Molecular cloning of o2-m5

The Opaque-2 (O2) locus of maize represents one of the repertoire of regulatory loci affecting the accumulation of zein polypeptides in developing kernels. Because the molecular cloning of the O2 locus and the analysis of its structure and expression may contribute to shed light on the nature of its regulatory effects, we developed an indirect strategy for cloning the O2 locus based on its association with the transposable element Ac. In order to induce instability at the O2 locus in the wx-m7 strain two experiments were performed in the summer nursery in 1985 and 1986 by crossing homozygous wx-m7 plants to a stable o2 tester line. Approximately 760,000 (1985) and 250,000 (1986) F1 kernels were obtained by hand-pollination. In the two experiments 451 and 235 kernels showing somatic instability at the O2 locus (i.e., with vitreous and opaque sectors) were identified. These kernels were sown and the resulting plants selfed. The majority of these plants segregated normal and opaque seeds in a 3:1 ratio, suggesting that the somatic instability observed in the F1 endosperms was not inherited. In fourteen cases, however, the variegated phenotype was transmitted to the next generation. The frequency of induction of mutable alleles at this locus was 1.4 x 10-5, a value consistent with that of I X 10-5 reported for the induction of unstable mutants by maize transposable elements. Genetic analysis of one of these alleles, i.e. o2-m5 showed that it contains an autonomous Ac element. For the cloning of o2 -m5 allele, we used a central segment of Ac as a probe. We identified two cloned fragments that contained a sequence that resembles the Ac element in structure. A DNA sequence flanking the autonomous Ac insertion was found to be o2 specific and provided a probe for the molecular analysis of the O2 locus.

F. Salamini, M. Motto, G. Ponziani, M. Maddaloni, C. Soave, N. Di Fonzo, R. Thompson and R. Marotta

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