Some thoughts on the nature and utilization of the Mutator system
--Donald S. Robertson

Because I have retired and my health will not permit me to continue my research, this will probably be the last article to be included in the Newsletter. Thus, I think it would be helpful to take this opportunity to summarize various miscellaneous aspects of the Mutator system that have not been reported before or to emphasize certain observations previously made and, where appropriate, comment on their significance for future research.

1. Tests for Mutator activity. The standard test I have used for the presence of an active Mutator has included the following elements: a) Self-pollinate the Mutator parent. b) Outcross the Mutator plant to a standard line (or any non-Mutator stock). c) If possible self-pollinate the second ear of the outcross parent. d) Seedling test the selfed ears of both parents to determine that neither is segregating for a new mutant. e) Seedling test the progeny of self-pollinated ears of 50 plants of the outcross progeny and score for new seedling mutations expected if the Mutator parent was active. Instead of seedling tests, some investigators have used the segregation of defective kernels on the self-pollinated ears of the outcross plants. This is probably an acceptable technique but I think there is a higher risk of error in scoring a given plant as having an active Mutator system than when seedling tests are used. I have found that spontaneous defective kernel mutants occur with a relatively higher frequency than seedling mutants and that environmental factors also can result in the production of the defective kernel phenotype. An added advantage of the seedling test is the opportunity to observe instability (mutability) in the mutants, which is seldom possible if only the defective kernel phenotype is utilized. The presence of mutability is a sure indicator that a Mutator-induced mutation has occurred.

2. Somatic mutability is an unreliable indicator of Mutator activity. In my research I have extensive data demonstrating that there is no correlation between somatic instability and Mutator activity (i.e., the ability to induce a high frequency of new mutants) of a plant from a mutable kernel or a mutable plant. From what now is known about the molecular basis of the Mutator system, such a lack of correlation is not surprising. All that is required for somatic mutability is a MuDR element at the mutant locus, or one of the receptor elements at the locus in addition to a MuDR element elsewhere in the genome. Thus, there can be somatic mutability with only one or two elements in the genome, while active Mutator stocks usually possess numerous elements. It is the large number of elements in the genome of active Mutator plants that is responsible for their high mutation frequencies. This does not mean that plants from mutable kernels or mutable plants with only one, two, or just a few elements are incapable of producing new mutations. However, the mutation frequency will be much lower in such plants than in plants with numerous elements.

3. The effects of an inbred condition on germinal and somatic mutability. Generally, the more inbred a Mutator stock the less likely it is to have Mutator activity in both the germ line and the soma. This is true whether the inbred condition is due to inbreeding per se or is the result of crossing the germinal Mutator system or an unstable Mutator-induced mutant into an inbred line. In some genetic backgrounds, this loss of activity may happen in fewer generations than others, but I have not observed any background where eventually an inbred condition will not eliminate both types of Mutator activity.

4. Mutator-induced mutants. Mutator-induced mutants and their storage location are summarized in the accompanying table..

5. The response of the Mutator system to ultraviolet light irradiation. When mature pollen from active Mutator plants was irradiated with U. V. for 30, 35, 40, 45 seconds a synergistic affect was observed. If these observations are valid (they need to be repeated), they suggest that the mechanism involved in repairing U.V. damage to DNA might create a situation that is amenable to the transposition of Mutator elements.

