The complete chloroplast genome of Dendrobium nobile, an endangered medicinal orchid from north-east India and its comparison with related Dendrobium species.

The medicinal orchid genus Dendrobium belonging to the Orchidaceae family is a huge genus comprising about 800-1,500 species. To better illustrate the species status in the genus Dendrobium, a comparative analysis of 33 available chloroplast genomes retrieved from NCBI RefSeq database was compared with that of the first complete chloroplast genome of D. nobile from north-east India based on next-generation sequencing methods (Illumina HiSeq 2500-PE150). Our results provide comparative chloroplast genomic information for taxonomical identification, alignment-free phylogenomic inference and other statistical features of Dendrobium plastomes, which can also provide valuable information on their mutational events and sequence divergence. ©2019 Konhar et al.

Introduction

Dendrobium is a huge genus of the tribe Dendrobieae (Orchidaceae: Epidendroideae) that was established by Olof Swartz in 1799. It includes approximately 800–1,500 species and occurs in diverse habitats throughout much of Southeast Asia, including China, Japan, India, and the Philippines, Indonesia, New Guinea, Vietnam, Australia and many of the islands in the Pacific (Wood, 2006).

Many species and cultivars of this genus are well-known floral motifs and have featured in artwork. Dendrobium orchids are popular not only for their visual appeal in cut flower market, but also for their herbal medicinal history of about 2,000 years in east and south Asian countries (Bulpitt et al., 2007). Many species in this genus have been extensively used as herbal medicines for several hundreds of years in treating diseases like kidney and lung ailments, gastrointestinal problems, lumbago and arthralgia. The plant extracts are also used as tonic for strengthening body’s immunity and improving sexual potency. However, many Dendrobium species in the wild face an extreme threat of extinction due to their low germination and slow growth rate, habitat decline and over exploitation arising out of anthropogenic activities  (Kong et al., 2003).

Dendrobium orchids have overwhelmed researchers because of their high economic importance in global horticultural trade and in Asian traditional medicine leading to extensive plant systemic studies particularly in species identification, novel marker development, breeding and conservation. In the past two decades, promising advances have been made in areas of molecular taxonomy, plant systematics and selective breeding of Dendrobium species by intensive use of molecular markers. Recently, a variety of molecular markers like microsatellite (SSR), Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) markers including several other DNA barcode markers from different loci of nuclear and chloroplast (cp) regions have been developed to study Dendrobium diversity. However, these species are notoriously difficult to identify (Teixeira da Silva et al., 2016).

The complete chloroplast genome usually contains a uniparentally inherited DNA, a feature which makes it an obvious choice for plant taxonomical analyses, phylogenomics and phylogeographic inferences at different taxonomic levels. One such classic example is the study of phylogenetic relationships among all families in the Order Liliales, based on 75 plastid genes from 35 species in 29 genera and 100 species spanning all monocot and major eudicot lineages, where underlying results were calibrated against 17 fossil dates to redefine the monocot evolutionary timelines (Givnish et al., 2016b). The significance of plastome-scale data was very well demonstrated in another study that highlighted a new functional model for understanding monocot evolution and some of their derived morphological features by way of convergent evolution from submersed aquatic ancestors (aquatic Hydatellaceae) (Givnish et al., 2018). The evolution of orchids, the largest and most diverse family of flowering plants second only to Asteraceae on Earth has long puzzled Charles Darwin and many other scientists. Recent advances in chloroplast genomics are giving researchers insights into the evolutionary history of these plants. One such study hypothesizes orchids to have arisen in Australia 112 Ma followed by migration to the Neotropics via Antarctica by 90 Ma. With the use of a combination of plastid genes, it was established that orchids and epidendroids exhibited maximally accelerated net diversification in Southeast Asia and the Neotropics respectively (Givnish et al., 2016a).

Studies pertaining to plastome genome sequences are useful in investigating the maternal inheritance in plants, especially those with polyploid species, owing to their high gene content and conserved genome structure (Birky, 1995; Soltis & Soltis, 2000; Song et al., 2002). Many species of orchids and other flowering plants exhibit rapid evolution and diversity. One of the main reasons for such diversity can be attributed to allopolyploidy or genetic redundancy, in which there are more than one gene involved in performing a particular task. In cases of useful mutation, plants evolve into new species. Hybridization and polyploidy are the decisive forces behind evolution and speciation. In the past there have been studies where a combination of AFLPs, cpDNA markers and flow cytometry was harnessed to investigate the evolutionary outcomes of hybridization between two endemic Ecuadorian species of Epidendrum (Orchidaceae) in three hybrid zones. The outcome of this study highlights the importance of hidden hybrid genotypes and their frequency which could help unravel the mysteries behind orchid evolution (Marques et al., 2014). The advent of high-throughput sequencing technologies has enabled a rapid increase in the rate of completion of cp genomes with faster and cheaper methods to sequence organellar genomes (Saski et al., 2007; Cronn et al., 2008). At the time of writing this manuscript, cp genomes from 33 Dendrobium species have been reported as per NCBI Organellar genome records (https://www.ncbi.nlm.nih.gov/genome/browse#!/organelles/dendrobium).

D. nobile Lindl. is one of the many highly prized medicinal plants in the genus Dendrobium. It is an endangered medicinal orchid listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II that demands immediate attention for its protection and propagation. Here, we report the first complete cp genome of D. nobile from north-east India based on next-generation sequencing methods (Illumina HiSeq 2500-PE150) and further compare its structure, gene arrangement and microsatellite repeats with 33 existing cp genomes of Dendrobium species. Our results provide comparative chloroplast genomic information for taxonomical identification, phylogenomic inference and other statistical features of Dendrobium plastomes. These can give further insights into their mutational events and sequence divergence. The availability of complete cp genome sequences of these species in the genus Dendrobium will benefit future phylogenetic analyses and aid in germplasm utilization of these plants.

