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Journal of Medicinal Plants Research Vol. 3(3), pp. 166-170, March, 2009 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875© 2009 Academic Journals

Full Length Research Paper

Genetic variation and polymorphism in the Himalayan nettle plant Urtica dioica based on RAPD marker

Vishal Bharmauria*, Navjyoti Narang, Vivek Verma and Shalini Sharma

Department of Biotechnology Shoolini Institute of Life Sciences and Business Management, Himachal Pradesh University, Solan, 173212, India.

Accepted 31 January, 2009

Urtica dioica is a medicinal plant and is distributed throughout the ranges of Himachal Pradesh. The Genetic variation was found in U. dioica plant samples, with respect to plant distribution in middle and lower Himalayas. Genetic variability was based on change in altitude. Examination of random amplified polymorphic DNA (RAPD) markers from six plant samples collected at different heights from sea level indicated that genetic variation was appreciable, as samples from lower altitudes showed low genetic similarity with samples collected from higher altitudes. Eight Random decamer primers were used for RAPD amplification. The RAPD products ranged from 500 - 2800 bp. A total number of 134 RAPD markers were obtained from 8 primers, out of which 27 were polymorphic (20.2%) and the rest were monoporphic (79.8%). The polymorphism ranged from 4 - 50%. Key words: Himachal Pradesh, medicinal plant, RAPD, PCR. INTRODUCTION Nettles (Genus: Urtica), are herbaceous perennial plants but some are annual and few are shrubby. There are 30 45 species of flowering plant of the genus urtica in the family Urticaceae, with a cosmopolitan though mainly temperate distribution. The most prominent member of the genus is the stinging nettle Urtica dioica, native to Europe, Africa, Asia, and North America. The plant is evenly distributed in Himalayas especially in middle and lower zone between 450 to 3500 m from sea level. U. dioica leaves have stinging hairs. So far very little work has been done on the molecular aspects of this plant. The only work that has been done is on the U. dioica agglutinin; UDA previously found in root and rhizomes as UDA-isolations has been shown to be composed of two hevein domains and is processed from a precursor protein through protein and cDNA sequencing (Mirjam et al., 1999). Genomic fragments encoding precursors for UDAIsolectins amplified by 5 independent PCR on genomic DNA. One amplified gene was completely required to show two base pair substitutions, two introns located at the same positions as in other plant chitinases, partial sequence analysis of 40 amplified genes, 16 different genes were identified which encoded seven putative UDA isolectins (Mirjam et al 1999). Urtica dioca contains various substrates (Adamski et al., 1984), water soluble glycoproteins (Anderson et al., 1978), choline acetyltransferase (Barlow et al., 1973), and water soluble acids (Bakke et al., 1878). The mannose specific plant lectins from Urtica dioica are potent and selective inhibitors of human immunodeficiency virus (Balzarini et al., 1992). Its medicinal properties have been shown by Chaurasia et al., 1987.Six basic UDA isolectin have the same molecular structure with identical agglutination properties due to identical amino acid at Carbohydrate-binding site (Van Damme et al., 1999). A great deal of work has been done on biochemical aspect of this plant due to its medicinal properties. Several pharmacological properties of nettle which have been reviewed like, anti-inflammatory activity by Caffeoylmatic acid which inhibits 5 lipoxygenasederived biosynthesis of leukotrine by 20.8 and 68.2% and inhibits the synthesis of cycloosygenose derived prostaglandins also reduces tumor-necrosis-factor (TNF) and Interlectin IB (IL-IB) in mouse L929 fibro sarcoma cells (Reichmann et al., 1999). It is suggested that nettle enhances the effectiveness of diclofenac in rheumatic con-

*Corresponding author. E-mail: [email protected] Abbreviations: CTAB, cetyltrimethylammoniunbromide; IAA, isoamylalcohol; PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA.

