Goel et. al. / JPBMS, 2011, 5 (21) Available online at www.jpbms.info ISSN NO- 2230 - 7885 Original Research Article JPBMS JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES Molecular Characterization of the Nettle Plant Urtica parviflora Based On RAPD Marker Goel Chirag, Verma Pankaj, Ahmad Naseer, Nailwal K Tapan* Department of Biotechnology, Kumaun University, Nainital, Campus, Bhimtal - 263136, Uttarakhand, India. Abstract : Urtica parviflora is considered as an important Medicinal plant, due to its various ethanomedical uses. Here, we analyze the Genetic Variation in U.parviflora, with respect to plant distribution in Kumaun hills based on change in altitude. Examination of Random amplified Polymorphic DNA (RAPD) markers from four 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. A total of 70 scorable bands were produced in four samples with 8 primers. The average number of bands per primer was 8.75. Out of 70 bands, 48 bands were polymorphic (68.75%) noted in the present investigation. The dendrogram of the samples showed two major clusters. The samples of Mukteshwar, Nainital and Bhowali are in one cluster and Bhimtal in other cluster. Key words : Genetic Diversity, Primers, RAPD analysis, Taq DNA polymerase. Introduction: The plant Urtica parviflora Roxb. (Urticaceae) commonly importance, little information is available on the called as stinging nettle, is a plant usually 1 to 2 m tall in phylogenetic relationships among the Urtica parviflora height . The plant is evenly distributed in the eastern Asian plant samples in Kumaun region. RAPD technique is one of Himalayas at a height of about 1700-2800 meters [6] . It is the most frequently used molecular methods for an important medicinal plant of Kumaun region as taxonomic and systematic analyses of various organisms revealed by its various ethanomedical uses such as, the [2] .RAPD markers were used for evaluation of genetic leaves and fresh roots of Urtica parviflora are used for the diversity in many plant species such as Urtica diocia [3] , treatment of fracture, dislocation of bones, boils, and Soybean [19] , Tobacco [7] , Lycopersicon [12] , Sugarcane [14] , decoction of herb is used as a febrifuge [16] .The leaves are Solanum [18] . Among local Kumaun people especially of also used in dysentery, joint pain and liver disorders [8] . Nainital and Mukteshwar districts in Uttarakhand there is The inflorescences are used as a cleansing agent after a general belief that nettle species found in higher parturition and in the treatment of dermatitis in the alpine Himalayan zone have high medicinal value compared to region of central and eastern Himalayas [16] . So far as no species found in lower Himalayan zone especially in foot work has been done on the molecular aspects of this plant. hills of Himalayas. RAPD is a common molecular approach The only work has been done on pharmacological aspect in DNA fingerprinting analysis for genotypic of this plant due to its medicinal properties. The differentiation, molecular taxonomy and other methanolic leaf extract of Urtica parviflora plants shown to applications [13] . Keeping in mind the above notion our aim have significant wound healing activities in excision, was to study the genetic variation among U. parviflora incision and dead space models. Histopathological plant samples collected from different heights from sea examination confirmed the mechanism of wound healing level in Himachal Pradesh based on RAPD markers as by increased deposition of collagen, fibroblast on the RAPD provides a quick approach to evaluate genetic granulation tissue and neovascularization [11] . The markers that are simple to evaluate for constructing ethanolic leaf extract of Urtica parviflora has shown the genetic linkage maps and can be used as a premise for hepatoprotective activity evaluated by the assay of liver further genetic analyses with advanced technology. We function, biochemical parameters such as Aspartate used RAPD markers for several reasons: (1) they reveal aminotransaminase (AST), Alanine aminotransaminase even small genetic differences and are a means of (ALT), alkaline phosphatase (ALP), total bilirubin serum assessing polymorphisms at a wide range of loci, since a protein and by the study of histopathology of the livers [10] . large part of the nuclear genome can be scanned [5] , (2) it In spite of its economic, medicinal and scientific is an efficient and inexpensive technique without requiring prior knowledge of the genome [4] , (3) RAPD *Corresponding Author assay has the advantage of being easy to use, requiring Dr.Tapan K Nailwal., very small amount of genomic DNA without the need for Assistant Professor, Department of Biotechnology, blotting and radioactive detection [1] , and are moderately Kumaun University, Nainital, Campus Bhimtal-263136 reproducible, and (4) there are not specific microsatellite India. primers