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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.
DNA Purification:
Results:
DNA Extraction:
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.
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.
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
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, the study can be used as a premise for advanced genetic analyses and molecular studies for novel purposes.
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