The complete chloroplast genome sequence of Calanthe sieboldii (orchidaceae)

Abstract

Calanthe sieboldii Decne. ex Regel is a terrestrial orchid with high ornamental and commercial value. In the present study, the chloroplast genome of C. sieboldii was characterized using Illumina technology. The chloroplast genome is 158,345 bp in length with a total AT content of 63.28%. There are 127 genes, comprising 37 tRNA genes, 82 protein-coding genes, and 8 rRNA genes. Phylogenetic relationship analysis was performed using common protein-coding genes extracted from 13 chloroplast genomes of Orchidaceae. It was revealed that C. sieboldi was sister to C. hancockii and closely clustered with C. aristulifera and C. henryi. These findings provide valuable genomic resources that are helpful for further phylogenetic and evolutionary studies of Calanthe.

Introduction

Calanthe sieboldii Decne. ex Regel is a terrestrial orchid with bright yellow flowers emitting fragrance and has been cultivated as an excellent breeding parent with high ornamental and commercial value (Huang et al. 2022). It has a synonym of Calanthe striata. The native range of this species is China (Hunan), S. Korea, W. Central & S. Japan to SW. Taiwan (https://powo.science.kew.org/). It grows in the mountainous forests at altitudes from 1200 to 1500 m (Chen et al. 2009). The wild populations of C. sieboldii are tiny, which was categorized as a critically endangered species (National Forestry and Grassland Administration 2021). It has been classified as a national first-level key protected wild plant in China and has been registered in the Information System of Chinese Rare and Endangered Plants (ISCREP) (http://www.iplant.cn/rep). The chloroplast genome has been extensively studied in plant molecular evolution and systematics due to its small size and highly conserved genomic structure. Recent years, chloroplast genomes of Calanthe were sequenced for phylogenetic analysis (Chen et al. 2019; Miao et al. 2019; Zhong et al. 2019; Zhang et al. 2020). However, the chloroplast genome of C. sieboldii has not been reported yet. In this study, we used Illumina high-throughput sequencing technique to obtain the chloroplast genome of C. sieboldii and compared to other species in Calanthe. Our primary objectives were to provide a valuable genomic resource to study the features of the chloroplast genome of C. sieboldii and to determine its phylogenetic position.

Materials and methods

Plants of C. sieboldii were grown in Jindong, Qiyang, Yongzhou, Hunan, P. R. China (26°17′56.62″ N, 112°05′35.84″ E) (Figure 1). We rinsed the leaves quickly twice with sterile distilled water, then chilled them in a dry ice-ethanol bath. The voucher specimen (accession no. JDQ_2022_QYZ_CasHu) was deposited in a chest freezer at Hunan Academy of Forestry (HAF, Website: http://www.hnlky.cn/, Contact: Xujun Wang, E-mail: xjwang0514@sina.com).

Figure 1.

The morphological characteristics of C. sieboldii. (A) Habit, (B) Inflorescence. The photograph was taken by Xujun Wang from Jindong, Qiyang, Yongzhou, Hunan, P. R. China (26°17′56.62″ N, 112°05′35.84″ E). The most characteristic feature of the specimen: flowers bright yellow, large, slightly fleshy; dorsal sepal elliptic; petals narrowly elliptic; lip adnate to entire length of column wings, yellow, mottled red at base.

We extracted total genomic DNA (gDNA) from 400 mg of leaf samples using the CTAB protocol (Doyle and Doyle 1987). The concentration, integrity and purity of the extracted gDNA was assessed using Qubit^® RNA Assay Kit in Qubit^® 3.0 Flurometer (Life Technologies, Carlsbad, CA, USA), agarose (1%) gel electrophoresis and NanoDrop 8000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Sonication was utilized to cleave the qualified gDNA into 350 bp fragments (Covaris, Woburn, MA, USA). The final library was obtained after DNA purification, terminal repair, A-tailing, adapter ligation and PCR amplification. Subsequently, a sequencing run was conducted on an Illumina HiSeq X platform (Illumina, San Diego, CA, USA). We got 90,472,192 clean reads and aligned the reads to the reference chloroplast genome of C. hancockii (GenBank: NC_064068). Finally, we assembled the chloroplast genome of C. sieboldii with the software SPAdes v3.11.1 (Bankevich et al. 2012) and annotated it with both of DOGMA (Wyman et al. 2004) and CPGAVAS (Liu et al. 2012) softwares. We extracted common protein-coding genes from the 13 complete chloroplast sequences through PhyloSuite (Zhang et al. 2020), then aligned them using MAFFT v7.313 (Katoh and Standley 2013) plugin integrated into PhyloSuite v1.2.1. A maximum likelihood phylogenetic tree was reconstructed using IQ-TREE v1.6.8 (Nguyen et al. 2015) under the GTR + F + I + G4 model for 5000 ultrafast bootstraps. Finally, the phylogenetic tree was visualized using FigTree v1.4.4.

