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Biodiversity of Six Varieties of Mangifera indica
Using RAPD
Hanumanth Kumar Gurijala Department of Biotechnology, Sri Venkateswara University, Tirupati, AP, India
Dileep Reddy Rampa
Stem Cell Culture Research Laboratory, Stanley Medical College, Chennai, TN, India
Pramoda Kumari Jasti Department of Microbiology, Sri Venkateswara University, Tirupati, AP, India
Email: [email protected]
Abstract—The objective of our study was to identify the
genetical diversity of six populations of Mangifera indica
using Random Amplified Polymorphic DNA (RAPD)
markers. Genomic DNA was extracted, subjected to PCR
with the primers OPAB01, OPAB 04, OPAB 05, OPAB 06,
OPAB 08, OPAB 09, OPAB 12, OPAB 14, OPAB 15, OPAB
19, OPAB 20, A2, A3, A 10, A 18, P10 and W18.Out of 17
RAPD primer tested, 15 showed polymorphism in the six
populations of Mangifera. The means of each population
contained in average 16.58 was found. From 15 primers 277
bands were found, out of them 94 bands shown
polymorphism at the rate of 31.23%. Phylogenetic
variability among all plants samples evaluated that
Thothapuri was the most prominent resembling species with
all of the remaining species following Malgoba, Rasapuri,
Neelam, Badhami and Sendura.
Index Terms—mangifera, RAPD, genetical diversity,
polymorphism
I. INTRODUCTION
The mango is a fleshy stone fruit belonging to the
genus Mangifera, consisting of numerous tropical fruiting
trees in the flowering plant family Anacardiaceae. The
mango is native to South Asia, It is the national fruit of
India. As a consequence, though the geographical
distribution of the mango under cultivation is vast, the
genetic variation may not be so well distributed [1].
Until now, the identification and characterization of
plants or varieties was essentially based on external
characteristics such as the morphological traits or
productivity. Such classical phenotypic features are still
extremely useful but can be widely influenced by
environmental conditions such as climate, temperature,
humidity, soil, etc. The morphology of the same plant
may be extremely variable depending on the external
growth conditions. Fundamental genetic characters may
Manuscript received March 3, 2015; revised May 1, 2015.
be masked and give the identification very difficult.
Moreover, this visual identification is time consuming
because it requires that the plant be grown to a suitable
developmental stage before certain characteristic can be
scored, so there is an urgent need to identify varieties of
mango species using species based specific molecular
assays [2].
Mango gene pool has attracted lot of interest for
molecular diversity analysis through several markers [3].
In the last 10 to 15 years, various molecular techniques
have been successfully applied in determining the
genotypic profiles of individuals and/or populations of
numerous wild and cultivated plant species, Moreover
DNA markers, such as restriction fragment length
polymorphism (RFLP), amplified fragment length
polymorphism (AFLP) and randomly amplified
polymorphic DNA (RAPD), offer significant advantages
in comparison with morphological and isozyme markers
because they can be readily obtained in large numbers,
can provide greater discrimination of cultivars, and are
unaffected by environmental factors [4]. In this context,
RAPD has been employed extensively not only for the
determination of genetic variability within plant
taxonomic groups but also as an auxiliary tool in breeding
programs and in obtaining genetic maps [1], [5].
Additionally, the RAPD technique is fundamental in
developing specific sequence-characterized amplified
region (SCAR) markers for use in the assisted selection
of crops [6].
Recent findings of improved molecular markers are co-
dominant, specific and highly variable, rendering them
highly suitable to study diversity in supposedly related
populations or cultivars [7]. Understanding distribution of
genetic variations within and among different populations
can help in crop breeding, mapping and germplasm
management. RAPD is considered to practical use over
the other markers since the DNA required is lower, it is
less costly, does not require blotting and data can be
collected quickly.
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 100
II. MATERIALS AND METHODS
A. Plant Material
Leaf samples of 6 varieties of Mangifera species were
collected from different agroclimatic zones of southern
region of India were selected for the present study.
