Molecular characterization of selected cultivars of rice, Oryza sativa L. using Random Amplified Polymorphic DNA (RAPD) markers

Abstract

Random Amplified Polymorphic DNA (RAPD) analysis was performed to assess the genetic diversity in ten selected cultivars of rice, Oryza sativa L. using 30 decamer random primers. Out of 30, 25 RAPD primers revealed polymorphism while the remaining 5 primers showed no reaction. The primers produced a total of 428 bands of which 363 were polymorphic (85.02%). The number of polymorphic fragments for each primer varied from 7 to 23 with an average of 14 polymorphic fragments. The primer OPB-17 produced the maximum number of polymorphic bands. The RAPD data was analyzed to determine the genetic similarity coefficients which ranged from 0.46 to 0.81. Cluster analysis was performed using Unweighted Paired Group of Arithmetic Means (UPGMA) using the Jaccard's similarity coefficient. The UPGMA dendrogram resolved the selected rice cultivars into two major clusters.

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*Corresponding author.
Email: deepundd@gmail.com
International Food Research Journal 20(2): 919-923 (2013)
Journal homepage: http://www.ifrj.upm.edu.my
1Rajani, J., 2*Deepu, V., 3Nair, G. M. and 1Nair, A. J.
1Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India
2Crop Improvement and Biotechnology Division, Centre for Medicinal Plants Research
(CMPR), Arya Vaidya Sala, Changuvetty, Kottakkal 676 503, Malappuram, Kerala, India
3School of Biosciences, Central University of Kerala, Kasaragod, Kerala, India
Molecular characterization of selected cultivars of rice, Oryza sativa L. using
Random Amplied Polymorphic DNA (RAPD) markers
Abstract
Random Amplied Polymorphic DNA (RAPD) analysis was performed to assess the genetic
diversity in ten selected cultivars of rice, Oryza sativa L. using 30 decamer random primers.
Out of 30, 25 RAPD primers revealed polymorphism while the remaining 5 primers showed no
reaction. The primers produced a total of 428 bands of which 363 were polymorphic (85.02%).
The number of polymorphic fragments for each primer varied from 7 to 23 with an average of 14
polymorphic fragments. The primer OPB-17 produced the maximum number of polymorphic
bands. The RAPD data was analyzed to determine the genetic similarity coefcients which
ranged from 0.46 to 0.81. Cluster analysis was performed using Unweighted Paired Group
of Arithmetic Means (UPGMA) using the Jaccard’s similarity coefcient. The UPGMA
dendrogram resolved the selected rice cultivars into two major clusters.
Introduction
Rice (Oryza sativa L.) is one of the most
important crops that provide food for more than half
of the world population (Malik et al., 2008). India
has a long history of rice cultivation and stands
rst in rice area and second in rice production, after
China. Approximately 90% of the world’s rice is
grown in the Asian continent and constitutes a staple
food for 2.7 billion people worldwide (Salim et al.,
2003; Paranthaman et al., 2009). The genus Oryza
contains 25 recognized species, of which 23 are wild
species and the remaining two are O. sativa and O.
glaberrima which are cultivated species (Brar and
Khush, 2003; Chang, 2003). O. sativa is the most
widely grown worldwide including in Asian, North
and South American, European Union, Middle
Eastern and African countries.
The world’s rice production has doubled during
last 25 years, largely due to the use of improved
technology such as high yielding varieties and better
crop management practices (Byerlee, 1996). Demand
for rice is growing every year and it is estimated that
in 2025 AD the requirement would be 140 million
tones. The land available for cultivation is decreasing
due to continuous urbanization and inappropriate
land use (Khush, 1997; Fischer et al., 2000). To
sustain present food self sufciency and to meet
future food requirements, India has to increase its rice
productivity by 3 per cent per annum (Thiyagarajan
and Selvaraju, 2001).
Further scope of crop improvement depends on
the conserved use of genetic variability and diversity
in plant breeding programmes and use of new
biotechnological tools. Molecular characterization
can reveal the maximum genetic variation or genetic
relatedness found in a population (Xu et al., 2000).
