The complete chloroplast genome sequence of an Endangered orchid species Dendrobium huoshanense (Orchidaceae) and phylogenetic analysis with related species

Abstract

The family Orchidaceae, including many medicinal plants, is a famous ornamental plant. While, Dendrobium huoshanense is an endangered Orchidaceae species mainly distributed in Huoshan county, Anhui province, China. Here, using the program MITObim v1.7, we assembled the complete chloroplast genome sequence of D. huoshanense, which consists of 153,188 bp, and a pair of inverted repeats (IR) of 26,525 bp. The genome also includes one small and one large single-copy region (SSC and LSC) of 14,524 bp and 85,614 bp, respectively. GC content of the genome is 40.0%, and it contains 129 genes coding for 76 proteins. Codon usage analysis showed that a total of 23,448 codons were identified, and the simple-sequence repeat sequences mainly contain A/T mononucleotide. Phylogenetic analyses revealed that D. huoshanense belongs to the Dendrobium of the Orchidaceae family, which is in accordance with the traditional classification. The research finding in this paper will be beneficial for further investigations on Dendrobium huoshanense from the aspect of evolution, and chloroplast genetic engineering.

Introduction

Dendrobium huoshanense is a most familiar traditional Chinese medicines, which belongs to the Dendrobium of the highly diverse Orchidaceae family. Due to its extremely demanding for growth environment, the D. huoshanense in wild is rare and endangered. Thus, D. huoshanense has become an endangered medicinal plant with an enormous pharmaceutical and medicinal value (Silva et al. 2015). So far, more and more complete chloroplast genome of the Dendrobium plants such as Dendrobium pendulum, Dendrobium monilforme, and Dendrobium bellatulum have been reported (Gao et al., 2017; Wang et al., 2017; Zhang et al., 2018). Whereas, the information about D. huoshanense chloroplast genome content is limited.

In plant cells, the chloroplast is a very important organelle, and take a significant role in the process of photosynthesis, and also has a vital function in the biosynthesis of fatty acids and starch (H. et al., 2000). Through photosynthesis, the atmospheric CO₂ was converted into carbohydrates, and the light energy is stored as chemical energy in this process. Thus, the chloroplast is also called “energy converters” (Raveendar et al. 2015). Additionally, the plant chloroplast genome is also named chloroplast DNA, which is circular in shape, and contains double strands. Moreover, it encodes genes that are photosynthesis and specific essential metabolic pathways (Kong and Yang 2015).

In 1986, the complete chloroplast genomes of tobacco and liverwort were firstly published (Shinozaki et al. 1986). And then, with the development of molecular biological techniques and cost-effective next-generation sequencing approaches, more and more complete chloroplast genomes from different species have been reported. Moreover, in 2009, the Consortium for the Barcode of Life’s Plant Working Group recommended the combination of two-locus chloroplast genes rbcL + matK as the plant “barcode” (Group 2009). Furthermore, the chloroplast genome was used as DNA barcoding (Provan et al. 2001) for phylogenetic analyses on account of the smaller and more conserved genomic sequence (Qian et al., 2013). Thus, in recent studies, the complete chloroplast genome was widely used as a barcode for taxonomy and identification of plant systems (Hollingsworth et al., 2011; Xiwen et al., 2014).

Here, the complete chloroplast genome of D. huoshanense was reported. Then, the codon usage, simple sequence repeats and IR junctions of D. huoshanense were analyzed. And then, the complete chloroplast genome comparison of five Dendrobium species was analyzed. We also analyzed the phyloevolutionary relationship between D. huoshanense and other Orchidaceae categories. The major purpose is to contribute to furnishing resourced organelle genome of Dendrobium, which will be beneficial for species identification, germplasm diversity, and genetic engineering.

Materials And Methods

DNA extraction and sequencing

The fresh leaves of D. huoshanense for the total genome DNA extraction were collected in the glasshouse of National Engineering laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China (108°53´30"E, 34°9´14"N). The D. huoshanense total genomic DNA was extracted, and then was used to construct shotgun libraries with the NEBNext^→ Ultra™ DNA Library Prep Kit for Illumina^Ⓡ. The whole-genome sequencing was executed by Shanghai Genesky Biotechnologies Inc. (Shanghai, China) with an Illumina Hiseq 2500 Sequencing System (Illumina, San Diego, CA, USA).

