Complete chloroplast genome of a rare and endangered plant species Phalaenopsis zhejiangensis: genomic features and phylogenetic relationship within Orchidaceae

Abstract

Phalaenopsis zhejiangensis is a rare and endangered plant species with extremely small populations. The complete chloroplast (cp) genome of P. zhejiangensis was assembled, its structural organization was described and comparative genomic analyses was carried out. The cp genome of P. zhejiangensis is 143,547 bp in length, with a GC content of 37.2%, which includes a pair of inverted repeats (IRs) of 24,464 bp separated by a small single-copy region of 10,764 bp and a large single-copy region of 83,856 bp. The cp genome contains 126 genes, consisting of 80 protein-coding genes, 38 transfer RNAs, and eight ribosomal RNAs. Six protein-coding genes, including ψndhB (two copies), ψndhD, ψndhG, ψndhK, and ψndhI, are identified as pseudogenes. Another six ndh genes, ndhA, ndhC, ndhE, ndhF, ndhH, and ndhJ, are missing from the plastid genome. A total of 41 cp simple sequence repeats (SSRs) were identified, including 40 mono-nucleotides and one di-nucleotides. Phylogenic analysis revealed P. zhejiangensis was nested inside the Phalaenopsis species and sister to P. wilsonii. The assembly and analysis of P. zhejiangensis cp genome will provide essential data for further study of taxonomy and systematics of Orchidaceae.

Keywords:

• Phalaenopsis zhejiangensis
• rare and endangered species
• chloroplast genome
• phylogenetic analysis

Introduction

Orchidaceae, a family of monocotyledonous plants, is one of the most successful and highly evolved monocots, comprising 763 genera with approximately 28,000 species (Hadi et al. 2015; Christenhusz and Byng 2016). The genus Phalaenopsis belongs to tribe Vandeae, which is commonly known as moth orchids, including more than 70 species widely distributed across Southeast Asia to northern Australia, with the majority in Indonesia and the Philippines (Ko et al. 2017; Gogoi and Rinya 2020; Lee et al. 2020). There are 12 Phalaenopsis species distributed in China, among which four species are endemics (Wu et al. 2009). Phalaenopsis zhejiangensis is a small epiphytic plant with slightly flattened roots, very short stems, basal leaves, and thinly textured flowers (Wu et al. 2009). P. zhejiangensis was transferred from Doritis zhejiangensis, while the latter was renamed from Nothodoritis zhejiangensis (Yukawa and Kita 2005), which was first found on a trunk of Podocarpus macrophyllus in 1970 at Xitianmu Mountain in Linan County, Zhejiang Province (Tsi 1989; Schuiteman 2012). P. zhejiangensis is a rare and endangered plant species in China, which is narrowly distributed in provinces of Zhejiang Province, Gansu, Anhui, and Shanxi, growing on the bark of trees or rock surfaces, with extremely small populations. As a result of habitat loss, P. zhejiangensis is listed as threatened with extinction. Fortunately, Zeng et al (2011) have established an effective propagation protocol for large-scale propagation of this endangered orchid species. In recent years, several chloroplast (cp) genomes of Phalaenopsis plants, including P. aphrodite, Phalaenopsis ‘Tiny Star’, P. lowii, P. lobbii, and P. mannii, were sequenced and assembled (Chang et al. 2006; Kim et al. 2016; Wang et al. 2019; Chen et al. 2020; Zhang et al. 2020). However, the cp genome of P. zhejiangensis has not yet been assembled. In our study, the complete cp genome sequence of P. zhejiangensis was assembled to provide new insights into taxonomy and systematics of Orchidaceae.

Materials and methods

Plant material and DNA extraction

Fresh leaves were collected from a plant nursery (28°65.762′N, 121°46.976′E) in Jiaojiang, Zhejiang Province, China. A voucher specimen was deposited at the Molecular Biology Laboratory in Taizhou University (http://www.tzc.edu.cn, Dr. Huijuan Zhang, [email protected]) under a collection number of CHS2020029. Fresh leaves were ground into a fine powder in liquid nitrogen with a mortar and pestle, and genomic DNA was extracted following the CTAB-based protocol as described by Doyle and Doyle (1987).

