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Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
Copyright Institute of Research Engineers and Doctors. All rights reserved.
ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04 Analysis of molecular diversity among Zygophyllum, Tribulus and
Fagonia species by PCR-RAPD in Saudi Arabia
Khulood Mohammad Al-arjany1, Najat Abdul wahab Bukhari*
1Mohamed M. Ibrahim1,
Abstract Zygophyllum species are succulent plants that are regions of the country are rich in native plant flora of drought resistant and/or salt tolerant, growing under severe, dry cultivated crops and medicinal plants (Anonymous, climatic conditions. There is little information concerning molecular 1995). Around 300 species of medicinal plants are used variations among species of this genus. Genetic diversity was assessed, using RAPD primers, of 6 populations collected from in traditional medicine in Saudi Arabia (Abbas, 2002). various locations in Saudi Arabia. Yong leaves were used for DNA Our approach involved the collection of Zygophyllum extraction. Molecular taxonomy for these studied plants were simplex, Zygophyllum migahidii, Tribulus terrestris, performed by Polymerase Chain Reaction- Random Amplification of Tribulus macropterus, Fagonia glutinosa and Fagonia Polymorphic DNA (PCR-RAPD). RAPD reactions are PCR indica from Zygophyllaceae from Saudi Arabia and at reactions, but they amplify segments of DNA which are essentially unknown to the scientist (random). PCR was used to amplify a the time of collection, a pressed specimen will be known sequence of DNA. Thus, we choose the sequence we wants to prepared and retained in the Herbarium of the amplify, then designed and makes primers which will anneal to Department of Botany and Microbiology, King Saud sequences flanking the sequence of interest. Thus, PCR leads to the University. The classical taxonomic parameters of these amplification of a particular segment of DNA. The obtained dendrogram from our results was based on Nie's UPGMA cluster analysis of the genetic differences. These results revealed that the morphometric variations between the studied species. obtained dendrogram based, Z. simplex represents the closest Molecular techniques based on the polymerase chain relationship with Z. migahidii. Phylogenetic distances show that F. reaction (PCR) can be achieved to differentiate between glutinosa and F. indica are far from T. terrestris and T. macropterus the selected species. Also extraction and identification of and also, has completely different pattern of RAPD profiles compare to other samples. phytochemical compounds derived from native plants belonging to family Zygophyllacea in Saudi Arabia. Key wordsZygophyllum species, PCR-RAPD, Molecular studies, Plants are collected primarily from variant extreme climatic conditions habitats. The aim of the present study was to investigate the molecular taxonomy of selected plants in Saudi Arabia by Polymerase Chain Reaction- Zygophyllaceae (Caltrop family) is a family of Random Amplification of Polymorphic DNA (PCR- approximately 25 genera and 240 species adapted to semi desert and Mediterranean climates [Hammoda et al 2013]. Zygophyllum belongs to the Zygophyllaceae Materials and methods
family Species belonging to this genus represent a group of succulent plants that are drought resistant and/or salt tolerant, living under severe, dry climatic conditions. DNA-RAPD analysis The abundance of species related to this genus could be DNA was extracted from the plant species using the kit attributed to their high tolerance to environmental method and used for the PCR-RAPD analysis. stresses in addition to their unpalatability. The growth Herbarium specimens from the plants were used for and distribution of Zygophyllum species are attributed to DNA extraction, frozen in liquid nitrogen, and stored at their dependence on the chemical nature of the soil of –80°C until extraction. Total genomic DNA was isolated their habitats [Hammad et al 2010]. Saudi Arabia using a CTAB method (Saghai-Maroof, 1984) with possesses a unique genetic diversity in the form of minor modifications. DNA quantity and quality were ecotypes of tree species. The western and southwestern measured with a UV spectrophotometer (ultrospec 2100 pro-Amersham Biosciences, England). RAPD assay 1.Department of Botany and Microbiology, Faculty of were performed using 25 random 10-mer oligonucleotide Science, King Saud University, Riyadh, Saudi Arabia. primers from Integrated DNA technologies (Belgium). After screening 25 primers, RAPD analysis was Corresponding author conducted using 8 most polymorphic primers tabulated Dr. Najat Abdul wahab Bukhari Email id: najatab@ksu.edu.sa Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
Copyright Institute of Research Engineers and Doctors. All rights reserved.
ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04 in Table 1. PCR (Amersham- Pharmacia Biotech, France) reaction were carried out in a 25 μL reaction mix containing approximately 15 ng template DNA, 2.5 pmol Saudi Arabia is lightly forested with less than 1% of of each dNTP (Amersham-Gene), 5 pmol of a single 10- forest cover with good resources of medicinal and mer primer, 1 unit of Taq DNA polymerase (Amersham- aromatic plants. The use of herbal medicine has been Gene). The buffer [10 mm Tris-HCl, PH 8.8 at 25 C, 1.5 increasing in the country since the 1990s. The mm MgCl , 50 mm KCl, 0.1% Triton X-100] used as per government stresses the safety, efficacy and registration instructions of the manufacturer. The PCR reaction were of traditional medicine. Traditional medicine in Saudi run in a Eppendorf thermal cycler (Eppendorf, Germany) Arabia is based on herbal remedies and spiritual healing. programmed for an initial denaturation step of 3 min at It has been mainly inherited from old tribes and still continues to flourish despite widespread availability of allopathic medicine. and 1.5 min at 72 C. A final elongation step of 5 min at There are many studies in medicinal plants of Saudi 72 C was included. The PCR products were separated Arabia, since the introduction of allopathic medicine in on 1.5% agarose gel run in 1 X TBE and stained with 1940, the health authorities have worked for its ethidium bromide. The gels were visualized with a UV development and today the country enjoys a sound trans-illuminator. All PCR reactions were run in modern health care system. Many higher plants are duplicate and only reproducible and clear bands were pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides (Balandrin & Klocke, 1988). The search for new plant derived chemicals should thus Molecular characterization
be a priority in current and future efforts toward The preceding methods used for distinguishing our sustainable conservation and rational utilization of studied species were mainly morphological and biodiversity (Phillipson, 1990). In the search for biochemical. However, these traditional methods of alternatives to production of desirable medicinal characterization compounds from plants, biotechnological approaches, identification of species. So, it is necessary to obtain a specifically, plant tissue cultures, are found to have better understanding of species diversity, molecular potential as a supplement to traditional agriculture in the methods should be adopted for species identification. In industrial production of bioactive plant metabolites this study, analysis of six species by RAPD using (Ramachandra Rao & Ravi Shankar, 2002). Cell oligonucleotide primers was made. PCR amplifications suspension culture systems could be used for large scale with some primers indicated that each primer-template culturing of plant cells from which secondary yielded district, easily detectable bands of variable metabolites could be extracted. intensities (Figure 1). The bands used for fingerprinting Advances in tissue culture, combined with improvement were those reproducible over repeated runs with in genetic engineering, specifically transformation sufficient intensity to detect presence or absence with technology, has opened new avenues for high volume production of pharmaceuticals, nutraceuticals, and other Pair-wise comparison of all RAPD profiles revealed a beneficial substances (Hansen & Wright, 1999). The similarity matrix. Simple matching coefficient (Ssm) and active principles of many drugs are found in plants or are genetic distance (d), derived from RAPD banding produced as secondary metabolites. The remarkable patterns, are shown in the dendrogram. The genetic contribution of plants to the drug industry was possible, distance between the two samples of Z. simplex and Z. because of the large number of phytochemical and migahiddi was considered to be short (0.839) and their biological studies all over the world. Herbal remedies RAPD banding pattern were quite similar to each other. used in folk medicine provide an interesting and still Also there is a close relationship between these two largely unexplored source for the creation and samples of Zygophyllum with Tribulus and Fagonia development of potentially new drugs for chemotherapy (0.854 and 0.849, respectively). The dendrogram which might help overcome the growing problem of constructed base on genetic distances, derived from resistance and also the toxicity of the currently available RAPD analysis, shown in Figure 2. Clustering analysis commercial antibiotics. Therefore, it is of great interest was based on UPGMA. to carry out a screening of these plants in order to validate their use in folk medicine and to reveal the Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
Copyright Institute of Research Engineers and Doctors. All rights reserved.
ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04 active principle by isolation and characterization of their and genetic diversity among barley cultivars with known origin. Theor Appl Genet 104: 845–851 constituents. The fruits of Tribulus terrestris, is a famous traditional Chinese medicine (2013) Chemical constituents from Tribulus terrestris Tribulus terrestris is also reported to have antimicrobial and screening of their antioxidant activity Phytochemistry 92:153-159 [8] (2010) Genetic diversity among Zygophyllum (Zygophyllaceae) population based on RAPD analysis. Genet Mol Res In this investigation, RAPD markers showed a high level [9] Hansen G, Wright MS (1999) Recent advances in the transformation of plants. Trends Plant Sci 4: 226-231 of polymorphism and a high number of clearly amplified [10] Hilu KW, Stalker HT (1995) Genetic relationships between peanut and bands in Figure 3. Extensive DNA polymorphism has wild species of Arachis sect. Arachis (Fabaceae): evidence from RAPDs. 98: 167–178 been reported using RAPD markers in several other [11] Hou YC, Yan ZH, Wei YM, Zheng YL (2005) Genetic diversity in barley crops plants (Hilu & Stalker, 1995; Iruela, 2002; Hou, from west china based on RAPD and ISSR analysis. Barley Genet Newl 2005). The RAPD-based dendrogram of different [12] Iruela MJ, Rubio JI, Cubero J, Gil, Milan T (2002) Phylogenetic analysis genotypes displayed the genetic relationships between in the genus Cicer and cultivated chickpea using RAPD and ISSR these accessions, which accorded with previous studies markers. Theor Appl Genet 104: 643–651 on different plant (Ahmad, 1992; Tayyar & Waines, [13] Jit S and Nag TN (1985) Indian journal of pharmaceutical sciences 47: 1996; Iruela, 2002). It was evident from the results that [14] Loarce Y, Gallego R, Ferre E (1996) A comparative analysis of genetic the dendrogram based on RAPD-markers was accord relationship between rye cultivars using RFLP and RAPD markers. Euphytica 88: 107–115 with the dendrogram based on morphological traits, as [15] Phillipson JD (1990) Plants as source of valuable products. In B.V. reported for other crops (Loarce, 1996; Fernandez, Charlwood, and M.J.C. Rhodes (eds.), Secondary Products from Plant Tissue Culture. Oxford: Clarendon Press 7:1-21 [16] Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol Adv 20: 101-153 [17] Saghai Maroof MA, Solima KM, Jorgenson RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian Molecular taxonomy was carried out for studied plants PCR- inheritance, chromosomal location and population dynamics. Proc. Natl RAPD finger print. The obtained dendrogram from our results Acad Sci USA 81: 8014–8018 was based on Nie's UPGMA cluster analysis of the genetic [18]HC, Norula JL, Varadarajan R (1977) Chemical Era, 13, 261. [Clinical Abstracts 89: 314h (1977)] differences. These results revealed that the obtained [19] Tayyar RI, Waines JG (1996) Genetic relationships among annual species dendrogram based, Z. simplex represents the closest of Cicer (Fabaceae) using isozymes variation. Theor Appl Genet 92:245– relationship with Z. migahidii. Phylogenetic distances show that F. glutinosa and F. indica are far from T. terrestris and T. [20 (1987) Farmastiya 37: 40 [Clinical Abstracts 108: 1614096d] [21]ZF, Huang XK (1988) Dictionary of Traditional Chinese Medicine, macropterus and also, has completely different pattern of Commercial Press, Ltd, Hong Kong, 205-208. RAPD profiles compare to other samples. This research project was supported by a grant from the "Research Center of the Female Scientific and Medical colleges", Deanship of Scientific Research, King Saud [1] Abbas, B, Al-Qarawi, A. A. and Al-Hawas, A (2002) The ethno- veterinary knowledge and practice of traditional healers in Qassim Region. Saudi Arabia. Journal of Arid Environments. 