Ethnobotanical Leaflets 12:
1129-36. 2008.
Molecular Analysis in Rauvolfia tetraphylla L. using RAPD Markers
R. Mahesh*, N. Nirmal
Kumar and R. Mary Sujin
Plant
Molecular Biology Research Unit,
PG
& Research Department of Plant Biology and Biotechnology,
St.Xavier's
College, (Autonomous). Palayamkottai - 627 002, Tamilnadu, India.
*e-mail:
rmaheshbio@gmail.com., Tel: 0091- 462 4264374., Fax: 0091- 462-2561765
Issued
ABSTRACT
����������� The Present
study reveals the molecular variations in different accessions of Rauvolfia tetraphylla L. a medicinally
important plant collected from five locations of the Tirunelveli hills in
Tamilnadu, India. Moleuclar analysis was carried out using RAPD markers. Out of
the five primers screened, a total of 27 scorable polymorphic markers were
generated. The genetic distance between the population ranged from 0.0770 to
0.3514 and the genetic identity ranged from 0.7037 to 0.9259. The overall
observed and effective number of alleles is about 0.5007 and 0.3690 respectively.
Nei (1978) overall genetic diversity is 0.2034. It is clear that there is
distinct genetic variability in R.
tetraphylla L.
Key words: Gene diversity, RAPD-
INTRODUCTION
Rauvolfia tetraphylla L. (Apocynaceae) is
commonly known as �Sarpagandha� a small ever green woody shrub. It is native to
West Indies and introduced to India where it is cultivated in gardens in
Uttarpradesh, West Bengal, Tamilnadu and Kerala.� It has become naturalized in many localities
and is distributed in moist habitats in Tamilnadu. Pharmacologically the roots
are important although they reportedly contain low concentrations of the
alkaloids like Reserpine, Ajmalicine and Tetraphyllicinine possesses
hypotensive property and can cure many other diseases (Faisal et al., 2005).
�Medicinally the plant extract mixed with
castor oil is used in treatment of refractory ailments and bark decoction is
used for chronic refractory skin diseases and destroys Parasites (Parrotta,
2001). The root is also used to stimulate uterine contractions and is
recommended for use in difficult childbirth cases (Villar et al., 1998).
To
understand the effective management of plant genetic diversity from a
conservation point of view it is essential to consider variation as richness
and distribution at intra and interspecific levels. Depending on the state of
our heritable understanding of taxon genetic diversity may be considered at
different organizational levels: the genopool, population, individual genome,
locus and
�
Randomly
amplified polymorphic
In
the present study was concentrated to determine the genetic variability of R. tetraphylla. L. collected from five
locations in Tamilnadu has been carried out using RAPD technique.
MATERIALS
R.
tetraphylla L.
samples were collected from five locations in Tirunelveli hills (Table 1.). Five plant specimens from each
location were collected. The plant materials were transferred to plastic bags
for transport from field to laboratory. Permanent storage was at -70� C.�
Table 1. Area of the study
of� R. tetraphylla. L.
|
Pop
ID |
Area
of the study |
|
1. |
Manjolai |
|
2. |
Oothu |
|
3. |
Kothayar |
|
4. |
Kuthiraivetti |
|
5. |
Yanai Satumbaloodai |
The amount of
Table 2. The reagents used to
amplify the genomic
|
S.No |
Constituent |
Quantity |
|
1. |
Sterile water |
13.3�l |
|
2. |
dNTPs |
4.0
�l |
|
3. |
MgCl2 |
0.3
�l |
|
4. |
|
2.5
�l |
|
5. |
Primer |
2.4
�l |
|
6. |
Taq polymerase |
0.5
�l |
|
7. |
Plant |
2.0
�l |
|
Total |
25.0
�l |
|
Earlier, eight primers had been tested
and five primers which produced reproducible bands were selected. The experiments
were repeated three times and confirmed the reproducibility of bands. The
reaction steps (Table 3.).
