Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.

Introduction

Commiphora Jacq., (family Burseraceae) is a genus of about 185 species distributed in tropical and subtropical regions (Byng, 2014). Several species of the genus Commiphora produce fragrant resins used for incense, perfume and also used medicinally in the diseases of liver, gastrointestinal disorder, urinary tract, rheumatism, scurvy and jaundice (Abdul-Ghani and Amin, 1997, Al-Howiriny et al., 2004, Al-Howiriny et al., 2005, Hanuša et al., 2005, Shen et al., 2008, Iluz and Hoffman, 2010), cancer (Hartwell, 1982), respiratory, kidney, muscular, and in kidney complaints (Tejero et al., 2008); and thus gum/resins produced by the different species of Commiphora have high commercial value (Chaudhary, 2001). In Saudi Arabia, the genus Commiphora is represented by seven species i.e. C. erythraea, C. gileadensis, C. habessinica, C. kataf, C. myrrha, C. opobalsamum and C. quadricincta (Chaudhary, 2001). The systematic understanding of the genus Commiphora at species level has been hindered because of the life history which includes deciduous habit, a predominantly dioecious breeding system, and a tendency to produce flowers prior to developing leaves (Weeks and Simpson, 2007). Infrageneric groups of Commiphora have been proposed but limited to northeastern and tropical East Africa species (Wild, 1959, Gillett, 1991, Vollesen, 1986). The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups (Chen et al., 2010, Poczai and Hyvönen, 2010, Yao et al., 2010, Ali et al., 2014). Hence, the present study attempts to establish ITS sequence of nrDNA based species-specific molecular signature for the genus Commiphora distributed in Saudi Arabia.

Materials and methods

Sampling of the plant materials and sequencing of ITS gene

The leaf materials of a total number of seven species of the genus Commiphora distributed in Saudi Arabia (Table 1) were collected from herbarium specimens housed at the Herbarium, Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia (KSUH). The taxonomic identification of specimens were confirmed through the consultation of Flora of Saudi Arabia (Chaudhary, 2001). The extraction of total genomic DNA were performed using Qiagen kit and then it was subjected to polymerase chain reaction for the amplification of ITS gene and the amplified product were further used for DNA sequencing following previously described method (Ali et al., 2010).

Table 1

Taxon sampling information and GenBank accession number for the sequences used in the molecular phylogenetic analysis.

S. No.	Taxon	Locality	Voucher	GenBank Accession No.
1.	Commiphora erythrea (Ehrenb.) Engl.	Abha, Saudi Arabia	Al-Farhan and Masin 1755 (KSUH)	MH522401
2.	Commiphora gileadensis (L.) C.Chr. [Syn = Commiphora opobalsamum (L.) Engl.]	Farasan, Saudi Arabia	R.A. Baseni 3159 (KSUH)	MH522402
3.	Commiphora habessinica (O.Berg) Engl.	Jabal Shada, Saudi Arabia	A.R. Al-Zaudi 21191 (KSUH)	MH522403
4.	Commiphora kataf (Forssk.) Engl.	Jabal Shada,Saudi Arabia	A.R. Al-Zaudi 21211 (KSUH)	MH522404
5.	Commiphora madagascariens Jacq. {[synonym Commiphora roxburghii Alston], [Illegitimate], http://www.theplantlist.org/tpl1.1/record/kew-2733654}	Jabal Fayfa, Saudi Arabia	M.I. Hassan 4092 (KSUH)	MH522405
6.	Commiphora myrrha (Nees) Engl.	Abha, Saudi Arabia	Al-Farhan and Masin 1786 (KSUH)	MH522406
7.	Commiphora quadricincta Schweinf.	Jabal, Saudi Arabia	sn 19278 (KSUH)	MH522407

Molecular phylogenetic analysis

The similarity of the ITS sequences of nrDNA generated in the present study were matched using BLAST (Basic Local Alignment Search Too) search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) by NCBI server (Altschul et al., 1990). The ITS sequences of nrDNA of 14 species of Commiphora (Table 2) were retrieved from GenBank (www.ncbi.nlm.nih.gov). The ITS sequences of nrDNA of Bursera attenuate (GenBank accession number AF445871) was used as outgroup in the phylogenetic analyses. The sequence included in the molecular phylogenetic analyses were aligned using CLUSTAL X v.1.81 (Thompson et al., 1997). The ITS sequences of nrDNA generated in the present study were submitted to GenBank (Table 1). The aligned sequences were saved in nexus format. The nexus file of aligned sequences were imported in to MEGA 5 software (Tamura et al., 2011), and phylogenetic analyses were performed using Maximum Parsimony (MP) method (Tamura et al., 2011).

Table 2

The ITS sequences of nDNA retrieved from GenBank, and used in the molecular phylogenetic analysis of Commiphora.

