Prakash et al.

for 10 minutes and finally once again extracted with SEVEG. The supernatant was

transferred to sterile glass corex tubes and DNA was precipitated with 2.0 volume

of chilled ethanol. The DNA was recovered by spooling. The recovered DNA was

washed with 70% ethanol and air dried and resuspended in 500 µl of TE-buffer

[10mM Tris-HCl (pH-8.0),1nM EDTA] and stored at 4°C until used. The DNA

was quantified both by spectrophotometric method and on 0.8% agarose gel. The

stock solution was diluted to uniform concentration of 10ng/µl.

DNA marker analysis

PCR reactions were performed according to the protocol of Williams et al., (1990).

The PCR amplification was carried in a 0.2 ml tube in GeneAmp 9700 PCR system

(Applied Biosystems, U.S.A.) with 20µl reaction volumes containing 20 mM Tris-

HCl (pH-8.4), 50mM KCl, 2mM MgCl _2, 0.2 µM primer, 0.1 mM of each of dATP,

dTTP, dCTP, dGTP, 0.5U of taq DNA. The random polymerase (Bangalore Genei)

and 20ng of template DNA was used. Five random primers covering most of the

chromosomes were obtained from Operon Technologies Ltd., Almada, U.S.A.

(Table 1). Amplification reactions were carried out by following cycle profiles:

1 cycle at 93°C for 2 min followed by 45 cycles at 93°C for 1min. 35°C for

1 min., 72°C for 2 min. and a final 7 min. extension at 72°C. PCR products were

electrophoresed on 1.5% agarose gel (Sambrook et al., 1989) in 1x TAE, stained

in ethidium bromide and the gel image was recorded using the gel-documentation

system.

table 1: List of RaPD primers used in the study and marker polymorphism

No. of

total no. of

% Poly-

Primer

Sequence (5’ – 3’)

Polymorphic

amplicons

morphism

amplicons

OPA – 01

CAGGCCCTTC

8

3

37.5

OPA – 02

TGCCGAGCTG

7

2

28.6

OPA – 03

AGTCAGCCAC

8

4

50.0

OPA – 04

AATGGGGCTG

6

3

50.0

OPA – 07

GAAACGGGTG

4

1

25.0

total

33

13

39.39

Inheritance and diversity analysis

The bending patterns generated by RAPD markers were scored as ‘1’ for the

presence of amplicons and ‘0’ for the absence. All RAPD markers were performed

twice and only reproducible bands in the range of 3500 to 500 were scored. A

similarity matrix was generated using the equation s=2Nxy/(Nx+Ny), where Nxy

is the number of shared amplications between x and y entries. ‘Nx’ is the total

26

Int. J. Bioinformatics and Biological Sci.: v.2 n.1&2, p. 23-39. March & June 2014