Fig. 1. Genomic Southern blot analysis of mutant genes at the wx locus of rice using the maize waxy gene as probe.
1) Institute of Radiation Breeding, Omiya-machi, Ibaraki-ken, 319-22 Japan
2) National Institute of Aerobiological Resources, Kannondai, Tsukuba City, 305 Japan
The waxy gene is a structural gene coding for glucosyl transferase which catalyses the synthesis of amylose in the endosperm of rice. There are at least three alleles among rice cultivars, two dominant (active) alleles Wxa and Wxb and a recessive (inactive) allele wx (Sano 1984). Amylose contents of 16 to 25% are controlled by the dominant alleles.
Mutant genes have been obtained at this locus, which bring about a range of amylose content from zero to the level of non-glutinous cultivars. Mutants with no amylose (100% amylopectins) are glutinous, while others may be called "leaky glutinous".
To examine alteration to DNA structure of these mutant genes, their DNAs were analyzed by Southern hybridization using pWX5-8, a cloned DNA of maize waxy gene (Shure et al. 1983) as the probe. Genomic Southern blot analysis for EcoRI digestion is shown in Fig. 1. A major band appeared at about 5.5 kb. Only a single hybridization band appeared even after digestion with BAMHI, BgIII, EcoRV, or Sal1. Among cultivars examined, the dominant (Wx) and recessive (wx) genes showed the same banding pattern and the same intensity of hybridization. These observations altogether suggest that both active (Wx) and inactive (wx) genes of rice had sequences homologous to that of the maize waxy gene, and that only a single gene copy exists for the waxy locus.
Fig. 1. Genomic Southern blot analysis of mutant genes at the wx locus
of rice using the maize waxy gene as probe.
Most of the wx mutants which were generated by chemical mutagen or gam- ma-ray irradiation, gave the same major band as the wild type, even though their phenotypes were glutinous or "leaky." One exception was KURwx4N1 induced by thermal neutron, which lacked the major band (Fig. 1). Another mutant 78KURwx2, induced by thermal neutron, expressed a "leaky " phenotype and showed an additional band with a high molecular weight beside the 5.5 kb band.
The Southern blot analysis we attempted was not so successful in detecting differences in the wx locus. There may be two possibilities. One is that the structural alterations of induced mutant genes are too small to be detected by this method. The other is that DNA alterations induced by mutagenesis exist outside the coding region.
References
Sano, Y., 1984. Differential regulation of waxy gene expression in rice endosperm. Theor. Appl. Genet. 68: 467-473.
Shure, M., S. Wesseler and N. Fedoroff, 1983. Molecular identification and isolation of the waxy locus in maize. Cell 35: 225-233.