Chloroplast genome assembly and annotation

After the chloroplast genome is sequenced, the whole raw reads were quality trimmed using CLC Genomics Workbenchv. 7.5 under default parameters (Patel and Mukesh 2012). Then, the complete chloroplast genome was constructed with the trimmed reads using the program MITObimv. 1.7 (Christoph et al., 2013), and the Dendrobium catenatum chloroplast genome (accession number: KC771275) was used as the initial reference. And then, the program GENEIOUS R8 (Kearse et al., 2012) was used to annotate the assembled sequence by comparing it with the initial reference.

Codon Usage, simple sequence repeats and comparative genome analyses

With the CodonW software, the distribution of codon usage with the RSCU ratio was detected (Sharp and Li 1987). Meanwhile, with the MISA software(Sebastian et al. 2017), the simple sequence repeats (SSRs) were investigated with the corresponding parameters (Li et al. 2013a). Additionally, with the mVISTA tool (Sebastian et al. 2017) and the Shuffle-LAGAN alignment algorithm, the whole-genome alignment for the chloroplast genomes of the four species including Dendrobium strongylanthum (GenBank:KR673323), Dendrobium pendulum (GenBank:KT695604), Dendrobium officinale (GenBank:KJ862886) and Dendrobium huoshanense (GenBank:KT630834) were performed and plotted with the Dendrobium catenatum (GenBank: KC771275) chloroplast genomes the initial reference.

Phylogenetic analyses

To determine the phylogenetic positions of D. huoshanense, we obtained 21 Orchidaceae species (Supplementary Table S1) complete chloroplast genomes sequences from the NCBI database, firstly. Then, we constructed a phylogenetic tree with the chloroplast genomes protein-coding genes of those species using the MEGA6 (Tamura et al., 2013) program under the neighbor-joining (NJ) method.

Results

The chloroplast genome characteristics of D. huoshanense

Once the D. huoshanense chloroplast genome was sequenced and annotated, we submitted it to the NCBI database, and got the GenBank number KT630834. The length of D. huoshanense chloroplast genome is153,188 bp, and it has an 85,614 bp LSC (large single-copy) region and a 14,524 bp SSC (small single-copy) region, respectively. It also has a pair of 26,525 bp IR (inverted repeat) regions (Fig. 1). In addition, the D. huoshanense chloroplast genome nucleotide composition is asymmetrical including 30.0% A, 20.1% C,22.9% G and 27.0% T, and the GC contents is 37.5% (Table 1). Meantime, the highest values of CG contents in the IR regions are 43.4%, and the values of CG contents in the LSC and SSC regions are 35.0% and 30.5%, respectively.

Table 1

Length of the whole chloroplast genome of *D. huoshanense* and its base composition
Regions	T(U) (%)	C (%)	A (%)	G (%)	Length (bp)
LSC	33.2	17.9	31.8	17.2	85,614
SSC	36.3	15.9	33.2	14.6	14,524
IRa	28.3	22.4	28.3	21.0	26,525
IRb	28.3	22.4	28.3	21.0	26,525
Total	31.8	19.0	30.7	18.5	153,188

Furthermore, we found 129 genes (111 species), including 76 protein-coding genes (69 species) in the D. huoshanense chloroplast genome. It also contains 38 transfer RNA genes (30 species) and eight ribosomal RNA genes (four species) (Table 2). We also found 17 genes have introns, including atpF, ycf3,clpP, petB, petD ,rpl2, ndhB, rps16, rps12, rpoC1 rpl16 and 6 tRNA genes (Table 3). Otherwise, we found that 14 genes only have a single intron, and rps12, clpP and ycf3 contain 2 introns (Table 3).

Table 2

Gene contents in the chloroplast genomes of *D. huoshanense*
No	Group of Genes	Gene names	Total
1	ATP synthase	atpA,atpB,atpE ,atpF ,atpH,atpI	6
2	NADH dehydrogenase	ndhA, ndhB (x2) ,ndhE, ndhF(x2), ndhG, ndhH ,ndhJ	9
3	Cytochrome b/f complex	petA, petB,petD ,petG, petL, petN	6
4	Photosystem I	psaA, psaB, psaC, psaI, psaJ	5
5	Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	15
6	RubisCO large subunit	rbcL	1
7	Ribosomal proteins (LSU)	rpl14,rpl16, rpl2(x2),rpl20,rpl22,rpl23(x2),rpl32, rpl33, rpl36	11
8	RNA polymerase	rpoA,rpoB,rpoC1,rpoC2	4
9	Ribosomal proteins (SSU)	rps11,rps12(x2),rps14,rps15,rps16,rps18,rps19(x2) ,rps2,rps3,rps4, rps7(x2),rps8	15
10	Ribosomal RNAs	rrn16(x2),rrn23(x2) rrn4.5(x2), rrn5(x2)	8
11	Transfer RNAs	38tRNAs	38
12	Proteins of unknown function	ycf1, ycf2(x2) ycf3,ycf4	5
13	Other genes	accD, ccsA,cemA,clpP, infA, matK	6

