Seventeen accessions of bread wheat (T. aestivum) and ten of durum wheat (T. turgidum) were obtained from National Bureau of Plant Genetic Resources (NBPGR), New Delhi, used for the present study (Table 1). DNA was isolated from 8 days old seedlings using a method of Dellaporta et al. (1983) with the modification of replacing TE and T50E10by 10 mM Tris-HCl.

PCR amplification was performed at 10 ng/mul, 20 ng/mul and 50 ng/mul with primer UBC-350 and UBC- 386, and accessions IC-82198 and IC-82199 combinations to optimize the concentration of template DNA- PCR amplification was performed in a volume of 25 mul volume containing 200 muM dNTPs, 11.25 unit Taq DNA polymerase (Bangalore Genei Limited, India), 10 ng primers, 1.5 mM of MgCl2 and 10x PCR assay buffer (supplied by the manufacturer of Taq polymerase) at a final concentration of 1x.
A set of ten decanucleotide RAPD primers was used for PCR amplification. The sequences of these primers were selected from literature (Table 2). The mixture was gently mixed and centrifuged for 10 seconds. The PCR amplification was conducted in Biometra thermal cycler programmed as initial denaturation at 94° C for 5 min; remaining 45 cycles with 94° C denaturation, 35° C annealing and 72° C polymerization temperature. Final extension was given at 72° C for 5 min.

Amplification products obtained were electrophoresed on 1.5% agarose gel (Sambrook et al. 1989). The gel image was viewed and stored in gel documentation system. Photographs from ethidium bromide stained gel were used to score the data manually and independently for RAPD analysis. Presence of amplified product was scored as 1 and its absence as 0. These data matrices were then entered into NTSYS-PC (numerical taxonomy and multivariate analysis system program) (Rohlf 1992). The data were analyzed by SIMQUAL (similarity for qualitative data) routine to generate Jaccard's similarity coefficients (Sokal and Sneath 1963). The similarity coefficients were then used to construct dendrogram using the UPGMA (unweighted pair-group method with arithmetic average).

Results and discussion

Optimization of template DNA concentration was done and 20 ng/mul concentration was found optimum with primer UBC-350 and UBC-386 and accessions IC-82198 and IC-82199 combination, respectively.

PCR amplification of DNA extracted from 27 genotypes was performed following the same protocol for all the ten primers. The number of RAPDs generated by the ten primers with 27 genotypes is presented in Table 3. The total number of bands amplified from the ten polymorphic primers was 103. This gave an average of 10.3 bands per primer. Out of the 103 bands, 82 (79.6%) were polymorphic. The size of the amplified products ranged between 0.3 to 3.0 kb. This gave an average of 8.2 polymorphic and 2.1 monomorphic bands per primer. The highest number of polymorphic bands was obtained with primer UBC-535 and the lowest with UBC-532. The primers UBC-18, UBC-337, UBC-534, UBC-535, UBC-572 and UBC-600 were found to give distinguishable number of unique RAPD markers (1, 1, 2, 4, 1 and 1 bands, respectively).

The RAPD amplification within 17 hexaploid wheat genotypes resulted in 98 amplified products. Of the total amplified products, 64 (65.3%) were polymorphic. Of the polymorphic hexaploid bands, the highest number of bands was obtained by primers UBC-535, and UBC-552, while the lowest by UBC-532. The size of amplified hexaploid products ranged between 0.3 and 3.0 kb. This gave an average of 6.4 polymorphic and 3.4 monomorphic bands per primer.

RAPD amplification with 10 tetraploid wheat genotypes showed 103 amplified products, of which 78 (75.7%) were polymorphic. The highest number for polymorphic bands was obtained by primer UBC-535 and UBC-552 (Fig. 1), while the lowest by primer UBC-532. Ten primers gave an average of 7.8 polymorphic and 2.5 monomorphic bands per primer. Association among the 27 genotypes revealed by UPGMA cluster analysis is presented in Fig. 2. The dendrogram readily separated the 27 genotypes into two clusters (duster I and cluster II). Cluster I comprised of 2 sub-clusters, of which sub-cluster I consisted of 6 hexaploid accessions with similarity coefficient of approximately 0.75. In this sub-cluster IC-82199 and IC-82202 showed higher genetic similarity followed by accessions IC-78754 and IC-78868. Sub-cluster II could be classified into 2 groups. Group I was further divided into 2 sub-groups of hexaploid wheat accessions having 3 and 8 accessions per sub-group. Group II included 7 tetraploid wheat accessions and in turn it could be divided into 2 subgroups having 2 and 5 accessions per sub-group, respectively. Cluster II comprised of 3 unique tetraploid wheat accessions and could be divided into 2 sub-clusters. Accessions IC-35720-D is in one sub-duster and the other two accessions IC-35177-D and IC-35161-D are in the other cluster.

Data of RAPD markers scanned from the 27 genotypes of wheat with 10 RAPD primers was used to generate similarity coefficients. The similarity coefficient among hexaploids and tetraploids ranged from 0.361 and 0.828. The two accessions of tetraploid wheat (IC-35107-D and IC-35144-D) were highly related as indicated by high value of similarity coefficient (0.966), followed by two other accessions of hexaploid wheat (IC-82280 and IC- 82526) with similarity coefficient of 0.952. Accession IC-35177-D (tetraploid) and IC-78868 (hexaploid) were highly unrelated having a low value of similarity coefficient (0.361). The similarity coefficient among hexaploid wheat genotypes ranged from 0.630 to 0.952, whereas among tetraploids it ranged from 0.400 to 0.966. It clearly showed that tetraploid genotypes had more wider genetic distance value than hexaploid genotypes with relatively narrow genetic distance value.