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dition (Chrubasik et al., 1997). CNS depressant activity has been reported. It leads to inhibition of drug­induced convulsions of body temperature (Sharma et al., 1983). Research has shown that U. dioca holds promise in the treatment of benign prostate hyperplasia (Dvorkin and Song, 2002; Dreikorn, 2007) and prostate cancer (Konrad and Mueller, 2000). Water extract of nettle plays role in antioxidant, antimicrobial, antiulcer and analgesic activities (Gulcin et al., 2004). Isolectins isolated from the rhizome are reported to cause non specific agglutination of erythrocytes to induce the synthesis of interferon by human lymphocytes (Shibuya et al., 1980) L-1210. Among local Himalayan people especially of Solan and Shimla district in Himachal Pradesh there is a general belief that nettle species found in higher Himalayan zone have high medicinal value compared to species found in lower Himalayan zone especially in foot hills of Himalayas. RAPD is a common molecular approach in DNA fingerprinting analysis for genotypic differentiation, molecular taxonomy and other applications (Lin et al., 1996). Keeping in mind the above notion our aim was to study the genetic variation among U. dioica plant samples collected from different heights from sea level in Himachal Pradesh based on RAPD markers as RAPD provides a quick approach to evaluate genetic markers that are simple to evaluate for constructing genetic linkage maps and can be used as a premise for further genetic analyses with advanced technology. U. dioca a useful medicinal plant and its molecular studies can be used to cure various diseases especially prostate cancer. Different samples of U. dioica were collected from two districts, that is, Shimla and Solan. Difference in altitude was taken as the parameter to study the variation in plant samples. The DNA was isolated by standardization of the protocol of Yadav (2003).

MATERIALS AND METHODS Sample Collection: Leaf Samples were collected from different sites (Kasauli [monkey point] 2010 m; Sanwara 1369 m; Kasauli town 1927 m; Solan 1450 m; Shimla 2310 m; Shoghi 1851 m) varying from 1369 to 2310 m from sea level in Himachal Pradesh and name and coded as 771, 773, 774, 776, 777 and 778 respectively. The leaves were brought in aluminium foil in an ice box and stored at 20° C.

of DNA) of Hoechst dye was added accordingly. Hoechst dye, 10 mg dye was dissolved in 10 ml Milli-Q water. It was stored in amber coloured bottle. 10X TNE Buffer (1000 ml Milli-Q Water) 100 mM Tris; 10 mM EDTA, 2 M NaCl. DNA extraction procedure Modified CTAB method (Yadav, 2003) was used for DNA isolation. The sequential steps followed are listed below: For DNA isolation of fresh leaf samples of U. dioica were collected, about 3 g of samples were weighed, sterilized with distilled water three times to remove pigments and dust. The samples were dried with blotting paper. They were then transferred to mortar and pestle with the help of spatula and ground into fine powder in liquid nitrogen. Proper grinding was done because it would have affected the quantity of DNA isolated. The resulting powder was transferred to 50 ml centrifuge containing 15 ml pre-warmed DEB (DNA Extraction Buffer) at 65° C. -mercaptoethanol (300 l) was added to tubes just before addition of fine powder. Vigorous vortexing was done for 2 min and the centrifuge tubes containing the samples were transferred to water bath for incubation at 65° for 1 h. The tubes were mixed by gentle inversions for C at least three times after every 15 min. After incubation, 9 ml of 24:1 (chloroform: Isoamyl alcohol) was added up to the lid in each tube so that phenols are completely removed. The tubes were given gentle inversions for 20 min and then centrifuged at 20,000 rpm for 15 min at 25° Three layers were formed out of which the aqueous C. upper layer containing DNA was removed with the help of a broad mouth pipette tip and transferred to centrifuge tubes containing 9 ml Isopropanol for DNA precipitation. DNA appeared as cloudy precipitate at the interface. DNA from this layer was transferred to 2 ml eppendorf's using broad mouth pipette tips and centrifuged at 5000 rpm for 5 min. The supernatant was discarded and DNA pellet was given a washing with 70% ethanol at least three times with gentle tapping to remove salts. The tubes were sealed with parafilm; small holes were made with the help of disinfected needle and tubes were kept at 4° drying. On complete drying after 24 h, 500 l T10 C E1 buffer was added to dissolve the DNA. DNA purification RNase A (10 ­ 15 l; 50 mg/ml) was added into the DNA samples and incubated at 37° for 1 h to degrade RNA. Equal volume of C chloroform: Isoamyl alcohol (24:1) (pH 8.0) was added and gently mixed for 10 min and centrifuged at 15,000 rpm for 15 min. Three layers were formed, the upper layer was removed into 2 ml eppendorf and and 3 M Sodium acetate at 1/10th of DNA sample was added. Double volume of chilled 100% ethanol was added for DNA precipitation. DNA precipitates appeared as clumped. They were centrifuged at 10,000 rpm at room Temperature. DNA quantification

