The complete chloroplast genome of Limonium tetragonum (Plumbaginaceae) isolated in Korea

Article information

Korean J. Pl. Taxon. 2021;51(3):337-344
Publication date (electronic) : 2021 September 30
doi : https://doi.org/10.11110/kjpt.2021.51.3.337
Honam National Institute of Biological Resources, Mokpo 58762, Korea
1InfoBoss Inc. and InfoBoss Research Center, Seoul 06278, Korea
*Author for correspondence: starflr@infoboss.co.kr
Received 2021 June 27; Revised 2021 August 16; Accepted 2021 September 7.

Abstract

The chloroplast genome of Limonium tetragonum (Thunb.) Bullock, a halophytic species, was sequenced to understand genetic differences based on its geographical distribution. The cp genome of L. tetragonum was 154,689 bp long (GC ratio is 37.0%) and has four subregions: 84,572 bp of large single-copy (35.3%) and 12,813 bp of small single-copy (31.5%) regions were separated by 28,562 bp of inverted repeat (40.9%) regions. It contained 128 genes (83 protein-coding genes, eight rRNAs, and 37 tRNAs). Thirty-five single-nucleotide polymorphisms and 33 INDEL regions (88 bp in length) were identified. Maximum-likelihood and Bayesian inference phylogenetic trees showed that L. tetragonum formed a sister group with L. aureum, which is incongruent with certain previous studies, including a phylogenetic analysis.

Limonium, belonging to Plumbaginaceae family, is a cosmopolitan halophytic genus, and a few species inhabit alkaline soil away from the coastal area (Kubitzki, 1993; Morgan and Funnell, 2018). Limonium tetragonum (Thunb.) Bullock, distributed in coastal areas of Korea and Japan, is a biennial halophytic species with radical leaves (8–15 × 1.5–3 cm) and yellow corolla (Owhi, 1965; Park, 2007; Lee et al., 2011; Park et al., 2020a). The crude extracts and solvent-partitioned fractions of whole plants of L. tetragonum display antioxidant (Lee et al., 2011) and anti-cancer activities (Kong et al., 2008); ethyl acetate soluble fraction of aerial parts exhibits anti-liver fibrosis (Kim et al., 2016); that of whole plants shows anti-alcohol toxicity (Kim et al., 2015); and its methanol extracts of whole plants present hepatoprotective activities (Yang et al., 2014). We sequenced L. tetragonum from the western coastal area of Korea for investigating intraspecific variations on chloroplast genomes with chloroplast of L. tetragonum isolated in eastern seashore of Korea.

Materials and Methods

Plant material

We collected L. tetragonum in Aphaedo island in Shinan-gun, Jeollanam-do, Korea (34.8612N, 126.32629E). A voucher specimen and genomic DNA were deposited in the InfoBoss Cyber Herbarium (IN, the voucher number IB-00899).

DNA extraction and chloroplast genome determination

The total genomic DNA was extracted from fresh leaf by using a DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Genome sequencing was performed using HiSeqX at Macrogen Inc., Korea, and de novo assembly was done by Velvet v1.2.10 (Zerbino and Birney, 2008) and GapCloser v1.12 (Zhao et al., 2011). Assembled sequences were confirmed by BWA v0.7.17 (Li, 2013) and SAMtools v1.9 (Li et al., 2009). All bioinformatic analyses were conducted in the Genome Information System (http://geis.infoboss.co.kr/) utilized in the previous studies (Kim et al., 2018, 2019a, 2019b, 2019c, 2021; Bum et al., 2020; Park et al., 2021c).

Genome annotation was conducted based on another L. tetragonum chloroplast (MW085088.1) with Geneious R11 v11.0.5 (Biomatters Ltd, Auckland, New Zealand). A circular map of L. tetragonum chloroplast genome was drawn using OGDRAW v1.31 (Greiner et al., 2019).

Identification of intraspecific variations

Single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were identified using the ‘Find variations/SNPs’ function implemented in the Geneious R11 v11.0.5 (Biomatters Ltd, Auckland, New Zealand) based on the pairwise alignment of the two chloroplast genomes of L. tetragonum conducted by MAFFT 7.450 (Katoh and Standley, 2013). This method has been used in the previous studies (Kim et al., 2019a; Min et al., 2019a; Choi et al., 2021; Park et al., 2021b, 2021d). INDEL region was defined as the continuous INDELs.

