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Materials and methods

Plant materials
Seven bread wheat genotypes differing for protein content, leaf rust resistance, grain size and pre-harvest sprouting tolerance were used in the present study ( Table 1).

DNA isolation
DNA was isolated from young leaves collected from 30 day old field grown plants using a modified CTAB method (Weising et al. 1995).

DAF primers
A total of 20 DAF primers (prCured from G. Caetano-Anolles and P.M. Gresshoff, University of Tennessee, USA) including 10 unstructured (linear) (Table 1) and 10 mini-hairpin primers were used for DNA amplification ( Table 2).

DNA amplification and electrophoresis
DNA amplification was performed in 20 microliter volume containing 3 microM primer, 0.15U/microliter (in case of linear primers) and 0.2U/microliter (in case of mini-hairpin primers) of Taq DNA polymerase (Bangalore Genei Limited, India), 0.2 ng/microliter template DNA, 200 microM each of dNTPs, 1.5 mM Of MgCl2 and 10x PCR buffer (supplied by the manufacturer of Taq polymerase) at a final concentration of 1x. Amplification was conducted in a Perkin-Elmer DNA Thermal Cycler for 35 cycles using temperature profiles of 96C for 30 sec, 30C for 30 sec and 72C for 30 see with a ramp time of 3 min from 30C to 72C in case of linear primers. In case of mini-hairpin primers, the temperature profiles included 95C for 30 sec, 55C for 2 min and 72C for 30 sec. The final extension in both the cases was 5 min at 72C. Amplification products obtained with linear primers were electrophoresed on 7% polyacrylamide-7M urea denaturing gel applying 7.5 V/cm for 12 h while amplification products primed with mini-hairpin primers were resolved on 10% polyacrylamide supergel (Gresshoff et al. 1997). Silver staining of gel was done following Tegelstrom (1992).

Evaluation of fragment patterns
Amplified fragments in each primer-genotype combination were scored in two ways. Firstly, for each primer, the number of polymorphic fragments included all those fragments which were absent in at least one genotype. Secondly, polymorphism was examined between each pair of the genotypes having contrasting phenotypes for a single trait (e.g. protein content, leaf rust resistance, etc.). Polymorphism in these cases was scored on the basis of presence or absence of fragments between the two genotypes. The amplification fragment(s) present in only one genotype and absent in the remaining genotypes was classified as unique band(s).


Results and discussion

Nine out of ten linear primers tested using seven genotypes of bread wheat, gave characteristic fingerprinting patterns. The DAF profiles contained 20-35 scorable bands (<2kb) in all the seven bread wheat genotypes examined (Fig. 1, Table 1). These results were utilized for a comparison of the DAF profiles with the GC/AT ratio in the sequences of the corresponding DAF primers. For this purpose, it may be noted from Table 1, that in five primers the GC/AT ratio is 5: 3, in three other primers, the ratio is 6: 2, in one primer, the ratio is 7: 1, while the remaining one primer has a GC/AT ratio of 8: 0. When these GC/AT ratios are compared with DAF profiles, it becomes apparent that the primers with low GC content gave better DAF profiles with distinct bands and low background smear (Fig. 1). For instance, the primer 8-47 with all GC gave smear with faint non-scorable bands. The average number of DAF products (25.4 to 34.7) with primers having 6: 2 and 7: 1 GC/AT ratio was higher in comparison to that (22.0 to 27.2) obtained with the primer having 5 : 3 GC/AT ratio (Table 1). However, the utility of a primer depends not so much on number of distinct products it gives, but on the degree of polymorphism, it detects. In this connection, in general, the primer with 5: 3 GC/AT ratio proved to be more useful giving more polymorphic DAF products than the remaining primers with high GC ratios (Table 1). For instance, the primers 8-8, 8-10, 8-4 and 8-5 having 5 : 3 GC/AT ratio respectively gave 21, 15, 14 and 13 polymorphic products. Out of the remaining primers with high GC content, only one primer (830) gave comparable number of polymorphic DAF products (Table 1 ). Therefore, with the limited number of linear primers used in this study, it may be concluded that the DAF primers with low GC content (60%) maybe more suitable for revealing polymorphism in bread wheat.

The above results differ from some earlier reports, which suggested that the GC content of the primer had no correlation with either the amplification itself or with the total number of distinct DAF products obtained or with the frequency of polymorphism detected (Caetano-Anolles and Gresshoff 1994; Prabhu and Gresshoff 1994). However, the present results do support another earlier study involving Staphylococcus aureus, soybean and Caucasian human (Caetano-Anolles et al. 1991), where it was suggested that the number of DAF products depended both on the genome size of the species being examined and on the GC content of the primer used. In the present study, it is possible that a large genome of bread wheat and 100% GC content of the primer 8-47 contributed to the smear obtained. A few unique bands (1-5 per primer) were obtained in one or the other genotype tested with all the primers except with the primer 8- 36 (Table 1). These specific bands may help in cultivar identification.

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