Reagents and chemicals CTAB (10% [w/v]), 1 M Tris HCl (pH 8), 4 M NaCl, 70% ethanol, chloroform-IAA (24:1[v/v]), mercaptoethanol (Sigma): 0.2% per sample, absolute alcohol, 0.5 M EDTA, Isopropanol (Sigma), 3 M sodium acetate. Extraction Buffer (100 ml) 0.5 M EDTA (pH 8), 4 ml; 1 m Tris HCl (pH 8),10 ml; 4 M NaCl, 35 ml; 10% CTAB, 20 ml; 29 ml distilled water and beta ­mercaptoethanol (300 µl). TE Buffer: 10 mM Tris-Cl buffer (pH 8), 1 mM EDTA (pH 8) Taq DNA polymerase enzyme, dNTP mixture (A, T, G, C). Assay buffer: 10 ml 10X TNE was added in 90 ml of Milli-Q water. The 10 l (for 100 ­ 500 ng of DNA) or 100 l (for more than 500 ng

The DNA Fluorometer (DNA Quant 200 Fluorometer from Hoefer, Amersham) was first calibrated using 2 ml assay buffer with 2 l Calf thymus DNA (50 ng/ l Conc.) added in it. The equipment exhibited 100 ng reading. Then the different samples were analysed for DNA quantification. PCR amplification Master Mix was prepared for 6 PCR reactions with eight different primers of Operon series (OPG-02, OPG-03, OPG-04, OPG-05, OPG-06, OPG-07, OPG-08, and OPG-09) and accordingly different PCR components were taken. The reaction components were added to PCR tubes kept on ice. The master mix was dispensed into

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Figure 1. DNA fingerprinting of Urtica dioica samples with primers OPG-02 and OPG-05.

Figure 4. DNA fingerprinting of Urtica dioca with OPG-08 and OPG-09.

thin walled 0.2 ml PCR tubes at 23 l volume per tube. After dispensing the master mix, 2 l DNA (25 ng / l) from each sample was dispensed. The final reaction volume had 1X PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl), 200 M dNTP, and 10 moles of random primers of Operon technologies Inc., USA, 0.75U of Taq DNA polymerase and 50 ng/ l of genomic DNA per PCR reaction. PCR tubes were placed inside the thermal cycler (MJ Research Inc., USA, and model PTC-100) with heat lid technology and the PCR machine was run at the following parameters. 95° C for 3 min; 40 cycles at 94° for 1 min, 36°C for 1 min, and 72° for C C 2 min; and a final extension at 72° for 8 min. PCR products were C subjected to agarose gel (1.5% [w/v]) electrophoresis in 0.5X TAE buffer, along with DNA markers (Banglore Genie, India) as size markers. DNA was stained with Ethidium Bromide and photographed under UV light.

Figure 2. DNA fingerprinting of Urticia dioca with primers with OPG-04 and OPG-05.

