International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Comparison of Genetic Variability of Plant Cultivars Using
RAPD Markers
I. D. Patil #1, Sheetal B. Gawande *2Jayant P. Parpalliwar*3,
2,3
Assistant Professor, 1 Professor and Head, 2 Research Student
Department of Biotechnology,
SSBT’s College of Engineering & Technology,
Bambhori, Jalgaon.
Maharashtra, India
Abstract: RAPD (Random amplification of different genetic qualities (they can be dominant or
polymorphic DNA) is a genetic marker which plays codominant, can amplify anonymous or characterized
a vital role for comparing the genetic variability of loci, can contain expressed or nonexpressed sequences,
living entity. Present investigation is focused on a etc.). A molecular marker can be defined as a genomic
comparative studies of DNA level of five different locus, detected through probe or specific starter
varieties of Moringa oliefera viz., DHANRAJ, JAFNA, (primer) which, in virtue of its presence, distinguishes
PKM 1,GKVK 1 by using OPL-6, OPL-8, OPL-10, unequivocally the chromosomic trait which it
OPL-11 primers, variation is occurred in isolated represents as well as the flanking regions at the 3′ and
DNA sample of leaves and pods of Moringa oliefera.
[7].
Molecular markers may or may not
Average kilo base pairs size of all investigated DNA is 5′ extremity
varies from variety to variety and part to part. It is correlate with phenotypic expression of a genomic
concluded that all tested varieties of Moringa oliefera trait. They offer numerous advantages over
bears same morphological characters but differs in conventional, phenotype-based alternatives as they are
molecular and genetic level; that affects yield of plant stable and detectable in all tissues regardless of
and also economic of agriculture. Presence of genetic growth, differentiation, development, or defense status
variability in crops is essential for its further of the cell. Rapid amplified polymorphic DNAs
improvement by providing options for the breeders to (RAPDs) were the first of PCR-based markers and are
develop new varieties and hybrids.
produced by PCR machines using genomic DNA and
arbitrary (random) primers which act as both forward
Keywords— Moringa, oliefera, DNA, RAPD, Genetic and backward primers in creation of multiple copies of
variability, PCR, Yield of plant.
DNA strands [8,9]. Moringa olieferaact or English
common name Drum stick tree is commonly
I.
Introduction:
cultivated species of genus Moringa. It have many
Genetic variability is the tendency of the individual functional roles for human being and few of them are
genotype to vary from the population. Genetic in nutrition, medicine, food. Regarding plants family,
variation generated continuously by the mutations. if a group of plants which are belong to the same
Genetic variability makes individual more susceptible genus, they will show all the characters such as
to the adverse environment condition. Genetic branching pattern, fruit , flowers, colour etc. which
diversity is the key pillar of biodiversity and diversity will be resemble to their parents. So the group of
within species, between species, and of ecosystems plants which are similar to their morphological
which was defined at the Rio de Janeiro Earth Summit. features are called as species. So different plant
However, the problem is that modern crop varieties, species grows in different areas with different
especially, have been developed primarily for high environmental conditions such as temperature,
yielding potential under well endowed production climatic conditions, resistance capacity against toxic
conditions. Such varieties are often not suitable for chemicals and pathogens. Though these species are
low income farmers in marginal production morphologically same but genetically they are
environments as they are facing highly variable stress different. Inorder to compare their genetic variability
conditions4. Most widely used genetic marker type is genetic markers can be used.
molecular markers, comprising a large variety of DNA
A genetic marker is a gene or DNA sequence
molecular markers, which can be employed for
with a known location on a chromosome and
analysis of genetic and molecular variation. These
associated with a particular gene or trait. These
markers can detect the variation that arises from
molecular markers are those which show the
deletion, duplication, inversion, and/or insertion in the
variations at DNA level of the different plant species.
chromosomes. Such markers themselves do not affect
With the advent of molecular markers, a new
the phenotype of the traits of interest because they are
generation of markers has been introduced over the
located only near or linked to genes controlling the
last two decades, which has revolutionized the entire
traits. These markers are inherited both in dominant
scenario of biological sciences. Molecular markers
and codominant patterns. Different markers have
have very rapidly complemented the classical
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strategies. Molecular markers include biochemical
constituents (e.g. secondary metabolites in plants)
and macromolecules, proteins. These metabolites
which are being used as markers should be ideally
neutral to environmental effects or management
practices. Hence, amongst the molecular markers
used, DNA markers are more suitable and ubiquitous
to most of the living organisms.
For the first time Williams et al in 1991
developed RAPD technique. It is a PCR based
technique that is widely used in molecular biology.
In case of PCR it amplifies the specific fragments of
DNA of species. In RAPD technique the primer
which designed randomly that it amplifies the
segment of DNA with respect to their
complementary sequences. Genomic DNA of two
individuals produces different RAPDs. RAPD stands
for Random Amplification of Polymorphic DNA. It
is a type of PCR reaction, but the segments of DNA
that are amplified are random. The scientist
performing RAPD creates several arbitrary, short
primers (8-12 nucleotides), then proceeds with the
PCR using a large template of genomic DNA, hoping
that fragments will amplify. By resolving the
resulting patterns, a semi-unique profile can be
gleaned from a RAPD reaction. No knowledge of the
DNA sequence for the targeted gene is required, as
the primers will bind somewhere in the sequence, but
it is not certain exactly where. The standard RAPD
technology utilizes short synthetic oligonucleotides
(10 bases long) of random sequences as primers to
amplify nanogram amounts of total genomic DNA
under low annealing temperatures by PCR.