DNA sequencing and sequence assembly

Construction of a genomic DNA library was carried out according to the manufacturer’s instructions, and high-throughput sequencing was then performed using the Illumina Hiseq X Ten system (Illumina, San Diego, CA). Approximately, 3.64 Gb raw data of 150 bp paired-end Illumina reads were generated, and 3.63 Gb high-quality clean reads were harvested by using the NGS QC Toolkit v2.3.3 (Patel and Jain 2012). Geneious Prime 2019 (Biomatters, Auckland, New Zealand) was employed to assemble the complete cp genome.

Chloroplast genome annotation

Annotation of the complete cp genome was performed with Geneious Prime 2019 by using P. lobbii as a reference genome (NCBI accession number: MT830847) (Wang et al. 2019), and the borders of each gene were manually inspected and corrected where necessary. The circular cp genome map of P. zhejiangensis was drawn using the online program OrganellarGenomeDRAW (OGDRAW, http://ogdraw.mpimp-golm.mpg.de) with default settings (Lohse et al. 2013).

Simple sequence repeat analysis

Perl scripts of MIcroSAtellite Identification Tool (MISA) were used to identify and locate the potential simple sequence repeats (SSRs) loci in complete cp genome sequence of P. zhejiangensis (Thiel et al. 2003). To determine the presence of SSRs, 1–6 bp nucleotide motifs were considered, and the minimum repeat numbers were set as 10 repeat units for mono-nucleotides, six for di-nucleotides, and five for tri-, tetra-, penta-, and hexa-nucleotides.

Phylogenetic analysis

The complete cp genome sequences of 39 Orchidaceae species downloaded from NCBI were used for phylogenetic analysis, which included eight Phalaenopsis plants, six Holcoglossum species, and five Cleisomeria plants. The complete cp genome of Coix lacryma-jobi (FJ261955) was applied as an outgroup. A total of 41 genome sequences were aligned with a multiple sequence alignment program MAFFT v7.388 plugin in Geneious Prime 2019 under default settings (Katoh and Standley 2013). The best-fit evolutionary model of DNA substitution for maximum-likelihood (ML) analysis was determined by using jModelTest 2.1.9 under the Akaike information criterion (AIC) (Darriba et al. 2012). A phylogenetic tree was generated by PhyML 3.1, with 1000 bootstrap replicates (Guindon et al. 2010).

Results

Genome sequencing and assembly

More than 12 million paired-end clean reads were obtained, and a circular cp genome was assembled with Geneious Prime. The complete cp plastome of P. zhejiangensis is 143,547 bp in length which includes a pair of inverted repeats (IRs) of 24,464 bp separated by a large single-copy (LSC) region of 83,855 bp and a small single-copy (SSC) region of 10,764 bp (Figure 1). The overall guanine–cytosine (GC) content of P. zhejiangensis cp genome is 36.8%, while the corresponding values of LSC, SSC, and IR sequences are 34.0%, 28%, and 43.5%, respectively.

Figure 1. The chloroplast genome of Phalaenopsis zhejiangensis. From the center going outward, the four circles indicate scattered forward and reverse repeats, tandem repeats, microsatellite sequences identified, and gene structure of the plastome.

Genome features of Phalaenopsis zhejiangensis

The complete cp plastome of P. zhejiangensis is comprised of 126 genes, including 74 protein-coding genes, 38 tRNA genes, eight rRNA genes, and six pseudogenes. All the six pseudogenes are ndhs, including ψndhD, ψndhG, ψndhI, ψndhK, and two copies of ψndhB. Additionally, other six ndh genes have been lost in the plastome, these are ndhA, ndhC, ndhE, ndhF, ndhH, and ndhJ. Six tRNA genes (trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC) and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23) are present in two copies. Among the protein-coding genes, eight genes including rpl16, rps16, rpoC1, petB, petD, atpF, and two copies of rpl2 contain a single intron, while three genes, rps12, ycf3, and clpP, contain two introns. Moreover, six tRNA genes, trnA-UGC, trnG-GCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC, harbored a single intron have been identified (Table 1).

Table 1. List of genes in the chloroplast genome of Phalaenopsis zhejiangensis.

