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
called as stinging nettle, is a plant usually 1 to 2 m tall in
height. The plant is evenly distributed in the eastern Asian
Himalayas at a height of about 1700-2800 meters [6]. It is
an important medicinal plant of Kumaun region as
revealed by its various ethanomedical uses such as, the
leaves and fresh roots of Urtica parviflora are used for the
treatment of fracture, dislocation of bones, boils, and
decoction of herb is used as a febrifuge [16].The leaves are
also used in dysentery, joint pain and liver disorders[8].
The inflorescences are used as a cleansing agent after
parturition and in the treatment of dermatitis in the alpine
region of central and eastern Himalayas [16]. So far as no
work has been done on the molecular aspects of this plant.
The only work has been done on pharmacological aspect
of this plant due to its medicinal properties. The
methanolic leaf extract of Urtica parviflora plants shown to
have significant wound healing activities in excision,
incision and dead space models. Histopathological
examination confirmed the mechanism of wound healing
by increased deposition of collagen, fibroblast on the
granulation tissue and neovascularization[11]. The
ethanolic leaf extract of Urtica parviflora has shown the
hepatoprotective activity evaluated by the assay of liver
function, biochemical parameters such as Aspartate
aminotransaminase (AST), Alanine aminotransaminase
(ALT), alkaline phosphatase (ALP), total bilirubin serum
protein and by the study of histopathology of the livers [10].
In spite of its economic, medicinal and scientific
*Corresponding Author
Dr.Tapan K Nailwal.,
Assistant Professor, Department of Biotechnology,
Kumaun University, Nainital, Campus Bhimtal-263136
India.
Contact no.: +919412986483
Email: tapannailwal@gmail.com.

importance, little information is available on the

phylogenetic relationships among the Urtica parviflora
plant samples in Kumaun region. RAPD technique is one of
the most frequently used molecular methods for
taxonomic and systematic analyses of various organisms
[2].RAPD markers were used for evaluation of genetic
diversity in many plant species such as Urtica diocia [3],
Soybean [19], Tobacco [7], Lycopersicon [12], Sugarcane [14],
Solanum [18]. Among local Kumaun people especially of
Nainital and Mukteshwar districts in Uttarakhand 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 [13]. Keeping in mind the above notion our aim
was to study the genetic variation among U. parviflora
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. We
used RAPD markers for several reasons: (1) they reveal
even small genetic differences and are a means of
assessing polymorphisms at a wide range of loci, since a
large part of the nuclear genome can be scanned [5], (2) it
is an efficient and inexpensive technique without
requiring prior knowledge of the genome [4], (3) RAPD
assay has the advantage of being easy to use, requiring
very small amount of genomic DNA without the need for
blotting and radioactive detection [1], and are moderately
reproducible, and (4) there are not specific microsatellite
primers developed for Urtica. Difference in altitude was
taken as the parameter to study the variation in plant
samples Therefore, the objective of the present work was
to identify genetic similarity and diversity between

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Goel et. al. / JPBMS, 2011, 5 (21)

DNA was extracted by Plant DNA Isolation Kit (Hi-Media,

India) according to the manufacturers instructions. These
DNA samples were further diluted and incubated at 4C.

The amplification products along with 2 l of loading dye

(bromophenol blue) were separated on 1.2 per cent
agarose gel at 80 volts using 1X TAE buffer of pH 8.0
containing Ethidium bromide (0.5 l 1/10 ml of gel). The
gel were viewed under UV transilluminator and
photographed for documentation. Out of 20 primers used
8 primer produced recognizable bands. Scorable bands for
a primer in each sample were compared and allotted 0
(absence) or 1 (presence) values. Band patterns (0, 1
matrix) were tabulated for individual primers separately
and the data pooled to obtained a combined matrix for
four samples off 8 primers. Diversity coefficient for each
primer (number of polymorphic bands/total number of
bands) and pair-wise genetic similarity were calculated
using NTSYS-PC (version 2.11a) software (Applied
Biostatistics, USA).

DNA Purification:

Results:

samples from four geographical heights by RAPD-PCR and

by means of the analysis of bandsharing values.

Materials and Methods:

Sample Collection: The leaves of Urtica parviflora were
collected from four different geographical regions of
Kumaun (Uttarakhand, India): Mukteshwar (2800m),
Nainital (2200m), Bhowali (1600m) and Bhimtal (1300m)
varying from 1300 to 2800 m from sea level in
Uttarakhand. The leaves were brought in aluminium foil in
an ice box and stored at - 20C.

