Prajapati et al.

tolerate these biotic stresses and have greater recovery

DNA. Amplification were carried out in a thermo-cycler

from damage. Genomic tools especially molecular

(Applied BiosystemVeriti, CA, USA) programmed for

markers have facilitated breeding in many cereal crops

40 cycles with an initial denaturation at 94°C for 4 min

leading to development of several improved cultivars/

followed by cycling conditions of denaturation at 94°C for

varieties with enhanced resistance / tolerance to biotic

1 min, annealing at 1min at 38°C and extension at 72°C

or abiotic stesses (Varshney et al., 2006). Molecular

for 2 min. After 40 cycles, there was a final extension

markers, such as RAPD, SCAR, SSR, RFLP, AFLP

step of 7 min at 72°C. The amplicons were analyzed on

etc., have been used to assess genetic variations at DNA

1.6% agarose gels and detected by staining with ethidium

level. RAPD, being a multi locus marker (Karp et al .,

bromide. UV trans- illuminated gels were photographed

1997) with simplest and fastest detection technology,

with gel documentation system (SYNGENE, USA).

have been successfully employed for determination of

genetic diversity in several grain legumes. This paper

Data collection and analysis

reports characterization of seventeenpigeonpea cultivars

Clear and distinct bands amplified by RAPD and SSR

for fusarium wilt.

primers were scored for the presence andabsence of the

corresponding band among the genotypes.The scores 1 and

Materials and Methods

0 indicates the presence or absence of bands respectively.

Plant material

The softwares used for the analysis of the scored data

were NTSYSpc version 2.02 (Rholf1994). The molecular

Seventeen pigeonpea genotypes viz ., GT-1, GT-100,

weight of the PCR products were estimated by Alpha

GT-101, GT-102, BANAS, AVPP-1, AGT-2, T-15-15,

EaseFC4.0.0 software (Alpha Innotech Corporation,

LRG-41, C-11, BDN-2, ICPL-87, ICPL-87119, ICPL-

USA) for each primer to analyze allele range.Coefficients

84060, ICP-8863, BSMR-853 and WRGE-119 showing

of similarity were calculated by using Jaccard’s similarity

variable resistance / susceptible reaction against fusarium

coefficient by SIMQUAL function and cluster analysis was

wilt were collected from Agricultural Research Station,

performed by agglomerative technique using the UPGMA

Dahod and Pulse Research station, Vadodara, Anand

(Un-weighted Pair Group Method with Arithmetic Mean)

Agricultural University, Anand and used in present study.

method by SAHN clustering function of NTSYSpc.

Plants were grown in pots and leaf samples of all plants

of each cultivar werecollected in labeled bags and stored

Results and Discussion

in -40°C freezer prior to genomic DNA isolation.

The data collected from random amplification of

Genomic DNA isolation

polymorphic DNA with 20 arbitrary primers produced

179 total loci and the total bands produced were 1942.

Extraction of DNA from seedlings was done using

Out of the 179 loci produced, 123 were polymorphic and

modified Cetyl-Trimethyl Ammonium Bromide (CTAB)

hence the total polymorphism percentage was found to be

method (Murray and Thompson, 1980) with some minor

65.42 %, whereas Malviya et al., (2010) and Choudhury

modifications.

et al., (2008) obtained 80% and 74.7% polymorphism,

respectively. Very low level of polymorphism in cultivated

PCR amplification using RAPD primers

pigeonpeagermplasm had been earlier reported which

Twenty oligonucleotides primers were selected from

was corroborated by many pigeonpea workers indicating

available literature (Table 1). Out of which were

the narrow genetic base existing in this crop (Odeny et

synthesized from MWG biotech, Germany and the rest

al . 2007). The average PIC (Polymorphism Information

were Operon series primers. PCR was carried out in 25μl

content) values for RAPD was 0.84. Average number

reaction volumes containing 2.5 μl of 10 x Taq assay

of loci per primer was found to be 8.95 and average

buffer (Tris with15mM MgCl ₂ ), 10 mM of each dATP,

number of polymorphic loci obtained per primer (Assay

dCTP, dGTP and dTTP, 5U Taq polymerase (BioLabs,

Efficiency Index) was found to be 6.15. The molecular

UK), 10picomole of primer and approx. 50ng of template

size of the amplified PCR products ranged from 185 bp

460