Table 3. The RAPD -
The amplification products were separated
by electrophoresis in agarose gel (1.4%). The gel was visualized by 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
RESULTS
Genetic relationship in R. tetraphylla L. in different locations
has been carried out using RAPD markers. Five primers generated reproducible,
informative and easily scorable RAPD profiles (Table 4.). These primers produced
multiple band profiles with a number of amplified
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. The
result obtained was analyzed in the
Table 4. Oligonucleotides used
as random primers and their sequences.
|
Primers |
Sequence 5�- |
Number of Polymorphic
bands |
|
OPA
02 |
TGCCGAGCTG |
6 |
|
OPA
06 |
AGGGGTCTTG |
5 |
|
OPA
08 |
GTGATCGCAG |
7 |
|
OPA
19 |
TCTGTGCTGG |
5 |
|
OPA
20 |
AGGTGACGCT |
4 |
The populations of R. tetraphylla L. from the pop 1 and pop 5 and the pop 3 and pop 4
are said to be formed in a separate clad where as other pop 2 get diverged from
pop 3 and pop 4. These variations show that the dispersion of the plant has
many mysteries in it. The lowest length between the pop 4 and pop 3 is 3.2910.
The highest length between the pop 3 and pop 2 is 10.2397 are shown in the
(Table 6). From this we can conclude that the population from pop 3 and pop 4
is more diversified where as the population from the pop 1 and pop 2 and also
pop 5 are closely related in their dimensions. In the dendrogram based on Nei's
genetic distance (UPGMA) obtained (Figure1), the populations were highly
differentiated by their own genetic distance.
The clustering results of different
accessions suggest that R. tetraphylla
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.
����������� Genetic diversity is a resource for the species own
survival and future evolution, it also promotes selective breeding. The global
pool of genetic diversity represents all the information pertinent to all
biological structures, functions and processes on this planet. (John De Britto
and Mary Sujin 2008). Apart from genetic drift, inbreeding depression may also
be one of the factors, which may lead to genetic variation (Sherwin and Moritz,
2000). An understanding of these genetic processes is required in order to
fully evaluate the consequences of fragmentation and its relationship to
genetic variation. Inbreeding is avoided in all the accessions of����������������� R. tetraphylla L. because the plants are dioecious, although
within-population gene exchange between plants is unavoidable.
����������� The wide range of variation observed might also be due to
two evolutionary forces, which include pollen flow and local selection
pressures. Pollen can be dispersed over large distances; this long-term
reciprocal movement of pollen must also have contributed to the variation.
Recent experiments using pollen traps have shown that oak pollen can migrate at
seven kilometers (Lahtinen et al.,
1996). The local selection pressures may be due to the effects of environmental
factors and due to struggle for existence in nature. The wide spread occurrence
of the wind pollination and breeding systems that promotes out crossing may
lead to higher genetic diversity.
����������� It is believed that mutations, genetic drift due to
finite population size, and natural selection will lead to the genetic
diversification of local populations and that the movement of gametes and
individuals (gene flow) will oppose that diversification. The lack of gene flow
and the effect of genetic drift due to restricted population size might have
caused the accessions of�� R.tetraphylla L. to differentiate genetically
among themselves and it is a recently categorized as endangered due to many
environmental and anthropogenic influences (Faisal and Anis, 2002).
Although
the results of the present study provide evidence for genetic loss, information
is required on the implications of reduced genetic variation for survival and
fertility. The high degree of genetic variation or differentiation recorded by
the transfer of germplasm between different locations should be avoided, to
ensure that the genetic material is adapted to local conditions (Ennos, 1998).
The genetic analyses presented here could be used for the development of
conservation strategies for the species, for example through the definition of
appropriate units of management (Newton et al., 1999). In the case where gene
flow between neighboring populations is not limited, populations of longer
geographical distances will generally show greater diversification.