Group of genes	Name of genes	Total number
Large subunit of ribosomal proteins	rpl2 (×2)*, rpl14, rpl16*, rpl20, rpl22, rpl23 (×2), rpl32, rpl33, rpl36	11
Small subunit of ribosomal proteins	rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 (×2)**, rps14, rps15, rps16*, rps18, rps19(×2)	15
DNA-dependent RNA polymerase	rpoA, rpoB, rpoC1*, rpoC2	4
Ribosomal RNA genes	rrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2)	8
Transfer RNA genes	trnA-UGC (×2)*, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC*, trnG-GCC, trnH-GUG (×2), trnI-CAU (×2), trnI-GAU (×2)*, trnK-UUU*, trnL-CAA (×2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU (×2), trnP-UGG, trnQ-UUG, trnR-ACG (×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (×2), trnV-UAC*, trnW-CCA, trnY-GUA	38
Photosystem I	psaA, psaB, psaC, psaI, psaJ, ycf3**	6
Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	15
Cytochrome b6/f complex	petA, petB*, petD*, petG, petL, petN	6
NADH dehydrogenase	ψndhB (×2), ψndhD, ψndhG, ψndhI, ψndhK	6
ATP synthase	atpA, atpB, atpE, atpF*, atpH, atpI	6
Rubisco	rbcL	1
Translational initiation factor	infA	1
Maturase	matK	1
Protease subunit P	clpP**	1
Envelop membrane protein	cemA	1
Subunit acetyl-CoA carboxylate	accD	1
c-type cytochrome synthesis gene	ccsA	1
Conserved open reading frames	ycf1, ycf2 (×2), ycf4	4
Total		126

(ψ) pseudogene; *one intron; **two introns.

IR expansion and contraction

The IR region of P. zhejiangensis is 24,464 bp in length, while the other eight Phalaenopsis IRs included in our study are longer than P. zhejiangensis, they range from 24,719 bp to 25,846 bp (Table 2). Comparisons of LSC, IRB, SSC, and IRA junction boundaries among nine Phalaenopsis cp genomes were performed and presented in Figure 2. In Phalaenopsis plastome, LSC/IRB boundary junctions lie within rpl22 gene except P. mannii, which locates in the intergenic region between rpl22 and rps19. Eight rpl12 genes extend 31 bp into IRB regions, while the rpl12 in P. wilsonii extends only one base pair into its IRB region (Figure 2). The IRB/SSC junctions of P. mannii, P. equestris, and P. lobbi locate within the ψycf1 pseudogenes; however, no ψycf1 is identified in other six Phalaenopsis cp genomes including P. zhejiangensis. The SSC/IRA junctions of P. wilsonii, P. mannii, P. equestris, and P. lobbii lie within ycf1 genes, which extend 0 bp, 132 bp, 9 bp, and 108 bp into IRA regions, respectively while the SSC/IRA junctions of P. zhejiangensis, P. lowii, P. japonica, P. aphrodite subsp. formosana, and the hybrid ‘Tiny Star’ locate between ycf1 and trnN-GUU.

Figure 2. Structural variations at inverted-repeat and single-copy borders in nine Phalaenopsis chloroplast genomes. The figure features are not to scale regarding sequence length.

Table 2. Details of nine Phalaenopsis chloroplast genomes.

Plant species	Accession number in NCBI	Genome length (bp)	GC contents (%)	LSC (bp)	SSC (bp)	IR (bp)
Phalaenopsis aphrodite subsp. formosana	AY916449	148,964	36.7	85,957	11,543	25,732
Phalaenopsis equestris	JF719062	148,959	36.7	85,967	11,300	25,846
Phalaenopsis hybrid ‘Tiny Star’	NC_025593	148,918	36.7	85,885	11,523	25,755
Phalaenopsis japonica	NC_046808	146,942	36.8	84,882	10,568	25,746
Phalaenopsis lobbii	MT830847	144,607	36.6	83,482	11,687	24,719
Phalaenopsis lowii	NC_050652	146,834	36.9	84,616	10,473	25,633
Phalaenopsis mannii	NC_050940	148,596	36.7	85,300	11,640	25,828
Phalaenopsis wilsonii	MW218959	145,373	36.9	84,995	10,668	24,855
Phalaenopsis zhejiangensis	MZ326749	143,574	38.7	83,854	10,764	24,464

SSR analysis

Totally, P. zhejiangensis cp genome contains 41 SSRs (Table 3). Among the SSRs identified herein, A/T mononucleotide repeats account for the largest proportion of 97.56%. Only one di-nucleotide repeat was observed, and no tri-, tetra-, penta-, or hexa-nucleotides presented in the plastome. Most SSRs are distributed in LSC (70.73%), followed by SSC (24.39%), and the proportion in IRs is less than 5%. Among 41 SSRs, 25 loci lie in intergenic regions, four in introns, and 12 in coding regions or pseudogenes.

