The cp genome characterization of Adenium obesum: Gene content, repeat organization and phylogeny.

Adenium obesum (Forssk.) Roem. & Schult. belonging to the family Apocynaceae, is remarkable for its horticultural and ornamental values, poisonous nature, and medicinal uses. In order to have understanding of cp genome characterization of highly valued medicinal plant, and the evolutionary and systematic relationships, the complete plastome / chloroplast (cp) genome of A. obesum was sequenced. The assembled cp genome of A. obesum was found to be 154,437 bp, with an overall GC content of 38.1%. A total of 127 unique coding genes were annotated including 96 protein-coding genes, 28 tRNA genes, and 3 rRNA genes. The repeat structures were found to comprise of only mononucleotide repeats. The SSR loci are compososed of only A/T bases. The phylogenetic analysis of cp genomes revealed its proximity with Nerium oleander.

Introduction

Adenium obesum (Forssk.) Roem. & Schult. (family Apocynaceae), the ‘Desert Rose’ is a poisonous, medicinal plant, distributed from Africa to Arabia, and is used traditoinally in the treatment of various ailments e.g. skin diseases, wounds, muscle pain, joint pain, venereal diseases, tooth decay, septic wounds, and nasal infections (Dimmitt and Hanson, 2002, Mouza and Hossain, 2015, Hossain et al., 2017). It is also used as a pesticide (Versiani et al., 2014), arrow poison for hunting in Africa (Oyen, 2008) and fish toxin (Wiseman, 2009). The A. obesum plant extract reported to possess cytotoxic (Almehdar et al., 2012), antimicrobial (Hossain et al., 2017) and anti-influenza (Kiyohara et al., 2012) activities. The phytochemical compounds identified from A. obesum include cardiac glycosides (cardenolides), pregnanes, triterpenes, flavonoids, and acetyldigitoxigenin (Versiani et al., 2014). The molecular docking of acetyldigitoxigenin elucidates the plausible mechanisms underlying the anticancer properties (Gurung et al., 2020).

The recent development in plastome or chloroplast (cp) genomics due to massive progress in the next-generation sequencing (NGS) platforms (Eid et al., 2009, Rothberg et al., 2011, Pattnaik et al., 2014, Jain et al., 2016, Shendure et al., 2017) and bioinformatics tools (Mavromatis et al., 2007, Knudsen et al., 2010, Huang et al., 2012, McElroy et al., 2012, Shendure and Aiden, 2012, Yang and Rannala, 2012, Caboche et al., 2014, Shcherbina, 2014, Kwon et al., 2015, Langmead and Nellore, 2018) have greatly impact on biotechnology application (Spök et al., 2008, Zhang et al., 2015, Daniell et al., 2016). We herein for the first time report the cp genome characterization of highly valued medicinal plant A. obesum, and discuss its structure including gene content, repeat organization, and phylogeny.

Materials and methods

DNA sequencing, assembly and annotation

The fresh leaves of A. obesum were collected from the wild condition of desert habitat near to Riyadh, Saudi Arabia. The total genomic DNA was isolated using QIAGEN DNeasy DNA extraction kit. The de novo sequencing base calling was performed using the Illumina Pipeline 1.3.2 (Nie et al., 2012). The raw reads were filtered using FastQC to obtain the high-quality clean data by removing adaptor sequences using trimmomatic and low-quality reads with Q-value ≤ 20. The filtered reads were assembled using Spades (Bankevich et al., 2012), and annotated using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (Tillich et al., 2017, Hansen et al., 2007). Further downstream analysis from the assembled cp genome included the repeat structure (Benson, 1999, Timme et al., 2007) and small inversion (Nagano et al., 1991, Yang et al., 2010, Doorduin et al., 2011, Castro et al., 2013, Beier et al., 2017).

Comparison of cp genome and phylogenetic analysis

The cp genome of A. obesum were plotted using the mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) program with a total number of nine complete cp genomes of Apocynaceae [i.e. (1) Asclepias nivea Forssk., (2) Carissa macrocarpa (Eckl.) A. DC., (3) Catharanthus roseus (L.) G. Don, (4) Cynanchum auriculatum Buch.-Ham. ex Wight, (5) Echites umbellatus Jacq., (6) Nerium oleander L., (7) Oncinotis tenuiloba Stapf, (8) Pentalinon luteum (L.) B.F. Hansen & Wunderlin, and (9) Rhazya stricta Decne.] (Table 1).

Table 1

The ingroup and outgroup taxon with their classification and GenBank accession number included in the phylogenetic analyses. The GenBank accession number marked with * were included in the mVISTA alignment.

