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Ghatole Ketaki, et al.; International Journal of Advance Research, Ideas and Innovations in Technology
© 2019, www.IJARIIT.com All Rights Reserved Page |417
ISSN: 2454-132X
Impact factor: 4.295
(Volume 5, Issue 5)
Available online at: www.ijariit.com
Molecular marker analysis of Oryza sativa using RAPD markers
Ketaki Ghatole
98ketaki@gmail.com
Ramaiah Institute of Technology,
Bangalore, Karnataka
Shruthi Mohan
shruthibgr@gmail.com
Ramaiah Institute of Technology,
Bangalore, Karnataka
Hashvitha R.
r.hashvitha@gmail.com
Ramaiah Institute of Technology,
Bangalore, Karnataka
Meghana K. J.
meghanamanavi@gmail.com
University of Agricultural Sciences, GKVK,
Bangalore, Karnataka
Suresh H. Antre
suresh.antre@gmail.com
University of Agricultural Sciences, GKVK,
Bangalore, Karnataka
ABSTRACT
Rice is one of the most important crops that provide food for more than half of the world’s population and hence their cultivation
is expanding drastically due to the increasing demand. There are different varieties of rice distinguished based on their
physiological characteristics and geographical area. Identification of these genotypes is important during the genetic breeding
programs. Molecular markers play a crucial role in studying genetic variation among different species. The analysis of this
diversity among six varieties of rice: Sahabagi Dhan, IR-64, MGD-101, BPT-5204, Azucena, Doddiga was performed by
extracting DNA and using 8 RAPD markers on them. The random DNA sequences amplified by PCR are compared and analyzed
by the process of electrophoresis. The binary of the gel images is used for analysis of genetic variation between the cultivars. It
is observed that the number of polymorphic fragments for each primer varied from 3 to 7 with an average of 4. The average
percentage polymorphism was calculated as 77.573%. The values of similarity co-efficient ranged from 0.41 to 0.85. The values
of similarity co-efficient ranged from 0.41 between MGD-101 and IR-64 to 0.85 between BPT-5204 and Azucena and an average
of 0.71. Cluster analysis showed that IR-64 and MGD-101 are bilious and Azucena is simplifolious indicating that it is a wild
variety and is substantially different from all others. Major Allele Frequency ranges from 0.61 to 0.90 with a mean of 0.79, Gene
Diversity values range from 0.17 to 0.46 with a mean of 0.29 and PIC values range from 0.14 to 0.36 with a mean of 0.23. The
information obtained in this study helps to understand genetic diversity. It can be used to emphasize conservation and
propagation of selected natural varieties and to select diverse parents to broaden the germplasm of rice.
Keywords— Oryza sativa, RAPD markers, PCR, Polymorphism, Genetic diversity
1. INTRODUCTION
Rice (Oryza sativa L.) is the most important cereal crop being grown (over 144.641 million ha) with a production of over 468.275
million tons in the world. It is also probably the world’s most versatile crop that grows at more than 3000 m elevations in the
Himalayas and at sea level in the deltas of the great rivers of Asia. It feeds more than half the world’s population. (1) White rice is a
good source of magnesium, phosphorus, manganese, selenium, iron, folic acid, thiamine, and niacin. (3) There are thousands of
varieties of cultivated rice. International Rice Gene Bank has developed more than a thousand improved varieties of rice and it has
over 90,000 samples of cultivated rice and wild species are stored making it the world's largest repository of rice. (4) Limitations in
conventional breeding arise because of the lack of resistance genes in cultivated rice germplasm and inadequate understanding of
phenotypic variability. Therefore, transgenic research offers unique opportunities to overcome these problems and to produce
improved varieties with reduced yield gaps. (2)
Molecular markers provide information that helps in deciding the distinctiveness of species and their ranking according to the
number of close relatives and phylogenetic positions. (5) Several types of molecular markers available for evaluating the genetic
variations in rice are RFLP, AFLP, RAPD, SSR, etc. Of all these, RAPD markers are being employed in genetic research due to its
rapid processing and simplicity. (6) It also allows the examination of genomic variation without prior knowledge of DNA sequences.