developed for Urtica . Difference in altitude was Contact no.: +919412986483 taken as the parameter to study the variation in plant Email: [email protected]. samples Therefore, the objective of the present work was to identify genetic similarity and diversity between 1 Journal of Pharmaceutical and Biomedical Sciences (JPBMS ), Vol. 05, Issue 05 Goel et. al. / JPBMS, 2011, 5 (21) samples from four geographical heights by RAPD-PCR and The amplification products along with 2 μl of loading dye by means of the analysis of bandsharing values. (bromophenol blue) were separated on 1.2 per cent agarose gel at 80 volts using 1X TAE buffer of pH 8.0 Materials and Methods: containing Ethidium bromide (0.5 μl 1/10 ml of gel). The Sample Collection: The leaves of Urtica parviflora were gel were viewed under UV transilluminator and collected from four different geographical regions of photographed for documentation. Out of 20 primers used Kumaun (Uttarakhand, India): Mukteshwar (2800m), 8 primer produced recognizable bands. Scorable bands for Nainital (2200m), Bhowali (1600m) and Bhimtal (1300m) a primer in each sample were compared and allotted 0 varying from 1300 to 2800 m from sea level in (absence) or 1 (presence) values. Band patterns (0, 1 Uttarakhand. The leaves were brought in aluminium foil in matrix) were tabulated for individual primers separately an ice box and stored at - 20°C. and the data pooled to obtained a combined matrix for four samples off 8 primers. Diversity coefficient for each DNA Extraction: primer (number of polymorphic bands/total number of DNA was extracted by Plant DNA Isolation Kit (Hi-Media, bands) and pair-wise genetic similarity were calculated India) according to the manufacturer’s instructions. These using NTSYS-PC (version 2.11a) software (Applied DNA samples were further diluted and incubated at 4’C. Biostatistics, USA). DNA Purification: Results: RNase A (10 – 15 µl; 50 mg/ml) was added into the DNA A total of 70 scorable bands were produced in four samples and incubated at 37°C for 1 h to degrade RNA. samples with 8 primers (Table 1) . This data was utilized Equal volume of chloroform: isoamyl alcohol (24:1) (pH for further computations. The average no. of bands per 8.0) was added and gently mixed for 10 min and primer was 8.75. Out of 70 bands, 48 bands were centrifuged at 15,000 rpm for 15 min. Three layers were polymorphic (68.75) noted in the present Investigations. formed, the upper layer was removed into 2 ml eppendorf Data matrices were prepared in which the presence of a and and 3 M sodium acetate at 1/10th of DNA sample was band was coded as 1 and absence as 0. The data matrices added. Double volume of chilled 100% ethanol was added were analyzed by the SIMQUAL programme of NTSYS-PC [17] for DNA precipitation. DNA precipitates appeared as (version 2.11a) , and similarity between accessions was [9] clumps. They were centrifuged at 10,000 rpm at room estimated using Jaccard coefficient . The similarity index temperature. values obtained for each pair wise comparison among U. parviflora samples from four different regions are shown in (Table 2). The similarity index based on eight RAPD DNA Quantification: markers ranged from 46% to 69%. The maximum Purity and concentration of genomic DNA was estimated similarity was observed between the sample pairs of by calculating the ratio of the optical density measured at Nainital and Bhowali (69%) followed by Mukteshwar and 260–280 nm with a spectrophotometer (Thermo Bhowali (51%), followed by Mukteshwar and Nainital Scientific Type UV1, England). (49%). The minimum similarity was observed between the sample pairs of Mukteshwar and Bhimtal (46%).The PCR Amplification: cluster analysis based on UPGMA and SAHN is depicted RAPD analysis was done individually with 20 random vide (Figure 1). A perusal of the UPGMA dendrogram decamer Primers. The thermal cycling conditions were; based on Jaccard’s similarity coefficients. The dendrogram Denaturation : 94 0C for 1 min. has grouped all the four samples into two major clusters A Primer annealing: 36 0C for 1 min. and B. Cluster A was further classified into two minor Primer extension: 72 0C for 2 min. clusters A1 (Mukteshwar) and A2 (Nainital,Bhowali). The The PCR reaction was carried out in a final volume of 25µl major cluster A is comprised of 3 samples, while the reaction containing 50ng of template DNA, dNTPs (200 cluster B (Bhimtal) is comprised of one sample . The Sub µM), Taq DNA polymerase (0.5 U), MgCl 2 (2.5mM) and 10 cluster A2 comprised of 2 samples which are Nainital and pMoles of primer in 25µl of 1X PCR buffer. The reaction Bhowali. components were added to PCR tubes kept on ice. Table 1: Scorable DNA bands generated by different random decamer primer through PCR S.no Primer (5N →3N) Total number of loci Number of Number of polymorphic loci Polymorphism monomorphic loci (%) 1. GAGCCCTCCA 12 5 7 58.33 2.