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Malaysian Journal of Science 29(1) : 37 - 40 (2010) Rapid Method for Extraction of Genomic DNA From Vitex negundo L. R. Mary Sujin1, A. John De Britto2*, R. Mahesh3 and K. Dharmar4 1234 Plant Molecular Biology Research Unit, St.Xavier'sCollege, (Autonomous) Palayamkottai - 627 002, Tamilnadu, India. *[email protected] (corresponding author ).Tel: 0091- 462 4264374, Fax: 0091- 462-2561765. Received on 28 August 2009, accepted in revised form 16Th March 2010 ABSTRACT DNA extracted from plants which contains rich amount of polyphenols and or polysaccharides are often problematic when subjected when mature tissues are used for DNA extraction. In order to overcome these problems, we develope the first reliable and efficient method for isolating vitex negundo L. genomic DNA that is free from solubilising polysaccharides and polyphenols. This protocol uses NaCl, PVP, mercaptoethanol, SDS and incubation at 68°C for 1 hour. Mild temperature conditions during extraction and precipitation were also recognized as important parameters. The quantity of isolated genomic DNA was confirmed by means of spectrophotometic analysis (260/280=1.6-1.8) routinely yielding 250-500 ng/µl per gram of leaf material and it was proved through RAPD-PCR analysis. (Keywords: DNA extraction, RAPD-PCR analysis, Vitex negundo L.) achieved by using detergents such as sodium dodecyl sulphate (SDS) or cetyltrimethylammonium bromide (CTAB). This released DNA should be protected from endogenous nuclease. The initial DNA extracts often contain large amounts of RNA, proteins, polysaccharides, tannins and pigments, which may interfere with the extracted DNA and are difficult to separate. Most proteins are removed by means of denaturation and precipitation from the extract with chloroform and /or phenol. Polysaccharides contaminates are particularly problematic [6].They can also coprecipitate with DNA after alcohol addition during DNA isolation to form highly viscous solutions [7]. INTRODUCTION Vitex negundo L. (Local Name-Nochi) belongs to the family Verbenaceae. It is a small tree credited with innumerable medicinal activities like anti-cancer, analgesic, anti-inflammatory, anticonvulsant, antioxidant, bronchial relaxant, hepatoprotective, etc. The ethanolic extract of leaves has been found safe as LD50 dose (by oral route) of it was recorded in non-toxic dose range [1]. The roots, fruits, flowers, leaves and bark of V. negundo species are used for medicinal purposes like foot and mouth diseases, impaction with fever [2]. Juice extracted from 100gm leaves is consumed to cure Dyspnoea (Shwasam mutt) [3]. Leaves are smoked in headache. Flowering tips are chewed for curing ulcers. Twigs are used as toothbrush. Leaf paste is applied for curing wounds [4]. Antioxidants are commonly used to address problems related to phenolics. Examples include the use of β-mecaptoethanol, ascorbic acid, bovine serum albumin (BSA), Sodium azide and polyvinylpyrrolidine (PVP) [8] [9]. Phenol extraction coupled with SDS is also helpful. However SDS tends to produce low DNA yields of plants rich in polyphenlics [10]. Isolation of plant nucleic acids for use in PCR, RFLP, RAPD, AP-PCR, DAF and genomic library construction is one of most important and time-consuming steps. The degree of purity and quantity varies among applications. A good extraction procedure for the isolation of DNA should yield adequate and intact DNA of reasonable purity. Dangerous chemicals should not be used, if possible, and the procedure should be simple, inexpensive and quick [5]. The optimised protocol described here is specifically designed to isolate genomic DNA of Vitex negundo L. and amplification detecting through RAPD-PCR techniques. MATERIALS AND METHODS Plant materials The leaves sample of Vitex negundo L. were used to determine the extracting sufficient amount of DNA. The leaves were collected during the summer season (April). The collected The extraction process involves breaking or digestion away cell walls to release the cellular constituents. This is followed by disruption of the cell membranes to release DNA into the extraction