Many mutable aleurone mutants induced by Mu have been obtained in our research program. These have included the loci of a1, a2, c2, bz1. To date, the a1-Mum mutants (Mu-induced mutable a1 mutants) that have been studied in the greatest detail are a1-Mum1, a1-Mum2 and a1-Mum3. These mutants all have the same pattern of mutability in that only small (late) revertant spots are observed. Considerable variation, however, in the intensity of spotting has been observed in both outcrosses and selfs of these mutants, varying from very intensely mutable to stable mutant phenotypes. The following scale is used in classifying seeds for intensity of mutability: Class 1 = stable, Class 2 = low mutability, Class 3 = medium mutability, Class 4 = high mutability and Class 5 = stable purple.
In the analysis of most of the early crosses of these mutable mutants, no discernible Mendelian ratios could be observed. However, as outcrossing continued with these stocks seemingly Mendelian ratios occasionally were found. In the 1985-86 nursery, three outcross ears (two of a1-Mum2, and one of a1-Mum-3), which segregated for putative 1:1 ratios in outcrosses to a1 sh2 tester, were selected for further analysis. The percentages of stable seeds observed among the a1-Mum seeds on these ears were 47.5 1 % (n = 301), 50.00% (n = 60), and 47.75% (n = 222). The first two crosses involved a1-Mum2 and the latter was an a1-Mum,3 cross.
Stable a1-Mum (= a1-Mum -stable, i.e., a stable derivative of a mutable a1-Mum mutant) seeds from these outcross ears were sown and the resulting plants crossed with plants from non-a1-Mum sibling seeds from the same ears. If the 1:1 ratios are the result of the segregation of a single dominant controlling element (a regulator element), half of these crosses should result in ears that again give 1:1 ratios and the other half would have only stable seeds. One of these crosses produced three ears, two of which gave mainly stable seeds or an occasional seed with a few spots. One ear, however, had 49 stable plus Class 2 seeds (53.26%) and 43 mutable (33 Class 2 and 10 Class 4). The second cross resulted in six ears that had only stable or stable and few low mutable seeds and two ears that segregated for mutability and stable. One of the latter two ears had 30 stable plus low mutables (14.78%) to 173 medium and high mutable. The second segregated for 32 stable plus low mutable (48.48%) and 34 medium plus high mutable. From the third cross only two ears were recovered, one segregated primarily stable seeds and a few seeds with an occasional spot while the other segregated 1:1 for stable versus mutable (i.e., 69 stable and low mutable (43.95%) and 88 medium and high mutable).
In 1986, mutable and stable seeds from these winter nursery outcross ears were sown. The plants from the mutable seeds were outcrossed to standard a1 sh2 stocks that had never been crossed with Mutator. The plants from the stable seeds were selfed.
Three plants from Class 4 seeds of the winter ear with 14.78% stables were crossed to a1 sh. One of the outcross ears had a low frequency of stables (i.e. 6.84%) like the ear from which it came. The second cross gave predominantly stable and low mutables (83.58%). However, if the low mutables are included with the medium and high mutables the stables are 59.38%; giving an approximately 1:1 ratio for mutable to stables (Note: see a later statement about how low mutables are usually counted and why). A self was obtained on the mutable parent of this first cross and it had predominantly mutable seeds of Classes 3 and 4. The third outcross ear gave 30.0% stable seeds. The self of the mutable parent in this cross had only mutable seeds of Class 4. It is possible that this line has three copies of a regulatory element although the inheritance patterns are not consistent.
One plant was obtained from a Class 4 seed from a winter outcross ear with 44.48% stable seeds. This plant on outcrossing to a1 sh2 gave an ear that was segregating 1:1 for mutable and stable seeds (56.78% stable).
Four plants were obtained from Class 2 seeds from the winter outcross ear with 53.26% mutability. Three of the four plants when crossed with a1 sh2 gave ears with some mutable seeds (predominantly of the low mutable class). The percent of stable seeds in these ears was 46.64%, 65.88%, and 71.69%. The fourth outcross ear had only stable seeds.