Table 3

Genes with introns in the chloroplast genomes of *D. huoshanense* as well as the lengths of the exons and introns.
Gene	Location	Exon I(bp)	Intron I(bp)	Exon II(bp)	Intron II(bp)	Exon III(bp)
trnK(uuu)	LSC	35	2815	37
trnG(ucc)	LSC	31	671	59
trnL(uaa)	LSC	35	800	50
trnV(uac)	LSC	37	587	39
trnI(gau)	IR	37	944	35
trnA(ugc)	IR	38	801	35
rps16	LSC	248	892	40
atpF	LSC	410	924	145
rpoC1	LSC	1611	762	453
ycf3	LSC	153	723	230	747	124
clpP	LSC	231	966	292	672	71
petB	LSC	6	732	642
petD	LSC	8	862	484
rpl16	LSC	399	1197	9
rpl2	IR	431	714	385
ndhB	IR	756	699	777
rps12	LSC	138	-	229	547	26

Codon Usage Analysis

According to the protein-coding genes sequences, we estimated the codon usage frequency of D. huoshanense chloroplast genome with the CodonW software. The result showed that there are 64 codons encoding 20 amino acids and 23,448 codons identified in the protein-coding genes of the D. huoshanense chloroplast genome (Supplementary Table S2). The results also indicated that in D. huoshanense chloroplast genome, the most universal amino acids is 2,369 encode leucine, and the least universal amino acid is 276 encode cysteine (Supplementary Table S2). In addition, the results revealed that the value of relative synonymous codon usage (RSCU) increased with the quantity of codons that code for a specific amino acid, such as leucine, serine and arginase (Supplementary Table S2). Furthermore, in addition to the tryptophan and methionine, most of amino acid codons have preferences (Supplementary Table S2).

Simple sequence repeats analyses

Simple sequence repeats (SSRs), generally including 1–6 nucleotide repeat units, also named as microsatellites, are tandem repeated DNA sequences, which are ubiquitous among the genomes (Powell et al. 1995). SSRs are universally used for molecular markers in species identification, phylogenetic investigations and population genetics for the high level of polymorphism (Jiao et al. 2012; Xue et al. 2012; Yang et al. 2011). Using the microsatellite identification tool (MISA), there were 165 SSRs identified, and the largest SSRs in number is mononucletide throughout these SSRs, a total of 105 were found (Table 4). A/T mononucleotide repeats (96.1%) were the most common SSRs, and the majority of dinucleotide repeat sequences were the AT/TA repeats (55.6%) (Table 3). These results are in accord with the previous studies where proportions of polythymine (polyT) and polyadenine (polyA) were higher than polyguanine (polyG) and polycytosine (polyC) within chloroplast SSRs in many plants (Kuang et al. 2011; Zhou et al. 2017).

Table 4

**Types and amounts of SSRs in the** D. huoshanense **chloroplast genomes**
SSR Type	Repeat Unit	Amount	Ratio(%)
Mono	A/T	105	96.3%
Mono	C/G	4	3.7%
Di	AC/GT	3	5.6%
	AG/CT	21	38.9%
	AT/AT	30	55.5%
Tri	AAC/GTT	8	11.3%
	AAG/CTT	28	39.4%
	AAT/ATT	12	16.9%
	ACC/GGT	2	2.8%
	ACT/AGT	4	5.6%
	AGC/CTG	4	5.6%
	AGG/CCT	6	8.5%
	ATC/ATG	7	9.9%
Tetra	AAAG/CTTT	3	50.0%
	AATT/AATT	1	16.7%
	ACAG/CTGT	1	16.7%
	AGAT/ATCT	1	16.7%
Penta	AATCC/ATTGG	1	50.0%
Penta	ATATC/ATATG	1	50.0%
Hexa	ACAGAT/ATCTGT	1	100%

IR junctions and comparative genome analyses

Based on the previously study, the contraction and expansion at the borders of the inverted repeat regions are widely events during evolution, and stand for the primary reasons for size variation of the chloroplast genome (Yang et al. 2010; Raubeson et al. 2007). We compared the junctions of the LSC and IR regions of four species including Nicotiana tabacum (GenBank: NC_001879), Arabidopsis thaliana (GenBank: NC_000932), Dendrobium huoshanense and the reference Dendrobium catenatum. The results showed that the SSC/IRa border was situated in the CDS of the ycf1 gene in the four chloroplast genomes, and the trnH genes were not located in the LSC regions in D. huoshanense and D. catenatum (Fig. 2). These results are in line with those described for members of Orchidaceae (Jing et al. 2014).

