biodiversity of six varieties of mangifera indica using rapd · biodiversity of six varieties of...

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



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



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

[1] S. Singh, A. B. Gaikwad, and J. L. Karihaloo, “Morphological and

molecular analysis of intracultivar variation in Indian mango

(Mangifera indica L.) cultivars,” Acta Horticulturae, vol. 29, pp. 205-212. June 2010.

[2] I. G. B. Souza, S. E. S. Valente, F. B. Britto, V. A. B. de souza,

and P. S. C. Lima, “RAPD analysis of genetic diversity of mango (Mangifera indica) germplasm in Brazil,” Genetics and Molecular

Research, vol. 10, pp. 3080-3089, Dec. 2011. [3] A. D. Ramessur and V. M. S. Ranghoo, “RAPD marker assisted

identification of genetic diversity among mango (Mangifera indica)

varieties in Mauritius,” International Journal of Agriculture and Biology, vol. 13, pp. 167-173, May 2011.

[4] Z. A. Bhat, W. S. Dhillon, R. Rashid, J. A. Bhat, W. Alidar, and M. Y. Zenaie, “The role of molecular markers in the improvement of

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identification of cultivar species in subtribe cucumerinae (Cuccurbitaceae) using RAPD and SCAR markers,” American

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[6] M. Marieschi, A. Torelli, F. Poli, and A. Bianchi, “Quality control of commercial Mediterranean oregano: Development of SCAR

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998-1003, Apr. 2010.

[7] H. Begum, T. M. Reddy, S. Malthi, B. P. Reddy, S. Archack, J. Nagaraju, et al., “Molecular analysis for genetic distinctiveness

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Biotechnology, vol. 6, pp. 24-37, Feb. 2010.

[8] S. Dellaporta, L. J. Wood, and J. B. Hicks, “A plant DNA minipreparation: Version II,” Plant Molecular Biology Reporter,

vol. 1, pp. 19-21, Sep. 1983. [9] J. Sambrook, E. F. Fritschi, and T. Maniatis, Molecular Cloning:

A Laboratory Manual, New York: Cold spring harbor laboratory

press, Nov. 1989.

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.



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