Molecular analysis of unstable alleles at the o2 locus
--Detlef Michel, Francesco Salamini, Mario Motto and Hans-Peter Döring

Transposon tagging, i.e., insertional mutagenesis using transposons, has proved to be a powerful technique for the isolation of maize genes. We have recently used the transposable element Ac to tag and clone the opaque2 (o2) gene, a positive trans-acting transcriptional activator of zein gene expression. The source of the Ac system of controlling element was a strain bearing the wx-m7 mutation. Homozygous plants from variegated wx-m7 kernels were crossed to a stable o2 tester line according to the following mating scheme: wx-m7 Ac O2 x Wx o2 R.

Among a population of about 1,000,000 kernels, variegated F1 kernels, i.e. kernels showing vitreous and opaque sectors, were selected from mature ears. Plants originating from these kernels were selfed to confirm the presence of variegated phenotypes in the F2 generation. In eight cases the variegated phenotype observed in the F1 kernels was heritable, giving rise to the mutable alleles termed o2-m5 to o2-m15. The frequency of induction of mutable alleles at the o2 locus was approximately 1.2 x 10-5, a value consistent with that reported for the induction of mutable alleles by maize transposable elements (Döring, Maydica 34:73-88, 1989). One of these mutations, the o2-m5 allele, was caused by the insertion of an Ac element into the o2 locus (Motto et al., Mol. Gen. Genet. 212:488-494, 1988). To establish if the remaining 7 unstable mutable o2 alleles (o2-m7, 8, 9, 10, 11, 12, and 13) were also generated by Ac elements, plants derived from o2 mutable kernels were crossed to a C-I wx Ds tester strain to evaluate the presence of endosperm variegation due to chromosome breakage (BFB cycle) and crossed to an o2 tester line. The presence of variegated o2 kernels did not induce Ds breakage due to an Ac element. These results excluded the presence of a functionally active Ac element in the autonomously controlled mutable alleles o2-m7, 8, 9, 10, 11, 12, 13.

The seven unstable o2 alleles were subsequently examined by Southern restriction mapping for the presence of insertions. No Ac or Ac-like elements were found. However, we found in six mutable alleles an insert which has a restriction map identical to the known map of the Spm/En element. In one allele we found an insertion which is very similar though not identical to an autonomous Bg element. The insertion points of the elements are distributed over the whole o2 locus. This finding indicates that the wx-m7 strain which was used for tagging mutagenesis carried not only an active Ac element, but also active Spm/En and Bg elements. A similar observation was also reported by Patterson and coworkers (Genetics 127:205-220, 1991) who used maize strains with active Mutator elements for transposon mutagenesis, but found two mutable alleles of the B locus which were caused by Spm-related insertions. 

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