Chakravarthi and Naravaneni (2006) reported the
usefulness of preservation and conservation of
genetic resources since genetic diversity provides
information to monitor germplasm and prediction
of potential genetic gains. Information regarding
genetic variability at molecular level could be used
to help, identify and develop genetically unique
germplasm that compliments existing cultivars (Ni
et al., 2002; Ravi et al., 2003; Chakravarthi and
Naravaneni, 2006). DNA based molecular markers
have proven to be powerful tools in the assessment
of genetic variation and in the elucidation of genetic
relationships within and among the species of rice
(Ragunathanchari et al., 1999, 2000; Shivapriya and
Hittalmani, 2006). The present investigation was
Keywords
Genetic variation
molecular markers
Oryza sativa
UPGMA
Article history
Received: 6 August 2012
Received in revised form:
4 October 2012
Accepted: 5 October 2012

920 Rajani et al./IFRJ 20(2): 919-923
undertaken for the assessment of genetic diversity
among the selected rice cultivars with the help of
RAPD markers.
Materials and Methods
Plant materials and genomic DNA isolation
The plant materials selected for the present study
were ten different cultivars of rice (Table 1). The
seeds of four varieties (GOURI-MO20, BHADRA-
MO4, PAVIZHAM-MO6 and JYOTHI-PTB39) were
collected from Rice Research Station, Mankomb,
Kerala, India. The seeds of remaining six varieties
were collected from traditional farmers of Palakkad,
Kerala, India.
Healthy seeds of each variety were sowed in
soil pots containing water under appropriate growth
conditions for getting fresh leaves. DNA extraction
was carried out from the fresh leaves collected from
tillers following cetyl trimethyl ammonium bromide
(CTAB) method (Doyle and Doyle, 1987) with some
modications. Freshly germinated 500 milligrams of
young leaves were ground to a very ne powder in
liquid nitrogen and dispersed in 3mL of pre-warmed
(65oC) CTAB DNA extraction buffer (2% CTAB;
1.4 M NaCl; 100 mM Tris-HCI, pH 8.0; 20 mM
EDTA, p.H. 8.0; 0.2% - mercaptoethanol (added just
before use). Oakridge tubes containing samples were
incubated at 65oC for 30 min in a water bath. The
samples were swirled every 10 min. After incubation
the mixture was cooled down to room temperature
and emulsied with an equal volume of chloroform:
isoamyl alcohol (24:1) and centrifuged at 8000 rpm
for 10 min. Following centrifugation, the aqueous
phase was collected and nucleic acid was precipitated
by mixing with equal volume of chilled isopropanol.
The precipitated nucleic acid was centrifuged at
12000 rpm for 10 min and the pellet was washed with
70% ethanol. The DNA pellet obtained was dried and
stored in 400 µL TE buffer.
Purication of DNA
The RNA was removed by RNase treatment at
37oC for 30 min in a water bath. After incubation,
DNA solution was extracted with an equal volume
of chloroform: isoamyl alcohol (24:1). The upper
aqueous phase was collected after centrifugation at
8000 rpm for 10 min and mixed with 50 µL of 3M
sodium acetate. DNA was precipitated by adding two
volumes of chilled absolute alcohol. The DNA pellet
was air dried and dissolved in 100 µL TE buffer.
Two µL of genomic DNA isolated was subjected to
electrophoresis on 0.8% agarose gel containing 1 mg/
mL ethidium bromide and the quantity of genomic
DNA was assessed using undigested lambda DNA as
control. For further use in PCR the DNA was diluted
to a concentration of approximately 25 ng/µL.
RAPD analysis
For the RAPD analysis of rice cultivars thirty
deca-nucleotide primers of Operon Technology Inc.
(Alameda, CA, USA) were used. The reaction was
carried out in 25 µL reaction volume containing 25
nanogram genomic DNA, 2.5 µL 10X PCR buffer, 2
µL 25 mM MgCl2, 2.5 µL 2.5 mM dNTPs, 0.4 µL Taq
DNA polymerase and 2 µL primer. All the reaction
chemicals except primers were procured from M/s.
Genei, Bangalore, India.
RAPD amplication procedure
Samples for amplication were carried out using
the method stipulated by Williams et al. (1990) with
some modications of thermal cycles. Amplication
was performed in a thermal cycler with an initial
denaturation of 94oC for 5 min followed by 35 cycles
which contains denaturation at 94oC for 1 min followed
by annealing in which the annealing temperature
was adjusted based on the Tm value of each primers
and nally extension at 72oC for 2 min. After 35
cycles, there was a nal extension step at 72oC for 10
min. All the reactions were amplied in a Gradient
Palmcycler (Corbett Research, CG-96, Australia).