�Hence the grouping of these accessions is
independent of the geographical distance. It proved that the accessions
collected from different locations exhibited similarities (leaf morphology and
length, color of the petiole and midrib, flower morphology and fruit
morphology) but their RAPD fingerprinting differed markedly. The more
variation, the better the chance that at least some of the individuals will
have an allelic variant that is suited for the new environment, and will
produce offspring with the variant that will in turn reproduce and continue the
population into subsequent generations. In order to prevent depletion of
biodiversity due to man-made efforts or otherwise, it is necessary to
understand how the diversity of life particularly in the genetic level is
maintained under natural conditions. Based on this knowledge one can suggest
appropriate strategies and policies for the conservation of biodiversity
�An
understanding of the genetic diversity responsible for individual species�� adaptations��
and�� responses�� to��
their environment (intra specific diversity) is a foundation for
understanding almost all ecological and evolutionary processes. Further
analysis is necessary to find out the individual polymorphism in each
population and to be the best and this data may be correlated with other
population and the superior population can be identified. Therefore the
difference found in the dendrogram could be partially explained by different
number of loci and there coverage of overall genome in obtaining reliable
estimation of genetic relationships among the plant species of R. tetraphylla L.
Table 5. Nei's Original Measures
of Genetic Identity and Genetic distance.��������������������� .
|
Pop ID |
1 |
2 |
3 |
4 |
5 |
|
1. |
**** |
0.7407 |
0.7037 |
0.7037 |
0.8519 |
|
2. |
0.3001 |
**** |
0.8148 |
0.8148 |
0.7407 |
|
3. |
0.3514 |
0.2048 |
**** |
0.9259 |
0.8519 |
|
4. |
0.3514 |
0.2048 |
0.0770 |
**** |
0.8519 |
|
5. |
��� 0.1603 |
0.3001 |
0.1603 |
0.1603 |
**** |
Nei's
genetic identity (above diagonal) and genetic distance (below diagonal)
Figure 1.� Dendrogram Based on Nei's (1972) Genetic distance:
Method = UPGMA
(Modified
from NEIGHBOR procedure of PHYLIP Version 3.5).
�������������������������������������������������������������������������������������������������������������������������������������������
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�������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������
Table 6. The length between the
populations.
|
Between |
And |
Length |
|
4 |
2 |
5.51362 |
|
2 |
Pop1 |
8.01713 |
|
2 |
Pop5 |
8.01713 |
|
4 |
3 |
3.29103 |
|
3 |
Pop2 |
����� 10.23972 |
|
3 |
1 |
6.39167 |
|
1 |
Pop3 |
�������
3.84805 |
|
1 |
Pop4 |
�������
3.8480 |
CONCLUDING REMARKS
����������� Therefore analysis of
RAPD data could be useful to detect genetic differentiation between six
districts of Tamilnadu. However detailed study is desirable to understand all the
aspect related to variations. Hence further information is required on patterns
of gene flow within and between population, and its effects on reproductive and
demographic processes, to assess its impact on population viability.
� The high degree of genetic variation recorded
that transfer of germplasm between different are as should be avoided, to
ensure that the genetic material is adapted to local conditions.
��������������������� It is important to point
out that the genetic variation that a population of organisms possesses is the
fuel that allows them to be able to change or evolve in response to changing
environmental conditions. Molecular markers are extensively use in genome
mapping, diagnosis of diseases and molecular systematic. These are the tools
which reveal the mystery of genomes. RAPD markers have recently caught the
fancy of many individuals in the field of applied plant breeding. This
molecular marker is based on the
���� �����������Therefore analysis of RAPD data
could be useful to detect genetic difference between accessions of R. tetraphylla L.� However detailed study is desirable to
understand all the aspects related to variations.� Hence further information is required on
patterns of gene flow within and between accessions to protect and select the
superior variety of the species.
ACKNOWLEDGEMENTS
����������� The authors are grateful
to the Ministry of Environment and Forests, Government of India,
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Plate 1. RAPD banding pattern
(OPA Primers) in R. tetraphylla L.