Materials and Methods

Sample collection, DNA extraction and sequencing

Fresh leaves of D. nobile were collected from plants growing in greenhouses of National Research Centre for Orchids, Sikkim, India and voucher specimen was deposited in Botanical Survey of India as well as in the Department of Botany, North-Eastern Hill University, Shillong. The high molecular weight cpDNA was extracted using a modified CTAB buffer, and treated according to a standard procedure for next generation sequencing on Illumina HiSeq 2500-PE150. The quality and quantity of the genomic DNA was assessed through agarose gel electrophoresis, Nanodrop and Qubit detection method. The experiments included both paired-end and mate-pair libraries. Tagmentation was carried out with ∼4 µg of Qubit quantified DNA and the tagmented sample was washed using AMPURE XP beads (Beckman Coulter #A63881) and further exposed to strand displacement. The strand-displaced sample of 2–5 kb and 8–13 kb gel was size selected and taken for overnight circularization. The linear DNA was digested using DNA Exonuclease. Further the circularized DNA molecules were sheared using Covaris microTUBE, S220 system (Covaris, Inc., Woburn, MA, USA) for obtaining fragments in the range 300 to 1,000 bp. M280 Streptavidin beads (ThermoFisher Scientific, Waltham, MA) was used to cleanse the sheared DNA fragments with biotinylated junction adapters. The bead-DNA complex was subjected to End Repair, A-Tailing and Adapter ligations.

Data processing

The data quality assessment for Illumina WGS raw reads was carried out using FastQC tool. Perl scripts were written for adapter clipping and low quality filtering. Chloroplast genomes of D. officinale, D. huoshanense and D. strongylanthum retrieved from NCBI-RefSeq database was used as reference for the assembly. BWA-MEM algorithm with default parameter settings was used for aligning the adapter clipped and low quality trimmed processed reads with the Dendrobium cp genomes (Li & Durbin, 2009). SPAdes-3.6.0 program was used for k-mer based (k-mer used 21, 33, 55 and 77) de-novo assembly with the aligned reads and the quality of the assembled genome was gauged using Samtools and Bcftools (read alignment and genome coverage calculation)  (Bankevich et al., 2012) (https://samtools.github.io/bcftools/bcftools.html). The cp genome of D. nobile was also generated through reference-assisted assembly using the high quality paired-end libraries by NOVOPlasty (Dierckxsens, Mardulyn & Smits, 2017) for further validation. It is specifically designed for de novo assembly of mitochondrial and chloroplast genomes from WGS data with the aid of a reference or seed sequence. The seed sequence can correspond to partial or complete sequence of chloroplasts of closely to distantly related species. The cpDNA RefSeq sequence of D. officinale was used as a seed sequence to perform reference-assisted assembly.

Genome annotation and codon usage

Basic Local Alignment Search Tool (BLAST; BLASTN, PHI-BLAST and BLASTX)  (Altschul et al., 1997), chloroplast genome analysis platform (CGAP) (Cheng et al., 2013) and Dual Organellar GenoMe Annotator (DOGMA) (Wyman, Jansen & Boore, 2004) was used to annotate protein-coding and ribosomal RNA genes. The boundaries of each annotated gene with putative start, stop, and intron positions were manually determined by comparison with homologous genes from other orchid cp genomes. Further tRNA genes were predicted using tRNAscan-SE  (Lowe & Eddy, 1997) and ARAGORN (Laslett & Canback, 2004). RNA editing sites in the protein-coding genes (PCG) of D. nobile were predicted using Plant RNA Editing Prediction & Analysis Computer Tool (PREPACT) (http://www.prepact.de). For this analysis, D. nobile cp genome was BLAST aligned against Nicotiana tabacum, Oryza sativa Japonica Group, Phalaenopsis aphrodite subsp. Formosana, Physcomitrella patens subsp. patens and Zea mays with a cutoff E-value set to 0.08. The circular genome map was drawn in OrganellarGenomeDRAW (Lohse et al., 2013) followed by manual modification. The sequencing data and gene annotation were submitted to GenBank with accession number KX377961. MEGA 7 was used to analyze and calculate GC content, codon usage, nucleotide sequence statistics and relative synonymous codon usage (RSCU) (Kumar, Stecher & Tamura, 2016).

Gene Ontology annotation and assignment of GO IDs

Gene Ontology (GO) annotation of D. nobile chloroplast genes was carried out in Blast2GO (Conesa et al., 2005) by blast aligning the gene sequences from the GenBank annotation files to Orchidaceae sequences in non-redundant (nr) database with an e-value cutoff of 1e−5 and queried in InterProScan (Jones et al., 2014). GO mapping and annotation of genes followed this from blast results and were subsequently merged with GO IDs from InterProScan. The merged GO annotations were validated based on True-Path-Rule by removing redundant child terms for each gene sequence. The GO annotations were slimmed down using plant-slim option.

Simple sequence repeats analysis

MISA (http://pgrc.ipk-gatersleben.de/misa/misa.html), a tool for identification and location of perfect microsatellites and compound microsatellites was used to search for potential simple sequence repeats (SSRs) loci in the cp genomes of different Dendrobium species. The threshold point for SSRs identification was set to 10, 5, 4, 3, and 3 for mono-, di-, tri-, tetra-, and penta-nucleotides SSRs, respectively. All the repeats found were manually curated and the redundant ones were removed.

Phylogenetic reconstruction with whole genome alignment and rearrangement analysis

For phylogenetic reconstruction, we included D. nobile cp genomes from India and China along with 32 other Dendrobium cp genomes retrieved from GenBank. Four Goodyera species were taken as outgroup. The cp genome sequences were aligned with MAFFT v7.0.0 (Katoh & Standley, 2013) and manually curated by visual inspection. PCGs as well as whole cp genomes were used for Bayesian phylogenetic reconstruction using MRBAYES 3.2.6 (Huelsenbeck & Ronquist, 2001). To further validate our results we employed “k-mer Based Tree Construction” in CLC Genomics Workbench that uses single sequences or sequence lists as input and creates a distance-based phylogenetic tree. For visualization and testing the presence of genome rearrangement and inversions, gene synteny was performed using MAUVE as implemented in DNASTAR 12.3 with default settings. Comparative analysis of intra nucleotide diversity (Pi) within the Dendrobium cp genomes was performed using MEGA 7.

Single nucleotide polymorphism identification and phylogenetic analysis without genome alignment

Phylogenetic tree was constructed based on the Single Nucleotide Polymorphisms (SNPs) identified in the whole cp genomes using kSNP3.0 with default settings except for k-mer size (Gardner, Slezak & Hall, 2015). SNPs were identified with k-mer size set to 23, based on which, approximately 79% of the k-mers generated from median-length genome were unique.