6. Mutator activity in the early development of the embryo. The mutants segregating in the progeny of many self-pollinated ears occur in less than the 3:1 ratio, which is expected if the mutant allele was carried by the pollen grain or the egg responsible for the self-pollinated plant. One of the possible explanations for this phenomenon is that instead of the mutant being carried in the gamete from the Mutator parent, it was induced early in the development of the embryo of the plant to be self-pollinated. A mutation occurring in a cell before the cell lineage of the tassel and ear separated, and whose descendent cells made up a portion of the meristems of both inflorescences, could account for such non-Mendelian ratios. Another evidence of mutations occurring in the early development of the embryo was the observation that in some tests for Mutator activity self-pollinated ears of Mutator plants, which were outcrossed, would not segregate for a new mutant. However, half or less than half, but a good portion, of self-pollinated ears from the plants of the outcross progenies would segregate for a new mutant. Such an observation suggests that a mutation occurred during the development of the embryo in the cell lineage giving rise to the tassel after the tassel and ear cell lineages had separated. Preliminary tests support the occurrence of both of these kinds of events, but much more data is needed to confirm this conclusion.
Storage location
vp5-Mum (Several independent isolates.) X
vp5-Mu3076-36 X
vp9-Mum (Several independent isolates.) X X
vp9-Mum2(3111-5) X
a1-Mum (5 independent isolates.) X X
a1-Mus (When first isolated mutants were stable. 4 independent isolates.) X
bz1-Mum (17 independent isolates.) X X
bz1-Mus (When first isolated mutants were stable. 10 independent isolates.) X X
a2-Mum (4 independent isolates.) X X
a2-Mus (When first isolated mutants were stable. 3 independent isolates.) X X
wx1-Mum (15 independent isolates.) X X
wx1-Mus (When first isolated mutants were stable. 3 independent isolates.) X X
sh1-Mu (7 independent isolates.) X
bt2-Mu1(9626-11) X
Mutator-induced opaques (8 independent isolates, not o2.) X
bt1-Mu4206 X
fl2-9234 X
o2-Mum1 X
o2-Mum3b X
vp1-Mum1 X
vp1-Mum3 X
c2-Mum1 X
Dap1 X
Dap2 X
Dap-py X
yg2-Mum (13 independent isolates.) X X
lw1-Mum3108 X
l10-Mus1359 (When first isolated mutant was stable.) X

7. Mutator-induced deletions. Deletions have been demonstrated in progenies of Mutator plants for the distal portion of both the short arm and the long arm of chromosome 9. However, genetic evidence for deletions in interstitial regions is meager. One has been found involving the a1 sh2 region of chromosome 3 and a putative deletion linked to a1 also has been found. These were just found incidental to other studies. A systematic search for such deletions may reveal whether they are a common phenomenon of the Mutator systems or not. The following regions would lend themselves to such studies: Chrom. 1 - an1 bz2, Chrom. 3 - a1 sh2, Chrom. 5 - pr1 gl8, Chrom. 9 - c1 sh1 bz1.

8. The presence of Spm in I.S.U. Mutator stocks. Dr. Vicki Chandler's laboratory has reported the presence of Spm in active Mutator stocks. I have attempted to test several I.S.U. stocks for the presence of Spm. A heterozygous Spm stock segregating for mutable and stable c2-m2, wx1-m8 kernels was obtained from another laboratory. This stock was supposed to carry all the genes other than c2-m2 necessary for aleurone color. Stable kernels were selected and the plants from these kernels were crossed to five different purple aleurone Mutator stocks, three a1-Mum2 mutable aleurone stocks, and four different Mu2 stocks. Unexpected results were obtained in the crosses to the Mu2 stocks. All of the F1 ears lacked kernels with aleurone pigmentation. Because our Mu2 stocks are A1 A1, A2 A2, C2 C2, c1 c1, r1 r1 and the Spm was supposed to be homozygous for the aleurone genes, these F1 ears should have all had mottled kernels. However, if the Spm stock was segregating for c1 and/or r1, the stable kernels selected for a source of plants used in these tests could have been stable because they were homozygous for c1 and/or r1 and not because they lacked Spm. Such kernels would have been classified as lacking Spm when in reality they could have carried Spm, which would not be detected because they lack a C1 and/or a R1 allele. Some F1 plants from all these tests, when backcrossed to plants from stable kernels of the Spm stock, did indeed segregate for mutable kernels, indicating the presence of a Spm. However, it can not be determined whether this controlling element came from the original Spm stock or the Mutator stocks. Unfortunately I was not able to repeat these experiments before I had to cease my research. Thus, the Spm status of the I.S.U. Mutator lines is undetermined as yet and, unless someone is inclined to make the appropriate tests in the future, it may never be known. (Note: I have included this information here so that workers in other laboratories, who were informed about our preliminary observations, suggesting the widespread presence of Spm in our stocks, will be aware that the prevalence of Spm in I.S.U. stocks is yet to be determined.) 

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