Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species, comparative analysis and phylogenetic relationships.

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.

Introduction

Cleomaceae Bercht. et J. Presl (1825) family include 18 genera and 150–200 species (Patchell et al., 2014), are distributed in the tropics and subtropics areas, and widely uses as ornamentals (Heywood et al., 2007, Fay and Christenhusez, 2010) and a hypothesized origin in central Asia (Feodorova et al., 2010). Cleomaceae are herbs or shrubs; leaves usually palmately compound; fruits capsules, nutlets, or schizocarps, absent septum; and seeds with a testa, usually not arillate (Hall et al., 2002, Iltis et al., 2011).

Until recently, the Cleomaceae has been thought to be closely related with Capparaceae based on the morphological and chemical data (Hall et al., 2002, Iltis, 1957, Rodman et al., 1993, Rodman et al., 1996, Rodman et al., 1998, Rollins, 1993). Some authors merge Brassicaceae, Capparaceae and Cleomaceae into Brassicaceae (Angiosperm Phylogeny Group, 1998, Angiosperm Phylogeny Group, 2003, Judd et al., 1994, Judd et al., 2007). Pax and Hoffmann, 1936, Melchior, 1964 classified the family Capparaceae into two major subfamilies, Capparoideae and Cleomoideae. Recently, the two subfamilies have been elevated to familial rank, which had been previously proposed and believed by earlier taxonomists (Airy Shaw, 1965, Hutchinson, 1967). The phylogeny analysis of the chloroplast genome data strongly supports that the Cleomaceae is a monophyletic family, also the chloroplast sequences data highly supports that Cleomaceae is sister to Brassicaceae (Hall et al., 2002, Hall et al., 2004, Simpson, 2006, Heywood et al., 2007, Hall, 2008, Martín-Bravo et al., 2009, Angiosperm Phylogeny Group, 2009, Angiosperm Phylogeny Group, 2016, Alzahrani et al., 2020). In this study, the Cleomaceae family is represented in two genera: Cleome (Cleome chrysantha) and Dipterygium (Dipterygium glaucum).

Dipterygium glaucum Decne. is a monotypic genus with one species, a medicinal herb, widespread in the tropical and subtropical regions such as Saudi Arabia, Egypt, Sudan, and Pakistan (Ahmad et al., 2014, Mehmood et al., 2010). It is one of the medicinal plants that has multiple uses, it is commonly used for its treatment of miss‑breathing troubles as trachea dilating agent (Moussa et al., 2012), to treat jaundice, blood purifier, psoriasis, and ringworm infestation and as an antiasthma drug (Ahmad et al., 2014, Rahman et al., 2004). It has been stated that the herb of D. glaucum contains significant antioxidant, cytotoxic, and antimicrobial activities (Shaheen et al. 2017). Previous studies demonstrated that D. glaucum plants includes vital phytochemical compounds such as alkaloids, cardiac glycoside, bound anthraquinones, saponins, terpenoids, and sterols (Mehmood et al., 2010, Abdel-Mogib et al., 2000). Hutchinson, 1967, placed genus Dipterygium in Brassicaceae, while Pax and Hoffmann 1936 and Hedge et al., 1980 sited the genus into the Capparaceae family. Based on the evedince of presence of methyl-glucosinolate, some authors have placed the genus Dipterygium within the Capparaceae family, subfamily Dipterygioideae (Hedge et al., 1980, Luning et al., 1992). Presence of six stamens of equal length and a short gynophore are the main floral features that located the genus Dipterygium in Cleomoideae. Patchell et al., 2014 reported Dipterygium belongs to Cleomaceae based on three cpDNA (ndhF, matK, ycf1), one mtDNA (rps3), and one nrDNA (ITS) regions. The result of the phylogenetic analysis in Alzahrani et al. 2020 showed D. glaucum and Tarenaia hassleriana from Cleomaceae in one clade, which confirms placement of D. glaucum in Cleomaceae and is sister to T. hassleriana.

Cleome L. is the largest genus of the subfamily Cleomoideae, Cleomaceae, including 180 to 200 species of herbaceous annual or perennial plants, widespread distribution worldwide in tropical and subtropical areas (Abdullah et al., 2016). Various studies have reported diverse pharmacological activities of plants of the genus Cleome including antidiabetic, anticancer, antibacterial, anti-inflammatory, analgesic, antidiarrheal and antimalarial, as a result of the chemical compounds present in different parts of the Cleome plants such as essential oils, terpenes, flavonoids, glucosinolates and alkaloids (Tripti et al., 2015, Abdullah et al., 2016).

The complete chloroplast genomic has provided large genetic information and molecular markers that are useful for resolve obscure phylogenetic relationships in seed plants (Luo et al., 2014). The majority of the chloroplast genomes of land plants range from 120 to 160 kb and possess their own genomes is rich of evolutionary and phylogenetic information (Raubeson and Jansen, 2005, Yap et al., 2015). At present, more than 3000 complete chloroplast genomes are available in the NCBI database (https://www.ncbi.nlm.nih.gov/genome/GenomesGroup.cgi?taxid = 2759&opt = plastid) (Li et al., 2019). However, there is only two sequence from the chloroplast genome of Cleomaceae species in GenBank.

In this study, we reported the characteristics of the complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha for the first time. We also compared the cp genomes of four Cleomaceae species to investigate the diversity among chloroplast genomes, and SSR was used as a tool to facilitate the assessment of molecular diversity and to identify related species. To understand the relationships of the D. glaucum and C. chrysantha with other species in related families, we constructed the phylogenetic tree using their fully sequenced chloroplast genome sequences.

Materials and methods

Plant material and DNA extraction

Fresh young leaf materials for D. glaucum and C. chrysantha were collected through field survey in Makkah region, Saudi Arabia. Total genomic DNA was extracted from the samples using Qiagen genomic DNA extraction kit according to the manufacturer’s protocols.

Library construction, sequencing and assembly

A total amount of 1.0μ g DNA was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext DNA Library Prep Kit following manufacturer’s recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350 bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulting libraries were analyzed for size distribution by Agilent 2100 Bio analyzer and quantified using real-time PCR. The qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume. The raw reads were filtered to get the clean reads (5 Gb) using PRINSEQlite v0.20.4 (Schmieder and Edwards, 2011) and were subjected to de novo assembly using NOVOPlasty2.7.2 (Dierckxsens et al., 2017) with kmer (K-mer = 33) to assemble the complete chloroplast genome from the whole genome sequence. Finally, for each species one contig containing the complete chloroplast genome sequence was generated.