buffer. This is normally 37 Malaysian Journal of Science 29(1) : 37 - 40 (2010) leaves were placed in plastic bag and stored in a deep freezer at -70°C. Reagents and chemicals • • • • • An extraction buffer consisting of 2% SDS, 800 mM NaCl, 25mM Tris/HCl(pH 8.0), 25mM EDTA 2% PVP and 0.8% β-mecaptoethanol was prepared. Chlorform-isoamylalcohol (24:1) Wash solution: 15mM potassium acetate, 75% ethanol TE buffer: 10mM Tris-HCl (pH 8), 1mM EDTA (pH 8) 0.5V of isopropanol DNA extraction protocol • • • • • • • • • Plant tissues (200 mg leaf) were quickly frozen in liquid nitrogen, powdered with motor and pestle and transferred into 1000µl of extraction buffer. The extract was slightly mixed and incubated in a waterbath at 68°C for 1 hour. Then 15mM potassium acetate was added followed by 30 minutes incubation on ice and centrifugation (12000rpm, 10 minutes, 4°C). The supernatant was transferred into a new tube, mixed 0.5 vol isopropanol by inverting the tubes three times and incubated on ice for 10 minutes. After another centrifugation (12000rpm, 10 minutes, 4°C) the supernatant was discarded completely. The pellet was dried under vaccum, dissolved in 20 µl 1×TE and extracted once with 500 µl of Chlorformisoamylalcohol (24:1). After centrifugation (12000rpm, 10 minutes, 4°C) the aqueous phase was transferred and nucleic acids were precipitated in 0.5V of isopropanol(20°C, 15 minutes). During this time the tubes were gently inverted at least ten times. Finally the tubes centrifuged again (12000rpm, 10 minutes, 4°C) The pellet was washed in 75% ethanol, dried under vaccum and dissolved in 20 µl 1×TE. Store the DNA at -2°C for long-term storage. If needed, treat the DNA solution with RNase before use. 38 DNA obtained with this technique constantly gives a 260/280 absorbance ratio of 1.6-1.8 indicating high quality intact DNA [11]. All of these DNA were used for RAPD-PCR analysis using the method [12]. Arbitrary decamer primes successfully amplified DNA fragments from Vitex negundo L. RESULTS AND DISCUSSION Medicinal plants have large amounts of secondary metabolites. These secondary metabolites make hindrance in DNA isolation and isolated DNA is not suitable for PCR amplification and restriction digestion. We followed the protocol described by [13]. Midiprep method for the isolation of DNA from plants with a high content of polyphenolics. DNA extracted, however was very viscous and could not be resolved on the agarose gel. The other problems encountered were low DNA yields and poor PCR amplification as well as restriction digestion. We increased the concentration of PVP, β-mercaptoethanol and NaCl and incubation temperature from 65°C to 70°C temperature. These modifications helped in the extraction of DNA of high purity and high quantity. We also eliminated the use of phenol for the purification of extracted DNA, which makes our method less hazardous. We also used 2 µl of RNase per 20 µl DNA samples for removal of RNA. The DNA yields and resolution on agarose gel however, were much higher and better from both fresh and dried root samples of medicinal plants with the use of our protocol. Sufficient amount of highly purified clean genomic DNA was obtained when the optimised protocol was described by us. All of these DNAs were used for RAPD-PCR analysis. Arbitrary decamer primers successfully amplified DNA fragments from 5 accessions of Vitex negundo L. The DNA sample was extracted using this procedure and assayed for RAPD-PCR using the primer OPX17(CAGACAAGCC) by using a TGradient thermal cycler. Each PCR 25 µl reaction mixture consisted of 4.0 µl dNTPs, 0.3 µl MgCl2, 2.5 µl PCR Buffer, 2.4 µl Primer, 0.5 µl Taq polymerase, 13.3µl Sterile water, 2.0 µl template DNA. After the initial denaturation 94°C for 5min, 35 PCR cycles were performed with 35 sec at 93°C, 55 sec at 53°C and 45 sec at 72°C,. The DNA extracts as well as the amplification products were run in 2% agarose gels repectively, stained with ethidium bromide and visualized under UV light. Malaysian Journal of Science 29(1) : 37 - 40 (2010) The different protocol are described earlier for isolation of genomic DNA from medicinal plants such as protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion [14], DNA protocols for plants [15], DNA isolation methods for medicinal and aromatic plants [16] and optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India [17]. Fig1. shows the results of PCR amplification using the primer OPX 17 of Vitex negundo L. This DNA preparation method yielded a prdominance of high molecular wieight DNA reliably produced PCR amplification products. Considering all factors involved, this method appears to be the most efficient, reliable and labor-effective DNA isolation procedure from plants containing a high content of polyphenolics. Figure 1: Showing the Genomic DNA and PCR- amplification product using RAPD OPX- 17 primer of Vitex negundo L. This method is a rapid, reliable and cost efficient method for the isolation of the PCRquality DNA from the Vitex negundo L. The DNA solutions are stable at 4°C for atleast two months and can be kept frozen at -20°C for longer storage. We anticipate that adoption of this protocol among the vitex species will speed genotyping, the mapping of mutants, transgenic studies and other PCR based applications. 2. Ambika Nag, Praveen Galav and Kalewa, S.S. (2007). Indigenous animal health care practices from Udaipur District, Rajasthan. Indian Journal of Traditional Knowledge. 6(4): 583-588. 3. Arun Vijayan, Liju, V.B. Reena John, J.V. Parthipan, B. and Renuka, C. (2007). Traditional remedies of Kani tribes of Kottoor Reserve forest, Agasthyavanam, Thiruvananthapuram, Kerala. Indian Journal of Traditional Knowledge. 6(4): 589-594. 4. Saroj Verma and Chauhan, N.S. (2007). Indigenous medicinal plants knowledge of Kumhar forest division district Solan. Indian Journal of Traditional Knowledge. 6 (3):494-497. 5. Daneshwar Puchooa and Sher Ullah Shahnoo Sulliman Khoyratty (2004). ACKNOWLEDGMENTS The authors thanking the International Council of Medicinal Research, Government of India, New Delhi (Ref: 59/12/2006/BMS/TRM dt.) for financial support. REFERENCES 1. Vishal Tandon R. (2005). Medicinal uses and biological activities of Vitex negundo. Natural Product Radiance. 4:162-165. 39 Malaysian Journal of Science 29(1) : 37 - 40 (2010) Genomic DNA extraction from Victoria amazonica. Plant Molecular Biology Reporter. 22: 195a-195j. and J. P. W. Young (eds.), Molecular Techniques in Taxonomy. NATOASI Series H, Cell Biology. 57:283-293. 6. Scott, K.D. and Playford, J. (1996). DNA extraction technique for PCR in rain forest plant species. Biotechnique.s 20:974-978. 7. Do, N. and Adams, R.P.(1991). A simple technique of removing plant polysaccharides contaminants from DNA. Biotechniques. 10:162-166. 16. Anna Maria Pirttilä, Merja Hirsikorpi, Terttu Kämäräinen, Laura Jaakola and Anja Hohtola (2001). DNA Isolation Methods for Medicinal and Aromatic Plants. Plant Molecular Biology Reporter 19: 273a–f. 8. Dawson, C.R. and Magee, R.J. (1995). Plant tyrosinase (polyphenol ozidase) In: Colowick SP and Kaplan NO (eds). Methods in Enzymology. 2: 817-827. 9. Clark, M.S. (1997). In: Plant Molecular Biology-A laboratory Manual. Springer-Verlog Berlin Heidelberg, New York. 305-328. 17. Padmalatha, K. and Prasad, M.N.V. (2006). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. African Journal of Biotechnology. 5 (3):230-234. 10. Rezaian, M.A. and Krake, L.R. (1987). Nucleic acid extraction and virus detection in grape vine. J Virol Meth. 17:277-285. 11. Sambrook, J. and Russel (2000). Molecular Cloning: A laboratory manual 3rd edn. Cold Spring Harbor Laboratory Press. New York: 78-85. 12. Williams, J.G.K. Kubelik, A.R. Livak, K.J. and Rafalski, J.A. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research. 18: 6531 6535. 13. Uta Pich and Ingo Schubert (1993). Midiprep method for isolation of DNA from plants with a high content of polyphenolics. Nucleic Acids Research. 21(14): 3328. 14. Salim Khan, M., Irfan Qureshi, Kamaluddin, Tanweer Alam and Abdin, M. Z. (2007). Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology. 6 (3):175178. 