Sl. No.	Taxon	Order	Family	Subfamily	Tribe	Subtribe	GenBank
Ingroup
1.	Adenium obesum (Forssk.) Roem. & Schult.	Gentianales	Apocynaceae	Apocynoideae	Nerieae	Neriinae	MN765097*
2.	Asclepias nivea Forssk.	Gentianales	Apocynaceae	Asclepiadoideae	Asclepiadeae	Asclepiadinae	NC_022431.1*
3.	Cynanchum auriculatum Buch.-Ham. ex Wight	Gentianales	Apocynaceae	Asclepiadoideae	Asclepiadeae	Cynanchinae	NC_029460.1*
4.	Carissa macrocarpa (Eckl.) A. DC.	Gentianales	Apocynaceae	Rauvolfioideae	Carisseae		NC_033354.1*
5.	Catharanthus roseus (L.) G. Don	Gentianales	Apocynaceae	Rauvolfioideae	Vinceae	Catharanthinae	NC_021423.1*
6.	Rhazya stricta Decne.	Gentianales	Apocynaceae	Rauvolfioideae	Amsonieae		NC_024292.1*
7.	Echites umbellatus Jacq.	Gentianales	Apocynaceae	Apocynoideae	Echiteae	Echitinae	NC_025655.1*
8.	Pentalinon luteum (L.) B.F. Hansen & Wunderlin	Gentianales	Apocynaceae	Apocynoideae	Echiteae	Pentalinoninae	NC_025658.1*
9.	Nerium oleander L.	Gentianales	Apocynaceae	Apocynoideae	Nerieae	Neriinae	NC_025656.1*
10.	Oncinotis tenuiloba Stapf	Gentianales	Apocynaceae	Apocynoideae	Baisseeae		NC_025657.1*
11.	Anethum graveolens L.	Apiales	Apiaceae				NC_029470.1
12.	Ilex delavayi Franch.	Aquifoliales	Aquifoliaceae				KX426470.1
13.	Helianthus annuus L.	Asterales	Apocynaceae				NC_007977.1
14.	Viburnum betulifolium Batalin	Dipsacales	Adoxaceae				NC_037951.1
15.	Eucommia ulmoides Oliv.	Garryales	Eucommiaceae				NC_037948.1
16.	Gentiana tibetica King ex Hook. f.	Gentianales	Gentianaceae				NC_025319.1
17.	Iodes cirrhosa Turcz.	Icacinales	Icacinaceae				NC_036254.1
18.	Premna microphylla Turcz.	Lamiales	Lamiaceae				NC_026291.1
19.	Iochroma australe Griseb.	Solanales	Solanaceae				NC_029833.1
Outgroup
20.	Cornus controversa Hemsl.	Cornales	Cornaceae				MG525004.1

The cp sequences of 48 genes [e.g. ATP synthase genes (atpA, atpB, atpE, atpF, atpH, and atpI), c-type cytochrome synthesis gene (ccsA), envelope membrane protein gene (cemA), Maturase gene (matK), cytochrome b6/f genes (petA, petB, petD, petG, and petN), Photosystem I genes (psaA, psaB, psaC, and psaJ), Photosystem II genes (psbA, psbC, psbE, psbH, psbI, psbJ, psbK, psbN, and psbT), Rubisco gene (rbcL), Large-subunit ribosomal protein genes (rpl14, rpl2, rpl20, rpl32, rpl33, and rpl36), RNA polymerase subunit genes (rpoB, rpoC1, and rpoC2), Small-subunit ribosomal protein genes (rps14, rps15, rps18, rps19, rps2, rps3, rps4, rps7, and rps8), Genes of unknown function (ycf3, and ycf4)] were retrieved from 19 ingroup taxon comprising 10 species of the family Apocynaceae, the representative of the family Apiaceae, Aquifoliaceae, Apocynaceae, Adoxaceae, Eucommiaceae, Gentianaceae, Icacinaceae, Lamiaceae, Solanaceae, and the outgroup from the family Cornaceae (Table 1), and aligned using Clustal X (Thompson et al., 1994), and the molecular phylogenetic analysis was performed by Maximum Evolution method (Rzhetsky and Nei, 1992) using in MEGA X (Kumar et al., 2018).

Results and discussion

The present study reports assembly of the complete cp genome map as a conserved circular structure comprising a total length of 154,437 bp (including LSC, SSC, IRa, and IRb), with an overall GC content of 38.1% (Fig. 1). The results revealed the gene contents, orientation, and the conservation as well as polymorphisms were found in the chloroplast genome as similar to those of other cp genome of angiosperms (Daniell et al., 2016). A total numner of 127 genes were annotated including 96 protein-coding genes, 28 tRNA genes, and 3 rRNA genes (NCBI GenBank accession number: MN765097).