Gene annotation

Genes were annotated using DOGMA (Dual Organellar GenoMe Annotator, University of Texas at Austin, Austin, TX, USA) (Wyman et al., 2014). The positions of start and stop codon were adjusted manually. tRNA genes were identified by the trnAscan-SE server (http://lowelab.ucsc.edu/tRNAscan-SE/) (Schattner et al., 2005). The circular chloroplast genome maps were drawn using OGDRAW (Organellar Genome DRAW) (Lohse et al., 2007). The sequences of the chloroplast genome were deposited in the GenBank database: D. glaucum (MT041700) and C. chrysantha (MT948188).

Sequence analysis

The relative synonymous codon usage (RSCU) values, base composition and codon usage were analysed using MEGA 6.0 software. Potential RNA editing sites present in the protein coding genes were predicted by the PREP suite (Kurtz et al., 2001) with the cutoff value set to 0.8

Repeat analysis in chloroplast genome

The online software MIcroSAtellite (MISA) (Thiel et al., 2003) was used to identify Simple Sequence Repeats (SSRs) in the chloroplast genome with the following parameters: eight, five, four and three repeats units for mononucleotides, dinucleotides, trinucleotides and tetra, penta, hexa nucleotides SSR motifs respectively. The REPuter software (https://bibiserv.cebitec.uni-bielefeld.de/reputer) (Kurtz et al., 2001) was used with default settings to detect the size and location of the long repeats in the two Cleomaceae species.

Genome comparison

The chloroplast genome of D. glaucum, C. chrysantha, C. lutea (NC_049613) and T. hassleriana (NC_034354) were compared using mVISTA program (Mayor et al., 2000) and the annotation of D. glaucum used as reference in the Shuffle-LAGAN mode (Frazer et al., 2004). Comparison of the large single copy (LSC), inverted repeat (IR), small single copy (SSC), and inverted repeat (IR) boundaries among the four species of Capparaceae plastomes.

Characterization of substitution rate

The nonsynonymous (dN) and synonymous (dS) substitution rates were calculated using the DNAsp v5.10.01 (Librado and Rozas 2009). The plastome of D. glaucum was compared with the plastome of C. chrysantha, C. lutea and T. hassleriana to identify the genes that are under selective pressure. Geneious software v. 8.1.3 (Biomatters, Ltd., Auckland, New Zealand) was used to align the individual protein coding genes separately, the aligned sequences were then translated into protein sequence.

Phylogenetic analysis

The analysis was conducted based on the complete chloroplast genome sequences of four Cleomaceae species (D. glaucum, C. chrysantha, C. lutea and T. hassleriana), four Capparaceae species, eight species of Brassicaceae and two species of Malvaceae that were used as outgroup. All of the sequences were aligned using MAFFT (Katoh and Standley 2013) with default settings. The phylogenetic trees were reconstructed based on Maximum Parsimony (MP) method using PAUP version 4.0b10 (Felsenstein 1978) and a using heuristic search strategy of 1000 random sequence addition replicates with tree bisection– reconnection (TBR) branch swapping, saving a maximum 100 trees pear replicate, with MulTrees on, gaps were treated as missing data. statistical support was assessed for clades with nonparametric bootstrap analysis using 1000 bootstrap replicates. A 50% majority-rule consensus tree was calculated from all the most parsimonious trees. Bayesian inference (BI) analyses were performed in MrBayes version 3.2.6 (Fredrik et al., 2012) the best models were selected using jModelTest version 3.7 (Posada 2008).

Results

Characteristics of two chloroplast genome

The complete plastome sequence has a circular and quadripartite structure, the total length of the D. glaucum genome is 158,576 bp (Alzahrani et al., 2020), 158,111 bp in C. chrysantha. The plastome has four distinct regions in D. glaucum and C. chrysantha which are Large Single Copy (LSC) length is 87,738 bp and 87,184 bp respectively, Small Single Copy (SSC) length is 18,420 bp and 18,425 bp respectively and a pair of Inverted repeats (IRa and IRb) length is 26,209 bp and 26,251 bp respectively which separates the LSC and SSC regions (Fig. 1; Table 1). The region coding for genes in D. glaucum and C. chrysantha respectively is 76, 598 bp – 76,905 bp in length which constitutes 48.30% − 48.63% of the genome, the remaining 72, 286 bp – 71,042 bp is the non-coding region which includes intron and intergenic spacer (45.58% − 44.93%). The plastome sequence has GC of 35.74% − 35.96% and AT content of 64.23% − 64.02% respectively (Table 1). The LSC regions possessed GC content of 33.27% − 33.59% and the SSC regions content of 28.76% − 28.97%, while the inverted repeats IRa and IRb have 42.34% − 42.32%. The chloroplast genome sequences were deposited in the GenBank: Accession Number for D. glaucum is (MT041700) and for C. chrysantha is (MT948188).

Fig. 1

Chloroplast genome maps of the two Cleomaceae (A) D. glaucum and (B) C. chrysantha. Genes inside of the circles are transcribed clockwise, while genes outside circles are transcribed counterclockwise. The light grey inner circle and the dark grey corresponds to the AT and GC content respectively. The different colours represent the different functional groups of the genes.

Table 1

Bases content in the D. glaucum and C. chrysantha chloroplast genomes.

Species	D. glaucum	C. chrysantha
Genome size (bp)	158,576	158,111
IR (bp)	26,209	26,251
LSC (bp)	87,738	87,184
SSC (bp)	18,420	18,425
Total number of genes	136	136
rRNA	4	4
tRNA	31	31
Protein-coding genes	80	80
T (U) %	32.57	32.48
C %	18.2	18.32
A %	31.66	31.54
G %	17.54	17.64

Result of the genes annotation revealed a total of 136 in the two species, 117 are unique; the remaining 19 are duplicated in the inverted region. The plastome contained 80 protein coding genes, 31 tRNA genes and four rRNA genes (Fig. 1 and Table 2). The inverted repeat region contained eight protein coding genes, seven tRNA in D. glaucum and eight tRNA in C. chrysantha and four rRNA in the single copy region; the LSC contained 61 protein coding genes and 23 tRNA genes, the remaining 13 protein coding genes and one tRNA are situated within the SSC region.

Table 2

Gene contents in the chloroplast genomes of D. glaucum and C. chrysantha.