15. Doyle, J. J. (1991). DNA protocols for plants. in: G. Hewitt, A. W. B.Johnson, 40 Madras Agric. J., 96 (7-12): 293-295, December 2009 Detection of DNA Polymorphism by RAPD-PCR Fingerprint in Plumbago zeylanica L. from Western Ghats A. John De Britto*, R. Mahesh, R. Mary Sujin and K. Dharmar PG & Research Department of Plant Biology and Biotechnology, St.Xavier'sCollege (Autonomous), Palayamkottai-627 002 Studies were undertaken for detection of DNA polymorphism through Random Amplified Polymorphic (RAPD) DNA fingerprint in Plumbago zeylanica L. collected from ten locations in Western Ghats. A total of 27 DNA fragment (bands), ranging from 4 to 6 was observed. The genetic distance between the population ranged from 0.1178 to 0.8109 and the genetic identity ranged from 0.4444 to 0.8889. The overall observed and effective number of alleles is 0.3958 and 0.3599 respectively. Over all genetic diversity is 0 .1817 and percentage of polymorphic loci was 81.48. The genetic similarity analysis was conducted on the basis of presence or absence of bands which revealed a wide range of variability within the populations. The cluster analysis clearly showed that there was high degree of diversity within the population. The closest genetic distance existed within population. Thus these RAPD markers have the potential for conservation of identified clones and characterization of genetic relatedness among the population. Key words: Gene diversity, RAPD-PCR analysis, Plumbago zeylanica Plumbago zeylanica L. is an important medicinal herb. It has high medicinal properties. It belongs to the family Plumbaginaceae. Local name: Vellai sidhirai mulam and Kotivelli. Plumbago zeylanica, a rambling subscandent perennial herb or under shrub with green branches, stems somewhat cylindrical, spreading, terate, striate, glabous. Leaves opposite/alternate, ovate or oblong, petiole narrow, amplexicaul at the base and often dialted into stipule like auricles. Flowers white, in axillary and terminal elongated spikes, bisexual. Calyx densely covered with stalked, sticky glands, corolla white, very slender, tubular, stamens 5, free, ovary superior, 5-gonous, one celled, and ovule single and basal (John De Britto and Mahesh 2007). The fruit, leaves, root of Plumbago zeylanica L. have high medicinal and economic value (Rajendran and Agarwal, 2007). It is used against constipation (Pande et al, 2007). The root of Plumbago zeylanica has numerous therapeutic uses. The root is known to be abortifacient and to have vesicant properties. It is used as appetizer and expectorant and used for dysentery, diarrhoea, diuretic and peptic ulcers. The root paste is applied externally for filarial diseases. It is used externally for early maturation, rupture and healing of abscess. The root powder taken orally along with honey gradually reduces hypercholostraemia and improves blood formation (anaemia). It is used to reduce obesity, vitiligo, splenomegaly, hepatomegaly and ascitis. It is also used to relieve coryza (running nose), hoarseness of voice and sore throat. It is *Corresponding author email: [email protected] used in the form of local applications for leucoderma, scabies, psoriasis, symptoms of leprosy and allied skin diseases. The decoction of the root is useful in checking and preventing spermatorrhoea (Madhava Chetty et al, 2006). The conservation of plant population and species is mostly concerned with the number of individuals present in the populations in order to assess factors such as genetic drift, inbreeding depression and lack of mates in self compatible species (Barrett and Kohn, 1991). Molecular techniques help researches not only to identify the genotypes but also in assessing and exploiting genetic variability through molecular markers. Random Amplified Polymorphic DNA (RAPD) analysis has proved for estimating genetic diversity particularly to assist in the conservation of rare species and plant genetic resources. RAPD analysis in particular has proven to be a rapid and efficient means of genome mapping and is well suited for the genetic resource characterization (Williams et al, 1990). The DNA fingerprinting generated by the polymerase chain reaction using arbitrary primers has provided a new tool for DNA polymorphism in number of herb species (Padmalatha and Prasad, 2006). In the present study the detection of DNA polymorphism in P. zeylanica L. collected from ten locations of Western Ghats has been carried out using RAPD markers. 