50: 367-379 [2] Ahmad F, Slinkard AE (1992) Genetic relationships in the genus cicer L. as revealed by polyacrylamide gel electrophoresis of seed storage proteins. Theor Appl Genet 84:688–692 [3] Anonymous (1995) Saudi Arabia: Country report to the FAO International Technical Conference on Plant Genetic Resources. National Agriculture and Water Research Centre, Riyadh 16: 21-35 [4] Balandrin MJ, Klocke JA (1988) Medicinal, aromatic and industrial materials from plants. In Y.P.S. Bajaj (ed.), Biotechnology in Agriculture and Forestry. Medicinal and Aromatic Plant 4:1-36 [5] Bose BC, Saifi AQ, Vijayvargiya R, Bhatnagar JN (1963) Some aspects of chemical and pharmacological studies of Tribulus terrestris Linn. Indian J Med Res 17:291-293. [6.] Fernandez MN, Figueiras AM, Benito C (2002) The use of ISSR and RAPD markers for detection DNA polymorphism, genotype identification Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
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ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04 Table 1: Oligonucleotide decamer primers along with their sequences used in the study. The most of the primers gave unique bands in all species. The primers, OPA04, OPA05, OPC15, OPC16, OPD11and OPD13discriminated species by the presence or absence of unique bands, which act as markers for species confirmation.


Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
Copyright Institute of Research Engineers and Doctors. All rights reserved.
ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04
Figure 1: RAPD analysis carried out with different oligonucleotide primer on genomic DNA isolated from lane 1= Zygophyllum simplex; lane
2= Zygophyllum migahidii; lane 3= Tribulus terrestris; lane 4= Tribulus macropterus; lane 5= Fagonia glutinosa and lane 6= Fagonia indica collected from Saudi Arabia


Proc. of the Intl. Conf. on Advances In Bio-Informatics And Environmental Engineering-ICABEE 2014.
Copyright Institute of Research Engineers and Doctors. All rights reserved.
ISBN: 978-1-63248-019-4 doi: 10.15224/ 978-1-63248-019-4-04 Figure 2: RAPD analysis carried out with mixed oligonucleotide primer (OPD11 and OPD 13) on genomic DNA isolated from; Lane M= 100-bP molecular weight marker Lane 1= Zygophyllum simplex; lane 2= Zygophyllum migahidii; lane 3= Tribulus terrestris; lane 4= Tribulus macropterus; lane 5= Fagonia glutinosa and lane 6= Fagonia indica collected from Saudi Arabia.

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Organic Process Research & Development 2003, 7, 983−989 Phase Transitions in Supersaturated Drug Solution Ste´phane Veesler,*,‡ Laurent Lafferre re,‡ Eric Garcia,§ and Christian Hoff Centre de Recherche sur les Me´canismes de la Croissance Cristalline, CRMC2† - CNRS, Campus de Luminy,Case 913, F-13288 Marseille Cedex 09, France, Sanofi-Synthe´labo Recherche, F-78440 PorcheVille, France, andSanofi-Synthe´labo Chimie, F-30390 Aramon, France

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Volume 01 Number 02 Page 51 - 99 December 2011 ISSN 2012 – 9238 Sri Lanka Journal of Indigenous Medicine Peer reviewed research publication of the INSTITUTE OF INDIGENOUS MEDICINE University of Colombo, Rajagiriya, Sri Lanka Sri Lanka Journal of Indigenous Medicine (SLJIM)