Sample 1 – Neelam;
Sample 2 – Thothapuri;
Sample 3 – Rasapuri;
Sample 4 – Sendura;
Sample 5 – Malgoba;
Sample 6 – Badhami.
B. DNA Extraction and Amplification
The DNA was extracted using modified cetyl trimethyl
ammonium bromide (CTAB) method from 2 g of leaf
tissue as described by Dellaporta et al. (1983) [9]. and
purified by RNase treatment followed by extraction using
phenol and chloroform. The DNA samples were checked
both qualitatively as well as quantitatively by nanodrop
(Thermoscientific, 2000c) followed by agarose gel
electrophoresis according to Sambrook et al. (1989) [10].
The PCR reactions were carried out on the total genomic
DNA in a final volume of 25µl reaction mixture with the
17 RAPD primers (Table I).
TABLE I. LIST OF PRIMERS USED IN THE PRESENT STUDY
S.No Primers used
1 OPAB01
2 OPAB 04
3 OPAB 05
4 OPAB 06
5 OPAB 08
6 OPAB 09
7 OPAB 12
8 OPAB 14
9 OPAB 15
10 OPAB 19
11 OPAB 20
12 A2
13 A3
14 A 10
15 A 18
16 P10
17 W18
C. PCR Amplification
A set of 17 primers was used for amplification. PCR
reaction mixture (20 μl) contained 20 ng/μl DNA, 10 ×
PCR buffer, 10 mmol /L dNTPs, 50 mmol /L MgCl2, and
10 μmol /L each of forward and reverse primers. The
amplification was carried out in a thermal cycler (Bio-
Rad C-1000) using a program configured with a
denaturation step of 5 min at 94°C followed by 40 cycles
of 30s at 94°C, 30s at an appropriate annealing
temperature, and 1 min at 72°C. The program ended with
one final extension at 72°C for 8 min. The amplified
products were separated by electrophoresis on a 3%
agarose gel containing ethidium bromide.
III. RESULTS AND DISCUSSION
Among the PCR based DNA marker systems, RAPD is
the most commonly and extensively used tools for
assessment of variability in crops. These marker systems
are efficient due to the ease, rapidity and reliability, for
analysis of molecular differentiation and for resolving
taxonomic identification problems in plants [2].
Genomic DNA, extracted was subjected to PCR with
the primers OPAB01, OPAB 04, OPAB 05, OPAB 06,
OPAB 08, OPAB 09, OPAB 12, OPAB 14, OPAB 15,
OPAB 19, OPAB 20, A2, A3, A 10, A 18, P10 and W18.
Of 17 RAPD primer tested, 15 showed clear reproducible
band patterns and were chosen for the study (Table II).
OPAB 05 and OPAB 12 do not show any polymorphism.
TABLE II. PRIMERS SHOWN AND NOT SHOWN POLY MORPHISM IN
PCR AMPLIFICATION
Primers shown polymorphism in PCR amplification
Primers not shown polymorphism in PCR
amplification
A2 OPAB 05
A3 OPAB 12
A10
A18
OPAB 01
OPAB 04
OPAB 06
OPAB 08
OPAB 09
OPAB 14
OPAB 15
OPAB 19
OPAB 20
P13
W19
The base pairs of size ranging from 200 to 3500 were
identified among obtained 277 total fragments. Out of
these 277 bands, 94 bands shown polymorphic loci and
183 does not shown polymorphism (Fig. 1). The number
of polymorphic fragments for each primer varied from 0
(OPAB 05) to 43 (OPAB 09) with an average of 16.58
bands (31.02%). The band data were used for generating
the distance similarity between the species. Our findings
suggested that phenotypical similarity was seen in
between the primers of OPAB15, A18, P-13 and W-19 of
which the plants may be genetically closely related.