Phylogenetic analysis

Maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees were constructed based on the multiple sequence alignment of all available nine Plumbaginaceae chloroplast genomes and the seven chloroplast genomes of non-core Caryophyllales clade (Crawly and Hilu, 2012; Yao et al., 2019) by MAFFT v7.450 (Katoh and Standley, 2013). The chloroplast genome of Nepenthes graciliflora Elmer (1912) (Yao et al., 2019) was marked as outgroup species. The ML tree was reconstructed in IQ-TREE v1.6.12 (Nguyen et al., 2015) with 1,000 bootstrap repeats. In the ML analysis, a heuristic search was used with nearest-neighbor interchange branch swapping, GTR+F + R4 model determined as the best fit model by the ModelFinder implemented in IQ-TREE, and uniform rates among sites. All other options used the default settings. The posterior probability of each node was estimated by the BI using MrBayes v3.2.6 (Huelsenbeck and Ronquist 2001). The HKY85 model with gamma rates was used as a molecular model. A Markov chain Monte Carlo algorithm was employed for 1,100,000 generations, sampling trees every 200 generations, with four chains running simultaneously. Trees from the first 100,000 generations were discarded as burn-in.

Data availablity

Chloroplast genome sequence can be accessed via accession number of MN044572 in GenBank of NCBI at https://www.ncbi.nlm.nih.gov. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA737051, SAMN19678692, and SRR14793527, respectively.

Results and Discussion

The chloroplast genome of L. tetragonum (GenBank accession of MN044572) isolated in Korea is 154,689 bp long (GC ratio of 37.0%) and had four subregions: 84,572 bp of large single copy (35.3%) and 12,813 bp of small single copy (31.5%) regions are separated by 28,562 bp of inverted repeat (IR; 40.9%) (Table 1), which is similar to those of the previously reported L. tetragonum chloroplast genome (MW085088) (Table 1). It contained 128 genes (83 protein-coding genes [PCGs], eight rRNAs, and 37 tRNAs); 14 genes (five PCGs, four rRNAs, and five tRNAs) were duplicated in the IR regions, which is also same to those of the previously reported L. tetragonum chloroplast genome (MW085088) (Fig. 1, Table 1).

List of two available chloroplast genomes of Limonium tetragonum.

Fig. 1.

Circular map of chloroplast genome of Limonium tetragonum isolated in Korea. Genes shown outside are transcribed clockwise, and those inside the circle are transcribed counter clockwise. Genes are color-coded to distinguish different functional groups. The dark grey and the light grey plot in the inner circle correspond to the GC content and AT content, respectively.

Based on the pair-wise sequence alignment with the previously reported L. tetragonum chloroplast genome (MW085088) (Darshetkar et al., 2021), 35 SNPs and 33 INDELs regions (88 bp in total) were identified. The longest INDEL region was 18-bp located between trnT and trnL. The inserted sequence was 18-bp repetitive sequences. In addition, two chloroplast genomes of Limonium bicolor (Bunge) Kuntze (Darshetkar et al., 2021) display extremely divergent manner, no SNP and 116 INDEL regions (15,840 bp in length), which is similar to the case of Phedimus takesimensis chloroplast genomes (129 SNPs and 112 INDEL regions (8,506 bp in length) (Park et al., in preparation).

Numbers of these intraspecific variations of L. tetragonum were smaller than those of Pseudostellaria palibiniana (Takeda) Ohwi (84 SNPs and 125-bp INDELs), Pyrus ussuriensis Maxim. (1,221 SNPs and 781-bp INDELs), Goodyera schlechtendaliana Rchb. f. (200 SNPs and 511-bp INDELs), Gastrodia elata Blume (324 SNPs and 630-bp INDELs) isolated in Korea; while they were larger than those of Artemisia fukudo Makino (seven SNPs and 12-bp INDELs) (Min et al., 2019b), Fagus multinervis Nakai (two SNPs and 2-bp INDELs) (Park and Oh, 2020), Aconitum coreanum (H. Lév.) Rapaics (five to 19 SNPs and 52-bp to 950bp INDELs) (Kim et al., 2019d), Viburnum erosum Thunb. (16 SNPs and 50-bp INDELs) (Choi et al., 2020), and Veronica nakaiana Ohwi (seven SNPs and 4-bp INDELs) (Lee et al., 2021). In addition, seven PCGs contained at least one SNP or INDELs, among which two synonymous SNPs were found in psbA and psaA (Table 2) and four non-synonymous SNPs were identified in rpl20, rpl32, and ycf2 located in the IR region (Table 2). The ratio of synonymous to non-synonymous SNPs was 0.5, which is different from the normal ratio, like Chenopodium album L. (Park et al., 2021a). One INDEL region was identified in the genic region of rpoC2, resulting the extension of rpoC2 length in comparison to the previously sequenced chloroplast genome (MW085088) (Table 2).

