University of Florida

ATHENS, GEORGIA

University of Georgia

EMS-induced waxy alleles: treatment of pollen versus seed

--Ron J. Okagaki, Melanie Trull and Susan R. Wessler

The chemical mutagen ethylmethane sulfonate, EMS, is known to produce single base substitutions in a two step process. The O-6 position of guanine is first ethylated; then during DNA replication the ethylated nucleotide pairs with thymine. However, the characterization of EMS-induced mutations at the waxy (wx) locus indicates that EMS produces other lesions (Genetics 128:425-431, 1991). Large structural alterations were found in or near two EMS-induced wx alleles, wx-1240 and wx-1050, by Southern blot analysis. Cloning of wx-1240 revealed a small deletion within the gene and at least 3 additional changes upstream of the locus. Current work is focused on identifying the lesions in wx-1050 and other EMS-induced alleles.

Genetic fine structure mapping is being employed to determine the location of the mutation within wx-1050. wx-1050 was crossed with the seven wx alleles used to map the lesion in wx-1240. Pollen from the heterozygous double mutant plants has been collected, stained with iodine, and examined. Two plants from each cross were sampled, and at least 100,000 pollen grains per plant were classified. It was difficult to score the pollen, because the wx-1050 allele is slightly leaky. Nevertheless, recombinant, Wx, pollen was found in heterozygotes between wx-1050 and all wx alleles except wx-M and possibly wx-Stonor. This places the genetic lesion responsible for wx-1050 in a region on the physical map bounded by wx-B1 and wx-Stonor. This region will be cloned from wx-1050 and its progenitor allele by PCR and sequenced.

EMS-induced wx alleles isolated by E. Amano, E have been characterized by Southern blot analysis. These alleles were induced by treating mature seeds with EMS. In contrast wx-1240 and wx-1050, obtained from M. G. Neuffer, MG, were generated by treating pollen. DNA was isolated from the progenitor Wx allele and eight mutant alleles. Samples were digested with SalI, fractionated in 1% agarose gels, and blotted onto GeneScreen Plus. Filters were sequentially probed with four subclones that cover the Wx locus. Seven of the wx alleles were indistinguishable from the progenitor allele. The remaining allele had a single change that could be accounted for by the loss of a SalI restriction enzyme site. Analysis of these eight alleles suggests that point mutations were produced by EMS in this collection of alleles. Genetic fine structure mapping followed by PCR and sequencing will be used to identify the lesions in several of these alleles.

The interesting observation from this work is the difference between treating mature seeds with EMS and treating pollen. A trivial explanation for this result would be that wx-1240 and wx-1050 represent unusual events; this will be resolved by examining more alleles induced by pollen treatment. Additional mutant alleles obtained that were produced in this manner include a wx allele from M. G. Neuffer, MG, several c2 alleles from M. G. Neuffer and E. Coe, EH, and five R-catspaw alleles from J. Kermicle, J. The second possibility is that pollen treatment produces complex lesions at a high frequency. This possibility deserves consideration because DNA replication, normally a necessary step for EMS mutagenesis, is delayed in pollen until after fertilization. This may give DNA repair mechanisms sufficient time to replace alkylated bases. Therefore, these mutations would be produced by another mechanism perhaps through cleavage of the DNA backbone. It is also possible that the DNA in pollen may be particularly susceptible to damage by EMS. If either of the latter two possibilities is correct, then characterizing pollen derived alleles may describe a spectrum of EMS-induced lesions produced by unanticipated mechanisms.

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