Proximity Matrix of six varieties of Mangifera
represents correlation between different species (Table
III). The highest proximity was observed in between 4, 5
and 2 and lowest proximity was observed in between 6-1,
5-3, 6-4, 3-5 and 4-6. Hierarchical cluster analysis
method was employed for construction of a dendrogram
based on the presence and absence of band number. The
cluster analysis showed a significant genetic variation
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 101
among the mango genotypes studied with a similarity
coefficient showing Thothapuri was the most prominent
species resembling with all the species followed by
Malgoba, Rasapuri, Neelam, Badhami and Sendura (Fig.
2).
Figure 1. Polymorphism in 6 varieties of Mangifera.
TABLE III. PROXIMITY MATRIX OF SIX VARIETIES OF MANGIFERA
Proximity Matrix
Squared Euclidean Distance
Case 1 2 3 4 5 6
1 .000 6.000 5.000 6.000 7.000 4.000
2 6.000 .000 9.000 10.000 11.000 6.000
3 5.000 9.000 .000 7.000 4.000 9.000
4 6.000 10.000 4.000 5.000 .000 9.000
5 7.000 11.000 4.000 5.000 .000 9.000
6 4.000 6.000 9.000 4.000 9.000 .000
Figure 2. Dendrogram showing species similarity among six species
1. Neelam; 2. Thothapuri; 3. Rasapuri; 4. Sendura; 5. Malgoba; 6. Badhami
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 102
Polymorphism was obtained with all primers used
except OPAB 05 and OPAB 12. A10, OPAB 20, OP 01,
OPAB 08, OPAB 14, OPAB 06, OPAB 04, OPAB 19
being least discriminatory in contrast to OPAB 15, A 18,
P 13, W 19, A 10, A 2, A 3, OPAB 09. Compared to all
the primers OPAB09, W19 and A3 were more
polymorphic.
Genetic resources for potential crop improvement were
invaluable; hence their collection, evaluation, and
documentation were important in order to efficiently
maintain the germplasm collection. In the current study, a
close examination leads to an interesting assumption that
the Malgoba, Rasapuri, Neelam, Badhami and Sendura
might have derived from of Thothapuri. The present
results were also useful in conservation biology to
quantify relationships and differences among populations.
REFERENCES
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[8] S. Dellaporta, L. J. Wood, and J. B. Hicks, “A plant DNA minipreparation: Version II,” Plant Molecular Biology Reporter,
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Jasti. Pramoda Kumari, M.Sc, M.Phil,
Ph.D, Diploma in Yoga and Allied
Sciences, is currently working as an
assistant professor (Senior Scale) &
head in Dept. of Microbiology,
Coordinator of Industrial Microbiology,
S V University, Tirupati, India. She is
expertise in a wide spectrum of areas in
proteomics of probiotics, biodiversity
and DNA barcoded pyrosequencing. She has published 62
research papers in reputed national (35 with NAAS rating factor:
46.3) and international (32 with impact factor: 42.655) journals,
guided 3 Ph.D. and is guiding 5 Ph.D. candidates. She has
published a book on proteomics of Eschericia coli Nissle 1917
grown under heavy metal stress by Verlag publishers, AV
Akademiker verlag GmbH & Co.Kg, Germany. She is also
acting as a reviewer for multiple national and international
journals.
Gurijala Hanumanth Kumar is a Ph.D.
research scholar in the field of plant
physiology, Department of
Biotechnology, Sri Venkateswara
University, India. He received his merit
degree in Master of Science in
biotechnology from Periyar University,
India in 2008. He has supervised a
number of master students research
programs. Currently his research is focused on
phytoremediation, bioremediation and phytoextraction. To date,
he has published 8 international research articles related to
phytoremediation.
Rampa Dileep Reddy is working as
SRF in Stem cell culture Research
Laboratory, Stanly medical college,
Chennai, TN, India. He received his
master degree in Master of Science in
biotechnology from Periyar University
India in 2008. He has published 3
international research articles related
chronic respiratory disorders. Currently
his research is focused on WNT signaling and EPCAM
expression in human liver cancer.
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 103