With the mVISTA program, the complete chloroplast genome of D. huoshanense was compared to those of D. pendulum (Wang et al. 2017), D. strongylanthum (Jing et al. 2015), D. officinale (Yang et al. 2016) and D. catenatum (Jing et al. 2014) (Fig. 3). The finding revealed that the two IR districts were less conserved than the SSC and LSC districts, and the four rRNA genes were more conserved than other genes. In addition, the results also showed that coding districts appeared a higher conserved than non-coding districts, and the most divergent districts were localized in the intergenic spacers throughout the five chloroplast genomes (Fig. 3).

Phylogenetic analyses

Due to the quickly development of high-throughput sequencing technology, much more complete chloroplast plant genomes have been reported (Curci et al. 2015), and the plant chloroplast genomes provide abundant resources for the phylogenetic studies, taxonomic and evolutionary (Jansen et al. 2008; Moore et al. 2007; Qian et al. 2013). Furthermore, more and more phyloevolutionary relationships at almost any taxonomic level were successfully resolved, depending on the protein-coding genes or whole chloroplast genomes (Li et al. 2013b). Orchidaceae family is a largest family in the Kingdom Plantae, and Dendrobium is the most important medicinal plant of this family. According to the chloroplast sequences of Dendrobium plants, much information about the phylogenetic relationships of this category have been reported (Jing et al. 2015; Wang et al. 2017; Yang et al. 2016).

Therefore, to identify the phylogenetic positions of D. huoshanense within Orchidaceae family, 21 species from Phalaenopsis, Corallorhiza, Cymbidium, Dendrobium as well as Paphiopedilum were used to construct a phylogenetic tree with the neighbor-joining (NJ) method. The results showed that the bootstrap values for all of the clades subgroups were high, suggesting that genes in the same subgroups may share a similar origin, and D. huoshanense is the part of Dendrobium of Orchidaceae family, which is in accord with the traditional systematic plant classification (Fig. 4).

Discussion

With the development of Next Generation Sequencing (NGS), we could get more biological information for the species identification, molecular genetic markers and evolution within and between different species (W. et al., 1995; Grassi et al., 2002; Leonie et al., 2011; Straub et al., 2011). Therefore, the complete chloroplast genome also could furnish abundant genetic information and molecular markers that are valuable tools to solve obscure phylogenetic relationships among land plants (Luo et al., 2014; Alzahrani et al., 2021).

Here, using the Illumina sequencing platform and GENEIOUS program, we firstly sequenced and assembled the complete chloroplast genome of D. huoshanense. Reported Dendrobium complete chloroplast genomes range in size from 156,612 to 156,781 bp (Wang et al., 2017; Yan-xia et al., 2018; Zhang et al., 2018), and the size of assembled chloroplast genome of D.huoshanense is 153,188 bp, which is consistent with those reported previously in plants of the same species. The average GC content of the D. huoshanense chloroplast genome is 40.0%, similar to other Dendrobium species (Gao et al.; Wang et al., 2017; Zhang et al., 2018). It suggested that this method which was used for assembling the plant complete chloroplast genome is feasible. Furthermore this method is very more convenient and efficient than other methods.

In this newly determined chloroplast genome, 129 predicted genes were found. There are 42 genes are involved in photosynthesis, of which 6 encode different subunits of ATP synthase, 9 for the subunits of the NADH-oxidoreductase, 6 for the cytochrome b6/f complex, 5 for photosystem I, 15 for photosystem II, and 1 for the Rubisco. In addition, there are 38 tRNA genes, 36 ribosomal subunit genes (15 small subunits and 11 large subunits), 8 rRNA genes and 4 RNA polymerase genes. Furthermore, 5 genes encode miscellaneous proteins which were involved in different functions, and 6 genes were of unknown function (Table 5). Those results also showed that the genome organization appeared to be more conserved with unique gene sequence as discovered previously in Dendrobium species (Wang et al., 2017; Yan-xia et al., 2018; Zhang et al., 2018).

