chloroplast genomes of Viola species may improve the resolution (Cheon et al. 2019). The hidden violet, Viola verecunda A. Gray, is commonly found in wet environments of the mountainous areas of Northeast Asia. Species identity of this taxon and delimitation between V. verecunda and V. arcuate are debated among various researchers (Wang and Huang 1993;Akiyama et al. 2002;Chen et al. 2007;Lee and Yoo 2020). This violet can be distinguished from other Viola species by its distinct stems, ovate to cordate leaves, and white flowers. The purpose of this investigation is to assemble and analyze the complete chloroplast genome of V. verecunda to contribute to its phylogenetics, systematics and bioinformatics.
Viola verecunda plant materials were collected from its natural habitat in Oita Prefecture, Japan (33 8 0 N, 131 16 0 E) and the voucher specimen (no. NIBRVP0000736057) was deposited in the herbarium of the National Institute of Biological Resources (KB; www.nibr.go.kr, Myounghai Kwak, mhkwak1@korea.kr). Total genomic DNA was extracted from silica gel-dried leaf samples using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987). Whole-genome sequencing was performed using the Illumina Novaseq 6000 platform (DNA Link Inc., Seoul, Korea). A total of 5.69 Gb raw reads (150 bp paired-end reads) were retrieved and quality-trimmed using the Trimmomatic tool (Bolger et al. 2014). The de novo assembly was performed using 36,925,394 (5.51 Gb) reads with GetOrganelle v1.5 software (Jin et al. 2020) and the V. websteri chloroplast genome (GenBank accession no. MH229819) as a reference. Annotation of the chloroplast genome was conducted using the GeSeq online program (Tillich et al. 2017) and the annotated genome sequence was deposited in GenBank (accession no. MW586692).
To confirm the phylogenetic position of V. verecunda within Viola, seven complete Viola chloroplast genomes were downloaded from GenBank as well as Balanops balansae (Balanopaceae) as the outgroup. The nine complete chloroplast sequences were aligned using MAFFT v7.388 software (Katoh and Standley 2013). Phylogenetic trees were calculated using PhyML v3.0 software (Guindon et al. 2010) with a generalized time-reversible (GTR) sequence evolution model and nearest neighbor interchange for tree improvement. Branch support was evaluated with 1,000 bootstrap replicates and the phylogenetic tree was constructed using the maximum-likelihood (ML) method (Figure 1). The phylogenetic positions of the new V. verecunda sequences among Viola species are shown in Figure 1. Viola verecunda was found to be closest to V. raddeana in subsection Bilobatae under section Plagiostigma. In conclusion, the complete chloroplast genome of V. verecunda contributes to a better understanding of the phylogenetic relationships among Viola species, which will improve genetic diversity assessment and molecular identification within this taxon.

Disclosure statement
No potential competing interest was reported by the author.

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession no MW586692. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA742503, SRR15006027, and SAMN19957749, respectively.