Category	Group of genes	Name of genes
RNA genes	ribosomal RNA genes (rRNA)	rrn5, rrn4.5, rrn16, rrn23
	Transfer RNA genes (tRNA)	trnH-GUG, trnK-UUU+, trnQ-UUG, trnS-GCU, trnT-CGU+, trnR-UCU, trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC (A, B+,a), trnT-GGU, trnS-UGA, trnG-UCC, trnfM-CAU, trnS-GGA, trnT-UGU, trnL-UAA+, trnF-GAA, trnV-UAC+, trnM-CAU, trnW-CCA, trnP-UGG, trnP-GGG, trnI-CAUa, trnL-CAAa, trnV-GACa, trnI-GAU (A+,a,B), trnA-UGC+,a, trnR-ACGa, trnN-GUUa, trnL-UAG
Ribosomal proteins	Small subunit of ribosome	rps2, rps3, rps4, rps7a, rps8, rps11, rps12a, rps14, rps15, rps16+, rps18, rps19
Transcription	Large subunit of ribosome	rpl2+,a, rpl14, rpl16, rpl20, rpl22, rpl23a, rpl32, rpl33, rpl36
	DNA dependent RNA polymerase	rpoA, rpoB, rpoC1+, rpoC2
	Translational initiation factor	infA
Protein genes	Photosystem I	psaA, psaB, psaC, psaI, psaJ, ycf3++
	Photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunit of cytochrome	petA, petB, petD, petG, petL, petN
	Subunit of synthase	atpA, atpB, atpE, atpF+, atpH, atpI
	Large subunit of rubisco	rbcL
	NADH dehydrogenase	ndhA+, ndhB+,a, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	ATP dependent protease subunit P	clpP++
	Chloroplast envelope membrabe protein	cemA
Other genes	Maturase	matK
	Subunit acetyl-coA carboxylase	accD
	C-type cytochrome systhesis	ccsA
	Hypothetical proteins	ycf2a, ycf4, ycf15 (A++,a,B)
	Component of TIC complex	ycf1a

*(A) D. glaucum cp genome; (B) C. chrysantha cp genome.

Gene with one intron, ++ Gene with two intron and a Gene with copies.

The composition of A is 31.66% − 31.54%, C is 18.2% − 18.32%, T is 32.57% − 32.48% and G is 17.54% − 17.64% in D. glaucum and C. chrysantha respectively. Our results revealed that the majority of protein coding genes start with the typical ATG codon, which is responsible in the coding of methionine, while others begin with the codons ATC, GTG and ACG, as in most Angiosperms plant chloroplast genomes (Raman and Park, 2016, Park et al., 2017, Li et al., 2017). Intron is present in several of the protein coding and tRNA genes of the D. glaucum and C. chrysantha chloroplast genomes, similar to other chloroplast genomes of flowering plants (Raman and Park, 2016, Park et al., 2017).

There are 15 genes in D. glaucum and C. chrysantha that contain intron out of the total genes, among the 15 genes, nine are protein coding genes while the remaining six are tRNA genes. One gene has the intron namely ndhA located in SSC region, five genes namely rpl2, ycf15, ndhB, trnI-GAU and trnA-UGC are located in the IR region in D. glaucum and four genes namely rpl2, ndhB, trnE-GAU and trnA-UGC in C. chrysantha, while the remaining nine are located in the LSC region. Only three genes ycf3, clpP and ycf15 have two introns in D. glaucum and two genes ycf3 and clpP in C. chrysantha, the other 12 genes have only one intron. The tRNA, trnK-UUU has the longest intron of 2570 bp in D. glaucum and 2568 pb in C. chrysantha, this is as a result of the matK gene being located within the intron of the trnK.

Codon usage

Codon usage is vital in the influence of the chloroplast genome evolution. Scientists have stated that the evolutionary phenomenon occurs as a result of bias in mutation (Li et al., 2017). The nucleotides of protein coding and tRNA genes were used in computing the codon usage bias of the plastome. The nucleotides sequences in D. glaucum: 94,112 bp and in C. chrysantha: 89,715 bp. Supplementary Table A1, Table A2 present the relative synonymous codon usage (RSCU) of each codon in the genome, the result indicated that all the genes in D. glaucum are encoded by 31,366 codons, while in C. chrysantha they encoded by 26,475 codons, coding for the amino acids Leucine are the most frequent codons, this has been stated in other flowering plant genomes (Liu et al., 2018); 2,772 (8.83%) in D. glaucum and 2,831 (10.69%) in C. chrysantha (Fig. 2), whereas codons coding are the least in the genome for Tryptophan 659 (2.10%) in D. glaucum and 452 (1.70%) in C. chrysantha (Fig. 2). A- and T- ending are discovered to be less frequent than their counterparts G- and C-. Appendices Table A1, Table A2 presented the result of the analysis that indicates the codon usage bias is low in the chloroplast genome of Cleomaceae species. The RSCU values of 28 codons were greater than 1 and all having A/T endings, whereas other 28 codons were less than 1 and all end with G/C. The RSCU values of Tryptophan and Methionine amino acids 1, therefore they have only one codon.

Table A1

Codon – anticodon recognition patterns and codon usage of D. glaucum chloroplast genome.

Codon	Amino Acid	RSCU	tRNA	Codon	Amino Acid	RSCU	tRNA
UUU	Phe	1.3	trnF-GAA	UAU	Tyr	1.45	trnY-GUA
UUC	Phe	0.7		UAC	Tyr	0.55
UUA	Leu	1.76	trnL-UAA	UAA	Stop	1.08
UUG	Leu	1.15	trnL-CAA	UAG	Stop	0.7
CUU	Leu	1.28	trnL-UAG	CAU	His	1.5	trnH-GUG
CUC	Leu	0.52		CAC	His	0.5
CUA	Leu	0.86		CAA	Gln	1.52	trnQ-UUG
CUG	Leu	0.42		CAG	Gln	0.48
AUU	Ile	1.46	trnI-GAU	AAU	Asn	1.43	trnN-GUU
AUC	Ile	0.73		AAC	Asn	0.57
AUA	Ile	0.81	trnI-CAU	AAA	Lys	1.48	trnK-UUU
AUG	Met	1	trnM-CAU	AAG	Lys	0.52
GUU	Val	1.45	trnV-GAC	GAU	Asp	1.51	trnD-GUC
GUC	Val	0.73		GAC	Asp	0.49
GUA	Val	1.27		GAA	Glu	1.45	trnE-UUC
GUG	Val	0.54	trnV-UAC	GAG	Glu	0.55
UCU	Ser	1.44	trnS-GGA	UGU	Cys	1.2	trnC-GCA
UCC	Ser	0.92		UGC	Cys	0.8
UCA	Ser	1.3		UGA	Stop	1.22
UCG	Ser	0.69	trnS-UGA	UGG	Trp	1	trnW-CCA
CCU	Pro	1.4	trnP-UGG	CGU	Arg	0.83	trnR-ACG
CCC	Pro	0.78		CGC	Arg	0.32	trnR-UCU
CCA	Pro	1.19		CGA	Arg	1.12
CCG	Pro	0.63		CGG	Arg	0.49
ACU	Thr	1.29		AGA	Arg	0.99
ACC	Thr	0.86		AGG	Arg	0.65
ACA	Thr	1.23	trnT-GGU	AGU	Ser	2.07	trnS-GCU
ACG	Thr	0.61	trnT-UGU	AGC	Ser	1.18
GCU	Ala	1.62	trnA-UGC	GGU	Gly	1.09
GCC	Ala	0.65		GGC	Gly	0.51
GCA	Ala	1.24		GGA	Gly	1.54
GCG	Ala	0.49		GGG	Gly	0.86	trnG-UCC

Table A2

Codon – anticodon recognition patterns and codon usage of C. chrysantha chloroplast genome.