294 Materials and Methods The extensive field survey was carried throughout Western Ghats and ten accession of P. zeylanica L. were collected from Kannanur, Kalakad, Kothyar, Chunkankadai, Maruthamalai, Thiruparapu, Kollimalai, Aralvoimozhi, Karayar and Courtallum. Five plant samples from each location were collected. The plant materials were transferred to plastic bags for transport from field to laboratory. The samples were maintained in deep freezer at 70° C till the analysis was done. Young leaf tissues were used for extracting total genomic DNA, following protocol suggested by (Uta Pich and Ingo Schubert (1993). The amount of DNA and its quality were assessed by UV spectrophotometer. The DNA was pure enough (OD260/280 =1.68), (Sambrook and Russel, 2000) for RAPD- PCR analysis (Williams et al., 1990). Earlier, ten primers were tested and five primers OPX 03 (GGAGGGTGTT), OPX 04 (CCGCTACCGA), OPX06 (AGGGGTCTTG), OPX12 (TCGCCAGCCA), OPX19 (TCTGTGCTGG) which produced reproducible bands and were selected. The experiments were repeated three times and confirmed the reproducibility of bands. PCR reactions were carried out in a total volume of 20 µl at a final concentration of 1 mM MgCl2, 2 mM dNTP, Taq DNA polymerase enzyme (1ul/20 µl), with approximately 200 ng DNA as a template and a single random primer (0.2 mM). The PCR cycles were as follows: 94°C for 2 min, 94°C for 15 sec, 35°C for 15 sec, 72°C for 30 sec which were repeated in 35 cycles followed by 5 min-extension at 72°C. 8 µl of PCR product was subjected to electrophoresis at 80 V using 1.5 % agarose gel stained with ethidium bromide.The bands were viewed under UV transilluminator and photographed with gel documentation system Alpha Imager 1200. Based on the primary data (presence or absence of bands), pair wise genetic distance between samples was calculated using POP GENE package version 1.31. (Yeh et al., 1999). Results and Discussion Analysis of ten accession of P. zeylanica L. revealed 22 polymorphic loci. Five primers generated reproducible, informative and easily scorable RAPD profiles. A total of 27 bands were scored for the 5 RAPD primers out of which 115 bands polymorphic with number of bands ranging from 4 to 6 (Plate 1). The same type of bands occurred at different frequencies in all populations. There were many additional bands neglected which were not reproducible. The percentage of polymorphic loci was 81.48. The genetic distance between the population ranged from 0.1178 to 0.8109 and the genetic identity ranged from 0.4444 to 0.8889 which are shown in Table 1. As it was Plate 1. Plumbago zeylanica L. observed in the Popgene software results, the overall observed and effective number of alleles was 1.8 and 1.3 respectively and overall genetic diversity was 0 .2719. The obtained result was analyzed in the POP GENE package 1.31 and the dendrogram obtained clearly indicates three clusters (Nei, 1978) (Fig 1). Population 5 form a separate clade. Populations 1,6,2,4 and 7 form one clade and populations 3,9,8 and 10 form another clade. Three groups are distinctly formed in the first clade (populations 1 and 6,2 and 4 and 7). In the second clade again three groups are formed (populations 3 and 9, 8 and 10) (Fig 1). The utility of RAPD markers in estimating genetic divergence has been demonsrated in several studies. The similar study was done in RAPD in in vitro regenerated leaf explants of Plumbago zeylanica L. (Rout, 2002). Genetic diversity analysis in Rauvolfia serpentina and Rauvolfia tetraphylla L, using RAPD Markers. (Padmalatha and Prasad 2007; Padmalatha and Prasad, 2006). Recently studies on molecular analysis in Urginea indica Kunth collected from different location of Karnataka was reported. (Harini et al., 2008). The clustering results of different accessions suggest that Plumbago zeylanica L. undergoes major part of genetic variation by environmental factors. Genetic diversity refers to the variation at the level of individual genes (polymorphisms), and provides a mechanism