Fig. 1

The gene map and genes contained in the cp genome of A. obesum.

The sequence identity of A. obesum plotted with the nine different complete cp genomes from the family Apocynaceae e.g. A. nivea, C. macrocarpa, C. roseus, C. auriculatum, E. umbellatus, N. oleander, O. tenuiloba, P. luteum and R. stricta using the mVISTA revealed high similarities amongst them with few regions where the identities was below 90% (Fig. 2).

Fig. 2

The percent identity plot for comparison of cp genome of A. obesum with the other Apocynaceae genomes. Lane from up to down: A. nivea, C. macrocarpa, C. roseus, C. auriculatum, E. umbellatus, N. oleander, O. tenuiloba, P. luteum, and R. stricta.

Moreover, the present study depicted the distribution and location of repeated structures and microsatellites in the cp genome. The microsatellites or simple sequence repeats (SSRs) are tandem repeats which ranges from 1 to 6 bp and are present commonly in cp genomes (Meng et al., 2018). SSRs have been served as an important marker for molecular characterization of plant species. A total of 40 SSRs were predicted in A. obesum (Table 2) which were composed of a length of at least 10 bp, all of which were found to be homopolymers containing multiple A or T nucleotides at each locus. These reveal that SSR loci are rich in A–T content in the A. obesum cp genome which supports previous chloroplast SSRs reports (Li et al., 2017). Among these SSRs, four SSRs were situated in coding regions and 31 were located in the intergenic regions (Table 2). A total number of 19 genes including 11 protein-coding genes and 8 tRNA genes contained one or two introns (Table 3). Furthermore, five SSRs were found in intronic regions. Thus, most of the repeats were situated in the intergenic region. Tandem and dispersed repeats were analyzed for A. obesum cp genomes and a total of 25 tandem and 19 dispersed repeats were observed (Fig. 3).

Table 2

The SSR loci in the cp genome of Adenium obesum.

	Start	End	Repeat	Repeat length of consensus	Locus	Region
	2109	2189	(A)10(A)12	81	ycf1	CDS
	2914	2925	(A)12	12	ycf1	CDS
	9557	9566	(T)10	10	ndhI-ndhG	intergenic
	13,831	13,840	(A)10	10	ccsA-trnL-UAG	intergenic
	15,378	15,388	(T)11	11	rpl32-ndhF	intergenic
	15,614	15,624	(A)11	11	rpl32-ndhF	intergenic
	23,933	23,950	(T)18	18	rrn23-trnA-UGC	intergenic
	43,878	43,887	(A)10	10	rpl2-rps19-fragment	intergenic
	49,254	49,266	(T)13	13	rps16	intron
	52,132	52,141	(A)10	10	psbI-trnS-GCU	intergenic
	53,347	53,356	(T)10	10	trnG-GCC	intron
	53,607	53,620	(T)14	14	trnG-GCC-trnR-UCU	intergenic
	53,763	53,775	(T)13	13	trnR-UCU-atpA	intergenic
	55,426	55,435	(A)10	10	atpA-atpF	intergenic
	56,129	56,139	(T)11	11	atpF	intron
	57,865	57,874	(T)10	10	atpH-atpI	intergenic
	58,082	58,093	(T)12	12	atpH-atpI	intergenic
	60,138	60,147	(A)10	10	rps2-rpoC2	intergenic
	62,367	62,377	(T)11	11	rpoC2	CDS
	72,576	72,585	(T)10	10	trnC-GCA-petN	intergenic
	79,736	79,747	(T)12	12	psbC-trnS-UGA	intergenic
	88,261	88,272	(T)12	12	ycf3	intron
	95,456	95,465	(T)10	10	ndhC-trnV-UAC	intergenic
	96,134	96,143	(A)10	10	ndhC-trnV-UAC	intergenic
	97,206	97,257	(T)12, (T)13	52	trnM-CAU-atpE	intergenic
	104,329	104,341	(T)13	13	psaI-ycf4	intergenic
	105,304	105,315	(A)12	12	ycf4-cemA	intergenic
	105,629	105,642	(T)14	14	ycf4-cemA	intergenic
	109,707	109,720	(T)14	14	psbE-petL	intergenic
	109,940	109,949	(A)10	10	psbE-petL	intergenic
	111,353	111,366	(T)14	14	trnP-UGG-psaJ	intergenic
	113,000	113,009	(A)10	10	rps18-rpl20	intergenic
	115,212	115,222	(T)11	11	clpP	intron
	120,988	120,997	(A)10	10	petB-petD	intergenic
	122,446	122,455	(A)10	10	petD-rpoA	intergenic
	125,125	125,136	(T)12	12	rps8-rpl14	intergenic
	125,645	125,710	(A)10, (T)10	66	rpl14-rpl16	intergenic
	128,414	128,424	(T)11	11	rpl22	CDS
	128,795	128,804	(T)10	10	rps19-rpl2	intergenic
	148,732	148,749	(A)18	18	trnA-UGC-rrn23	intergenic

Table 3

The intron containing genes in the cp genome of Adenium obesum.