Codon	Amino Acid	RSCU	tRNA	Codon	Amino Acid	RSCU	tRNA
UUU	Phe	1.31	trnF-GAA	UAU	Tyr	1.6	trnY-GUA
UUC	Phe	0.69		UAC	Tyr	0.4
UUA	Leu	1.88	trnL-UAA	UAA	Stop	0.86
UUG	Leu	1.16	trnL-CAA	UAG	Stop	1.48
CUU	Leu	1.25	trnL-UAG	CAU	His	1.51	trnH-GUG
CUC	Leu	0.46		CAC	His	0.49
CUA	Leu	0.84		CAA	Gln	1.55	trnQ-UUG
CUG	Leu	0.41		CAG	Gln	0.45
AUU	Ile	1.48	trnI-GAU	AAU	Asn	1.54	trnN-GUU
AUC	Ile	0.61		AAC	Asn	0.46
AUA	Ile	0.92	trnI-CAU	AAA	Lys	1.49	trnK-UUU
AUG	Met	1	trnM-CAU	AAG	Lys	0.51
GUU	Val	1.42	trnV-GAC	GAU	Asp	1.61	trnD-GUC
GUC	Val	0.55		GAC	Asp	0.39
GUA	Val	1.43		GAA	Glu	1.47	trnE-UUC
GUG	Val	0.6	trnV-UAC	GAG	Glu	0.53
UCU	Ser	1.7	trnS-GGA	UGU	Cys	1.45	trnC-GCA
UCC	Ser	0.92		UGC	Cys	0.55
UCA	Ser	1.21		UGA	Stop	0.67
UCG	Ser	0.58	trnS-UGA	UGG	Trp	1	trnW-CCA
CCU	Pro	1.58	trnP-UGG	CGU	Arg	1.33	trnR-ACG
CCC	Pro	0.78		CGC	Arg	0.4	trnR-UCU
CCA	Pro	1.14		CGA	Arg	1.38
CCG	Pro	0.5		CGG	Arg	0.48
ACU	Thr	1.63		AGA	Arg	1.71
ACC	Thr	0.74		AGG	Arg	0.7
ACA	Thr	1.21	trnT-GGU	AGU	Ser	1.17	trnS-GCU
ACG	Thr	0.42	trnT-UGU	AGC	Ser	0.42
GCU	Ala	1.85	trnA-UGC	GGU	Gly	1.3
GCC	Ala	0.59		GGC	Gly	0.37
GCA	Ala	1.1		GGA	Gly	1.66
GCG	Ala	0.45		GGG	Gly	0.67	trnG-UCC

Fig. 2

Amino acids frequencies in D. glaucum and C. chrysantha chloroplast genome protein coding sequences.

RNA editing sites

RNA editing sites feature a set of processes that include inserting, deleting or modifying nucleotides which alter the DNA-encoded sequence of a transcribed RNA (Mower 2009), which saves a way to create transcript and protein diversity (Bundschuh et al., 2011). Some chloroplast RNA editing site are preserved in plants (Zeng et al., 2007). The PREP suite program was used to predict the RNA editing sites in the two species chloroplast genomes, the first codon position of the first nucleotide used in all the analysis. The results (Appendices Table A3, Table A4) indicates that the amino acid Serine to Leucine are the majority of the conversion in the codon positions, this conversion is found to occur more frequently (Luo et al., 2014). In all, the programme revealed 41 editing sites in the genome distributed among 16 protein coding genes in D. glaucum and 35 editing sites distributed among 14 protein coding genes in C. chrysantha. As reported in previous researches (Wang et al., 2017, Kumbhar et al., 2018, Park et al., 2018) in D. glaucum, the ndhB gene have the highest number of editing site (10 sites) followed by ndhD (7 sites) and accD, psaB, psbE, psbF, rpoC2, rps14, rps16 have the least one site each; in C. chrysantha the ndhD gene have the highest number of editing site (seven sites) followed by ndhB (five sites) and the following genes: psbE, psbF, rpoC1, rpoC2, rps14, rps16 have the least, one site each. Certain RNA sites amidst all the conversion in the RNA editing (modification) site changed the amino acid from Proline to Serine, one site in D. glaucum and two sites in C. chrysantha. RNA predicting site in the first codon of the first nucleotides are not present in the following genes atpA, atpB, atpF, atpI, ccsA, petB, petD, petG, psaI, psbB, psbL, rpl2, rpl20, rpl23, rpoA, rps2, rps8 and ycf3 among others, except the genes ndhA, petL and psaB which are only found in C. chrysantha. This result indicated that the preservation of RNA editing is fundamental (Magdalena et al., 2009, Huang et al., 2013).

Table A3

Predicted RNA editing site in the D. glaucum chloroplast genome.

Gene	Nucleotide Position	Amino Acid Position	Codon	Amino Acid	Score
accD	791	264	TCG => TTG	S => L	0.8
clpP	464	155	GCT => GTT	A => V	1
	493	165	CAT => TAT	H => Y	1
matK	706	236	CAT => TAT	H => Y	1
	1250	417	TCA => TTA	S => L	0.86
	1309	437	CAC => TAC	H => Y	1
ndhA	125	42	ACA => ATA	T => I	0.8
	341	114	TCA => TTA	S => L	1
ndhB	149	50	TCA => TTA	S => L	1
	467	156	CCA => CTA	P => L	1
	586	196	CAT => TAT	H => Y	1
	611	204	TCA => TTA	S => L	0.8
	746	249	TCT => TTT	S => F	1
	830	277	TCA => TTA	S => L	1
	836	279	TCA => TTA	S => L	1
	1255	419	CAT => TAT	H => Y	1
	1481	494	CCA => CTA	P => L	1
	1526	509	CCT => CTT	P => L	1
ndhD	65	22	TCT => TTT	S => F	0.8
	401	134	TCA => TTA	S => L	1
	692	231	TCG => TTG	S => L	1
	896	299	TCA => TTA	S => L	1
	905	302	CCC => CTC	P => L	1
	1328	443	TCA => TTA	S => L	0.8
	1423	475	CTT => TTT	L => F	0.8
ndhF	205	69	CAT => TAT	H => Y	0.8
	290	97	TCA => TTA	S => L	1
	586	196	CTT => TTT	L => F	0.8
ndhG	166	56	CAT => TAT	H => Y	0.8
	314	105	ACA => ATA	T => I	0.8
psaB	452	151	ACA => ATA	T => I	1
psbE	214	72	CCT => TCT	P => S	1
psbF	77	26	TCT => TTT	S => F	1
rpoB	338	113	TCT => TTT	S => F	1
	551	184	TCA => TTA	S => L	1
	2432	811	TCA => TTA	S => L	0.86
rpoC1	41	14	TCA => TTA	S => L	1
	1943	648	ACT => ATT	T => I	0.86
rpoC2	2335	779	GCC => GTC	A => V	0.86
rps14	80	27	TCA => TTA	S => L	1
rps16	176	59	TCA => TTA	S => L	0.83

Table A4

Predicted RNA editing site in the C. chrysantha chloroplast genome.