S. No.	Species	GenBank Accession No.
1	Commiphora africana(A.Rich.) Endl.	AF445873
2	Commiphora edulis (Klotzsch) Engl.	JF919026
3	Commiphora eminii Engl.	JN882700
4	Commiphora falcata Capuron	KF906076
5	Commiphora grandifolia Engl.	JN882671
6	Commiphora kataf (Forssk.) Engl.	JN882709
7	Commiphora kua (R.Br. ex Royle) Vollesen	JN882696
8	Commiphora leptophloeos (Mart.) J.B.Gillett	AF445875
9	Commiphora monstruosa (H.Perrier) Capuron	AF080004
10	Commiphora myrrha (Nees) Engl.	KC311151
11	Commiphora neglecta Verd.	JF919029
12	Commiphora orbicularis Engl.	JN882697
13	Commiphora schimperi (O.Bergman) Engl.	JN882702
14	Commiphora wightii (Arn.) Bhandari	EU419975

Results and discussion

Combined length of the ITS gene, GC content and BLAST

The combined length and GC content of ITS region (ITS1-5.8S-ITS2) in studied taxa ranges from 608 to 656 nucleotide base pairs (bp) and 65–67% respectively (length 620 bp and GC content 67% in C. erythrea, length 619 bp and GC content 67% in C. gileadensis, length 649 bp and GC content 66% in C. habessinica, length 608 bp and GC content 65% in C. kataf, length 646 bp and GC content 66% in C. madagascariens, length 651 bp and GC content 66% in C. myrrha, and length 656 bp and GC content 67% in C. quadricincta). The BLAST search of ITS sequence shows high similarity with GenBank sequences of C. myrrha, C. kataf, C. neglecta, C. gowlello, C. habessinica and C. kua [99% identity of sequence of C. gileadensis with C. myrrha (KC311151), 99% identity of sequence of C. kataf with GenBank sequence of C. kataf (JN882709), 100% identity of sequence of C. myrrha with GenBank sequence of C. myrrha (JN882706) and 96% identity with C. kua (JN882696), 97% identity of sequence of C. erythrea with C. neglecta (JF919029), 97% identity of sequence of C. habessinica with C. gowlello (JN882674), 96% identity of sequence of C. madagascariens with C. gowlello (JN882674), and 96% identity of sequence of C. quadricincta with C. habessinica (JN882673) and 96% identity with C. kua (JN882696)].

Molecular phylogenetic relationships

The molecular phylogenetic analysis of the ITS gene (data matrix- 513 positions, parsimony informative sites- 106) using MP method (Eck and Dayhoff, 1966, Felsenstein, 1985, Nei and Kumar, 2000, Tamura et al., 2011) resulted in 16 maximally parsimonious trees (length- 287, consistency index- 0.637, retention index- 0.741, and composite index- 0.540) (Fig. 1).

Fig. 1

The maximum parsimony tree (length- 287, consistency index- 0.637, retention index- 0.741, composite index- 0.540) recovered from the molecular phylogenetic analysis of ITS sequence of nuclear ribosomal DNA of 21 species of Commiphora; the number on the node indicated bootstrap support in 1000 bootstrap replicates.

The maximum parsimony tree recovered (Fig. 1) from the phylogenetic analysis clearly revealed the occurrence of the C. kataf and C. myrrha in Saudi Arabia; C. kataf and C. myrrha showed proximity [bootstrap support (BS 99%)] with the sequence of C. kataf and C. myrrha included in the analysis from GenBank (GenBank accession number JN882709 and KC311151) respectively. C. kataf clade with C. gileadensis-C. neglecta (BS 36%) and C. erythrea- C. eminii (BS 54%). Morphologically C. myrrha (fruit beaked, pseudoaril with broad triangular lobes) differs from C. gileadensis (fruit 4-valved, with prominent line) and C. erythrea (stout gnarled tree, fruit globose-ovoid, tomentose).

New distributional record of Commiphora madagascariens

C. quadricincta clade with C. africana, C. schimperi, C. myrrha, C, wightii, C. kua, C. madagascariens and C. habessinica (BS 95%). C. madagascariens (fruit beaked rounded oblong when viewed from above, with 2 small apical pits) is native to Tanzania, formerly cultivated in Mauritius, and surviving near Bhagalpur (India) on the Ganges as a relic of cultivation (http://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:127719-1)] showed close proximity with C. habessinica (fruit not beaked, pseudoaril with four parallel-sided arms) (BS 95%). At the molecular level, there were four specific nucleotide differences at the alignment position i.e. 99 (C → Gap), 207 (Gap → G), 482 (C → Gap and 483 (T → Gap) were noted in between C. habessinica and C. madagascariens. Thus the present study herein reports the occurrence of C. madagascariens for the first time from Saudi Arabia.

Species-specific molecular signature

The taxonomic identification of plants based on morphological characteristics depends on sufficient experience and expertise in plant taxonomy, and it is well known that the morphological characteristics can easily be varied by the geographical environment (Marcon et al., 2005, Rai et al., 2012); however, the DNA sequences is hardly influenced by environment, and remain same even in the developmental stages (Liu et al., 2011); and therefore, the DNA based species-specific molecular genotyping (DNA barcoding) is an effective tools to classical morphological methods (Hebert et al., 2003) which is being used for the species identification successfully across all the groups of live forms. Further, the tools and techniques of DNA barcoding has now been proven useful in assessment of biodiversity, forensics and in conservation genetics (Ali et al., 2014). The ITS sequences of nrDNA generated in the present study have been submitted to GenBank (Table 1), which may be used as species-specific molecular signature of Commiphora species of Saudi Arabia.