Gene	Location	Exon I bp	Intron I bp	Exon II bp	Intron II bp	Exon III bp
trnA-UGC	IR	35	818	38
trnI-GAU	IR	35	943	42
rps12*	LSC-IR	234	536	25		114
ndhB	IR	777	684	756
rpl2	IR	391	649	434
trnK-UUU	LSC	35	2476	37
rps16	LSC	226	843	41
trnG-GCC	LSC	23	691	37
atpF	LSC	411	706	144
rpoC1	LSC	1613	749	451
ycf3	LSC	155	773	228	738	124
trnL-UAA	LSC	37	491	50
trnV-UAC	LSC	37	586	38
clpP	LSC	229	657	291	746	71
rpl2	IR	434	649	391
ndhB	IR	756	684	777
rps12	IR	25	536	234
trnI-GAU	IR	42	943	35
trnA-UGC	IR	38	818	35

*rps12 is trans-spliced gene with 5′ end exon located in the LSC region and the duplicated 3′ end exon located in IR regions.

Fig. 3

(A-C). The repeat structure analysis in the cp genome of A. obesum. The cutoff value for tandem repeat is 15 bp and 30 bp for dispersed repeat. A. Frequency of repeats by length; B. Repeat type; C. The location distribution of all the repeats.

The phylogenetic relationships of a total number of 48 cp genes from the 19 cp genomes including the family Apocynaceae and the representative members of the family Apiaceae (Apiales), Aquifoliaceae (Aquifoliales), Adoxaceae (Dipsacales), Eucommiaceae (Garryales), Gentianaceae (Gentianales), Icacinaceae (Icacinales), Lamiaceae (Lamiales), Solanaceae (Solanales), and the outgroup at the family Cornaceae (Cornales) revealed the proximity of A. obesum (Subfamily Apocynoideae, Tribe Nerieae, Subtribe Neriinae) with Nerium oleander (Subfamily Apocynoideae, Tribe Nerieae, Subtribe Neriinae) (Fig. 4). The family Apocynaceae is one of the 10 largest angiosperm families with c. 4,500 species under c. 370 genera globally with the greatest diversity in the tropics and subtropics (Stevens, 2001, Endress et al., 2014, APG, 2016). Apart from the large number of molecular phylogenetic studies on the family Apocynaceae (e.g. Liede and Täuber, 2000, Liede and Täuber, 2002, Liede, 2001, Liede and Meve, 2001, Liede and Meve, 2002, Meve and Liede, 2001, Meve and Liede, 2002, Meve and Liede, 2004a, Meve and Liede, 2004b, Potgieter and Albert, 2001, Liede and Kunze, 2002, Liede et al., 2002a, Liede et al., 2002b, Verhoeven et al., 2003, Rapini et al., 2003, Rapini et al., 2004, Rapini et al., 2006, Rapini et al., 2007, Simões et al., 2004, Simões et al., 2006, Simões et al., 2007, Liede-Schumann et al., 2005, Venter et al., 2006, Endress et al., 2007, Goyder et al., 2007, Ionta and Judd, 2007, Lahaye et al., 2007, Livshultz et al., 2007, Meve and Liede-Schumann, 2007, Wanntorp and Forster, 2007), the family has also been intensely studied for their pollination biology, plant–herbivore interactions, and secondary chemistry (Wyatt and Broyles, 1994, Góngora Castillo et al., 2012, Courdavault et al., 2014, Agrawal et al., 2015). The phylogenetic nesting of the family Asclepiadaceae in Apocynaceae s.s. has been demonstrated repeatedly (Wanntorp, 1988, Judd et al., 1994, Sennblad and Bremer, 1996, Potgieter and Albert, 2001). The most recent classification of Apocynaceae (Endress et al., 2014) segregated the family into five subfamilies, two paraphyletic which correspond to the former Apocynaceae s.s. (Rauvolfioideae and Apocynoideae) and three monophyletic that relates to the former Asclepiadaceae (Periplocoideae, Secamonoideae, and Asclepiadoideae).

Fig. 4

The maximum likelihood tree inferred from the cp genome of A. obesum analyzed together with the members of the family Apocynaceae and Aquifoliaceae, Adoxaceae, Eucommiaceae, Gentianaceae, Icacinaceae, Lamiaceae, and Solanaceae.

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.