Gene	Nucleotide Position	Amino Acid Position	Codon	Amino Acid	Score
accD	791	264	TCG => TTG	S => L	0.8
	1400	467	CCT => CTT	P => L	1
clpP	167	56	GCT => GTT	A => V	1
	196	66	CAT => TAT	H => Y	1
matK	709	237	CAT => TAT	H => Y	1
	1253	418	TCA => TTA	S => L	0.86
	1312	438	CAC => TAC	H => Y	1
ndhB	149	50	TCA => TTA	S => L	1
	467	156	CCA => CTA	P => L	1
	586	196	CAT => TAT	H => Y	1
	611	204	TCA => TTA	S => L	0.8
	746	249	TCT => TTT	S => F	1
ndhD	20	7	ACG => ATG	T => M	1
	401	134	TCA => TTA	S => L	1
	692	231	TCA => TTA	S => L	1
	896	299	TCA => TTA	S => L	1
	905	302	CCC => CTC	P => L	1
	1328	443	TCA => TTA	S => L	0.8
	1423	475	CTT => TTT	L => F	0.8
ndhF	205	69	CAT => TAT	H => Y	0.8
	290	97	TCA => TTA	S => L	1
	586	196	CTT => TTT	L => F	0.8
ndhG	166	56	CAT => TAT	H => Y	0.8
	314	105	ACA => ATA	T => I	0.8
psbE	214	72	CCT => TCT	P => S	1
psbF	77	26	TCT => TTT	S => F	1
rpoB	338	113	TCT => TTT	S => F	1
	551	184	TCA => TTA	S => L	1
	566	189	TCG => TTG	S => L	1
	1981	561	CCC => TCC	P => S	0.86
	2434	812	TCA => TTA	S => L	0.86
rpoC1	1517	506	ACT => ATT	T => I	0.86
rpoC2	2342	781	GCC => GTC	A => V	0.86
rps14	80	27	TCA => TTA	S => L	1
rps16	176	59	TCA => TTA	S => L	0.83

Repeat analysis

Long repeats

Repeats sequences in the chloroplast genomes of the four species were determined by the REPuter programme with default settings; obtained results clearly show that forward, reverse, palindrome, and complemented repeats were detected in the cp genomes. The long repeats analysis showed in Dipterygium glaucum, Cleome chrysantha, Cleomella lutea and Tarenaya hassleriana: 20–22-16–16 palindromic repeats, 16–19-16–19 forward repeats, 11–6-16–14 reverse repeats and 2–2-1–0 complement repeat respectively (Fig. 3). Majority of the repeats size respectively are: between 20 and 29 bp (81.63%-89.79%-91.83%-59.18%), followed 30–39 bp (12.24%-6.12%-6.12%-24.48%), whereas 40–49 bp (2.04%-2.04%- absent-14.28%), followed 50–59 bp (4.08%-2.04%- absent-2.04%) and 60–69 bp present only in C. lutea (2.04%) (Appendices Table A5, Table A6). In total, the chloroplast genome of the four species there are 49 repeats. In the first location the codon region harbored 61.22% of the repeats in D. glaucum and 67.34% in C. chrysantha, tRNA contained 3 repeats (6.12%) in D. glaucum and 6 repeats (12.24%) in C. chrysantha, the remaining are located in the protein coding genes 8 repeats (16.32%) in D. glaucum and 5 repeats (10.20%) in C. chrysantha. The length of repeated sequences in four chloroplast genomes ranged from 10 to 69 bp, analogous to the lengths in other angiosperm plants (Li et al., 2017, Greiner et al., 2008, Song et al., 2017).

Fig. 3

Number of different repeats in the chloroplast genomes of the four Cleomaceae species. P = palindromic, F = forward, R = reverse and C = complement.

Table A5

Repeat sequences present in the D. glaucum chloroplast genome.

SN	Repeat Size	Repeat Position 1	Repeat Type	Repeat Location	Repeat Position 2	Repeat Location 2	E-Value
1	55	0	P	IGS	87,683	rps19	5.45E-24
2	52	30,403	P	IGS	30,403	IGS	3.49E-22
3	47	40,642	F	psaB	42,866	psaA	3.57E-19
4	39	45,750	F	ycf3 Intron	102,076	IGS	2.34E-14
5	39	45,750	P	ycf3 Intron	144,199	IGS	2.34E-14
6	34	9240	R	IGS	9240	IGS	2.40E-11
7	34	44,864	P	ycf3 Intron	44,864	ycf3 Intron	2.40E-11
8	31	9248	R	IGS	9248	IGS	1.53E-09
9	30	8750	P	IGS	8750	IGS	6.13E-09
10	29	30,292	F	IGS	30,321	IGS	2.45E-08
11	29	95,068	F	ycf2	95,086	ycf2	2.45E-08
12	29	95,068	P	ycf2	151,199	ycf2	2.45E-08
13	29	95,086	P	ycf2	151,217	ycf2	2.45E-08
14	29	151,199	F	ycf2	151,217	ycf2	2.45E-08
15	28	8507	P	trnS-GCU	46,871	trnS-GGA	9.81E-08
16	26	9240	F	IGS	9253	IGS	1.57E-06
17	26	10,325	P	IGS	10,357	IGS	1.57E-06
18	25	38,275	R	IGS	38,275	IGS	6.28E-06
19	25	128,760	R	ycf1	128,760	ycf1	6.28E-06
20	24	8760	R	IGS	8760	IGS	2.51E-05
21	24	32,513	P	IGS	32,513	IGS	2.51E-05
22	24	44,474	P	IGS	73,687	clpP Intron	2.51E-05
23	24	127,804	P	IGS	127,804	IGS	2.51E-05
24	23	3434	P	matK	91,728	ycf2	1.01E-04
25	23	3434	F	matK	154,563	ycf2	1.01E-04
26	23	8872	F	IGS	8895	IGS	1.01E-04
27	23	10,643	R	IGS	10,643	IGS	1.01E-04
28	23	30,247	F	IGS	30,268	IGS	1.01E-04
29	23	44,479	R	IGS	44,479	IGS	1.01E-04
30	23	92,588	F	ycf2	92,612	ycf2	1.01E-04
31	23	92,588	P	ycf2	153,679	ycf2	1.01E-04
32	23	92,612	P	ycf2	153,703	ycf2	1.01E-04
33	23	153,679	F	ycf2	153,703	ycf2	1.01E-04
34	22	10,745	F	IGS	10,767	IGS	4.02E-04
35	22	15,273	C	IGS	28,813	IGS	4.02E-04
36	22	43,907	P	IGS	96,899	ycf2	4.02E-04
37	22	43,907	F	IGS	149,393	IGS	4.02E-04
38	22	44,898	P	ycf3 Intron	44,898	ycf3 Intron	4.02E-04
39	22	48,054	F	IGS	48,074	IGS	4.02E-04
40	22	63,409	P	IGS	63,409	IGS	4.02E-04
41	22	103,201	R	IGS	103,201	IGS	4.02E-04
42	22	103,201	C	IGS	143,091	IGS	4.02E-04
43	22	113,539	P	ycf1	113,539	ycf1	4.02E-04
44	22	113,539	F	ycf1	132,753	ycf1	4.02E-04
45	22	132,753	P	ycf1	132,753	ycf1	4.02E-04
46	22	143,091	R	IGS	143,091	IGS	4.02E-04
47	21	8511	F	trnS-GCU	37,322	trnS-UGA	1.61E-03
48	21	8723	R	IGS	8723	IGS	1.61E-03
49	21	9240	R	IGS	9240	IGS	1.61E-03