Results

Genome organization and features

The complete cp genome of D. nobile was determined from the data generated out of a whole genome project initiative of the same species by Paired-end and Mate pair data from Illumina HighSeq with 150*2 and Illumina NextSeq500 with 75*2 respectively. Further the aligned Illumina reads were separated and assembled using CLC Main Workbench Version 7.7.1 into the single longest scaffold. The D. nobile cp genome is a typical circular double-stranded DNA with a quadripartite structure; it is 152,018 bp in size and consists of Large Single Copy (LSC) (1..84,944; 84,944 bp), Small Single Copy (SSC) (111,230..125,733; 14,504 bp), and two Inverted Repeat (IR) regions of 26,285 bp: IRA (84,945..111,229) and IRB (125,734..152018). In total 134 unique genes (79 PCGs, 8 rRNA genes, 7 pseudogenes and 38 tRNA genes) were successfully annotated, of which 12 genes {rps16, atpF, rpoC1, ycf3, rps12 (2), clpP, petB, rpl2 (2), ndhB (2)} are reported with introns (Fig. 1). We could identify a total of 20, 81 and 11 genes duplicated in the IR, LSC and SSC regions respectively in the D. nobile cp genome. There were a total of 49 RNA editing sites predicted in 23 genes of D. nobile cp genome. The whole chloroplast genome alignment included 34 Dendrobium species and four species from the genus Goodyera as outgroup. Each genome’s panel contained its name, sequence coordinates and a black coloured horizontal centre line with coloured block outlines appearing above and below it. Homology between the cp genomes is represented by each block with the genes, internally free from genomic rearrangement, connected by thin lines to similarly coloured blocks depicting comparative homology between the genomes (Fig. 2). The positions of LSC/IRA/SSC/IRB borders revealed similar structures at the IR/LSC junction in the overall alignment of Dendrobium whole cp genomes (Fig. 3).

Figure 1

Gene map of Dendrobium nobile chloroplast genome from north-east India.

Genes shown inside the circle are transcribed clockwise, and those outside are transcribed anticlockwise. Color coding indicates genes of different functional groups. A pair of inverted repeats (IRA and IRB) separate the genome into LSC and SSC regions.

Figure 2

Whole chloroplast genome alignment of 38 orchid species.

The whole chloroplast genome alignment includes 34 Dendrobium species and four species from the genus Goodyera as outgroup. Each genome’s panel contains its name, sequence coordinates and a black coloured horizontal centre line with coloured block outlines appearing above and below it. Each block represents homology with the genes, internally free from genomic rearrangement, connected by lines to similarly coloured blocks depicting comparative homology across genomes.

Figure 3

Comparison of the borders of LSC, SSC and IR regions across Dendrobium chloroplast genomes.

Gene ontology mapping and annotation

We further analyzed the D. nobile coding cp genome sequences using the Blast2GO suite and annotated the sequences for three GO terms (biological process, molecular function, and cellular component). In case of GO term there were a total of 231 annotations in biological process (P), molecular function (F) and cellular compartment (C) level. In the category of biological processes a large number of these sequences are annotated for translation, photosynthesis, metabolic processes, and ribosome biogenesis. Similarly, for the GO term molecular function, the top GO categories include functions related to structural molecule activity, catalytic activity, ion and rRNA binding, transporter and transferase activity. Finally, terms including membrane, ribosome and thylakoid were annotated GO categories for cellular compartment with most of the sequences. These results are summarized along with the information on RNA editing sites in Table 1.

Table 1

RNA editing sites predicted in Dendrobium nobile chloroplast genome along with its GO annotations.

D. nobile cp genome was BLAST aligned against reference datasets of Nicotiana tabacum, Oryza sativa Japonica Group, Phalaenopsis aphrodite subsp. Formosana, Physcomitrella patens subsp. Patens and Zea mays. Threshold for congruent prediction of RNA editing sites from the reference taxa was set to ≥3 (Count) and 75% (Percentage of prevalence). Count is in the form of (number of reference taxa against which editing site found)/(number of taxa with the homologous site). Further, the genes were exported to OMIX box, blast aligned and subsequently mapped and annotated with Gene ontology (GO) slim terms. Their corresponding GO ids and annotations are shown in the table.

