International Journal of Latest Research in Science and Technology
Volume 3, Issue 1: Page No.106-110 ,January-February 2014
http://www.mnkjournals.com/ijlrst.htm
ISSN (Online):2278-5299
STUDIES ON MOLECULAR MARKER BASED
GENETIC DIVERSITY IN Quercus SPECIES OF
NAINITAL, UTTARAKHAND
Sukesh Kumar , 2Shashi Shekhar Kumar
Department of Biotechnology, S. U College Hilsa, Nalanda-801302 India
2
Department of Biotechnology, Nalanda College Biharsharif, Nalanda-803101 India
1
1
Abstract- The temperate forests of Kumaun Himalayan region are usually distributed between 1200m and 3000m, which are represented
either by pure oak or oak-Rhododendron mixed forest. Four species of genus Quercus i.e Q. leucotrichophora, Q. floribunda, Q.
semecarpifolia and Q. lanuginosa were distributed at the different height of Kumaun Himalayan region, with partially overlapping
distributions. Within the area of overlap, there are localities where morphological intergradations occur. Random amplified polymorphic
DNA (RAPD) markers were used to measure the genetic diversity within these quercus species which occur at different heights of Kumaun
Himalayan region and collected from different locations of these regions. A total of sixteen primers were used, out of which six primers gave
positive results. A total of 71 amplified bands were scored from six random decamer primers and at a mean of 11.8 amplified bands per
primer. Genetic diversity estimates indicated that Q. leucotrichophora and Q. floribunda are 50% genetically similar. Similarly Q.
semecarpifolia and Q. lanuginosa are 25% different genetically or 75% genetically same. The results show that the genetic diversity of these
species is high possibly allowing it to more easily adapt to environmental variation. This distributive pattern of genetic variation of these
species provides important baseline data for conservation and collection strategies. It is suggested that most of these species should be
sampled in ex-situ protection as to retain as much genetic diversity as possible
Keywords - : Quercus species, DNA, RAPD
INTRODUCTION
Quercus is a widely distributed genus in the northern
hemisphere with 600 recorded species. It belongs to family
fagaceae. It is one of the characteristic tree species of the
Mediterranean ecosystem. They are the dominant species on
north-facing slopes in the Himalayas, from 2400-3600
meters. In China it grows right up to tree line, where it
becomes a thicket-forming shrub. In East Asia the genus is
native to Himalayas from Afghanistan to Nepal and China.
Mexico is considered the centre of diversity of Quercus in
the western hemisphere with an estimated total no. of species
around 135-150 (Nixon, 1993). Diverse topology, climate
and habitat probably exert an important influence in the
process of radiation and maintenance of Oak species
diversity. (Nixon, et. al, 1993).
Various changes in
Himalayan forests are appearing in their structure, density
and composition due to global warming (Gaur, 1982),
uncontrolled lopping and felling of trees for fuel wood,
fodder and grazing (Kumar et al, 2004).The study area
(Uttarakhand, Central Himalaya) is located between 79°23’
and 79°42’E, and 29°20’ and 29°30’N. The altitude ranges
between 1300 and 2600m.The morphological patterns of
variation between many oak species have been reported.
Paleobotanical evidence suggests that the cooler, drier, and
more variable climates that developed after the EoceneOligocene transition in North America encouraged the
evolution and migration of Quercus. Morphological and
genetic intermediacy represents the ancestral state, and
apparent parent are the result of divergence from a common
gene pool. Molecular markers can provide a large number of
ISSN:2278-5299
neutral and independent characters that are extremely useful
in the genetic analysis. RAPD markers indicates a shift in the
genetic composition of populations from one species to the
other along the macro geographic gradient, with genetically
intermediate populations situated in the area of overlap. The
particular goal of this report is to assess the degree of
congruence between morphological and molecular variation.
The species of genus Quercus show great genetic and
morphological variation.
This diversity gives a wide
tolerance to ecological factors such as climate, soils,
elevation etc. These species play a vital role not only in soil
and water management but also contribute significantly to the
sustenance of rural ecosystems.
Genetic markers have many potential applications in forest
genetics and tree breeding research, including genetic
diversity, population structure, phylogeny, mating systems,
and the tree classification. The lack of genetic markers has
hindered the direct analysis of genetic structures in plant
species. Isozyme have been useful to estimate genetic
diversity of plant populations (Hokanson et al., 1993). The
isozyme analysis has an advantage in its simplicity and
rapidity, however only a limited number of polymorphic loci
are available for use as genetic markers. A well known
method for comparing random amplified polymorphic DNA
(RAPD) has been successfully used to differentiate species,
varieties, cultivars and clones in many crop plants.
1) MATERIALS AND METHODS
Plant Material
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International Journal of Latest Research in Science and Technology.
The four plant species; Q. leucotrichophora,
Q.floribunda, Q.semecapifolia and Q.lanuginosa were
collected from hills of Kumaun Himalayan region. Out of
these Q.leucotrichophora and Q. floribunda were collected
from the hills behind the D.S.B campus, Kumaun University,
Nainital. Q.lanuginosa was collected from the Kilvari
(Nainital) region and Q.semecarpifolia from hill tops of
China peak (Nainital).
