A newly isolated unstable mutation wx-m32 was proven by genetic analysis to be caused by the insertion of the Bg autonomous transposable element into the waxy (Wx) gene. To identify the molecular structure of the wx-m32 allele, genomic Southern blots were performed using plant DNA extracted from a heterozygous wx-m32/wx plant and from the parental lines (A69Y A C R wx and A69Y o2 R Bg-m Wx). The DNAs were digested with several restriction enzymes and investigated at the molecular level by blot hybridization to Wx and Bg probes. The molecular probe of the Wx gene was a 2.0kb SalI fragment corresponding to the central region of the Wx gene, while as Bg probe we used a 4.0kb XhoI-EcoRI fragment corresponding to the Bg receptor of the o2-m(r) allele (Thompson et al., unpublished).
In the hybridization experiment shown in Fig. 1A, PvuI digested genomic DNAs were hybridized with the 2.0kb SalI Wx probe. It was clearly evident that the progenitor Wx allele and the stable recessive wx allele contain, respectively, a 3.1 and 5.8kb PvuI fragment. The wx-m32 allele, however, contains, in addition to the 5.8kb PvuI fragment of the stable wx allele, a new fragment of ~ 8.8kb in size, indicating an insertion of approximately 5.7kb into the 3.1kb PvuI fragment of the Wx wild-type allele. Moreover, in the same lane of the wx-m32 allele, a less intense band was visible, similar in size to the 3.1kb PvuI fragment of the Wx wild-type allele. This result can be explained considering that, if somatic reversion events take place, induced by the autonomous Bg element present at the locus, they should restore the wild-type fragment size of the parental Wx allele after excision of Bg. Because plant DNA of the wx-m32 allele was prepared from young leaves, we concluded that Bg activity is not restricted to the endosperm tissue.
When the same PvuI digests were hybridized with the Bg probe (Fig. 1B) it was clearly evident that a unique restriction fragment could be correlated with the mutable phenotype of the wx-m32 allele. This mutable allele contained a novel 8.8kb PvuI band absent in the parental lines. The band migrated in the gel at the same position as that of the PvuI fragment of 8.8kb lit up by the Wx probe. To prove whether the insertion is entirely contained in this PvuI fragment, digested genomic DNAs of the previous genotypes were also hybridized with different probes of the 5' and 3' region of the Wx locus. The resulting data revealed no apparent alteration of the expected 5' and 3' PvuI fragments. Therefore, the insertion is located in the PvuI fragment and its size is 5.7kb. The screening of a genomic library from the wx-m32 allele is now underway.
Figure 1. Southern blot analysis of the wx-m32 allele. Genomic DNAs, prepared from the leaves of plants with the genotypes indicated above each lane, were digested with PvuI. Each DNA sample (~12ug) was divided in two parts, electrophoresed through an 0.8% agarose gel in parallel experiments, Southern blotted and probed with the 2.0kb SalI, Wx probe (A) and Bg probe (B). Sizes are shown in kb.
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