Table 3. Repeat sequences and their distribution within Phalaenopsis zhejiangensis chloroplast genome.

Number	Motif	Size	Start	End	Location
1	(T)10	10	4245	4254	IGS
2	(A)10	10	7693	7702	IGS
3	(T)10	10	9184	9193	IGS
4	(T)10	10	9599	9608	IGS
5	(T)17	17	14,113	14,129	IGS
6	(T)12	12	17,053	17,064	IGS
7	(A)16	16	27,913	27,928	IGS
8	(T)10	10	28,756	28,765	IGS
9	(A)11	11	32,765	32,775	IGS
10	(T)10	10	33,072	33,081	IGS
11	(A)10	10	33,357	33,366	IGS
12	(A)10	10	37,057	37,066	IGS
13	(A)11	11	47,194	47,204	IGS
14	(A)10	10	47,875	47,884	IGS
15	(A)11	11	56,140	56,150	IGS
16	(T)12	12	62,492	62,503	IGS
17	(A)10	10	64,380	64,389	IGS
18	(T)10	10	64,612	64,621	IGS
19	(T)10	10	71,393	71,402	IGS
20	(T)10	10	79,492	79,501	IGS
21	(T)10	10	83,505	83,514	IGS
22	(T)10	10	98,214	98,223	IGS
23	(A)11	11	108,703	108,713	IGS
24	(T)10	10	113,355	113,364	IGS
25	(A)10	10	129,180	129,189	IGS
26	(A)10	10	111,423	111,432	ψndhD
27	(A)10	10	111,579	111,588	ψndhD
28	(TA)8	16	111,722	111,737	ψndhD
29	(T)10	10	49,573	49,582	ψndhK
30	(A)12	12	74,999	75,010	petB intron
31	(T)10	10	82,386	82,395	rpl16 intron
32	(A)11	11	23,345	23,355	rpoC1 intron
33	(T)11	11	69,574	69,584	clpP
34	(A)10	10	69,867	69,876	rps12
35	(T)10	10	9805	9814	trnG-GCC intron
36	(T)12	12	114,415	114,426	ycf1
37	(T)11	11	115,271	115,281	ycf1
38	(T)13	13	116,348	116,360	ycf1
39	(T)10	10	117,578	117,587	ycf1
40	(T)10	10	117,695	117,704	ycf1
41	(A)13	13	45,453	45,465	ycf3

IGS: intergenic spacer region.

Phylogenetic analysis

Statistical selection of the best-fit model of nucleotide substitution was carried out by the jModelTest program, and GTR + G+I was turned out to be the best-fitted. To evaluate the phylogenetic relationship of P. zhejiangensis within Orchidaceae, a phylogenetic tree using the complete plastome sequences of 41 plant species were generated (Figure 3). The resulting ML tree had very high bootstrap support for the majority of clades, and the phylogenetic tree was shown to be consistent with the traditional morphology-based taxonomy of Orchidaceae. Nine plants from the genus Phalaenopsis formed a well-supported monophyletic clade with 100% bootstrap value, and P. zhejiangensis was sister to P. wilsonii, with a support value of 97%.

Figure 3. The maximum-likelihood tree inferred from 41 Orchidaceae complete chloroplast genomes under GTR + G+I model with maximum-likelihood value (Llk)=–604333.07383, Akaike information criterion (AIC)=1208844.14766, and Bayesian information criterion (BIC)=1209758.57750.