Gene	GO IDs	GO slim annotation	Nucleotide position	Amino acid position	Triplet position within codon	Base conversion	Codon change	Amino acid conversion	Count	Percentage of Prevalence
matK	F: GO:0005198	F: structural molecule activity	1258 913	420 305	1 1	C→U C→U	CAC→UAC CAU→UAU	H→Y H→Y	4/5 4/5	80 80
		P: GO:0006412									P: translation
	C: GO:0005840; GO:0009507	C: ribosome; chloroplast
rps16	F: GO:0000166; GO:0005215	F: nucleotide binding; transporter activity	143	48	2	C→U	UCA→UUA	S→L	4/4	100
		P: GO:0006139; GO:0006810; GO:0009058									P: nucleobase-containing compound metabolic process; transport; biosynthetic process;
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
atpA	F: GO:0000166; GO:0005215	F: nucleotide binding; transporter activity	773	258	2	C→U	UCA→UUA	S→L	5/5	100
		P: GO:0006139; GO:0006810; GO:0009058									P: nucleobase-containing compound metabolic process; transport; biosynthetic process
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
atpF	F: GO:0005215	F: transporter activity	92	31	2	C→U	CCA→CUA	P→L	5/5	100
		P: GO:0006139; GO:0006810; GO:0009058									P: nucleobase-containing compound metabolic process; transport; biosynthetic process
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
atpI	F: GO:0005215	F: transporter activity	629	210	2	C→U	UCA→UUA	S→L	5/5	100
		P: GO:0006139; GO:0006810; GO:0009058	P: nucleobase-containing compound metabolic process; transport; biosynthetic process	428	143	2	C→U	CCU→CUU	P→L	5/5	100
	C: GO:0005886; GO:0009507; GO:0009579	C: plasma membrane; chloroplast; thylakoid
rpoC1	F: GO:0003677; GO:0016740	F: DNA binding; transferase activity	617	206	2	C→U	UCG→UUG	S→L	5/5	100
		P: GO:0006139; GO:0009058	P: nucleobase-containing compound metabolic process; biosynthetic process	488	163	2	C→U	UCA→UUA	S→L	5/5	100
		C: GO:0009507	C: chloroplast	182	61	2	C→U	UCU→UUU	S→F	5/5	100
	41			14	2	C→U	CCA→CUA	P→L	5/5	100
rpoB	F: GO:0003677; GO:0016740	F: DNA binding; transferase activity	2426	809	2	C→U	UCA→UUA	S→L	4/5	80
				623	208	2	C→U	CCG→CUG	P→L	4/5	80
		P: GO:0006139; GO:0009058	P: nucleobase-containing compound metabolic process; biosynthetic process	566	189	2	C→U	UCG→UUG	S→L	5/5	100
		C: GO:0009507	C: chloroplast	551	184	2	C→U	UCA→UUA	S→L	5/5	100
						473	158	2	C→U	UCG→UUG	S→L	5/5	100
	338			113	2	C→U	UCU→UUU	S→F	5/5	100
rps14	F: GO:0003723; GO:0005198	F: RNA binding; structural molecule activity	149	50	2	C→U	CCA→CUA	P→L	5/5	100
		P: GO:0006091; GO:0006412; GO:0015979									P: generation of precursor metabolites and energy; translation; photosynthesis
	C: GO:0009507; GO:0009579; GO:0016020; GO:0005840	C: chloroplast; thylakoid; membrane; ribosome
ycf3	F: GO:0005515	F: protein binding	191	64	2	C→U	CCA→CUA	P→L	5/5	100
		P: GO:0015979	P: photosynthesis	185	62	2	C→U	ACG→AUG	T→M	5/5	100
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane	44	15	2	C→U	UCU→UUU	S→F	5/5	100
atpB	F: GO:0000166; GO:0005215	F: nucleotide binding; transporter activity	1184	395	2	C→U	UCA→UUA	S→L	5/5	100
		P: GO:0006139; GO:0006810; GO:0009058									P: nucleobase-containing compound metabolic process; transport; biosynthetic process
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
accD	F: GO:0000166; GO:0016740	F: nucleotide binding; transporter activity	1184	395	2	C→U	UCA→UUA	S→L	4/4	100
		P: GO:0006139; GO:0006629; GO:0009058	P: nucleobase-containing compound metabolic process; lipid metabolic process; biosynthetic process	1412	471	2	C→U	CCA→CUA	P→L	3/3	100
	C: GO:0009507	C: chloroplast	1430	477	2	C→U	CCU→CUU	P→L	3/3	100
psaI	P: GO:0015979;	P: photosynthesis	80	27	2	C→U	UCU→UUU	S→F	5/5	100
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
psbF	F: GO:0003824; GO:0005488	F: catalytic activity; binding	77	26	2	C→U	UCU→UUU	S→F	5/5	100
		P: GO:0006091; GO:0015979;									P: generation of precursor metabolites and energy; photosynthesis
	C: GO:0005739; GO:0009507; GO:0009579; GO:0016020	C: mitochondrion; chloroplast; thylakoid; membrane
petL	F: GO:0003824	F: catalytic activity	5	2	2	C→U	CCU→CUU	P→L	5/5	100
	C: GO:0009579	C: thylakoid
rpl20	F: GO:0003723; GO:0005198	F: RNA binding; structural molecule activity	308	103	2	C→U	UCA→UUA	S→L	4/5	80
		P: GO:0006412; GO:0016043									P: translation; cellular component organization
	C: GO:0005840; GO:0009507	C: ribosome; chloroplast
clpP	F: GO:0016787	F: hydrolase activity	559	187	1	C→U	CAU→UAU	H→Y	5/5	100
		P: GO:0019538	P: protein metabolic process	82	28	1	C→U	CAU→UAU	H→Y	5/5	100
	C: GO:0009507	C: chloroplast
petB	F: GO:0003824; GO:0005488	F: catalytic activity; binding	611	204	2	C→U	UCA→UUA	S→L	5/5	100
		P: GO:0006091; GO:0015979									P: generation of precursor metabolites and energy; photosynthesis
	C:GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane
rpoA	F: GO:0003677; GO:0005515; GO:0016740	F: DNA binding; protein binding; transferase activity	830	277	2	C→U	UCA→UUA	S→L	4/4	100
		P: GO:0006139; GO:0009058	P: nucleobase-containing compound metabolic process; biosynthetic process	368	123	2	C→U	UCA→UUA	S→L	4/4	100
	C: GO:0009507	C: chloroplast	200	67	2	C→U	UCU→UUU	S→F	3/4	75
rpl2	F: GO:0003723; GO:0005198; GO:0016740	F: RNA binding; structural molecule activity; transferase activity	2	1	2	C→U	ACG→AUG	T→M	5/5	100
		P: GO:0006412									P: translation
	C: GO:0005840; GO:0009507	C: ribosome; chloroplast
ndhD	F: GO:0003824; GO:0005488	F: catalytic activity; binding	878	293	2	C→U	UCA→UUA	S→L	4/4	100
		P: GO:0006091	P: generation of precursor metabolites and energy	674	225	2	C→U	UCG→UUG	S→L	4/4	100
	C: GO:0009507; GO:0009579; GO:0016020	C: chloroplast; thylakoid; membrane	383	128	2	C→U	UCA→UUA	S→L	4/4	100
ndhA	F: GO:0003824; GO:0005488	F: catalytic activity; binding	473	158	2	C→U	UCA→UUA	S→L	4/4	100
		P: GO:0006091; GO:0015979									P: generation of precursor metabolites and energy; photosynthesis
	C: GO:0005886; GO:0009507; GO:0009579	C: plasma membrane; chloroplast; thylakoid
ndhB	F: GO:0003824; GO:0005488	F: catalytic activity; binding	149	50	2	C→U	UCA→UUA	S→L	4/4	100
						467	156	2	C→U	CCA→CUA	P→L	4/4	100
				586	196	1	C→U	CAU→UAU	H→Y	4/4	100
		P: GO:0006091; GO:0015979	P: generation of precursor metabolites and energy; photosynthesis	704	235	2	C→U	UCC→UUC	S→F	4/4	100
						737	246	2	C→U	CCA→CUA	P→L	4/4	100
				830	277	2	C→U	UCA→UUA	S→L	4/5	80
		C: GO:0005886; GO:0009507; GO:0009579	C: plasma membrane; chloroplast; thylakoid	836	279	2	C→U	UCA→UUA	S→L	4/5	80
	1481			494	2	C→U	CCA→CUA	P→L	4/4	100
rpl23	F: GO:0003723; GO:0005198	F: RNA binding; structural molecule activity	71	24	2	C→U	UCU→UUU	S→F	4/5	80
		P: GO:0006412									P: translation
	C: GO:0005840; GO:0009507	C: ribosome; chloroplast

Simple sequence repeat identification

SSRs were identified in MISA perl scripts with a minimum of 10 bp repeats among all the Dendrobium species. Of all the SSRs, the mononucleotide A/T repeat units occupied the highest proportion. A higher proportion of di-, tri- repeats are reported rather than tetra- and penta-nucleotide repeats across Dendrobium cp genomes (Fig. 4).