II.
Materials and Methods:
a) Sample collection
For comparing the genetic variability the plant species
called as Moringa oliefera was taken. Following are
the cultivars of Moringa oliefera.
1)
PKM 1
2)
GKVK-I
3)
GKVK-II
4)
Dhanraj
5)
Jafna.
b) Isolation of DNA
A standard protocol of isolation of plant DNA was
followed for the total genomic DNA isolation. 1-2 gm
of plant leaf was taken from the above mentioned
species to isolate the Genomic DNA. The DNA was
extracted following the protocol described by
Gentzbittel et al.(1992) with modification, from frozen
leaves stored at -80°C. The leaves cut into pieces were
completely homogenized in liquid nitrogen. Extraction
buffer (100 mM Tris HCl (pH 8.0), 20 mM EDTA, 1.4
mM NaCl, 2% CTAB per liter) was added in 50
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ml tubes filled with leaf powder to a volume of 15 ml
and mixed well. The tubeswere incubated at 65°C for
30 minutes with repeated shaking. Equal volume of
chloroform : isoamylalcohol mix (24:1) was added
and mixed thoroughly for 15 minutes, followed by
centrifugation at 4000 rpm for 30 minutes. Equal
volume of isopropanol was added to the supernatant.
DNA was hooked out after half an hour and washed in
70% ethanol and suspended in 500 μl of TE buffer
(pH 8.0). The DNA was incubated with 10-15 μl (10
μg/μl concentration) of RNAse for 30 minutes. To
this, equal volume of chloroform : isoamyl alcohol
was added and centrifuged at 12,500 rpm for 10
minutes. Twice the volume of absolute ethanol and
1/10th volume of 3 M sodium acetate were added to
the aqueous layer and incubated over-night. The
content was centrifuged at 12,500 rpm for 15 minutes
and supernatant was discarded. The pellet was washed
with 70% ethanol and air dried. Then, the pellet was
dissolved in 500 μl of TE and stored at -20°C. The
quality and quantity were checked through 0.8%
agarose gel by electrophoresis. DNA concentration for
PCR reaction was estimated, by comparing the band
intensity produced by the known dilution that gave
good amplification. DNA sample were quantified by
UV spectrophotometer. Qualitative and quantitative
analysis was also done on 1% agorose gel
electrophoresis. The electrophoresis banding pattern is
further used for PCR analysis for getting RAPD.
c)
PCR for RAPD
Initial denaturation:
Complete denaturation of template DNA at the start of
the PCR reaction is the key of the reaction.IIt is
generally performed at 95°c for 1-3 min depending on
the GC content of the template.
Denaturation:
Subsequent denaturation performed for a shorter
time30 sec to 1 min.
Annealing:
The optimal annealing temperature is generally at
55°c lower than the melting temperature of primertemplate DNA duplex, performed for 30 sec to 1 min.
Extension:
Primer extension resulting in synthesizing of new
DNA strand is carried out at 72°c which is optimal
temperature for Taq polymerase activity.
Final extension:
DNA synthesis proceeds it becomes less efficient as
most of the components get used up. Hence following
the last cycle enzyme is allowed to finish any
incomplete synthesis by carrying out a final extension
at 72°c for 5-15min.
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
TABLE I: PCR CYCLE:
2.RAPD by OLP-6
Sr.
Step
Temperature
Time
01
Initial denaturation
95°C
1 min
02
Denaturation
95°C
1 min
03
Annealing
50°C
30sec
04
Extension
72°C
30 sec
05
Final extension
72°C
5-15 min
no
TABLE II: PCR master mix
Sr.no
Reagents
Volume
01
Buffer
5 µl
02
dNTP
1µl
03
MgCl2
2µl
04
Miliq water
34µl
05
Taq polymerase
1µl
06
Primer F
1µl
07
Primer R
1µl
08
Sample DNA
5µl
Fig 2: RAPD pattern of 05 genotypes generated by primer OLP-6.
Lane M: Marker 1kb ladder, Lanes: 1-5
3.RAPD by OLP-8
TABLE III: Name and sequence of primers
Sr.no
Name of Primer
Sequence of 5’to 3’
01
OPL-6
GAGGGAAGAG
02
OPL-8
AGCAGGTGGA
03
OPL-10
TGGGAGATGG
04
OPL-11
ACGATGAGCC
Fig 2 : RAPD pattern of 05 genotypes generated by primer OLP-8.
4.RAPD by OLP-10
4. Results
1.Isolation of Genomic DNA.
Fig 3: RAPD pattern of 03 genotypes generated by primer OLP-10.
Fig 1:DNA banding pattern of different cultivars of Moringa
oliefera.
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International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
5.RAPD by OLP-11
Fig 4: RAPD pattern of 04 genotypes generated by primer OLP-11.
4. Conclusion:
By observing the bands polymorphism which are
separated on the basis of their molecular weightIn 1 %
agarose gel it concluded that the plant varieties shows
variation at their genetic level,that it though they are
belongs to the same species but their DNA sequences
are different. It means that different types of genes
may express which codes for different proteins with
different functions, if considered the central dogma of
molecular biology. The primer which is randomly
designed & amplified the fragment of DNA randomly
showed the polymorphism. Hence these chosen plant
varieties are different at their genetic level
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