Discussion

The classification of Orchidaceae is a very complex problem for its bewildering diversity and morphological parallelism, and also for botanical neglect (Dressler 1993). Orchidaceae were divided into five subfamilies, including Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae, and Apostasioideae are monophyletic and sister to other subfamilies (Dixon et al. 2003; Tsai et al. 2013). The subfamily Orchidoideae is comprised of a large group of orchids, including four tribes (Diurideae, Cranichideae, Codonorchideae, and Orchideae) with around 3600 plant species that largely share terrestrial habits (Bateman et al. 2003; Serna-Sánchez et al. 2021). The genus Phalaenopsis belongs to tribe Orchideae, which consists of about 62 genera (Inda et al. 2012). The morphology of P. zhejiangensis is similar to both Doritis and P. lowii, with the characteristic feature of pollinia similar to that of Doritis, and its shapes of petals and columns similar to P. lowii (Tsi 1999; Christenson 2001). P. zhejiangensis was initially named N. zhejiangensis based on morphology, and then renamed as D. zhejiangensis (Yukawa and Kita 2005; Schuiteman 2012). Molecular analyses using ITS, trnL intron as well as trnL-F spacer prove that N. zhejiangensis is species nested in genus Phalaenopsis (Deng et al. 2015). Our molecular phylogenetic analyses confirm its systematic position, which indicates that P. zhejiangensis is sister to P. wilsonii, with a high support value.

The sizes of previously published Phalaenopsis cp genomes ranged from 144,607 bp (P. lobbii) to 148,964 bp (P. phalaenopsis aphrodite subsp. formosana) (Chang et al. 2006; Kim et al. 2016; Wang et al. 2019; Chen et al. 2020; Zhang et al. 2020). In our present study, the complete plastid genome of P. zhejiangensis is only 143,574 bp in length, which turns out to be the smallest among all known cp genome sequences in genus Phalaenopsis. The cp genomes of most flowering plants range from 120 kb to 160 kb, and the difference in genome size is mainly caused by contraction and expansion of the IR region (Goulding et al. 1996; Ingvarsson et al. 2003; Yao et al. 2020). The IR sizes of published Phalaenopsis plastid genomes are 24,719 (P. lobbii) (Wang et al. 2019) to 25,846 bp (P. equestris) (Jheng et al. 2012). In our present study, the length of P. zhejiangensis IR sequence is 24,464 bp, which is smaller than that of P. lobbii.

Plastid gene loss and pseudogenization are common phenomena in parasitic, semi-parasitic, and saprophytic plants (Molina et al. 2014; Li and Zheng 2018; Nie et al. 2019). In semi-parasitic plants of Macrosolen cochinchinensis, M. tricolor, and M. bibracteolatus, the infA and all the ndh genes were lost among the three species, and two genes, ycf1 and rpl2, were found to be pseudogenes (Nie et al. 2019). Gastrodia elata is a saprophytic plant with extremely small cp genome in which many genes related to photosynthesis are missing, and it contains only 28 genes, including 20 protein-coding genes, three rRNAs, and five tRNAs (Park et al. 2020). However, cp gene loss and pseudogenization are rarer in photosynthetic species, because the plastid gene cannot simply be discarded (Magee et al. 2010; Wicke et al. 2011; Daniell et al. 2016). Interestingly, pseudogenization, truncation, and deletion of ndh genes in cp genomes are common in some cp genomes of photosynthetic orchid plants (Kim et al. 2015). Orchids like Apostasia odorata, Sobralia callosa, Paphiopedilum armeniacum, and Phragmipedium longifolium retain the complete set of ndh genes, whereas most of the ndh genes in P. equestris, Dendrobium officinale, and D. catenatum were found to be lost in their plastids (Kim et al. 2015; Lin et al. 2017). In this study, six ndh genes are pseudogenized, and another six ndh genes are missing from the plastid genome of Phalaenopsis zhejiangensis.

Authors contributions

Conceived and designed the experiments: MJ and HJZ. Performed the experiments: MJ, WQ, and ZY. Analyzed the data: HZW and LQP. Contributed to reagents/materials/analysis tools: MJ and WQ. Wrote the paper: MJ and HJZ.

Disclosure statement

The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/nuccore/MZ326749. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA734444, SRR14710950, and SAMN19491762, respectively.

Additional information

Funding

This work was supported by 211 Talent Training Fund of Taizhou under Grant number [2012].