Figure 4

SSR distribution among different Dendrobium plastomes.

The SSR were determined in MISA per scripts based on the comparison between plastomes of each tested Dendrobium species and D. nobile. Histograms with different color codes indicate the numbers of SSRs. The minimum number (thresholds) of SSRs was set as 10, 5, 4, 3, and 3 for mono-, di-, tri-, tetra-, and penta-nucleotides SSRs, respectively.

Phylogenetic analysis

Phylogenetic analyses of chloroplast PCGs from Dendrobium species were performed with or without partitions of sequences. Both Bayesian and K-mer based trees (Figs. 5 and 6) recovered a monophyly of the Dendrobium species, irrespective of whether or not the partitions of sequences were incorporated in the analysis supported by strong bootstrap values. The phylogenetic analyses based on complete cp genomes, suggested that five major subgroups within the genus Dendrobium evolved in a nested evolutionary relationship. D. aphyllum, D. parishii, D. loddigesii and D. primulinum are the most recently evolved species that nested into a single monophyletic sub group within the Dendrobium clade. D. chrysotoxum and D. salaccense were a bit primitive on the evolutionary ladder in the phylogenetic tree. Goodyera species emerged as the outgroup that claded separately in the over all tree topology. Similar results were also obtained in the alignment free phylogenetic tree with SNPs (Fig. 6).

Figure 5

Phylogenetic tree based on Bayesian inference from the whole genome alignment matrix of Dendrobium chloroplast genomes.

The tree yielded monophyletic groupings of the genus Dendrobium and Goodyera species emerged as outgroup with a separate clade. Posterior probability/bootstrap values are indicated on the internal nodes, which are highly supportive of the overall tree topology.

Figure 6

Alignment free phylogenetic tree reconstruction based on SNP identification.

The optimum kmer size for the dataset is determined that calculates FCK, a measure of diversity of sequences in the dataset (Kchooser) and a consensus of the equally most parsimonious trees are reported. The numbers at the nodes indicate the number of SNPs that are present in all of the descendants of that node and absent in others. The numbers within parentheses at the tips indicate the number of SNPs unique to each particular species.

Discussion

Potential RNA editing sites

RNA editing is involved in plastid posttranscriptional regulation and thus provides an effective way to create transcript and protein diversity (Chen & Bundschuh, 2012; Knoop, 2011). In Orchidaceae, RNA editing sites were identified in 24 protein-coding transcripts in P. aphrodite (Zeng, Liao & Chang, 2007). Earlier studies indicate RNA editing sites from the same subfamily to be more conserved than those from different subfamily (Luo et al., 2014). However, orchids and other angiosperms have relatively less common editing sites. For example, orchids and Cocos nucifera share 10 potential RNA editing sites; comparisons among Nicotiana tabacum, Arabidopsis thaliana and orchid RNA editing sites have shown low conservation of editing sites (one common editing site in rpo B) (Luo et al., 2014). Our studies congruently predicted all 49 RNA editing sites (Table 1) in 23 genes of D. nobile from at least 75% of the reference organisms (Nicotiana tabacum, Oryza sativa Japonica Group, Phalaenopsis aphrodite subsp. Formosana, Physcomitrella patens subsp. Patens and Zea mays) and resulted in amino acid substitutions. All the RNA-editing sites were non-silent and edited C to U. Of the 49 RNA editing sites 89.8% (44) editing sites appeared in the second position of triplet codon, 10.2% (five) editing sites appeared in the first position of triplet codon whereas no editing sites appeared in the third base of triplet codon. The genes ndhD, rpoB, rpoC1 had eight, six, and four RNA editing sites, respectively. All the 49 RNA editing sites led to changes in the amino acid. The most frequent amino acid conversion was hydrophilic to hydrophobic (S to L, 22 occurrences and S to F, eight occurrences), followed by hydrophobic to hydrophobic conversions (P to L, 12 occurrences). Seven conversions were found to be hydrophilic to hydrophilic (H to Y, five occurrences and T to M, two occurrences).

Comparison with other chloroplast genomes within the genus Dendrobium

We compared thirty-four chloroplast genomes representing different species within the genus Dendrobium (Table 2). The length of the Dendrobium species cp genomes ranged from 148,778 to 153,953 bp, with D. chrysotoxum being the largest cp genome and D. moniliforme the smallest. The cp genomes have acquired the familial angiosperm plastome organization comprising of a LSC, an SSC and a pair of IR regions each. Dendrobium cp genomes are also AT-rich (62.26–62.39%) quite similar to other orchid cp genomes (Zhitao et al., 2017). Differences in the cp genome size of these species are primarily due to the variations in the length of LSC, SSC and IR regions. Synteny comparison revealed a lack of genome rearrangement and inversions, thereby, substantiating for the highly conserved nature in the genomic structure, including gene number and gene order in these cp genomes. However, structural variation was predominant in the LSC/IR/SSC boundaries (Fig. 2), which could be harnessed for predicting potential biomarkers for species identification.

Table 2

Summary of characteristics in chloroplast genome sequences of thirty-four Dendrobium species and four Goodyera species (taken as outgroup).