Table A6

Repeat sequences present in the C. chrysantha chloroplast genome.

SN	Repeat Size	Repeat Position 1	Repeat Type	Repeat Location	Repeat Position 2	Repeat Location 2	E-Value
1	52	30,002	P	IGS	30,002	IGS	3.47E-22
2	47	40,405	F	psaB	42,629	psaA	3.55E-19
3	38	29,603	P	IGS	29,603	IGS	9.31E-14
4	34	32,963	P	IGS	32,963	IGS	2.38E-11
5	33	117,125	F	IGS	117,141	IGS	9.53E-11
6	29	92,074	F	ycf2	92,098	ycf2	2.44E-08
7	29	92,074	P	ycf2	153,124	ycf2	2.44E-08
8	29	92,098	P	ycf2	153,148	ycf2	2.44E-08
9	29	118,905	F	IGS	118,921	IGS	2.44E-08
10	29	153,124	F	ycf2	153,148	ycf2	2.44E-08
11	28	8401	P	trnS-GCU	46,575	trnS-GGA	9.76E-08
12	27	37,820	R	IGS	37,820	IGS	3.90E-07
13	26	79	P	IGS	79	IGS	1.56E-06
14	26	9949	P	IGS	9981	IGS	1.56E-06
15	26	116,265	P	IGS	116,265	IGS	1.56E-06
16	24	28,548	P	IGS	28,548	IGS	2.50E-05
17	24	32,078	P	IGS	32,078	IGS	2.50E-05
18	24	58,529	P	IGS	58,558	IGS	2.50E-05
19	24	76,983	P	IGS	77,008	IGS	2.50E-05
20	24	116,687	F	IGS	116,710	IGS	2.50E-05
21	23	4642	F	IGS	4708	IGS	9.99E-05
22	23	46,656	F	IGS	46,676	IGS	9.99E-05
23	23	97,249	F	IGS	97,271	IGS	9.99E-05
24	23	97,249	P	IGS	147,957	IGS	9.99E-05
25	23	97,271	P	IGS	147,979	IGS	9.99E-05
26	23	147,957	F	IGS	147,979	IGS	9.99E-05
27	22	38,011	P	IGS	38,011	IGS	4.00E-04
28	22	67,702	F	IGS	67,724	IGS	4.00E-04
29	22	113,002	P	ycf1	113,002	ycf1	4.00E-04
30	22	113,002	F	ycf1	132,227	ycf1	4.00E-04
31	22	117,470	R	IGS	117,470	IGS	4.00E-04
32	22	132,227	P	ycf1	132,227	ycf1	4.00E-04
33	21	314	P	IGS	363	IGS	1.60E-03
34	21	4878	R	IGS	4878	IGS	1.60E-03
35	21	5114	C	IGS	32,753	IGS	1.60E-03
36	21	5141	F	IGS	5166	IGS	1.60E-03
37	21	8068	C	IGS	30,261	IGS	1.60E-03
38	21	8405	F	trnS-GCU	37,002	trnS-UGA	1.60E-03
39	21	37,002	P	trnS-UGA	46,578	trnS-GGA	1.60E-03
40	21	38,404	F	trnfM-CAU	69,049	trnP-GGG - trnP-UGG	1.60E-03
41	21	56,602	F	IGS	56,623	IGS	1.60E-03
42	21	74,400	R	IGS	74,400	IGS	1.60E-03
43	21	117,116	R	IGS	117,116	IGS	1.60E-03
44	21	119,109	R	IGS	119,109	IGS	1.60E-03
45	20	160	F	IGS	8149	psbI	6.39E-03
46	20	4063	F	trnK-UUU Intron	23,615	rpoC1 Intron	6.39E-03
47	20	4063	P	trnK-UUU Intron	28,441	IGS	6.39E-03
48	20	4064	F	trnK-UUU Intron	111,518	IGS	6.39E-03
49	20	4064	P	trnK-UUU Intron	133,713	IGS	6.39E-03

Simple sequence repeats (SSRs).

The SSRs or microsatellites are a group of short repeat sequences of nucleotide series (1–6 bp), which are used as a tool that facilitates the assessment of molecular diversity (Kaila et al., 2017). The genetic variation within and among species with the valuable molecular marker of the SSRs are extremely important for studying genetic heterogeneity, and contributes to species recognition (Bryan et al., 1999, Provan, 2000, Ebert and Peakall, 2009). In this study, the microsatellites were found in plastid genome of D. glaucum is 323, C. chrysantha is 313, C. lutea is 258 and of T. hassleriana is 328 (Table 3). Majority of SSRs in the cp genome in D. glaucum, C. chrysantha, C. lutea and T. hassleriana are mononucleotide (90.24%-86.93%-84.88%-87.19%) respectively of which most are poly T and A (Fig. 4) Poly T (polythymine) constituted (51.21%-48.24%-49.18%-55.61%) whereas poly A (polyadenine) (37.56%-36.68%-31.89%-30.61%) respectively. Only two poly C (polycytosine) (0.97%) in D. glaucum, three (1.50%) in C. chrysantha and (1.53%) in T. hassleriana and five (2.7%) in C. lutea and only a single poly G (polyguanine) in three species (0.48% in D. glaucum, 0.50% in C. chrysantha, 0.54% in C. lutea) and absent in T. hassleriana. The dinucleotide AT/AT is found in the all genomes. Reflecting series complementary, three trinucleotide AAC/GTT, AAG/CTT and AAT/ATT, seven tetranucleotide AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AACG/CGTT, AATT/AATT, AGAT/ATCT and ATCC/ATGG, four pentanucleotide AAATG/ATTTC, AAATG/ATTTC, AATAT/ATATT and AATTC/AATTG and two hexanucleotide were discovered in the genome (Fig. 4). High richness in mono nucleotides poly A and T has been observed in most flowering plants cp genome (Li et al., 2017). The density of microsatellite in the intergenic spacer regions are significantly more (88.29%-86.39%) than the coding regions (11.70%-13.60%) in D. glaucum and C. chrysantha respectively (Fig. 5).