List of intraspecific variations identified from the two Limonium tetragonum chloroplast genomes.

Sixteen chloroplast genomes were used for constructing ML and BI phylogenetic trees. Both phylogenetic trees displayed that two L. tetragonum, collected from the East and West coastal areas of Korean peninsula, respectively, were clustered with high bootstrap value (Fig. 2). The sister species of L. tetragonum is controversial: L. tetragonum formed a sister group with Limonium aureum (L.) Mill. in both trees (Fig. 2), whereas it was clustered with L. bicolor in the previous study using chloroplast genomes (Darshetkar et al., 2021). The close relative of L. tetragonum was not clearly confirmed because the phylogenetic study using chloroplast and nuclear loci could not determine the relationship with Limonium sinense (Girard) Kuntze, Limonium tenellum (Turczaninow) Kuntze, and Limonium flexuosum (Linnaeus) Kuntze (Koutroumpa et al., 2018) and the study with the complete chloroplast genome of L. tetragonum displayed that L. bicolor was sister species (Darshetkar et al., 2021), however, number of taxa used in this study was limited due to lack of complete chloroplast genomes and L. bicolor was not included in the previous study. This incongruency of the phylogenetic relationship might be caused by different sequences in both studies. Moreover, the supportive value of the clade of L. tetragonum and L. bicolor was 73%, which was lower than those (100%) in this study (Fig. 2), suggesting that L. aureum might be a sister species of L. tetragonum. Most of Limonium phylogenetic studies were conducted with the lack of East Asian Limonium species (Lledó et al., 2005; Malekmohammadi et al., 2017; Koutroumpa et al., 2018), requiring the additional phylogenetic studies including East Asian Limonium species.

Fig. 2.

Maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees of 16 chloroplast genomes. Phylogenetic tree was drawn based on ML tree. The numbers above branches indicate support values of ML and BI trees, respectively.

Acknowledgements

This study was carried out with the support of the Honam National Institute of Biological Resources (HNIBR202101107).

Notes

Conflict of Interest

The authors declare that there are no conflicts of interest.

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Article information Continued

Fig. 1.

Circular map of chloroplast genome of Limonium tetragonum isolated in Korea. Genes shown outside are transcribed clockwise, and those inside the circle are transcribed counter clockwise. Genes are color-coded to distinguish different functional groups. The dark grey and the light grey plot in the inner circle correspond to the GC content and AT content, respectively.

Fig. 2.

Maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees of 16 chloroplast genomes. Phylogenetic tree was drawn based on ML tree. The numbers above branches indicate support values of ML and BI trees, respectively.

Table 1.

List of two available chloroplast genomes of Limonium tetragonum.

GenBank accession Length (bp) GC contents No. of genes



Whole LSC SSC IR Whole (%) LSC (%) SSC (%) IR (%) No. of PCGs No. of tRNAs No. of rRNAs
MN044572 154,689 84,572 13,013 28,562 37.0 35.3 31.5 40.9 83 37 8
MW085088 154,691 84,568 12,997 28,563 37.0 35.3 31.5 40.9 83 37 8

LSC, large single copy; SSC, small single copy; IR, inverted repeat ; PCG, protein-coding gene.

Table 2.

List of intraspecific variations identified from the two Limonium tetragonum chloroplast genomes.