The above 1986 tests were from a1-Mum2 stocks. The last test was with an a1-Mum3 stock, which in the winter nursery gave an outcross ear that segregated 43.95% stable seeds. Plants from four Class 4 seeds from the winter outcross ear when outcrossed to a] sh2 gave ears with the following percents of stable plus low mutable seeds: ratios 66.67%, 57.89% and 54.91% and 48.94%. Results from the foregoing 1985-86 and 1986 crosses are summarized in Table 1.
Stable seeds (a1-Mum stables) from each of the winter nursery ears tested in the above 1986 crosses gave plants with selfed ears that had predominantly stable seeds. On these ears, only an occasional seed was found with one or a few spots.
The results, so far presented, suggest that it is possible to derive stocks from Mu-induced aleurone mutants that have the classical two element pattern. Sibling seeds from the four winter nursery outcross ears from which the seeds for the 1986 outcross tests were selected were planted in the selfing block and selfed to determine if the a1-Mum parent plants in the 85-86 winter nursery had germinal mutator activity. Two of the plants did. One had 6.06% mutation frequency (n = 33) and the other had a 2.94% mutation frequency (n = 34). The other two winter nursery plants did not have mutator activity (i.e., no mutants were found, n = 28 and 43). These tests are very limited, thus it is impossible to generalize from them. However, the results from these tests suggest that there are different mechanisms responsible for regulating germinal Mutator activity as measured by induced mutants and somatic Mu activity as measured by germinal activity. This observation supports conclusions presented in last year's News Letter (MNL 60:8-9, 1986).
In the winter of 1985-1986, several new 1:1 ears were found among outcrosses of a1-Mum/a1 sh2 stocks again to a1 sh2. These were tested further in 1985 by crossing plants from a1-Mum-stable seeds to plants from non-a1-Mum seeds from the same ear (i.e. a1 sh2 segregants), as in the previous 85-86 tests. Also, in two instances, plants from mutable seeds of these 1:1 ears were crossed to standard a1 sh2 stocks. The results of these tests are given in Table 2. As indicated in a footnote the ratios have just been estimated at this time. Also as noted in a footnote, the percent stable on these 1:1 ears was calculated by combining Classes 1 and 2 into the stable class and Classes 3 and 4 into the mutable class. The Class 2 mutables were usually pooled with the stables because, frequently Class 2 seeds will give rise to plants that have predominantly stable seeds in the next generation. Also plants from stable seeds when selfed and/or outcrossed to a1 sh2 will frequently give ears with predominantly stable seeds plus, sometimes, a few Class 2 seeds as well. Thus Class 2 seeds seem to be incipient stables. It is obvious from Table 2 that mutability can be restored to a1-Mum-stable stocks by crossing to plants from sibling non-a1-Mum seeds from the same 1:1 ear. When this is observed, the ratio obtained is again 1:1. Approximately half of the crosses result in this 1:1 reactivation. These results are expected if a single regulator of a1-Mum mutability is segregating in these stocks. The fact that the same results are observed in all but two of the reciprocal crosses reinforces this conclusion, as does the 1:1 ratio observed in the outcrosses to a1 sh2 of the plants from mutable seeds of these 1:1 ears. Samples of seeds from these 1:1 ears were planted in the 1986 selfing block to screen for germline Mu activity in these stocks. As of this writing, we have the results from only three of these tests. Two had germinal activity, one did not. In both positive tests, the population size was 37 and the mutation frequency 2.70%. The population in the negative test was 36. These results provide additional support for the hypothesis that different mechanisms are responsible for germinal and somatic Mu activity.
In summary, the results reported here suggest that it is possible to derive an apparent two element system from Mu-induced mutable aleurone mutants. What role if any the putative regulator element plays in the regulation of germinal activity of the Mutator system is not known at this time. In fact, the evidence here agrees with that presented last year (MNL 60:8-9, 1986) and in other reports in this year's News Letter that would suggest somatic mutability and germline mutability are not necessarily correlated.
Table 1. Summary 1985-86 tests of putative 1:1 (mutable: stable) a1-Mum2 and a1-Mum3 outcross ears and the 1986 follow-up tests with a1 A2.
Table 2. Test for segregation of a single regulator element for somatic mutability in Mu induced a1 mutants. (Plants from stable a] derivatives from ears segregating 1:1 mutable: stable crossed to plants from non-a1-Mum sibling seed or mutable seeds from the same ear crossed to a standard a1 sh2 stock-See text for more details about these crosses.)
Donald S. Robertson
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