Table 3

The SSRs in four chloroplast genomes of Cleomaceae.

SSR type	Repeat unit	Species
		D. glaucum	C. chrysantha	C. lutea	T. hassleriana
Mono	A/T	277	268	212	282
	C/G	5	4	7	4
Di	AG/CT	1	1	1	0
	AT/AT	16	12	17	11
Tri	AAC/GTT	1	1	0	0
	AAG/CTT	0	1	0	1
	AAT/ATT	4	3	5	7
Tetra	AAAC/GTTT	0	0	0	1
	AAAG/CTTT	2	2	0	1
	AAAT/ATTT	11	12	10	14
	AACG/CGTT	1	0	0	1
	AATT/AATT	1	5	3	1
	AGAT/ATCT	1	1	1	1
	ATCC/ATGG	1	1	1	1
Penta	AAAAG/CTTTT	0	0	0	1
	AAATG/ATTTC	1	0	0	0
	AATAT/ATATT	1	0	0	1
	AATTC/AATTG	0	1	0	0
Hexa	AAAAAG/CTTTTT	0	0	1	0
	AAATTC/AATTTG	0	1	0	1

Fig. 4

Frequency of different SSR motifs in different repeat types in four chloroplast genomes of Cleomaceae.

Fig. 5

SSR Number in complete genome, protein coding regions, and noncoding genes: (A) D. glaucum, (B) C. chrysantha.

The result of comparative analysis of the simple sequence repeat between the chloroplast genome sequences of the four Cleomaceae species (Fig. 6) indicated that the more frequent occurrences are the mononucleotide repeats. The highest number of mononucleotide in T. hassleriana is 286. Pentanucleotide is not present in C. lutea and hexanucleotide is not present in D. glaucum however they are present in the other species.

Fig. 6

Number of different SSR types in the four-chloroplast genome of Cleomaceae.

Comparative analysis of the cp genome of the Cleomaceae species.

To analyse the DNA sequence divergence among the chloroplast genomes of the four Cleomaceae species: D. glaucum, C. chrysantha, C. lutea and T. hassleriana comparative analysis was done using mVISTA program to align the sequences. To understand the structural characteristics in the cp genomes, the annotation of D. glaucum used as reference. The alignment outcome reveals highly conserved genomes with few variations. As in many flowering plants cp genomes, the noncoding gene regions were less conserved than the coding regions (Fig. 7). Among the four cp genomes, the results showed that the following genes, rps16 trnQ, psbK-trnS, atpF-atpH, atpH-atpI, rpoB-trnC, psbM-trnD, trnD-trnY, trnE-trnT, trnT-psbD, trnT-trnL, trnM-atpE, rbcL-accD, petA-psbJ, psbE-petL, rbs16-rbs3, ndhF-rpl32 and trnV-rps12, were the most divergent non-coding regions. However, considerable slight variation was observed in four genes (atpF, rpoC2, rps19 and ycf1) of the four chloroplast genomes sequences.

Fig. 7

Comparison of four chloroplast genomes in Cleomaceae family using mVISTA. Annotation of D. glaucum was used as reference. The top arrows above the alignment indicate gene orientation. Blue bars represent protein coding, pink bars represent noncoding sequences (CNS) and light green indicates tRNAs and rRNAs. The horizontal axis indicates the coordinates in the cp genome, the vertical scale indicates the percentage identity between 50 and 100%.

In this current study, the IR-LCS and IR-SSC boundaries of the four Cleomaceae species genomes were compared. Even though the result showed that there are similarities among the compared cp genomes of all four species (Fig. 8), C. lutea has the smallest chloroplast genome (154,124 bp), whereas D. glaucum has the largest chloroplast genome (158,576 bp). The smallest IR region in T. hassleriana (25,804 bp) and the largest in C. chrysantha (26,251 bp). Furthermore, the lengths of LSC regions varied among the four Cleomaceae species, it is 87,738 bp in D. glaucum, 87,184 bp in C. chrysantha, 83,700 bp in C. lutea, and 87,509 bp in T. hassleriana. Also, comparative analysis of the cp genome of the four Cleomaceae species revealed that the location of the rpsl9 gene is between the LSC and IRb regions. The ycf1 gene was located in IRb regions and crossed the SSC/IRa region and extends into the SSC region by different lengths depending on the genome (D. glaucum 4,372 bp; C. chrysantha 4,385 bp; C. lutea 4,410 bp and T. hassleriana 4,436 bp); the IRb region includes 1,021; 1,027; 1,014 and 1,033 bp respectively of the ycf1 gene. The ndhF was found in the IRb/SSC to have 34 bp in D. glaucum, 64 bp in C. chrysantha and T. hassleriana and 36 bp in C. lutea in the IRb region and extends into the SSC region 2,207 bp in D. glaucum, 224 bp in C. chrysantha and T. hassleriana and 2,205 bp in C. lutea.

Fig. 8

Comparison of the IR, SSC and LSC junction positions among four chloroplast genomes of Cleomaceae.

Divergence of protein coding gene sequence

The rates of synonymous (dS) and nonsynonymous (dN) substitution and dN/dS ratio were calculated to detect the selective pressure among 80 protein coding genes in the cp genome of four Cleomaceae species. The results showed that the dN/dS ration is less than 1 in all of the paired genes except rps14, ycf1 and ycf15 in D. glaucum vs C. chrysantha having 1.19, 1 and 2.16 respectively, rps12, rps14 and ycf1 in D. glaucum vs C. lutea having 1.83, 1.03 and 1.16 respectively and psaI, rps7, rps16 and ycf2 in D. glaucum vs T. hassleriana having 1.04, 1.3, 1.33 and 1.7 respectively (Fig. 9). The synonymous (dS) values in all the genes ranges from 0 to 0.32 (Fig. 9).