Each amplication reaction for the screened primers
was replicated two times individually with the same
procedure in order to verify that the RAPD markers
were reproducible and consistent.
Electrophoresis and visualization of RAPD products
Amplied products were fractionated by 1.5%
agarose gel in 1X TBE buffer (pH-8.0) at 100 V for 2
h and stained with ethidium bromide. 1kb DNA ladder
was used as size marker. The gels were visualized
under a UV transilluminator and documented using
a digital camera. Total number of bands and number
of polymorphic bands present in each cultivar was
detected from the gels and scored manually. Each
polymorphic band was considered as binary characters
and was scored 1 (presence) or 0 (absence) for each
sample. Only those fragments with medium and high
intensity were taken into consideration.
Table 1. List of selected rice cultivars used in the genetic analysis
Sl. No.
Name of cultivars
Sou rc e
1
GOU RI -M O2 0
Rice Research Station, Mankomb, Kerala, India
2
PAV I ZH A M -MO 6
Rice Research Station, Mankomb, Kerala, India
3
BH A DR A-M O4
Rice Research Station, Mankomb, Kerala, India
4
JY OTH I -PTB 39
Rice Research Station, Mankomb, Kerala, India
5
AST 120
Chittoor, Palakkad, Kerala, India
6
JAYA
Chittoor, Palakkad, Kerala, India
7
JY OTH I
Chittoor, Palakkad, Kerala, India
8
KAN CH A NA
Chittoor, Palakkad, K e r a l a , I n d i a
9
PON M ANI
Chittoor, Palakkad, K e r a l a , I n d i a
10
SUJ AT H A
Chittoor, Palakkad, K e r a l a , I n d i a

Rajani et al./IFRJ 20(2): 919-923 921
Data analysis
The gel images were scored using a binary scoring
system that recorded the presence and absence of
bands as “1” and “0” respectively. From the binary
data, the similarity coefcient values between the
cultivars were derived based on the probability that
a particular character of one accession will also be
present in another with the Jaccard’s correlation
analysis using the statistical software “SPSS” version
7.5 for Windows. The statistical analysis is performed
using NTSYSpc version 2.1 (Rohlf, 1998). The data
matrix was used to construct a phenetic dendrogram
using UPGMA (unweighted pair group method of
arithmetic averages) (Sneath and Sokal, 1973) in
order to cluster the accessions.
Results and Discussion
The results of present study indicated a
considerable level of genetic diversity among the
cultivars selected. Among 30 primers used in this study,
results of 25 primers were taken into consideration
since they had given reproducible bands. Each
polymorphic RAPD marker was considered as a locus
so that every locus had two alleles, identied by the
presence and absence of the band. A total of 428 DNA
fragments were generated by 25 primers out of which
363 were polymorphic (85.02% polymorphism)
(Table 2). Out of 25 primers, only 16 primers exhibited
more than 80% polymorphism. The number of
polymorphic fragments for each primer varied from
7 to 23 with an average of 14 polymorphic fragments.
The primer OPB-17 produced the maximum
number of polymorphic bands. The percentage of
polymorphism was calculated as 85.02%. The size
of amplied fragments ranges between 250 bp to
2500 bp (Figure 1). It was observed that the level
of polymorphism with primers differed between
the cultivars. Similarity between the cultivars was
derived by Jaccard’s correlation coefcient (Jaccard,
1908). Correlation matrices obtained from all the
primers used were consolidated in one single matrix
and the mean values were presented (Table 3).
Jaccard’s pair-wise similarities computed between
the cultivars showed that SUJATHA and AST 120
were the closest (0.81). The greatest distance was
observed between the cultivars KANCHANA and
JYOTHI (0.47). RAPD data generated by twenty ve
primers were subjected to UPGMA cluster analysis
and the dendrogram was constructed (Figure 2).
Cluster analysis revealed the similarity between the
rice cultivars and it ranged from 52% to 81%. The
dendrogram classied the cultivars into two distinct
clusters. The rst cluster included three cultivars,
GOURI MO-20, PAVIZHAM MO-6 and BHADRA
MO-4. The second cluster included six cultivars
collected from Palakkad.