DNA isolation
DNA was isolated from Quercus gluca, Quercus
leucotrichophora, Quercus floribunda, Quercus lanuginosa
and Quercus semecarpifolia separately by using CTAB
method (Sambrook, 1989). Quantification of DNA was
determined by ratio of OD at 260/280nm. (If ratio of O.D at
260/280nm is between 1.6 to1.8 indicates DNA preparation
is pure, below 1.6 and above 1.8 indicates protein and RNA
contamination respectively in DNA sample.
Agarose gel Electrophorasis
Agarose gel 0.8%(w/v) was prepared using 1x TAE buffer
pH 8.0(for 50X TAE buffer, 242gm Tris-base, 37.2gm
EDTA, pH is adjusted with glacial acetic acid) by boiling for
homogeneity. Gel was placed in electrophoresis unit filled
with 1x TAE Buffer, DNA sample (10µl) was mix with 8µl
loading dye (50%glycerol, 1mM EDTA (pH8.0),
0.25%xylene cyanol and distilled water). Sample was loaded
in slots of agarose gel. The gel was run at 80Vfor 180min in
a Horizontal electrophoresis unit. After electrophoresis the
gel was visualized in UV tarns illuminator and photographed
in gel documentation system.
Quantitative analysis of DNA
The yield of DNA per gram of leaf tissue extracted was
measured using a UV spectrophotometer at 260nm
wavelength of light. It is calculated by formula.
Concentration of DNA= A260 × 50 × Dilution factor (µg/ml)
RAPD Analysis
RAPD is PCR based technology which is very
sensitive to changes in experimental parameters shows, a
total no.1 of 8 primers were initially screened against 4
selected species of Quercus. The effect of magnesium,
template DNA concentration, pH value and length of
denaturation stage for amplification were examined. When
trying to optimize annealing temperature, we run the test
reaction at 370C, 390C and 420C. The decamer primers can be
clearly amplified at 370C.
Primer No.
1.
2.
3.
4.
5.
6.
7.
8.
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After this these primers are analyzed based on the strong
amplification product and production of uniform
reproducible fragment between replicate PCR.
RESULTS & DISCUSSION
The RAPD markers were to measure the genetic diversity
of Q. leucotrichophora, Q. floribunda, Q. semecarpifolia and
Q. lanuginosa. RAPD provide useful tools for assessing
genetic diversity of rare endemic species and for resolving
relationship among populations. Comperative analysis of
inter and intraspecific variation in nuclear geome
composition was made using DNA samples from four species
of Quercus. Sixteen primers were used in the RAPD analysis.
Results of this analysis show many polymorphic and
monomorphic loci. However we could not find any speciesspecific band in this study. The following events were carried
out in the present study.
Extraction of genomic DNA
In the present study we had to optimize the protocol for
isolation of genomic DNA and PCR conditions for RAPD
leucotrichophora,
floribunda,
analysis
of
species
semecarpifolia and lanuginosa of genus Quercus. They have
high levels of polyphenols and secondary metabolites which
adversely affects the yield and purity of extracted DNA. The
DNA extraction was improved by modifying some steps in
CTAB method originally developed for others plants. The
extracted DNA was of high quality as it showed the optical
density or absorbance readings between1.6 to1.9, after
calculating the ratio 260/280 nm. The concentration of DNA
obtained (table no. 2) ranged from 1000ng/ìl to 1300ng/ìl
Concentration of DNA= A260 × 50 × Dilution factor (µg/ml)
A260= Absorbance at wavelength 260nm.
The purity of DNA isolated by this method has strong
and reliable amplification products showing its compatibility
for RAPD-PCR using random decamer primers. Polyphenols
should be removed since they are strong oxidizing agents and
binds to DNA covalently, making its usefulness for research
application, if there is presence of polyphenols reducing the
yield and purity (Peterson et.al., 1997; Porebaski et.al.,
1997). Tannin which are difficult to isolate from DNA and
polysaccharides are known to inhibit RAPD reaction by
distorting the result in many analytical application and
therefore lead to wrong interpretation (Kotchaoni et al.,
2003). Many DNA isolation procedures also yield large
amount of RNA (Doyle and Doyle, 1987; Mejjad et.al.,
1994).
Table no 1. Sequence of random nucleotide primers used
Name of Primer
Nucleotide sequence
P1
5’-GCGGCCCTCT-3’
P2
5’-GTGATCGCAG-3’
P3
5’-TCAGTCCGGG-3’
P4
5’-TCCATGCCGT-3’
P5
5’-TATGGTCCGG-3’
P6
5’-AGGAAGGTGC-3,
P7
5’-CTGGGAGTGG-3’
P8
5’-CACAACGGGT-3’
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International Journal of Latest Research in Science and Technology.