for populations to adapt to their ever-changing environment. The genetic variability in P. zeylanica L. may be partly explained 295 Table 1. Nei’s Original Measures of Genetic Identity and Genetic distance pop ID 1 2 3 4 5 6 7 8 9 10 1 **** 0.8519 0.7407 0.8148 0.5556 0.8519 0.8519 0.7778 0.7037 0.6667 2 0.1603 **** 0.6667 0.8889 0.6296 0.7778 0.77778 0.7037 0.7778 0.5926 3 0.3001 0.4055 **** 0.5556 0.4444 0.6667 0.6667 0.7407 0.8148 0.7778 4 0.2048 0.1178 0.5878 **** 0.7407 0.7407 0.8889 0.6667 0.6667 0.6296 5 0.5878 0.4626 0.8109 0.3001 **** 0.4815 0.6296 0.5556 0.4815 0.5185 6 0.1603 0.2513 0.4055 0.3001 0.7309 **** 0.7778 0.7778 0.7037 0.5185 7 0.1603 0.2513 0.4055 0.1178 0.4626 0.2513 **** 0.7778 0.7037 0.7407 8 0.2513 0.3514 0.3001 0.4055 0.5878 0.2513 0.2513 **** 0.7778 0.6667 9 0.3514 0.2513 0.2048 0.4055 0.7309 0.3514 0.3514 0.2513 **** 0.6667 John De Britto, A. and Mahesh, R. 2007. Evolutionary medicine of kani tribal's botanical knowledge in Agasthiayamalai Biosphere Reserve, South-India. Ethnobotanical Leaflets, 11: 280-290. Madhava Chetty, K., Sivaji, K., Sudarsanam, G. and Hindu Sekar, P. 2006. Pharmaceutical studies and therapeutic uses of Plumbago Zeylanica L. roots. Ethnobotanical Leaflets, 10: 294-304. Nei, 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89:583. Figure 1. Dendrogram Based Nei’s Genetic distance as a result of abiotic- geographical, hydrographical connections, climatic differentiations - annual rainfall, temperature, pH of the soil, humidity etc., and also the various biotic factors. Genetic variation in a population is measured by the heterozygosity or the degree of polymorphism. For the conservation of a species, genetic variability is of the utmost importance to preserve. Genetic variability among all species is important to maintain since it represents the 'blue print' for all of the living things on earth. It is desired to maximize the preservation of alleles. Geographical, climatic or reproductive variables explain the partitioning of the diversity observed which may aid in improving the strategies for maximizing the efficiency of germplasm collection and preservation. References Barrett, S.C.H. and Kohn, R. 1991. Genetic and evolutionary consequences of small population size in plants: Implication for conservation. In D.Falk and K. Holsinger (eds.). Genetic and conservation of rare plants. Oxford University Press. Oxford. UK. 3-10. Harini, S.S., Leelambika, M., Shiva Karmeswari, M.N. & Sathyanarayana, N. 2008. Optimization of DNA isolation and PCR-RAPD methods for molecular analysis of Urginea indica (Kunth). International J. Integrative Biol., 2:138-144. Padmalatha, K. and Prasad, M.N.V. 2007. Inter and Intrapopulation genetic diversity of Rauvolfia serpentine (L). Benth. Ex Kurz, an endangered medicinal plant, by RAPD analysis. Indian J. Biotech., 40: 18-22. Padmalatha, K. and Prasad, M.N.V. 2006. Genetic diversity in Rauvolfia tetraphylla L.f using RAPD markers. Plant Biotechnology, 8: 1-7. Pande, P.C., Lalit Tiwari and Pande, H.C. 2007. Ethno veterinary plants of Uttaranchal-A review. Indian J. Traditional Knowledge, 6: 444-458. Rajendran, S.M. and Agarwal, S.C. 2007. Medicinal plants conservation through sacred forests by ethnic tribals of Virudhunagar district, Tamilnadu. Indian J. Traditional Knowledge, 6: 328-333. Rout, G.R. 2002. RAPD from in vitro regenerated leaf explants of Plumbago zeylanica L. Annals of Applied Biology, 140: 305-313. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A laboratory manual 2nd edn. (Cold Spring Harbor laboratory Press. Cold Spring Harbor, New York. Uta Pich and Ingo Schubert 1993. Midiprep method for isolation of DNA from plants with a high content of polyphenolics. Nucleic Acids Research. 21: 3328. Williams, J.G.K., Kubelik, A.R., Livak, K.J. and Rafalski, J.A. 1990. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research.18: 6531-6535. Yeh, F.C., Boyle, T., Rongcai, Y., Ye, Z. and Xian, J.M. 1999. POPGENE Version 1.31. A Microsoft Window based freeware for population genetic analysis. University of Alberta Edmonton. Received: December 02, 2008; Revised: September 20, 2009; Accepted: November 20, 2009