It is especially useful for unzipping the variations in species with low genetic variability. RAPD markers are unbiased and neutral
for genetic mapping, taxonomy and genetic diagnostics. (7) RAPD is a single, short oligonucleotide primer, which binds to different
loci and is used to amplify random sequences from a complex DNA template. This means that the amplified fragment generated by
Ghatole Ketaki, et al.; International Journal of Advance Research, Ideas and Innovations in Technology
© 2019, www.IJARIIT.com All Rights Reserved Page |418
PCR depends on the length and size of both primer and target genome under the assumption that the given DNA sequence
(complementary to the primer) will occur in the genome, which is readily amplifiable by PCR. (8)
Genetic diversity that exists is the foundation of the genetic improvement of crops. Researchers are uncovering new genes and traits
in rice that will improve the yield and face challenges like adverse climatic conditions, pests, diseases, etc. Mutation and
recombination bring new variations to a population, whereas selection and genetic drift remove some alleles. (9)
Under the green revolution, we are aiming to develop tools to produce high yielding, drought tolerant, pest resistant, and good
nutritional value rice. So, in this study, an attempt is made to assess the molecular diversity using RAPD markers among some
common cultivars in Karnataka. Such information will have significance in providing the basis for selection of pre-breeding
material, conservation of resource material and useful for rice crop improvement program. (11)
2. MATERIALS AND METHODS
2.1 Plant Material and DNA Extraction
The seeds of Sahabagi Dhan, IR-64, MGD-101, BPT-5204, Azucena and Doddiga were selected based on their characteristics (Table
1) and germinated separately in plastic pots labeled with their names and grown in the greenhouse. The leaf samples were collected
after 15 days and DNA was extracted using CTAB method (Doyle and Doyle 1990)
2.2 Selection of primers and RAPD optimization
A total of eight RAPD primers were used to assess the genetic diversity between selected rice varieties. The sequences of the primers
used in this study are tabulated (Table2). The primers were obtained from Sigma Pvt. Ltd. The PCR reaction mixture and
composition used were 1µl of 1X TBE Buffer, 6.2 µl of Sigma water, 0.5 µl of dNTP, 0.7 µl of Primer 0.4 µl of Taq polymerase
and 1.2 µl Sample DNA.
The PCR amplification for each RAPD primer was performed at 95oC for 5 minutes (initial denaturation), 95oC for 1
minute(denaturation), 36oC for 1.5 minutes(annealing),72oC for 2 minutes(extension), 72oC for 8 minutes(final extension) in
Eppendorf mastercycler nexus gradient.
2.3 Agarose gel electrophoresis
The amplified product was subjected to 1.5% agarose gel electrophoresis with a 100bp ladder and the gel was stained with EtBr.
The gels are then analyzed using the gel documentation unit AlphaImager which consists of a UV transilluminator. Alpha imager
EP software was used for visualization.
2.4 Data Scoring
The RAPD banding pattern in each gel was subjected to scoring visually by marking the presence (1) or (0) polymorphic bands in
individual lanes. The scores with respect to each primer were tabulated and are analyzed using different statistical tools. The NTSYS-
PC software ver. 2.02j was used to estimate genetic similarities with the Jaccard coefficient. (12) The matrix of generated similarities
was analyzed by the UPGMA using SAHN clustering module. The cophenetic module is applied to compute a cophenetic value
matrix using the UPGMA matrix. (13) The values for major allele frequency, gene diversity and PIC were obtained using the
PowerMarker V3.25 (2001-2004 by Jack Liu). Major allele frequency refers to the frequency at which the most common allele
occurs in a population. Genetic diversity plays an important role in the populations to adapt to changing environments. (14) The
Polymorphism Information Content (PIC) value is often used to measure the informativeness of a genetic marker for linkage
studies. (15)
3. RESULTS AND DISCUSSION
3.1 Analysis of genetic variation
The bands obtained (figure 1 a-h) were analyzed and the following data was obtained with respect to the polymorphism between
the different varieties. It was observed that the number of polymorphic bands for each primer varied from 3 to 7. The primer OPA-
14 produced maximum polymorphic bands i.e. 7 bands whereas primer OPA-18 produced minimum polymorphic bands i.e. 3 bands.
It was also observed that the percentage of polymorphism ranges from 60% to 100%. Out of 8 primers, 5 primers exhibited more
than or equal to 80 % polymorphism. The reason for this high level of polymorphism can be due to intraspecies variation among
cultivars. It was observed that the level of polymorphism with primers differed between cultivars. (16)
3.2 Similarity Matrix
The genetic relatedness between the different rice varieties was determined using similarity matrix in the present study. (17, 18) The
values of similarity co-efficient ranged from 0.41 to 0.85 with an average of 0.71. MGD-101 and IR-64 were found to be the most
closely related genotypes with similarity index of 0.85. The least value of similarity co-efficient was 0.41 and it was observed
between BPT-5204 and Azucena. (Table 3)
3.3 Analysis of genetic analysis
Cluster analysis was performed based on similarity coefficients (19) in the UPGMA program. Based on the UPGMA dendrogram
(Fig. 2), the 6 varieties were grouped into two clusters. Cluster 1 had five varieties: Sahabagi Dhan, IR-64, MGD-101, BPT-5204,
and Doddiga. The IR-64 and MGD-101 were grouped into a single cluster indicating that both these varieties were more like each
other than they are with Sahabagidhan. While both BPT-5204 and doddiga formed an independent clade and were less similar or
related. Cluster 2 was a simplifolious clade with Azucena which was reported as a wild variety and is substantially different from
all others. (20).
Ghatole Ketaki, et al.; International Journal of Advance Research, Ideas and Innovations in Technology
© 2019, www.IJARIIT.com All Rights Reserved Page |419
3.4 Allele Frequency and PIC
The major allele frequency is the common frequency at which the most common allele occurs in a given population (table 4). In
this study, major allele frequency ranges from 0.61 to 0.90 with a mean of 0.79 and the gene diversity values range from 0.17 to
0.46 with a mean of 0.29.
