Molecular and functional analyses of the induced mutable gene slg in rice.

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A mutable slender glume gene slg was induced by gamma-ray irradiation to seeds of the rice variety Gimbozu. Occasionally reverting to its wild-type, slg is assumed to involve an active transposable element in it. To identify the molecular characteristics of this element, we attempted to clone slg with a positional cloning method. slg has already been mapped on the overlapping region of two YAC clones, Y1774 and Y3356. We first selected 209 candidate clones from the cDNA clone library of the rice variety Nipponbare using a YAC clone Y1774 as a probe, and then partially sequenced 54 clones. Based on the sequence data, 40 clones were found to be independent of each other. Using these 40 clones as probes, we finally selected out cDNA clones that hybridized with both YAC clones. But many of them were hybridized not only with both YAC clones but also with multiple regions of genomic DNA, indicating the occurrence of non-specific hybridization. Thus, it was difficult to identify the clones other than No.27 that was really located on both YAC clones. Southern analyses were also conducted to find out polymorphic bands among the original variety, mutant line (slg) and revertant (wild-type). Consequently, polymorphic bands were observed when using clone No.120, suggesting that this clone is tightly linked with slg locus. The effect of slg on the genomic methylation was also investigated because there was a possibility that the mutability of slg was caused by DNA methylation. The mutant line was significantly lower than the original variety in the relative amount of methylcytosine to total cytosine on MspI site. This indicates that slg may have a decreasing effect on the genomic methylation, resulting in the mutability of slg.