Table.2. Quality and concentration of DNA extracted from 1gm leaf of Q leucotrichophora, Q. floribunda, Q.
semecarpifolia and Q. lanuginosa
Species
OD
OD
OD260/280 nm
Concn(ng/ìl)
260nm
280nm
Q. leucotrichophora
0.023
0.014
1.643
1150
Q. floribunda
0.026
0.015
1.733
1300
Q. semecarpifolia
0.025
0.014
1.785
1250
Q. lanuginose
0.025
0.013
1.923
1250
RAPD amplification
Since RAPD PCR technology is sensitive to change in
experimental parameters. so its protocol was optimized by
testing the parameters like the concentration of template
DNA concentration of MgCl2, primers, taq polymerase,
annealing temperature and the time interval during the
denaturation, annealing and elongation. All these parameters
were optimized and a standard protocol for RAPD was
established. PCR amplification of DNA extracted from the
four species of Quercus that is leucotricophora, floribunda,
and semecarpifolia and lanuginose was done by using 8
random decamer primers, designed by the Operon
Technologies, USA. Out of these only six random decamer
primers showed the positive results. RAPD fragments were
separated electrophoretically on 1.5 % agarose gel in 1 X
TAE buffer stained with ethidium bromide and photographed
on a UV illuminator using a digital camera. The bands were
scored manually. The amplification profile generated by such
primers was compared and relative molecular size of each
band was examined by comparing the position of bands with
DNA marker. Amplification with each primer was repeated
three times and clearly resolvable and reproducible fragments
were considered for analysis. A total of 71 bands were
obtained from six RAPD random primers, corresponding to
an average of 11.8 bands per primers. But since different
primers binds to the different species,it is tedious to calculate
the polymorphic and monomorphic loci however out of
seventy one, 41 bands were polymorphic. So the percentage
of polymorphic bands out of 71 is 57.7%.
To calculate the genetic distance of each band we can use
the following simple equation:
D = 1- Nxy/ (Nx+Ny-Nxy)
Where:
D = The genetic distance between plant “X”and “Y”
Nxy = The no. of bands shared by plant “X” & “Y”
Nx = The no. of bands in plant “X”
Ny = The no. of bands in plant “Y”
From above formula we can calculate the genetic distance
between plants of Quercus species. For example, the genetic
distance between Q. leucotrichophora and Q. floribunda can
be calculated by counting the bands in the figure and so from
the formula,
D = 1- 3/ (6+3-3) = 1- 3/6 = 0.50 = 50%
For this very small no. of bands scored, the calculation of
genetic distance says that these plants are 50% different or
50% same genetically. Similarly, the genetic distance
between Q. semecarpifolia and Q. lanuginosa were
calculated 25%. The calculation of genetic distance says that
these plants are 25% different or 75% genetically same. The
results of this study support the occurrence of morphological
and genetic variation between Quercus species. This results
show that the genetic diversity of these species is high,
possibly allowing it to more easily adapt to environmental
conditions.
Though this study we have documented the genetic
relationship among four Quercus species evolved in different
region of the Kumaun Himalayas.
Table no. 3 Sequence of primers which gave positive results during RAPD analysis of DNA extracted from Q.
leucotricophora, Q. floribunda, Q. semecarpifolia and Q. lanuginosa
Primers
Q.leucotrichophora
Q.floribunda
Q.semecarpifolia
Q.lanuginosa
Sl.No
1.
5’-GCGGCCCTCT-3’
+
+
+
2.
5’-GTGATCGCAG-3’
+
3.
5’-TCAGTCCGGG-3’
+
+
+
+
4.
5’-TCCATGCCGT-3’
+
5.
5’-TATGGTCCGG-3’
+
+
+
+
6.
5’-AGGAAGGTGC-3,
+
+
+
7.
5’-CTGGGAGTGG-3’
+
+
8.
5’-CACAACGGGT-3’
+
-
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International Journal of Latest Research in Science and Technology.
Further this study revealed the considerable differences could
be observed between the results obtained from molecular
markers. These results demonstrated that RAPD markers are
a useful tools for measuring genetic variation of the genus
Quercus at intra and interspecific levels.
Table no.4 Parameters used for optimisation of PCR conditions for RAPD analysis of DNA extracted from Q.
leucotrichophora, Q. floribunda, Q. semecarpifolia and Q. lanuginose
PCR parameters
Tested range
Optimum condition
MgCl2 (mM)
dNTPs (mm)
Taq polymerase
Primers conc. (ìM)
Initial denaturation time interval (min.)
Annealing temperature(0C)
Time interval (sec)
Reaction volume (ìl)
No. of cycles
L1
L2
F1
F2
2, 2.5, 3
0.1, 0.2, 0.3
0.4, 0.5, 0.6
0.2, 0.3, 0.4
2.5Mm
0.2mM
0.6U
0.2ìM
2, 3, 4, 5
30, 35, 37,42
20, 40, 50, 60
15, 20, 25
35, 40, 45
5 min
370C
60 sec
25 ìl
40
S1
S2
I1
I2
Fig-1: Agarose gel electrophoresis of DNA extracted bymodified CTAB method
L1- L2 - Quercus leucotrichophora, F1- F2 - Quercus floribunda, S1-S2- Quercus
semecarpifolia, I1-I2- Quercus lanuginosa
Fig- 2: Agarose gel electrophoresis pattern of RAPD analysis under optimized condition of four
Quercus species
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International Journal of Latest Research in Science and Technology.
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