The Polymorphism Information Content (PIC) of a marker is defined as the probability of marker genotype of the offspring of a
heterozygous parent affected with a dominant disease that allows one to deduce the marker allele that was inherited by the offspring
from the parent. It represents the effectiveness of marker in linkage analysis. In this study, PIC values range from 0.14 to 0.36 with
a mean of 0.23
4. CONCLUSION
The present study revealed the levels of genetic differences between cultivars of rice-based on RAPD markers. We have determined
the genetic relationship and the degree of genetic diversity among the six varieties of rice. The data obtained in this study confirmed
the efficiency of RAPD markers in assessment of genetic variation in population. The polymorphism detected in this study is
necessary to ascertain the germplasm conservation and the development of improved rice genotypes with good quality traits through
various breeding programs Also, the estimation of genetic distance between genotypes can be used for selection of diverse parents
to perform appropriate crosses and broaden the germplasm during hybridization programs. The information on intraspecific variation
obtained from high level of polymorphism is useful in making decision for improvement of rice cultivars. However, in the present
study only six cultivars and ten primers were used in RAPD analysis hence the chance to obtain reliable knowledge about the genetic
structure of each variety is reduced. Further studies including large number of primers and cultivars can be conducted to obtain more
precise information.
5. ACKNOWLEDGEMENTS
We would like to thank the “Department of Plant Biotechnology”, University of Agricultural Sciences, Bengaluru for providing
guidance, support and laboratory facilities. We would also like to extend our gratitude to Dr. Sharath R for his constant guidance
during the editing of the article.
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APPENDIX
Table 1: Selected plant varieties with their special characteristics
S no.
Plant name
Characteristic.
1
Sahabagidhan
Drought-tolerant, high yield rice variety. (24)
2
IR-64
Early maturity, disease resistant, excellent cooking quality. (22)
3
MGD-101
Drought and blast resistant properties. (23)
4
BPT-5204
Early maturity, drought-tolerant, fine grains. (21)
5
Azucena
Symbiosis with arbuscular mycorrhizal (AM) fungi that help the plant to improve nutrient uptake. (26)
6
Doddiga
Tolerance to drought and low soil fertility(25)
Table 2: RAPD primers, their sequences, size and melting temperature
Table 3: Similarity matrix of rice varieties
Sahabagidhan
IR-64
MGD-101
BPT-5204
Azucena
Doddiga
Sahabagidhan
1
IR-64
0.79
1
MGD-101
0.77
0.85
1
BPT-5204
0.73
0.69
0.79
1
Azucena
0.42
0.42
0.46
0.41
1
Doddiga
0.58
0.54
0.57
0.54
0.52
1
Table 4: Summary Statistics of different primers
S no.
Marker
Sequence
Total number
of bands (loci)
No. of polymorphic
bands (loci)
% Polymorphic bands
(loci)
1
OPA-02
5’TGCCGAGCTG3’
6
5
83.33
2
OPA-05
5’AGGGGTCTTG3’
6
5
83.33
3
OPA-07
5’GAAACGGGTG3’
5
4
80
4
OPA-12
5’TCGGCGATAG3’
5
4
80
5
OPA-14
5’TCTGTGCTGG3’
7
7
100
6
OPA-18
5’AGGTGACCGT3’
5
3
60
7
OPA-20
5’GTTGCGATCC3’
8
5
62.5
8
OPK-11
5’AATGCCCCAG3’
7
5
71.43
Marker
Major Allele Frequency
Gene Diversity
PIC
Average
Highest
Lowest
Average
Highest
Lowest
Average
Highest
Lowest
OPA-02
0.69
1.00
0.50
0.36
0.50
0.00
0.28
0.38
0.00
OPA-05
0.83
1.00
0.67
0.24
0.44
0.00
0.20
0.35
0.00
OPA-07
0.77
1.00
0.67
0.32
0.44
0.00
0.26
0.35
0.00
OPA-12
0.77
1.00
0.67
0.32
0.44
0.00
0.26
0.35
0.00
OPA-14
0.76
0.83
0.67
0.35
0.44
0.28
0.28
0.35
0.24
OPA-18
0.90
1.00
0.83
0.17
0.28
0.00
0.14
0.24
0.00
OPA-20
0.79
1.00
0.50
0.25
0.50
0.00
0.20
0.37
0.00
OPK-11
0.88
1.00
0.83
0.20
0.28
0.00
0.17
0.24
0.00
Ghatole Ketaki, et al.; International Journal of Advance Research, Ideas and Innovations in Technology
© 2019, www.IJARIIT.com All Rights Reserved Page |422
(f)
(g)
(h)
Fig. 1: RAPD Pattern of the rice varieties with Primer
(a) OPA-02
(b) OPA-05
(c) OPA-07
(d) OPA-12
(e) OPA-14
(f) OPA-18
(g) OPA-20
(h) OPK-11
Fig. 2: UPGMA Dendrogram