The present investigation revealed the
effectiveness of RAPD in detecting polymorphism
among different cultivars of rice. The success of
RAPD analysis in O. sativa accessions were also
reported earlier (Muhammad et al., 2005; Rahman
et al., 2007; Malik et al., 2008). The percentage of
polymorphism was found to be 85.02%. One of the
reasons for this high level of polymorphism can be
due to intraspecic variation among the cultivars.
Information on intraspecic variation from the
present study might be useful in making decision for
Primer
name
Sequenc e (5 ’-3’)
Total
number
of bands
Numbe r o f
poly morphic
bands
Percentage of
poly mor phi sm
OPA-01
CAGGCCCTTC
11
11
100
OPA-04
AAT C GGGCTG
14
14
100
OPA-13
CAGCACCCAC
24
21
87.5
OPA-17
GAC C GC T TGT
15
15
100
OPA-18
AGGT GA CCGT
20
16
80
OPB -08
GTCC ACAC GG
21
20
95
OPB -12
CCTTGACGCA
16
13
81
OPB -17
AGGG AAC GA G
23
23
100
OPC -04
CCGCATCTAC
19
16
84
OPC -16
CACACTCCAG
13
13
100
OPC -20
ACTTCGCCAC
12
7
58
OPD-05
TGAGC GGAC A
18
17
94
OPD-06
AC CTGAAC GG
13
13
100
OPD-07
TT GGCA CGGG
17
10
59
OPD-08
GTGTGC GC C A
23
14
61
OPD-15
CATCCGTGCT
14
11
79
OPD-20
AC CCGGT C AC
20
16
80
OPE -01
CC C AAGGTC C
14
13
93
OPG-02
GGC AC T GAGG
21
20
95
OPG-04
AGC GT GT C TG
13
10
77
OPH -05
AGTCGTCCCC
19
15
79
OPK-16
GAGC GTC GAA
18
18
100
OPT -17
CC AAC GT C GT
12
9
75
OPX-11
GGAG CCTC AG
18
14
78
OPY-11
AGAC GATGGG
20
14
70
Total
428
363
85.02
Table 2. Sequence of 25 random primers with the number of scorable,
amplied and polymorphic bands
Table 3. Similarity matrix of ten rice cultivars based on Jaccard’s
similarity index
1) GOURI MO-20, 2) PAVIZHAM MO-6, 3) BHADRA MO-4, 4) JYOTHI PTB-39,
5) JAYA, 6) KANCHANA, 7) SUJATHA, 8) AST 120, 9) JYOTHI, 10) PONMANI

922 Rajani et al./IFRJ 20(2): 919-923
improvement of rice cultivars. Similarity level up to
50% in cluster analysis is indicative of plant derived
from interspecic hybridization (Marsolais et al.,
1993). Three cultivars (GOURI MO-20, PAVIZHAM
MO-6 and BHADRA MO-4) collected from Rice
Research Station, Mankomb were grouped in a
single cluster indicating more similarity among them
and expressed more diversity from all the cultivars
collected from traditional farmers of Palakkad. It is
interesting to note that JYOTHI PTB-39 collected
from Rice Research Station, Mankomb was totally
excluded from both the clusters. The ndings of
Reby Skaria et al. (2011) indicate that PAVIZHAM
MO-6 and JYOTHI PTB-39 are genetically distant.
The present ndings conrm that genetic diversity
of the plants is closely related to their geographic
distribution. It has been reported that species
with a wide geographic area generally have more
genetic diversity (Wilikie et al., 1993). The present
investigation reveals that RAPD is a valuable tool for
estimating the extent of genetic diversity as well as to
ascertain the genetic relationship between different
cultivars of Oryza sativa.
Conclusion
The present study revealed that the levels of
genetic differentiation between cultivars of O.
sativa increased with geographical distance. The
polymorphism detected among the accessions will
be helpful in selecting genetically diverse cultivars
in future breeding programme. However, there
were some precincts in the present study that only
ten cultivars and thirty primers were used in RAPD
analysis and hence reduce the chance to obtain
a reliable knowledge precisely about the genetic
structure of each cultivar of rice. Further studies
involving large number of accessions and primers need
to be conducted to get more precise information.
Acknowledgement
The authors gratefully acknowledge Rice Research
Station, Mankomb, Kerala, India for providing the
seed material of improved cultivars of rice.
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