Reversion frequencies of Mu-induced waxy alleles

At last count we had 13 independent Mu-induced mutable waxy alleles (wx-Mum). For the last few years we have been developing wx-Mum stocks that can be used to test the frequency of germinal revertants. To do this we have produced stocks of wx-Mum1, wx-Mum2 and wx-Mum4 that are homozygous for gl1 and y1 as well (contamination markers). This past summer we planted large amounts of these multiply marked stocks in an isolation plot. The wx-Mum lines were used as detasseled females and y1 y1 wx wx gl1 gl1 plants were used as the pollen source. Unfortunately a second isolation plot, in which the plants were heterozygous for y1, wx, and gl1, was planted too close to the wx-Mum plot for complete isolation (results from this block are described in our previous article on Mu activity in the male gametophyte.) By the time it was discovered that the two plots were insufficiently separated, they both had been planted. The wx-Mum block was planted three weeks before the other block and thus, if we were lucky, the wx-Mum block would have completed pollinating before the other block shed. Since contamination from the wx-Mum block would not affect the results of the other block, it was decided to proceed under these less than satisfactory conditions. The degree of contamination, if any, in the wx-Mum block could be determined by the frequency of yellow waxy kernels occurring upon wx-Mum ears.

For the wx-Mum1 allele, 40,485 kernels were scored. Nine starchy kernels and 2 intermediate staining kernels (i.e., stained blue but not as rapidly as expected for a Wx kernel) were found. If the intermediate kernels are counted as revertants, the frequency is 2.72 x 10-4. If the intermediates are excluded, the frequency of revertants would be 2.22 x 10-4. However, 2 yellow wx kernels were observed in this population, which probably were the result of contamination. In the block from which the contaminating pollen came, Y1 wx pollen was produced in the same frequency as y1 Wx pollen. Thus 2 of the 9 revertant kernels could be expected to result from contamination. If indeed there were only 2 contaminants among the starchy seeds, the revertant frequency would be 1.73 x 10-4. A chi-square test to determine if the 9 Wx kernels would all be expected to be contaminants, based on observation of the 2 known contaminants, gave a value of X2 = 4.4545, which is significant at the 5% level. Thus some of the starchy kernels are probably germinal revertants, but the exact frequency is yet to be determined.

The wx-Mum2 allele is quite different from the wx-Mum1 allele both in its phenotype and reversion frequency The somatic mutability pattern of wx-Mum1 is typical of most Mu-induced mutants (i.e., it reverts quite late in the development of a given cell lineage, resulting in revertant sectors of one or just a few cells). The revertant sectors of wx-Mum2 are quite variable in size, ranging from one cell to very large sectors easily visible with the naked eye. This is one of two mutable Mutator mutants with consistent large revertant patterns that we have observed; the other is vp1-Mum2. Dr. Vicki Chandler has seen a similar pattern in a couple of her Mu-induced B-Peru mutants.

The wx-Mum2 allele also differs from wx-Mum1 in that it has a much higher germinal reversion frequency. Out of a population of 642,933 kernels screened, 521 starch kernels were observed, giving a reversion frequency of 8.10 x 10-4. The wx-Mum2 allele also differs from wx-Mum1 in that, with the latter allele, no sectors of revertant kernels were observed on 133 ears scored. However, from the 1,899 ears scored of wx-Mum2, 21 had sectors of revertant seeds, indicating that some revertant events were premeiotic. There were 13 2-seeded, 3 3-seeded, 3 4-seeded, 15-seeded and 1 11-seeded sectors. These counts just represent the starchy revertant kernels observed in these sectors. The starchy kernels, of course, were interspersed with waxy non-revertant seeds. Each sector is the result of one reversion event and thus each was only counted as a single kernel in the above total of 521.

In the wx-Mum2 material, only 4 yellow waxy contaminant kernels were found (frequency = 6.22 x 10-6). If one assumes that 4 of the white starchy kernels were contaminants rather than revertants, the reversion frequency would be 8.04 x 10-4.

All wx-Mum kernels in this isolation plot came from crossing homozygous wx-Mum y1 gl1 plants. All kernels were screened before planting to be sure they were waxy, and all seedlings were checked for glossy in the field. These points are emphasized because in the wx-Mum2 material, 13 ears were observed that were heterozygous for starchy seeds. The frequency of such ears was 6.80 x 10-3 (13/1,912). These ears are expected if the kernels that produced these plants resulted from the functioning of a pollen grain in which a reversion had occurred in one of the sperm but not the other. A kernel in which the egg was fertilized by a sperm with the revertant allele would have a waxy endosperm because the sperm with the non-revertant allele would have had to fertilize the polar fusion nucleus. These heterozygous ears were expected because white starchy kernels (presumably kernels with revertant alleles) were observed on some of the ears used as the source of kernels for the isolation block. For this reason, the seeds planted were carefully screened to be sure no starchy kernels were planted. Seventeen plants from starchy (presumed revertant) kernels, from the ears providing the kernels for the isolation plot, were self-pollinated this past summer. Sixteen of these selfed ears segregated for starchy kernels, as expected, and one was homozygous waxy. The kernel that gave rise to this latter plant was probably the result of a fertilization involving a pollen grain with one wx-Mum2 sperm, which fertilized the egg, and one revertant sperm, which fertilized the polar fusion nucleus. This is the fertilization pattern reciprocal to that suggested to be responsible for the heterozygous starchy ears found in the isolation plot.

The wx-Mum4 block yielded 2 starchy revertant seeds but 2 yellow waxy contaminant seeds were also found; thus, nothing can be said at this time about the reversion frequency of this allele.

No starchy revertant or yellow waxy contaminant kernels were found on the ears of the y1 y1 wx wx gl1 gl1 plants of the pollen rows, which also served as controls for this experiment. There Were 171,277 kernels in these rows and since each kernel is the product of 2 y1 wx gl1 gametes this number of kernels is equivalent to testing 342,554 gametes.

The reversion frequencies for both wx-Mum1 and wx-Mum2 are minimal values because it is known that there were kernels with different levels of somatic mutability on the ears that provided the kernels for this test. Many of these ears had some stable derivative kernels as well. Thus all plants would not be expected to have the same frequency of reversion and some, those derived from stable kernels, might possibly have no reversions. A few kernels from all ears used as a source of kernels for the isolation plot were scored for mutability and only ears segregating mutable kernels were used. However, it was not practical to score each kernel planted or all the ears harvested for levels of mutability.

Donald S. Robertson and Philip S. Stinard

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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