Fig. 9

The synonymous (dS) and dN/dS ration values of 80 protein coding genes from four Cleomaceae cp genomes.

Phylogenetic relationships based on the Bayesian and Maximum Parsimony Analysis placed all samples into three main clades where the results match in the two analysis with strong support in all the nodes PP, 1.00, and MP, 100 (Fig. 10). The first clade contains species of Capparaceae family. The second clade comprised of Cleomaceae species while the third clade includes species from Brassicaceae family. The phylogenetic tree showed that the family Cleomaceae was separated from Capparaceae and becomes sister to Brassicaceae family which is consistent with some previous classifications of the order Brassicales (Angiosperm Phylogeny Group, 2009, Angiosperm Phylogeny Group, 2016).

Fig. 10

Phylogenetic tree reconstruction based on the complete chloroplast genome of sixteen taxa inferred from Bayesian Inference (BI) methods showing relationship within Cleomaceae and other families. Numbers in the clade represent posterior probability (PP) values.

Discussion

Next Generation Sequencing (NGS) provide sufficient information for molecular genetic markers, species identification, relationships and evolution within and between different species (Powell et al., 1995, Grassi et al., 2002, Doorduin et al., 2011, Straub et al., 2012). The complete chloroplast genome has provided large genetic information and molecular markers that are valuable tools to solve obscure phylogenetic relationships among land plants (Luo et al., 2014).

The chloroplast genomes of D. glaucum and C. chrysantha have similar roots to chloroplast genome of angiosperms (Raman and Park, 2016, Park et al., 2017, Chen et al., 2018). This study presents for the first time the characterization of complete chloroplast genomes of C. chrysantha and D. glaucum sequenced. The chloroplast genome size of D. glaucum is 158,576 bp and 158,111 bp in C. chrysantha (Fig. 1). The plastome sequence of D. glaucum and C. chrysantha has GC of 35.74% − 35.96% and AT content of 64.23% − 64.02% respectively (Table 1). The GC content in the IR region is higher (42.34% − 42.32%) than that of the LSC (33.27% − 33.59%) and SSC regions (28.76% − 28.97%).

Previous studies have shown that the plastomes of flowering plants are much conserved in structural organization and gene content; however, contraction and expansion do occur (Chang et al., 2006, Chen et al., 2018). There are 136 genes in the both genomes, which includes 80 protein coding genes, 31 tRNA genes and 4 rRNA genes. The IR region contained 8 protein coding genes, 7 tRNA in D. glaucum and 8 tRNA in C. chrysantha and 4 rRNA; the LSC contained 61 protein coding genes and 23 tRNA genes, the SSC region contained 13 protein coding genes and 1 tRNA. Intron is present in several of the protein coding and tRNA genes of the D. glaucum and C. chrysantha chloroplast genomes, similar to other chloroplast genomes of flowering plants (Raman and Park, 2016, Park et al., 2017, Chen et al., 2018). Out of the total genes in the cp genomes of both species, 15 genes are containing intron, nine of which are protein coding genes, whereas the remaining six genes are tRNA.

The results showed the genes in the plastome are encoded by 31,366 codons in D. glaucum and 26,475 codons in C. chrysantha, the most common codons are the coding for the amino acids Leucine, which has been previously stated in several cp genome of flowering plants (Liu et al., 2018). The Result of RNA editing sites revealed that most of the amino acid conversions in the codon positions were Serine to Leucine, presenting 41 editing sites in the genome distributed among 16 protein coding genes in D. glaucum and 35 editing sites distributed among 14 protein coding genes in C. chrysantha. The repeat sequence was identified in the chloroplast genomes of D. glaucum, C. chrysantha, C. lutea and T. hassleriana using default settings. The long repeats analysis showed 20–22-16–16 palindromic repeats, 16–19-16–19 forward repeats, 11–6-16–14 reverse repeats and 2–2-1–0 complement repeat respectively (Fig. 3). The length of repeated sequences in the four chloroplast genomes ranged from 10 to 69 bp, analogous to the lengths in other angiosperm plants (Greiner et al., 2008, Li et al., 2017, Song et al., 2017). Majority of SSRs in the cp genomes are mononucleotide, the largest number was found in D. glaucum of which most are poly T and A (Fig. 4) as in most flowering plants cp genome (Li et al., 2017). The highest constituted of Poly T (polythymine) in T. hassleriana whereas poly A (polyadenine) in D. glaucum. Only two poly C (polycytosine) in D. glaucum, three in C. chrysantha and T. hassleriana and five in C. lutea and only a single poly G (polyguanine) in three species (D. glaucum, C. chrysantha, C. lutea) and absent in T. hassleriana. The dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide are found in all four genomes (Fig. 4).

The degree of DNA sequence divergence was examined in the four-chloroplast genome. There were many different non-coding regions among the four cp genomes in addition to following genes atpF, rpoC2, rps19 and ycf1. Similarly, to the majority of the angiosperm cp genomes, the gene-coding regions are more highly conserved than those of their noncoding counterparts (Fig. 7). Among the four cp genomes, some divergent non-coding regions have been observed in addition to the following genes atpF, rpoC2, rps19 and ycf1. This study compared between IR-LCS and IR-SSC boundaries of the four cp genomes of Cleomaceae, the results showed that C. lutea has the smallest chloroplast genome from the four studied, while D. glaucum has the greatest chloroplast genome in size. The smallest IR region in T. hassleriana and the largest in C. chrysantha, the smallest LSC region in C. lutea and largest in D. glaucum and the smallest SSC region in C. lutea and largest in T. hassleriana. The results to rates of the selective pressure among 80 protein coding genes in the chloroplast genome of the four Cleomaceae species revealed that the dN/dS ration is less than 1 in all of the paired genes, yet there were a few exceptions (Fig. 7). The identical (dS) values in all of the protein coding genes ranges from 0 to 0.32 (Fig. 9). Phylogenetic relationships based on the Bayesian and Maximum Parsimony Analysis placed all samples into three main clades, where every family is in a separate clade (Fig. 10). The phylogenetic tree showed that the family Cleomaceae was separated from Capparaceae and becomes sister to Brassicaceae family which is consistent with some previous classifications of the order Brassicales (Angiosperm Phylogeny Group, 2009, Angiosperm Phylogeny Group, 2016).

Conclusion

This current study used the Illumina HiSeq 2500 platform to sequence the complete chloroplast genome of Dipterygium glaucum and Cleome chrysantha, which provided valuable plastid genomic resources for these medicinally important plants. We annotated the cp genome for both species, moreover, we identified the base composition, codon usage and RNA editing site, SSRs and Long repeat.

Data availability

The data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov, reference number (D. glaucum MT041700; C. chrysantha MT948188).

Declaration of Competing Interest

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