Organism	Accession number	Length	Weight (single-stranded) Mda	Weight (double-stranded) Mda
Dendrobium nobile	KX377961	152,018	46.932	93.912
Dendrobium officinale	NC_024019	152,221	46.995	94.038
Dendrobium strongylanthum	NC_027691	153,059	47.256	94.556
Dendrobium huoshanense	NC_028430	153,188	47.294	94.635
Dendrobium chrysotoxum	NC_028549	153,953	47.528	95.108
Dendrobium nobile (China)	NC_029456	153,660	47.453	94.927
Dendrobium pendulum	NC_029705	153,038	47.246	94.542
Dendrobium moniliforme	NC_035154	148,778	45.931	91.911
Dendrobium primulinum	NC_035321	150,767	46.545	93.14
Dendrobium aphyllum	NC_035322	151,524	46.779	93.607
Dendrobium brymerianum	NC_035323	151,830	46.873	93.796
Dendrobium denneanum	NC_035324	151,565	46.793	93.633
Dendrobium devonianum	NC_035325	151,945	46.909	93.867
Dendrobium falconeri	NC_035326	151,890	46.891	93.833
Dendrobium gratiosissimum	NC_035327	151,829	46.873	93.796
Dendrobium hercoglossum	NC_035328	151,939	46.908	93.864
Dendrobium wardianum	NC_035329	151,788	46.861	93.77
Dendrobium wilsonii	NC_035330	152,080	46.951	93.951
Dendrobium crepidatum	NC_035331	151,717	46.837	93.726
Dendrobium salaccense	NC_035332	151,104	46.648	93.347
Dendrobium spatella	NC_035333	151,829	46.872	93.796
Dendrobium parciflorum	NC_035334	150,073	46.331	92.711
Dendrobium henryi	NC_035335	151,850	46.88	93.809
Dendrobium chrysanthum	NC_035336	151,790	46.861	93.772
Dendrobium jenkinsii	NC_035337	151,717	46.839	93.726
Dendrobium lohohense	NC_035338	151,812	46.868	93.785
Dendrobium parishii	NC_035339	151,689	46.83	93.709
Dendrobium ellipsophyllum	NC_035340	152,026	46.935	93.917
Dendrobium xichouense	NC_035341	152,052	46.942	93.933
Dendrobium fimbriatum	NC_035342	151,673	46.825	93.699
Dendrobium exile	NC_035343	151,294	46.707	93.465
Dendrobium fanjingshanense	NC_035344	152,108	46.96	93.968
Dendrobium candidum	NC_035745	152,094	46.955	93.959
Dendrobium loddigesii	NC_036355	152,493	47.077	94.205
Goodyera fumata	NC_026773	155,643	48.048	96.151
Goodyera procera	NC_029363	153,240	47.306	94.667
Goodyera schlechtendaliana	NC_029364	154,348	47.648	95.351
Goodyera velutina	NC_029365	152,692	47.138	94.328

IR regions are generally considered to be highly conserved regions in the chloroplast genome. IR expansion or contraction is determined by the variability of genes flanking IR/SC junctions (Huelsenbeck & Ronquist, 2001). In the evolutionary ladder, SSC and IR border regions experience expansion and contraction that overall contribute to the variation in chloroplast genome length (Wang et al., 2008; Li et al., 2013). At the IR/LSC boundaries, most IRs of non-orchid monocots exhibit trnH-rps19 gene clusters, excluding Ψrpl22 genes, leading to more-progressive expansion of IRs compared to non-monocot angiosperms (Yang et al., 2010; Goulding et al., 1996). Contrarily, the orchid chloroplast genomes have distinct characteristics at the IR/SSC junction and are classified into four types based on the organization of genes flanking the IRB/SSC junction (JSB). In type I structure, JSB is located upstream of the ndhF-rpl32 cluster and is primarily seen in Cypripedium and Dendrobium species. Type II junction is found in Cymbidium species in which JSB is located within Ψycf1 and ndhF genes. Type III is reported in Oncidium, Erycina, and Phalaenopsis equestris, in which JSB is located inside the Ψycf1-rpl32 cluster, with the loss of ndhF gene. The type IV structure is characterized by the incorporation of the entire ycf1 into the SSC, with JSBinside trnN-rpl32 (Gardner, Slezak & Hall, 2015). In the present study, the positions of LSC/IRA/SSC/IRB borders were examined in the overall alignment of Dendrobium whole cp genomes and all of them were found to have similar structures at the IR/LSC junction akin to type I structure (Fig. 3). Previous studies emphasize that IR expansion or contraction may not correlate with the taxonomic relationships (Chen & Bundschuh, 2012). More molecular data is required for enhancing our present understanding of the genes flanking IR/SSC junctions and their underlying variations.

A comparative nucleotide sequence statistics (counts of annotations, AT/GC counts, nucleotide frequency in codon positions etc.) for all the Dendrobium species including representatives from outgroup are outlined in Tables 3, 4 and 5. The relative synonymous codon usage is given in parentheses following the codon frequency (averages over all taxa) involved (Table 6). Maximum Likelihood analysis of natural selection codon-by-codon was carried out. For each codon, estimates of the numbers of inferred synonymous (s) and nonsynonymous (n) substitutions are presented along with the number of sites that are estimated to be synonymous (S) and nonsynonymous (N) (Table S1). These estimates were calculated using the joint Maximum Likelihood reconstructions of ancestral states under a Muse-Gaut model (Muse & Gaut, 1994) of codon substitution and Felsenstein 1981 model  (Felsenstein, 1981) of nucleotide substitution. For estimating ML values, a tree topology was automatically computed. The test statistic dN-dS was used for detecting codons that have undergone positive selection, where dS is the number of synonymous substitutions per site (s/S) and dN is the number of nonsynonymous substitutions per site (n/N). A positive value for the test statistic indicates an overabundance of nonsynonymous substitutions. In this case, the probability of rejecting the null hypothesis of neutral evolution (p-value) was calculated  (Kosakovsky Pond & Frost, 2005; Suzuki & Gojobori, 1999). A value of p less than 0.05 was considered significant at a 5% level and was highlighted (Table S2). Normalized dN-dS for the test statistic is obtained using the total number of substitutions in the tree (measured in expected substitutions per site). The analysis involved 38 nucleotide sequences. Codon positions included were 1st+2nd+3rd+non-coding and all positions containing gaps and missing data were eliminated. There were a total of 108,594 positions in the final dataset.

Table 3

Summary features of chloroplast genome sequences of thirty-four Dendrobium species and four Goodyera species.