RESULTS Data matrices were prepared in which the presence of a band was coded as 1 and absence as 0. The data matrices were analyzed by the SIMQUAL programme of NTSYS-PC (version 2.08), Rolph (1998), and similarity between accessions was estimated using Jaccard coefficient (Jaccard, 1908). The RAPD products ranged from 500 ­ 2800 bp. The highest number of RAPD markers were obtained with the primer OPG-03(33) followed by OPG-02(25). The least number of RAPD markers were obtained from primer OPG-08(11) and OPG-09(11). A total number of 134 RAPD markers were obtained from 8 primers, out of which 107 were polymorphic (20.2%) and rest were monomorphic (79.8%). The polymorphism ranged from 4 - 50% (Figures 1 ­ 4). The similarity matrix varied from 0.052 to 1.000 with the average being 0.493 (Table 1). The least genetically similar sample was from monkey point Kasauli (6% genetic similarity). The cluster analysis based on UPGMA and SAHN is depicted vide Figure 5. A perusal of the UPGMA dendrogram based on Jaccard's similarity coefficients reveals that the samples from Shimla. BCS (DNA code-

Figure 3. DNA fingerprinting of Urtica dioica with primers OPG-06 and OPG-07.

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Table 1. Jaccard's Similarity Analysis.

Sample 771 773 774 776 777 778

771 1.000 0.0000000 0.0526316 0.2653061 0.5000000 0.3958333

773 1.000 0.0000000 0.0000000 0.0000000 0.0000000

774

776

777

778

1.000 0.1250000 0.0526316 0.0250000

1.000 0.2916667 0.2115385

1.000 0.5581395

1.000

Jaccard's similarity matrix based on eight RAPD primers in eight samples of Urtica dioica.

Figure 5. Dendrogram of Six Genotpes of Urtica dioca, based on RAPD.

777) and Shoghi (DNA code-778) had maximum similarity to extent of 55%. The sample from Kasauli town (DNA code-771) had maximum similarity with samples from BCS Shimla and Shoghi (DNA code-777, 778) that is, about 45%. Whereas samples from Solan (DNA code776) is also related to the extent of about 27% with samples from Kasauli town, Shimla BCS and Shoghi. The germplasm from Sanwara is not at all related to with other germplasm with 0% similarity. DISCUSSION Molecular characterization of germplasm is basic to the improvement of the species and can be done at the DNA level. The finding of present study suggests that the genetic variability present in U. dioica was quite appreciable due to change in height above sea level as sample from Sanwara showed low genetic similarity with samples

collected from higher altitudes. Among local Himalayan people there is a general belief that nettle species found in higher Himalayan zone have high medicinal value compared to species found in lower Himalayan zone especially in foot hills of Himalayas. Various reasons can be held responsible for this assumption, as, U. dioica leaves is known to be genetically variable in number of stinging hairs per unit area as well as other characters. Our findings also suggest that leave samples collected from lower altitudes have lesser number of stinging hairs than samples collected from higher altitudes. Overall variations in trichome numbers (Pollard and Briggs, 1982) accounted for difference in important clinical values associated with this plant with respect to plant distribution at different altitudes. Leave sample from lower altitude have lower number of stinging hair and believed to exhibit lower medicinal values than samples from higher altitudes with higher number of stinging hairs. Various environ-

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mental factors like rainfall, temperature, altitude and the dosage of the UV rays can be responsible for such appreciable variation in the species. Spatial and genetic variations are often assumed to result from environmental heterogeneity and different selection pressures. Simple sequence repeats (SSRs) are a group of repetitive DNA sequences that represent a significant portion of higher eukaryote genomes. They can serve as highly informative genetic markers, and in conjunction with the use of polymerase chain reaction (PCR) technology enable the detection of length variation. Polyploidy is an adaptive change which can be responsible for variation in a species. The isolation of DNA offered a great challenge as pvp was not used, and the plant was rich in phenolic compounds. DNA during isolation appeared to be yellowish green yellowish blue in colour, highly viscous, while pipeting it out formed a thick and sticky flow. Interestingly during purification, all the phenolics pigments were removed but there was just a slight improvement in decolourization of two samples from Sanwara (773) and Kasauli town (774), rest all the pigmented samples were discarded. 773 and 774 were selected for PCR to study the effect of this pigmentation in form of phenolic or something else at the genetic level. After the results of PCR were obtained on the gel documentation system it was observed that the Primers did not attach to the samples 773 and 774 indicating that there were no free sites available for the primer to attach. So it can be interpreted that the coloration had effect on the primer attachment sites. 25 RAPD markers were obtained with primer OPG02 with 4% polymorphic character whereas 33 RAPD were obtained with primer OPG-03 with 18% polymorphism indicating that these primer sequences were present in all most all the samples. Primer OPG-04 produced 50% polymorphic band but the primers OPG-05-OPG-09 produced slight polymorphism ranging from 12.5 - 30%. Conclusion In the present study only 8 primers were used which are insufficient to study the complete genetic variability in any species so more number of primers are required. More work needs to be done on genetic level to completely characterize this magnificent plant rich in numerous medicinal properties. Since the results with 8 primers showed appreciable variation in the species and keeping in mind the use of plant as a potent medicine for various diseases, especially prostate cancer, the study can be used as a premise for advanced genetic analyses and molecular studies for novel purposes.

