A dominant resistance gene for powdery mildew has been transferred from H. bulbosum into cultivated barley (Xu and Kasha, 1992). Plants with the gene show a hypersensitive resistance reaction to mildew isolates with virulence against resistance genes Ml-h and Ml-h + Ml-al and to a mixture of isolates. To assist in the incorporation of the gene, we screened for RAPD markers that might be linked to the resistance gene. Once established, such molecular markers would accelerate moving, mapping, or molecular cloning of the transferred H. bulbosum gene.
Genomic DNA for the amplification was extracted from leaf disks using the techniques of Edwards et al. (1991). One hundred sixty-one 10-nucleotide random primers (from J. Carlson, Biotechnology Laboratory, University of British Columbia) were tested in DNA pooled from mildew-resistant vs -susceptible BC2 plants derived from the interspecific cross of H. vulgare (2x) × H. bulbosum (4x). DNA bulked from 5, 10, or 15 plants-produced no difference in amplification patterns using the primer TGC CCC GAG C. The DNA pooled from 10 or 15 plants was routinely used in the experiment. The five-hour RAPD program of Williams et al. (1990) and a 2.5-hour program as modified by Yu and Pauls (1992) were initially tested using three primers TTA ACC CGG C, TGC CCC GAG C, and ATC GGG TCC G at concentrations of 0.24 µM, 0.48 µM, and 0.72 µM. The conclusions were: 1. Both programs produced identical RAPD patterns except that the longer program gave rise to stronger bands than shorter one. 2. A primer concentration of 0.24 µM produced fewer bands than concentrations of 0.48 µM and 0.72 µM. These additional bands were relatively small in size. The same RAPD pattern was amplified using 0.48 µM and 0.72 µM, indicating that 0.48 µM of primer was sufficient to saturate priming sites in the genome.
Of 161 primers tested, 37.3% produced either no amplification (19.9%, 32/161) or unscoreable products (17.4%, 28/161) seen as a smear or very weak bands. One hundred one (62.7%) of 161 primers produced scoreable amplification products ranging from 0.5 to 3.0 kb in size. The bands resolved in 1.5% agarose gels and stained with ethidium bromide vary in intensity and in number (1-13) with 7-9 bands being most frequent for the primers (40.6%) (Table 1). Seventeen (16.8%) of the 101 useful primers produced bands that were polymorphic between the DNA pools from resistance and susceptible plants. However, the polymorphism was not repeatable in further tests using either pooled or DNA from individual plants. The polymorphic bands, strong or weak, were either invisible or seen in both amplifications. No RAPD bands linked to the transferred resistance gene have been identified to-date because of the instability of the putative polymorphic bands.
While there are reports on successful application of RAPD markers (Williams et al., 1990) and more recently, bulked segregant analysis (Michelmore et al., 1991), there have also been concerns about repeatability of the molecular polymorphism at least in barley under the conditions used (Kleinhofs, personal communication). Among the possible reasons for the instability, one is the large number of bands. The observation that 78.2% of the useful primers produced more than four bands and that the bands differ in brightness in the gel implies that there are multiple priming sites present in the genome. These are likely to vary in homology to the primers. Imperfect or non-specific priming may contribute to the poor repeatability. Likely solutions to the problems include increasing hybridization stringency and the use of longer primers.
Edwards, K., C. Johnstone, and C. Thompson. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19-1349
Michelmore, R. W., I. Paran, and R.V. Kesseli. 1991. Identification of markers linked to disease-resistance gene by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA Vol. 88:9828-9832.
Williams, J. G. K., A. R. Kubelic, K. J. Livak, J. A. Rafalski, and S. V. Tingeny. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18(22):6531 -6535.
Xu, J., and K. J. Kasha. 1992. Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare). Theor. Appl. Genet. (In press).
Yu, K., and K. P. Pauls. 1992. Optimization of the PCR program for RAPD analysis. Nucleic Acids Res. 20:2606.