No. Type Changed bases Coordination Type Position SNP type


MN044572 MW085088 MN044572 MW085088
1 SNP G A 485 485 Genic psbA Synonymous SNP
2 INDEL - AA 4,316 4,316–4,317 Intergenic trnK-rps16
3 SNP G A 4,549 4,551 Intergenic trnK-rps16
4 INDEL T - 6,256 6,258 Intergenic rps16-trnQ
5 INDEL - AA 6,670 6,671–6,672 Intergenic trnQ-psbK
6 INDEL - AAA 7,214 7,217–7,219 Intergenic psbK-psbI
7 INDEL - T 7,866 7,872 Intergenic trnS-trnG
8 INDEL - CGGGTCA 7,966 7,973–7,979 Intergenic trnS-trnG
9 INDEL - A 8,224 8,238 Intergenic trnS-trnG
10 INDEL T - 15,561 15,576 Intergenic rps2-rpoC2
11 INDEL - T 15,898 15,912 Genic rpoC2 Frameshift
12 SNP T G 27,306 27,321 Intergenic rpoB-trnC
13 SNP T G 27,307 27,322 Intergenic rpoB-trnC
14 SNP C G 27,308 27,323 Intergenic rpoB-trnC
15 SNP C A 27,309 27,324 Intergenic rpoB-trnC
16 SNP C A 27,310 27,325 Intergenic rpoB-trnC
17 INDEL - A 28,271 28,286 Intergenic trnC-petN
18 INDEL A - 31,349 31,435 Intergenic trnE-trnT
19 INDEL - AA 31,472 31,487-31,488 Intergenic trnE-trnT
20 SNP C A 31,768 31,785 Intergenic trnE-trnT
21 SNP A G 32,417 32,434 Intergenic trnT-psbD
22 INDEL AAAG - 36,846–36,849 36,863 Intergenic psbZ-trnG
23 SNP G A 42,109 42,122 Genic psaA Synonymous SNP
24 SNP A T 42,931 42,944 Intergenic psaA-ycf3
25 SNP A T 42,932 42,945 Intergenic psaA-ycf3
26 INDEL ATATATTTATAT ATTATA - 47,679–47,696 47,692 Intergenic trnT-trnL
27 INDEL - TAAT 47,873 47,868–47,871 Intergenic trnT-trnL
28 INDEL ATTTAAA - 48,687–48,693 48,686 Intronic trnL
29 SNP A G 49,304 49,296 Intergenic trnF-ndhJ
30 INDEL - AAAA 55,296 55288–55,291 Intergenic atpB-rbcL
31 INDEL - T 55,474 55,470 Intergenic atpB-rbcL
32 INDEL - A 57,897 57,894 Intergenic rbcL-accD
33 INDEL A - 61,499 61,497 Intergenic ycf4-cemA
34 INDEL - TCTA 63,882 63,879-63,882 Intergenic petA-psbJ
35 SNP G T 64,137 64,138 Intergenic petA-psbJ
36 SNP A T 64,138 64,139 Intergenic petA-psbJ
37 SNP A T 64,139 64,140 Intergenic petA-psbJ
38 SNP T G 64,140 64,141 Intergenic petA-psbJ
39 SNP G T 64,141 64,142 Intergenic petA-psbJ
40 SNP A T 64,142 64,143 Intergenic petA-psbJ
41 SNP G T 64,143 64,144 Intergenic petA-psbJ
42 SNP A C 64,146 64,147 Intergenic petA-psbJ
43 SNP A T 64,147 64,148 Intergenic petA-psbJ
44 SNP A C 64,148 64,149 Intergenic petA-psbJ
45 SNP C A 64,149 64,150 Intergenic petA-psbJ
46 SNP A T 64,150 64,151 Intergenic petA-psbJ
47 SNP A T 64,151 64,152 Intergenic petA-psbJ
48 SNP A C 64,152 64,153 Intergenic petA-psbJ
49 INDEL T - 65,511 65,512 Intergenic psbE-petL
50 INDEL T - 66,150 66,150 Intergenic petL-petG
51 INDEL TA - 68,210-68,211 68,209 Intergenic rpl33-rps18
52 INDEL - C 68,295 68,292 Intergenic rpl33-rps18
53 SNP T G 69,126 69,124 Genic rpl20 Non-synomynous SNP
54 INDEL CTTT - 69,359-69,362 69,357 Intergenic rpl20-rps12
55 SNP C A 71,121 71,115 Intronic clpP
56 INDEL A - 75,382 75,376 Intergenic psbH-petB
57 INDEL A - 80,610 80,603 Intergenic rps8-rpl14
58 INDEL - AAAA 81,060 81,052–81,055 Intergenic rpl14-rpl16
59 SNP C A 86,943 86,939 Genic ycf2 Non-synomynous SNP
60 INDEL - G 92,158 92,154 Intergenic ycf2-trnL
61 SNP A T 106,466 106,463 Intergenic trnR-trnN
62 SNP A T 115,749 115,746 Intergenic ndhF-rpl32
63 SNP G A 116,280 116,277 Genic rpl32 Non-synomynous SNP
64 INDEL - T 116,808 116,805 Intergenic rpl32-trnL
65 INDEL - TTT 123,377 123,375-123,377 Intronic ndhA
66 SNP T A 132,796 132,797 Intergenic trnN-trnR
67 INDEL - C 147,096 147,097 Intergenic trnL-ycf2
68 SNP G T 152,319 152,321 Genic ycf2 Non-synomynous SNP

SNP, single nucleotide polymorphism; INDEL, insertions and deletion.