Organism	CDS	Exon	Gene	Misc. feature	Repeat region	rRNA	tRNA
Dendrobium nobile	79	22	132	2	2	8	38
Dendrobium officinale	76	0	129	0	0	8	38
Dendrobium strongylanthum	77	0	130	2	2	8	38
Dendrobium huoshanense	76	0	129	2	2	8	38
Dendrobium chrysotoxum	63	0	116	2	2	8	38
Dendrobium nobile (China)	77	0	130	2	2	8	38
Dendrobium pendulum	76	0	129	2	2	8	38
Dendrobium moniliforme	73	0	129	11	2	8	39
Dendrobium primulinum	72	0	132	16	2	8	38
Dendrobium aphyllum	72	0	132	16	2	8	38
Dendrobium brymerianum	72	0	132	16	2	8	38
Dendrobium denneanum	72	0	132	16	2	8	38
Dendrobium devonianum	72	0	132	16	2	8	38
Dendrobium falconeri	72	0	132	16	2	8	38
Dendrobium gratiosissimum	72	0	132	16	2	8	38
Dendrobium hercoglossum	72	0	132	16	2	8	38
Dendrobium wardianum	71	0	131	16	2	8	38
Dendrobium wilsonii	72	0	132	16	2	8	38
Dendrobium crepidatum	72	0	132	16	2	8	38
Dendrobium salaccense	72	0	132	16	2	8	38
Dendrobium spatella	72	0	132	16	2	8	38
Dendrobium parciflorum	72	0	131	16	2	7	38
Dendrobium henryi	72	0	132	16	2	8	38
Dendrobium chrysanthum	72	0	132	16	2	8	38
Dendrobium jenkinsii	72	0	132	16	2	8	38
Dendrobium lohohense	72	0	132	16	2	8	38
Dendrobium parishii	72	0	132	16	2	8	38
Dendrobium ellipsophyllum	72	0	132	16	2	8	38
Dendrobium xichouense	72	0	132	16	2	8	38
Dendrobium fimbriatum	72	0	132	16	2	8	38
Dendrobium exile	72	0	132	16	2	8	38
Dendrobium fanjingshanense	72	0	132	16	2	8	38
Dendrobium candidum	75	0	128	0	0	8	38
Dendrobium loddigesii	68	0	120	9	0	8	39
Goodyera fumata	87	0	133	0	0	8	38
Goodyera procera	80	0	127	0	0	8	39
Goodyera schlechtendaliana	81	0	129	0	0	8	40
Goodyera velutina	79	0	126	0	0	8	39

Table 4

Counts of nucleotides in the chloroplast genomes.

Nucleotide	Adenine (A)	Cytosine (C)	Guanine (G)	Thymine (T)	C + G	A + T
Dendrobium nobile	46576	28853	28039	48381	56892	94957
Dendrobium officinale	46743	28924	28107	48447	57031	95190
Dendrobium strongylanthum	46940	29147	28431	48541	57578	95481
Dendrobium huoshanense	47032	29111	28316	48729	57427	95761
Dendrobium chrysotoxum	47180	29400	28492	48881	57892	96061
Dendrobium nobile (China)	47118	28871	28748	48923	57619	96041
Dendrobium pendulum	46997	29122	28242	48677	57364	95674
Dendrobium moniliforme	45551	28339	27520	47368	55859	92919
Dendrobium primulinum	46191	28750	27909	47917	56659	94108
Dendrobium aphyllum	46417	28917	28057	48133	56974	94550
Dendrobium brymerianum	46509	28968	28123	48230	57091	94739
Dendrobium denneanum	46440	28913	28115	48097	57028	94537
Dendrobium devonianum	46615	28943	28108	48279	57051	94894
Dendrobium falconeri	46591	28911	28040	48348	56951	94939
Dendrobium gratiosissimum	46521	28954	28095	48259	57049	94780
Dendrobium hercoglossum	46592	28941	28131	48275	57072	94867
Dendrobium wardianum	46479	28955	28118	48236	57073	94715
Dendrobium wilsonii	46668	28948	28101	48363	57049	95031
Dendrobium crepidatum	46482	28951	28056	48228	57007	94710
Dendrobium salaccense	46493	28635	27735	48241	56370	94734
Dendrobium spatella	46524	28969	28091	48245	57060	94769
Dendrobium parciflorum	45941	28699	27829	47604	56528	93545
Dendrobium henryi	46550	28936	28093	48271	57029	94821
Dendrobium chrysanthum	46519	28939	28078	48254	57017	94773
Dendrobium jenkinsii	46497	28942	28105	48173	57047	94670
Dendrobium lohohense	46558	28928	28098	48228	57026	94786
Dendrobium parishii	46487	28924	28079	48199	57003	94686
Dendrobium ellipsophyllum	46690	28922	28091	48323	57013	95013
Dendrobium xichouense	46672	28937	28098	48345	57035	95017
Dendrobium fimbriatum	46483	28932	28094	48164	57026	94647
Dendrobium exile	46251	28937	28065	48041	57002	94292
Dendrobium fanjingshanense	46694	28947	28115	48352	57062	95046
Dendrobium candidum	46695	28914	28091	48394	57005	95089
Dendrobium loddigesii	46868	28934	28064	48627	56998	95495
Goodyera fumata	48186	29569	28447	49441	58016	97627
Goodyera procera	47095	29370	28303	48472	57673	95567
Goodyera schlechtendaliana	47822	29206	28146	49174	57352	96996
Goodyera velutina	47554	28694	27658	48786	56352	96340

Table 5

Counts of nucleotide frequency in codon positions across the chloroplast genomes.

Nucleotide per position	1 A	1 C	1 G	1 T	2 A	2 C	2 G	2 T	3 A	3 C	3 G	3 T
D. nobile	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. officinale	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. strongylanthum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. huoshanense	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. chrysotoxum	0.3	0.19	0.28	0.22	0.29	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. nobile (China)	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. pendulum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. moniliforme	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.17	0.38
D. primulinum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. aphyllum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. brymerianum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. denneanum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. devonianum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. falconeri	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. gratiosissimum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.17	0.38
D. hercoglossum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. wardianum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. wilsonii	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. crepidatum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. salaccense	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. spatella	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.31	0.14	0.17	0.38
D. parciflorum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.31	0.14	0.17	0.38
D. henryi	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. chrysanthum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. jenkinsii	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. lohohense	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. parishii	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.17	0.38
D. ellipsophyllum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. xichouense	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. fimbriatum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. exile	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.31	0.14	0.16	0.38
D. fanjingshanense	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. candidum	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
D. loddigesii	0.31	0.19	0.27	0.23	0.3	0.2	0.18	0.32	0.32	0.14	0.16	0.38
G. fumata	0.31	0.19	0.26	0.24	0.29	0.2	0.18	0.33	0.32	0.14	0.16	0.38
G. procera	0.31	0.19	0.26	0.24	0.3	0.2	0.17	0.33	0.32	0.14	0.16	0.38
G. schlechtendaliana	0.31	0.19	0.26	0.24	0.29	0.21	0.17	0.33	0.31	0.14	0.16	0.38
G. velutina	0.31	0.19	0.27	0.24	0.29	0.21	0.18	0.33	0.32	0.14	0.16	0.38

Table 6

Relative synonymous codon usage (in parentheses) following the codon frequency across the chloroplast genomes in the genus Dendrobium.

Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	2018.1	1.16	UCU(S)	1330	1.63	UAU(Y)	1371	1.38	UGU(C)	706.9	1.24
UUC(F)	1459.2	0.84	UCC(S)	882.8	1.08	UAC(Y)	621.4	0.62	UGC(C)	437	0.76
UUA(L)	918.4	1.14	UCA(S)	999.4	1.23	UAA(*)	970.5	1.05	UGA(*)	1065	1.15
UUG(L)	970.9	1.21	UCG(S)	576.9	0.71	UAG(*)	732.2	0.79	UGG(W)	691.4	1
CUU(L)	1068.9	1.33	CCU(P)	638	1.13	CAU(H)	919.7	1.43	CGU(R)	336.1	0.63
CUC(L)	629.2	0.78	CCC(P)	547.8	0.97	CAC(H)	369.3	0.57	CGC(R)	220.7	0.41
CUA(L)	762.8	0.95	CCA(P)	689.4	1.23	CAA(Q)	952.8	1.38	CGA(R)	545.2	1.02
CUG(L)	473.7	0.59	CCG(P)	375.4	0.67	CAG(Q)	423.2	0.62	CGG(R)	343	0.64
AUU(I)	1635.7	1.21	ACU(T)	646	1.21	AAU(N)	1580	1.39	AGU(S)	659.9	0.81
AUC(I)	1072.9	0.8	ACC(T)	530.8	1	AAC(N)	695	0.61	AGC(S)	435.8	0.54
AUA(I)	1337.4	0.99	ACA(T)	610.3	1.15	AAA(K)	1914	1.31	AGA(R)	1171	2.2
AUG(M)	891.4	1	ACG(T)	343.2	0.64	AAG(K)	1009	0.69	AGG(R)	576	1.08
GUU(V)	709.4	1.36	GCU(A)	467.5	1.29	GAU(D)	1038	1.43	GGU(G)	523.7	0.99
GUC(V)	366.7	0.7	GCC(A)	326.4	0.9	GAC(D)	413.9	0.57	GGC(G)	314.4	0.59
GUA(V)	647.8	1.24	GCA(A)	438.7	1.21	GAA(E)	1335	1.37	GGA(G)	754.1	1.43
GUG(V)	366.9	0.7	GCG(A)	221.5	0.61	GAG(E)	618.3	0.63	GGG(G)	521.8	0.99

Gene ontology analysis

The GO annotation revealed majority of the chloroplast genes are involved in the process of translation, photosynthesis, ion transport and transcription (Table 1). The molecular functions of the genes are majorly binding—RNA, metal ion, DNA, ion and electron transport, RNA polymerase activity and various other enzymatic activities. Enzyme classification showed seven genes to be translocases, four as transferases, two as oxidoreductases, and one each as hydrolase, lyase and ligase. A majority of the genes encode proteins localizing in chloroplast thylakoid membrane, ribosome and few are transported to the mitochondria. The ndhB gene is involved in photosynthesis, while rpoB and rpoC1 are involved in biosynthetic process.

Characterization of simple sequence repeats

Previous studies have documented prevalence of mononucleotide and dinucleotide SSRs in atleast 15 Dendrobium species from 92 syntenic intergenic and intronic loci. Of all these loci, 10(mutational hotspots: psbB-psbT, rpl16-rps3, trnR-atpA, trnL intron ndhF-rpl32, rpl32-trnL, trnT-trnL, clpB-psbB, rps16-trnQ and trnE-trnT) are reported to be the fastest evolving and are termed as top ten hotspots (Chen & Bundschuh, 2012). The SSRs lying in these regions could be further investigated for identifying potential markers that can aid in barcoding analysis.

Phylogenetic analyses

In the present study, we employed two different approaches for phylogeny reconstruction. First we aligned the whole cp genomes and exported the alignment matrices for creating a Bayesian tree (Fig. 5). Two independent MCMC chains were run with first 25% of the cycles removed as burn-in, coalescence of substitution rate and rate model parameters were also examined and average standard deviation of split frequencies was carried out and generations added until the standard deviation value was lowered to 0.01. Secondly we performed a phylogenetic tree construction using an alignment free approach. In this case we identified the SNPs from the cp genomes and utilised them in constructing the phylogenetic tree (Fig. 6). A total of 13,839 SNPs were identified in the 38 genomes analyzed, of which 2,203 were homoplastic SNPs i.e., SNPs that do not correspond to any node in the parsimony tree. The fraction of k-mers present in all genomes is 0.482. The numbers at the nodes in the phylogenetic tree indicate the number of SNPs that are present in all of the descendants of that node and absent in others (Fig. 6). The numbers at the tips indicate the number of SNPs unique to each particular species.

The two different methods that employed both alignment and alignment-free approach resulted in highly reliable identical phylogenetic trees within each data set. Different analyses based on the two datasets generated largely congruent topologies (Figs. 5 and 6) with Dendrobium species forming one clade and Goodyera species forming another clade as an outgroup.

Conclusions

This study provides the first comparative account on the complete chloroplast genome of D. nobile from north-east India with 33 other species from the genus Dendrobium that revealed higher sequence variation in SSC and LSC regions compared with IR regions in both coding and non-coding regions. The gene order, gene content and genomic structure were highly conserved with those of other sequenced Dendrobium species. However, IR contraction is observed within the genus and several SNPs identified from these cp genomes were quite instrumental in generating alignment-free robust phylogenetic trees that congrued with trees generated from aligned matrices of whole cp genomes. This gives an indication that the SNPs, insertions and deletions, LSC and SSC regions in the cp genomes of this medicinal orchid genus can be utilized for barcoding and biodiversity studies. Further, this would augment more and more plastome sequencing of Dendrobium species that are not reported in this study.

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Click here for additional data file.