The complete chloroplast genome of Viola grypoceras (Violaceae)

Abstract We constructed and characterized the chloroplast genome of Viola grypoceras via de novo assembly of Illumina data. The complete circular chloroplast genome is 158,357 bp long and contains four parts: a large single-copy (LSC) region of 86,764 bp, a small single-copy (SSC) region of 17,345 bp, and two inverted-repeat regions (IRa and IRb) of 27,124 bp each. Genome annotation predicted that this genome harbors 111 genes, comprising 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. Phylogenetic analysis demonstrated that V. grypoceras shares a close systematic relationship with V. mirabilis and V. websteri by forming a basal clade in the genus Viola.

The violet Viola grypoceras Gray 1857 is a Northeast Asian plant species, distributed in Korea, Japan, Taiwan, and mainland China (Akiyama et al. 2002;Chen et al. 2007; Lee and Yoo 2020). In Korea, this species occupies the southernmost parts of the Korean peninsula and Jeju Island. V. grypoceras plants have aerial stems and flowers with purplish to white petals with purple stripes. Plastid genomic information is available for only a few Viola species with aerial stems (Cheon et al. 2019;Kwak 2021). To explore the relationships among the Korean Viola species, especially caulescent species, the whole chloroplast genome sequence was studied based on next-generation sequencing (NGS).
In the present study, we generated the complete chloroplast genome sequence of V. grypoceras. Fresh leaf samples were collected for DNA extraction on the banks of Gangjeong-cheon stream in Jeju Island, South Korea (33 28 0 2.25 00 N, 126 30 0 56.55 00 E). A dried plant specimen was deposited in the Herbarium of the Nakdonggang National Institute of Biological Resources (NNH) under voucher number NNIBRVP90496 (https://fbp.nnibr.re.kr/portal/; contact: Jin Hee Park; parkjh23@nnibr.re.kr). Total genomic DNA was extracted from the leaf tissue using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The isolated genomic DNA was used to construct a paired-end library (PE) with a mean insert size of 500 bp by Theragen Bio (Suwon, South Korea), followed by sequencing on an Illumina HiSeq 2500 platform. A total of 25.37 Gb of 150-bp PE reads were obtained by Illumina HiSeq NGS and assembled using CLC Genomics Workbench (ver. 8.05 CLC Inc., Aarhus, Denmark) (Jeong et al. 2014). The chloroplast genome structure was verified using long PCR and Sanger sequencing (Lee et al. 2015). The assembled structure and the genes in the complete chloroplast genome were annotated using Sequin and were manually curated based on BLAST searches. The annotated genome was deposited in GenBank (accession no. OM055663).
The complete plastid genome of V. grypoceras is a quadripartite circular structure of 158,357 bp in length with a GC content of 36.2%. The genome is composed of four distinct regions: a large single-copy region of 86,764 bp, a small single-copy region of 17,345 bp, and a pair of identical invertedrepeat regions (IRa and IRb) of 27,124 bp each. We annotated 111 genes in the V. grypoceras chloroplast genome, which consists of 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. The V. grypoceras chloroplast genome contains 20 introns, with one group I intron and 19 group II introns. The group I intron is present in the gene trnL-UAA (Besendahl et al. 2000). Nineteen group II introns are distributed in 16 genes. Three genes including ycf3, clpP, and rps12 contain two group II introns. The first intron of rps12 is a trans-splicing intron (Hildebrand et al. 1988;Lee et al. 2015). Twelve genes contain a single group II intron: ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, and trnV-UAC. However, the chloroplast genomes of all Viola species including V. grypoceras lack the rps16 gene and its group II intron, which is rarely absent in angiosperm plastid genomes (Alqahtani and Jansen 2021).
We compared seven genes (psaA, psaB, psbA, psbB, psbC, psbD, and rbcL) in algae (Jeong et al. 2014) and angiosperms (Hong et al. 2017) to examine the phylogenetic relationships among Viola species. We inferred the evolutionary history of these species using the maximum-likelihood method and General Time Reversible model (Nei and Kumar 2000) with 1000 bootstrap replications in MEGA X (Kumar et al. 2018).
Twelve Viola species formed three major clades in the phylogenetic tree, with 99-100% bootstrap support, using Passiflora miniata of the Passifloraceae and Salix bicolor of the Salicaceae as the outgroups (Figure 1). V. grypoceras belongs to the basal-most clade in the tree, along with V. mirabilis and V. websteri. Seven Viola species without aerial stems form a derived clade. Two caulescent species, V. raddeana and V. verecunda, were placed between the two clades. As shown in Figure 1, five Viola species with aerial stems were placed in basal lineages of the genus Viola. The phylogenetic tree places V. grypoceras close to V. mirabilis. Further investigation of the caulescent Viola taxa at the interspecies and intraspecies levels would further our understanding of the relationships of Viola species.

Author contributions
JHP and JL conceived and designed the study. JHP and JL collected the plant material. JHP prepared herbarium specimen. ML performed the-¼ experiments. ML, JL, and YL analyzed the data. JHP and JL wrote the manuscript. All authors read and approved the final manuscript.

Ethical approval
No permission from UNESCO or Korean government was required to collect this plant, which occurs at a streamside of coastal area in Jeju Island. The authors comply with relevant institutional, national, and international guidelines and legislation for plant study.

Disclosure statement
No potential conflict of interest was reported by the authors.