Table 5

Genes present in the *D. huoshanense* chloroplast genome.
Group	Name of genes	Numbers
Photosystem I	psaA, psaB, psaC, psaI, psaJ	5
Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ	15
Cytochrome b6/f	petA, petB, petD, petG, petL, petN	6
ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI	6
NADH dehydrogenase	ndhA, ndhB (×2), ndhD, ndhE, ndhF, ndhG, ndhH, ndhJ	9
Rubisco	rbcL	1
RNA polymerase	rpoA, rpoB, rpoC1, rpoC2	4
Small subunits of ribosome	rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2), rps14, rps15, rps16, rps18,rps19(×2)	15
Large subunits of ribosome	rpl2(×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36	11
Other genes	accD, ccsA, cemA, clpP, infA, matK	6
Miscellaneous proteins coding gene	ycf1, ycf2(×2), ycf3, ycf4	5
tRNAs	trnA-UGC(x2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC, trnH-GUG(x2), trnI-CAU(x2), trnI-GAU(x2), trnK-UUU, trnL-CAA(x2), trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(x2), trnP-UGG trnQ-UUG, trnR-ACG(x2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-GAC, trnV-UAC, trnW-CCA, trnY-GUA	38
rRNAs	rrn4.5(×2), rrn5(×2), rrn16(×2), rrn23(×2)	8

Additionally, we also found that there are 23,448 codons encoding the genes in the D. huoshanense chloroplast genome, and the most universal codons are the coding for the amino acids Leucine, which has been previously reported in some complete chloroplast genomes of plants (Liu et al., 2018; Alzahrani et al., 2021). Furthermore, there are 165 simple sequence repeats (SSRs) markers found in the 153,188 bp sequence of the D. huoshanense chloroplast genome. In other words, the observed frequency of SSRs was approximately 1/928.4 bp of chloroplast genome. As previously study, the SSRs were also only observed in the non-coding region of the chloroplast genome (Raveendar et al., 2015).

With the mVISTA program, we examined the degree of DNA sequence divergence in the four-chloroplast genome. In addition to the genes of ycf1, rps19, rpoC2, and atpF, many different non-coding regions among the four chloroplast genomes. We also found that the gene-coding regions are more highly conserved than those of their non-coding districts, which was similar to the other Dendrobium chloroplast genome. Otherwise, the IR-LCS and IR-SSC boundaries of four chloroplast genome plants were compared. The results displayed that N. tabacum has the greatest chloroplast genome from the four plants, while, D. catenatum has the smallest chloroplast genome in size. The largest LSC region in N. tabacum and the smallest in A. thaliana, The largest SSC region in N. tabacum and the smallest in D. catenatum, the largest IR region in D. huoshanense and the smallest in N. tabacum.

Phylogenetic relationships based on the chloroplast genomes protein-coding genes placed all samples into five main clades, where every family is in a separate clade (Fig. 4). The results advised that all species in the same clade could be clustered into the same genus, which indicated that the relationships and phylogenetic positions of this family could be resolved by the chloroplast genome. Nevertheless, it should use different methods to analyze the phylogeny for accurately clarify the evolution of the Orchidaceae family (Fig. 4). This study could offer a reference for plant classification among Dendrobium, and other genus with using the chloroplast genome.

Conclusions

D. huoshanense is a traditional Chinese herbal medicine with important phamaceutical and economic value. Here, we assemble the complete chloroplast genome of D. huoshanense, and revealed the genome characteristics and gene content. These studies enhance the genomic information for Dendrobium plants, and contribute to the study of germplasm diversity. These data also provide a valuable resource of labels for future research on Orchidaceae family. In addition, the genome sequence in chloroplast also offers more information on functional protein variability about the chloroplast.

Declarations

AcknowledgementsThe authors are thankful to the National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, for providing plant material to carry out this research work.

Funding This study was carried out with the support of the Jiangxi province project Education Fund (Project no: GJJ170434 ),National Natural Science Foundation of China ( Project no: 21968001 and 41562021 )

Author Contributions BW initiated the project, and BW and JC conducted experiments and drafted the manuscript. Bioinformatic analyses were performed by ML.

Availability of Data and Materials All the data and plant material are available with the corresponding author.

Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interest The authors declare that they have no conflict of interest.

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