Results

The average coverage depth of the assembly of the C. sieboldii chloroplast genome turned out to be 1,461 bp (Figure S1). The entire chloroplast genome (GenBank: OP270615) is 158,345 bp in size (Figure 2). It has a typical quadripartite structure, containing 87,204 bp large and 18,429 bp small single-copy regions with AT contents of 65.70 and 70.28%, respectively. The small and large single-copy regions are divided by two inverted repeat regions of 26,358 bp. Within the chloroplast genome, there are in total 127 genes, comprising 37 tRNA genes, 82 protein-coding genes, and 8 rRNA genes.

Figure 2.

Circular map of the C. sieboldii chloroplast genome. Genes shown inside and outside the circle are transcribed counterclockwise and clockwise, respectively. Different colors represent genes with different types of function. The dark and light grey in the dashed area in the inner circle indicates the GC and at contents, respectively. LSC: large single-copy region; IR: inverted repeat; SSC: small single-copy region.

In total, we observed 18 intron-containing genes, comprising 16 genes (rps16, trnS-CGA, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, rpl2, ndhB, trnE-UUC, trnA-UGC, ndhA, trnA-UGC_copy2, trnE-UUC_copy2, ndhB_copy2, rpl2_copy2), each containing one intron, and two genes (ycf3 and clpP), each containing two introns (Figure S2). The single 5′-end of the trans-spliced gene rps12 is positioned in the large single-copy region, while repeated 3′-end exons are located in the two inverted repeat regions (Figure S3).

To determine the phylogenetic position of C. sieboldii, a phylogenetic analysis was conducted using common protein-coding genes extracted from chloroplast genomes of 11 Calanthe and two Phaius species (Table S1). Two Phaius species were choosen as the outgroup because they were the closest genetic relatives of the Calanthe. The results revealed that the C. sieboldi is sister to C. hancockii, and they cluster together in a clade. The clade is closely associated with C. henryi and C. aristulifera (Figure 3).

Figure 3.

Phylogenetic tree based on common protein-coding genes of 13 species of the family orchidaceae. The following sequences were used: C. sieboldii OP270615 (this study), C. hancockii NC_064068 (unpublished), C. aristulifera NC_046812 (Ou et al. 2022), C. henryi NC_054177 (Ou et al. 2022), C. reflexa NC_062707 (unpublished), C. davidli NC_037438 (Miao et al. 2019), C. sylvatica NC_044633 (Ou et al. 2022), C. herbacea NC_062706 (unpublished), C. triplicata NC_024544 (Miao et al. 2019), C. lyroglossa NC_050867 (Ou et al. 2022), phaius hainanensis NC_057607 (Ou et al. 2022), P. tancarvilleae NC_050866 (Wang et al. 2021), C. arcuata OK075084 (unpublished). Supports for nodes were calculated via 1000 standard bootstrap replicates. Scale bar = 0.003.

Discussion and conclusion

In this investigation, we successfully assembled the chloroplast genome sequence of C. sieboldii for the first time and provided annotations for it. It showed that the genome size, AT content and gene composition of the chloroplast genome sequence of C. sieboldii were similar to those of other species of the genus (Chen et al. 2019; Miao et al. 2019; Zhong et al. 2019; Zhang et al. 2020). Phylogenetic analysis demonstrated that C. sieboldi exhibited the closest relationship with C. hancockii. Overall, our study provides valuable genetic data for phylogenetic and evolutionary studies of the genus Calanthe.

Funding Statement

This work was supported by the Science and Technology Innovation Platform and Talent Plan of Hunan Province [2016TP2009].

Ethical approval

The material involved in the article does not involve ethical conflicts. This species is not collected from a natural reserve, so it did not need specific permissions or licenses. This work was approved by Jindong Forest Farm of Yongzhou City and complies with the Regulations of the People’s Republic of China on the Protection of Wild Plants, the International Union for Conservation of Nature (IUCN) policies research involving species at risk of extinction, the Convention on Biological Diversity and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Author contributions

CP and XW conceived the project; CP, DL and KL performed the experiments; XW and WG analyzed the data; CP, XW and WG wrote the paper. All authors agree to be accountable for all aspects of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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 accession no. OP270615. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA872222, SRR21168988, and SAMN30449765, respectively.