REFERENCES Adamski R, Beiganska J (1984).Studies on substrate present in Urtica dioica L. leaves .analysis for protein amino acids and nitrogen containing non-protein amino acids. Herba Pol 30: 17-26. Andersen S, Wold JK(1978). Water-soluble glycoprotein from Urtica dioica leaves. Phytochemistry 17: 1875-1877. Balzarini J, Neyts J (1992). The mannose specific plant lectins from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro. Antiviral Res. 18:2, 191-207 Barlow RB, Dixon ROD (1973). Choline acetyltransferase in the nettle Urtica Dioica L. Biochemistry 132: 15-18. Chaurasia N, Wichtl M (1987) Flavonolglykoside aus Urtica dioica. Planta Medica 53: 432-434. Chrubasik S, Enderlein W, Baeur R, Grabner W (1997). Evidence for antirheumatic effectiveness of herb Urtica dioica in acute arthrittis: a pilot study. Phytomedicine; 4: 105-108. Dreikorn K (2007). Phytotherapeutic agents in the treatment of benign prostate huperplasia.Current Urology Reports.1:103-109. Dvorkin L, Song KY (2002). Herbs for benign prostate hyperplasia. The Annals Pharmac. 36: 1443-1452. Gulcin I, Kufrevioglu I, Oktay M and Buyukokuroglu Me. (2004) antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (urtica diocia L). J. Ethnopharmacol.90:205-215. Konrad L, Mueller H H (2000) antiproliferative effect on human prostate cancer cells by a stinging nettle root (urtica dioca) extract. Plant Medica. 66 (1): 44-47. Lin JJ, Kuo J, Ma J, Saunders JA, Beard HS, Macdonald MH, Kenworthy W, Ude GN, Mathews BF (1996). Identification of molecular markers in soybean comparing RFLP, RAPD and AFLP DNA mapping techniques. Plant Mol Biol Rep 14: 156-69. Mirjam P Does, David K.Ng, Henk L. Dekker, Willy J. Peumans (1998). Characterization of urtica dioica agglutinin isolectin and the encoding gene family. Plant Mol. Biol. 39, 335-347 Pollard AJ and Briggs D (1982). Genecological studies of Urtica dioica L. The nature of interaspecific variation in Urtica dioica. New phytologist 92: 453-470. Reihemann K, Behnke B, Schulze-Osthoff K (1999). Plant extracts from stinging nettle Urtica dioica, an antirheumatic remedy, inhibit the proinflammatory transcription factor NF B. FEBS letter; 442: 89-94. Rohlf FJ (1998). NTSYS-PC Numerical Taxonomy and Multivariate Analysis System Version 2.1, Exeter Software, Appl. Biostat. New York. Van Damme EJM, Peumans WJ (1987). The Urtica dioica agglutinin is a complex mixture of Isolectins. Plant Physiol. 71: 328-334. Yadav MC (2003). Molecular breeding for development of genetically improved strains and hybrids of Agaricus bisporus. In: Current Vistas in Mushroom Biology and Production (R.C. Upadhyay, S.K. Singh and R.D. Rai, eds.) Mushroom Society of India, Solan. pp. 261-274, Yadav MC (2004). Molecular markers in genetics and breeding of mushrooms. In: Recent Advances in Biotechnology (N.C. Gautam and M.P. Singh, eds.) Shree Publishers & Distributors, New Delhi. pp. 89-104.

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