| Results and Discussion Unlabelled and 3H-labelled DNA's were isolated from sterile embryo cultures of common wheat cv. Chinese Spring, rye cv. Prolific and diploid A. elongatum according to a method described by DVORAK (1972a). DNA Base Composition of Wheat, Rye and Agropyron The mole percent of guanine plus cytosine (GC) of wheat, rye and Agropyron DNA's was determined from the thermal dissociation profiles in 0.1 SSC buffer (1 SSC is 0.15 M NaCl and 0.015 M Na citrate pH 7.0). The relative GC content was calculated from the midpoints of thermal dissociation profiles (Tm) according to a relationship proposed by SCHILDKRAUT et al. (1962). The base composition of A. elongatum DNA was also determined by equilibrium centrifugation in a CsCl density gradient. Its buoyant density was found to be 1.699 g/cm3 which, according to the reltionship of density to mole percent GC proposed by MANDEL et al. (1970) and SCHILDKRAUT et al. (1962), corresponds to a GC content of 40.4%. The absence of significant differences among the Tm's of wheat, rye and Agropyron DNA's (Table 1) indicates that the relative base composition of the DNA of wheat, rye and Agropyron has been stable during evolution. Hybridization among Wheat, Rye and Agropyron DNA's The homology of nucleotide sequences among wheat, rye and Agropyron DNA's was studied by DNA-DNA hybridization which was carried out according to a procedure described by GILLESPIE and SPIEGELMAN (1964). Twenty-seven microg of unlabelled, single-stranded wheat or rye or Agropyron DNA was immobilized on nitrocellulose filters (Schleicher and Schuell Co., B-6, 25 mm in diameter). The filters with immobilized DNA were incubated with 0.7 ml of solution containing 5 microg/ml of either homologous (same species) or heterologous (different species), single-stranded, sheared, 3H-labelled DNA in 1 SSC and 50% formamide at 43C. The selectivity of the reassociation of DNA from solution with filter-immobilized DNA decreases with an increase in the concentration of Na+ in the solution. However, it increases with an increase in temperature above the point at which the maximum rate of reassociation occurs. The use of a formamide solution makes it possible to carry out DNA-DNA hybridization at lower temperatures (BONNER et al., 1967; MCCONAUGHY et al., 1969) with increased selectivity. In a preliminary experiment it was found that the maximum rate of reassociation of wheat single-stranded DNA with filter-immobilized Triticale DNA occurred in 1 SSC and 50% formamide at 38C. The temperature chosen (43C) for the following DNA- DNA hybridization experiments makes the DNA reassociation highly selective, yielding only well matched DNA/DNA duplexes. One estimation of the relatedness of nucleotide sequences among the DNA's studied was based on the amount of reassociation between heterologous DNA's as compared to that between homologous DNA's. The less related the nucleotide sequences of heterologous DNA's are, the less reassociation is expected to occur between 3H-labelled DNA in solution and filter-immobilized DNA compared to the control in which both DNA's are from the same source. |
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