Early sectoring and germinal reversion in a Mutator line with the bz1-mu1 reporter allele

--Virginia Walbot

In MNL 64:107 I reported the isolation of a line carrying the bz1-mu1 reporter gene, an allele with a Mu1 insertion, that exhibits two properties atypical of Mutator lines: early sectoring and an increased germinal reversion rate. The early excision stock was grown in an isolation plot in 1990, and 750 ears have been analyzed from this population thus far. Germinal reversion is ~0.1%, occurring almost exclusively as single kernel events. Only a few ear sectors have been recovered: one encompassed half the ear, and the others contained only a few kernels. The events fit a Poisson distribution suggesting that reversion events are independent. This is consistent with the hypothesis that the majority of the events occur through the pollen. This has been confirmed in 1991 by reciprocal crosses between a bz1 deletion stock (gift of Hugo Dooner, HK) and the bz1-mu1 early excision line. Approximately 90% of the revertants recovered were from the pollen parent. Interestingly a similar frequency of germinal reversion (0.1%) was observed in a population of plants grown from bz1-mu1 kernels that had a large Bz1 sector (1/8 to 1/2 of the kernel) and from a population of kernels with the standard spotting pattern from the same parental ears. This suggests that genetic elements required for both large spotting (10-4 frequency of half-kernel sectors) and germinal reversion (10-3) are present in kernels with either the standard or the big spot phenotype.

To test the timing of germinal reversion, Bz1' germinal revertants from the open-pollination experiment were planted in 1991 and pollinated by the bz1 deletion stock; all 90 resulting ears transmitted the purple phenotype, indicating concordance between endosperm phenotype and embryo genotype. Consequently, Mu1 excision occurs before the second mitotic division in microgametogenesis. In examining a small population of early excision bz1-mu1 with R-r present, I found no sectors as large as an entire anther. In fact, the excision pattern of tiny dots appeared to be the same as found in the standard bz1-mu1 line in both the anthers and in the plant body (B pl). These data suggest that reversion is very late in the sporophyte and hence typical of standard Mutator lines.

What is different about the big spot line is that excision occurs at a higher frequency during microgametogenesis and that this behavior carries over into the first few mitotic divisions in the aleurone to produce some half, quarter and eighth kernel sectors. It is noteworthy that the next size classes of sectors are one to two orders of magnitude less frequent, until after cell division 10. In both standard and the bz1-mu1 big spot line Mutator is "activated" at this stage, producing a high frequency (up to the percent range) of sectors containing 1 - 128 cells.

At the molecular level, both the early excision and standard bz1-mu1 lines contain >50 copies of Mu1 and >20 copies of Mu9, the putative regulatory element for the Mutator family (Hershberger, RJ et al., PNAS 88:10198, 1991). The bz1-mu1 allele appears unchanged. Tests are in progress to determine whether early sectoring is dominant or recessive by crossing this stock to Mutator lines carrying other mutable alleles.

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