Characterization of the complete plastome sequence of Korean endemic, Cardamine glechomifolia H.Lév. (Brassicaceae, Brassicales)

Abstract In this study, we report the complete plastome sequence of Cardamine glechomifolia H.Lév. 1913 (NCBI acc. no. OP894664). This plastome shows typical quadripartite structure. The plastome size is 154,307 bp, which consists of 84,015 bp large single-copy (LSC), 17,690 bp small single-copy (SSC), and 26,301 bp inverted repeat (IR) regions. The plastome contains 112 genes, including 78 protein-coding, 30 tRNA, and four rRNA genes. The infA gene is pseudogenized. Sixteen genes contain one intron and two genes (clpP and ycf3) have two introns. The phylogenomic analysis conducted in our study reveals that the genus Cardamine, which encompasses C. glechomifolia, exhibits three distinct clades. In order to elucidate the interrelationship among the three clades, it is imperative to conduct additional investigations by augmenting the number of Cardamine samples.


Introduction
Cardamine Linnaeus is a large genus of Brassicaceae and includes about 200 species (Christenhusz et al. 2017).They are distributed all over the world except Antarctica.Cardamine glechomifolia H.L� ev.1913 is a plant species exclusively found in Korea.The distribution of this species is primarily concentrated in Jeju Island and the southern coastal region of South Korea.The study of endemic plant species holds significance in the context of safeguarding national control and conservation of biological resources.In fact, complete plastome sequences of many species belonging to the genus Cardamine have been reported (Hu et al. 2015;Dann et al. 2017;Ren et al. 2017;Ru et al. 2020;Walden et al. 2020;Raman et al. 2021;Huang et al. 2022;Raman and Park 2022;Wang et al. 2022;Xu et al. 2022).However, for the conservation and management of endemic plants, we report for the first time the complete plastome sequence of C. glechomifolia, a Korean endemic plant.

Materials and methods
The study utilized freshly harvested leaves sourced from Jeju Island in South Korea (33 � 20 0 79.30 00 N and 126 � 22 0 48.30 00E) at an altitude of 550 m (Figure 1).The voucher specimens were placed in the herbarium of the Korea Research Institute of Bioscience & Biotechnology (KRIB, acc.no.KRIB 0092706, Jin-Hyub Paik, jpaik@kribb.re.kr).Despite being an endemic species of Korea, C. glechomifolia does not possess the status of being endangered or protected.The study gained ethical approval from the Institutional Bioethics Committee of KRIBB.
The extraction of DNA from fresh leaves was performed using the DNeasy Plant Mini Kit manufactured by QIAGEN.The genomic DNAs are deposited in the International Biological Material Research Center (IBMRC acc.no.KRIB 0092706).Total raw reads (44,582,760) were generated using an Illumina NovaSeq platform (Illumina Inc., San Diego, CA).Total raw reads were assembled using NOVOPlasty 4.3.1.pl.(Dierckxsens et al. 2017).The complete plastome was annotated using the Geneious Prime v. 2022.2.2 (Biomatters Ltd., Auckland, New Zealand; Kearse et al. 2012), National Center for Biotechnology Information (NCBI) BLAST, Chloroplast Genome Viewer (CPGView) (Liu et al. 2023), and tRNAscan-SE 2.0 programs (Lowe and Eddy 1997).The circular plastome map was constructed by OrganellarGenomeDRAW (OGDRAW) (Greiner et al. 2019).For the phylogenomic analysis, we selected and downloaded 27 complete plastome sequences based on the APG IV system (APG IV 2016) from the NCBI database.The phylogenomic analysis was conducted on a dataset using RAxML v.8.2.12 on the CIPRES website, as described by Stamatakis (2014).The 28 plastome sequences, which had one inverted repeat (IR) sequence removed, were subjected to alignment using the MAFFT v.7.490 alignment programme in Geneious Prime v. 2022.2.2 (Biomatters Ltd., Auckland, New Zealand; Kearse et al. 2012).mVISTA analysis was performed to confirm the structural differences between C. glechomifolia and its relatives (Frazer et al. 2004).

Results and discussion
The length of the plastome of Cardamine glechomifolia is 154,307 bp.The structure of this entity conforms to a conventional quadripartite arrangement, as illustrated in Figure 2. It consists of four distinct sections: the large single-copy (LSC) region spanning 84,015 bp, the small single-copy (SSC) region spanning 17,690 bp, and two IR regions, namely IRa and IRb, each spanning 26,301 bp.The coverage of the plastome is 621.0�, as depicted in Figure S1.The plastome consisted of a total of 112 distinct genes, which can be further categorized into 78 protein-coding genes, 30 tRNA genes, and four rRNA genes.The IR regions contain duplicated copies of six protein-coding genes, seven tRNA genes, and four rRNA genes.The infA gene can be classified as a pseudogene.The average guanine-cytosine (G-C) level is 36.3%.There are a total of 16 genes that possess a single intron, whereas two specific genes, namely ycf3 and clpP, exhibit the presence of two introns (Figures S2 and S3).
In order to ascertain the phylogenomic relationships within the genus Cardamine in the Brassicaceae, a maximumlikelihood tree was generated.This was achieved by utilizing a dataset consisting of 24 Cardamine plastome sequences, along with four additional Brassicaceae sequences (serving as outgroup).Notably, one of the IR sequences was excluded from the analysis.The resulting alignment had a length of 137,087 bp.The tree analysis reveals that the genus Cardamine has given rise to three distinct clades, with C. glechomifolia positioned inside clade I (Figure 3).C. glechomifolia is considered a sister species to C. amara, as supported by a bootstrap value of 100% (Figure 3).The complete plastome structure was compared with clade I group (11 species) where C. glechomifolia is located.As a result, no structural differences between them were found (Figure S4).In this

Conclusions
This paper presents the complete plastome sequence of Cardamine glechomifolia, a plant species endemic to Korea.The study of endemic plant species holds significance in the context of safeguarding national control and ownership of biological resources.The plastome exhibits the characteristic structure typically observed in angiosperms.Our results revealed a close relationship between C. glechomifolia and C. amara as they nested together in our phylogenomic tree.We believe that our study holds significant importance in the resolution of Cardamine's confusing phylogenetic relationship.

Figure 1 .
Figure 1.Field photo of Cardamine glechomifolia H.L� ev.Species photo was taken by the author (Jinki Kim) in Jeju Island, South Korea, April 2020, without any copyright issues.