DNA Extraction:

RNase A (10 15 l; 50 mg/ml) was added into the DNA

samples and incubated at 37C for 1 h to degrade RNA.
Equal volume of 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
clumps. They were centrifuged at 10,000 rpm at room
temperature.

DNA Quantification:
Purity and concentration of genomic DNA was estimated
by calculating the ratio of the optical density measured at
260280 nm with a spectrophotometer (Thermo
Scientific Type UV1, England).

PCR Amplification:
RAPD analysis was done individually with 20 random
decamer Primers. The thermal cycling conditions were;
Denaturation : 940C for 1 min.
Primer annealing: 360C for 1 min.
Primer extension: 720C for 2 min.
The PCR reaction was carried out in a final volume of 25l
reaction containing 50ng of template DNA, dNTPs (200
M), Taq DNA polymerase (0.5 U), MgCl2 (2.5mM) and 10
pMoles of primer in 25l of 1X PCR buffer. The reaction
components were added to PCR tubes kept on ice.

A total of 70 scorable bands were produced in four

samples with 8 primers (Table 1). This data was utilized
for further computations. The average no. of bands per
primer was 8.75. Out of 70 bands, 48 bands were
polymorphic (68.75) noted in the present Investigations.
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 2.11a) [17], and similarity between accessions was
estimated using Jaccard coefficient [9]. The similarity index
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
markers ranged from 46% to 69%. The maximum
similarity was observed between the sample pairs of
Nainital and Bhowali (69%) followed by Mukteshwar and
Bhowali (51%), followed by Mukteshwar and Nainital
(49%). The minimum similarity was observed between
the sample pairs of Mukteshwar and Bhimtal (46%).The
cluster analysis based on UPGMA and SAHN is depicted
vide (Figure 1). A perusal of the UPGMA dendrogram
based on Jaccards similarity coefficients. The dendrogram
has grouped all the four samples into two major clusters A
and B. Cluster A was further classified into two minor
clusters A1 (Mukteshwar) and A2 (Nainital,Bhowali). The
major cluster A is comprised of 3 samples, while the
cluster B (Bhimtal) is comprised of one sample. The Sub
cluster A2 comprised of 2 samples which are Nainital and
Bhowali.

Table 1: Scorable DNA bands generated by different random decamer primer through PCR
S.no Primer (5N3N) Total number of loci
Number of
Number of polymorphic loci
monomorphic loci
12

Polymorphism
(%)

1.

GAGCCCTCCA

58.33

2.

GAACCTGCGG

66.66

3.

TCACGTCCAC

71.42

4.

AGGGCCGTCT

71.42

5.

CAGCTCACGA

14

12

85.71

6.

GGATGAGACC

71.42

7.

AGCGTCCTCC

10

70

8.

GGATGAGACC

25

Table 2: Jaccards Similarity Analysis of four altitudinal samples of U.parviflora as obtained from RAPD markers.

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Goel et. al. / JPBMS, 2011, 5 (21)

Rows/Cols

Mukteshwar

Nainital

Bhowali

Bhimtal

Mukteshwar

J000E + 000

Nainital

4.9019607

1.0000000

Bhowali

5.1020408

6.9230769

1.0000000

Bhimtal

4.6666666

4.8148148

4.7169811

1.0000000

Figure 1: Dendrogram of four altitudinal samples of U.parviflora (Mukteshwar, Nainital, Bhowali and Bhimtal.

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. parviflora was quite
appreciable due to change in height above sea level as
samples from Bhimtal region showed low genetic
similarity with samples collected from higher altitudes.
Among local Kumaun 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. parviflora 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 [15] 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 environmental 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. 14 RAPD markers were obtained
with primer 5 with 85.71% polymorphic character
whereas 12 RAPD markers were obtained with primer 1
with 58.3 % polymorphism indicating that these primer
sequences were present in almost all the samples. Primers
3, 4 and 6 produced 71.4 % polymorphic bands, whereas
the primers 2, 7 and 8 produced polymorphism 66.66 %,
70 % and 25% respectively.

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

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Goel et. al. / JPBMS, 2011, 5 (21)

showed appreciable variation in the species and keeping in mind the use of plant as a potent medicine for various
diseases, the study can be used as a premise for advanced genetic analyses and molecular studies for novel purposes.

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Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 05, Issue 05