Further evidence for Mutator activity in the male gametophyte and for differential activity of Mu in male and female germ lines

In last year's News Letter, we presented evidence that Mutator could induce mutants in the gametophyte (MNL 60:12-14, 1986). One line of evidence came from the frequency of discordant seeds (i.e., seeds in which the genotype of the embryo and endosperm differed) produced in reciprocal crosses. Mu-induced y1 mutants produced when Mu plants were used as females had a much lower frequency of discordant seeds than Mu-induced y1 mutants produced when Mu plants were used as males. To date, only white-seeded mutants have been scored for discordancy. There is a reciprocal discordant class expected (i.e., y1 y1 embryo and heterozygous yellow endosperm). This class is much more difficult to score, because it can not be distinguished by classifying the seeds but must be determined by scoring the ears on plants from the yellow seeds.

If mutants are induced during the development of the male gametophyte, it is possible to have pollen grains with one sperm carrying the nonmutated Y1 allele and the other sperm with a mutant y1 allele. If sperm from such pollen grains fertilizes an embryo sac of a y1 y1 plant, half the time the sperm nucleus with the mutant allele will be included in the triple fusion product that produces the endosperm while the other sperm nucleus (with the nonmutant Y1 allele) will unite with the egg nucleus, and a discordant seed with a white endosperm and a Y1 y1 embryo will be produced. If the Mu plant is the female parent, however, mutations in the gametophyte would not be expected to produce many, if any, discordant white endosperm seeds. Such an event would require two independent mutations in the two cell lineages giving rise to the polar nuclei. Also, the mutation in the cell lineage that produces the egg nucleus would have to take place after the cell lineage giving rise to the polar nucleus separates from the one that produces the egg. Such a combination of events is very unlikely. In 1985, we reported a high frequency of discordant seeds in crosses involving Mu2 plants as males and a low frequency of discordant seeds in crosses with female Mu2 plants, as expected if Mu is active in the gametophyte.

Additional studies of this phenomenon (i.e., the different frequency of discordant seed in reciprocal cross) were carried out in 1986. As in the 1985 studies, exact reciprocal crosses were made between Mu2 stocks and a y1 y1 wx wx gl1 gl1 stock (a MU2 line is the progeny of the cross between two standard Mu lines). Mu2 was used because of its high mutation frequency and the multiple y1 wx gl1 stock was used so that contaminants could be recognized when this stock was used as a female parent. The results from the two years of crosses are seen in Table 1. There are over twice as many white seeds found in the crosses of Mu2 as males than when the cross is in the reverse direction. Such a discrepancy is expected if gametophytic mutants are being produced.

To determine if putative discordant seeds, expected as a result of gametophytic Mu-induced mutants in the male gametophyte, could account for the difference in frequency of white seeds in these reciprocal crosses, 16 y1 mutant seeds obtained from Mu2 ears from 1985 crosses were planted and self-pollinated. All of the resulting ears were homozygous for the y1. Of thirty-five white seeds from the reciprocal cross (Mu 2 as male), twelve turned out to be discordant (i.e., these plants were Y1 y1) (frequency = 34.29%). Thus, we see that discordant seeds occur only in crosses in which Mu2 was the male parent. These results are in agreement with those predicted for Mu activity in the male gametophyte.

The results so far obtained from the reciprocal crosses only test for one class of discordant seeds. The reciprocal class of yellow endosperm-homozygous y1 embryo seeds cannot be scored. To do this we will plant a large sample of yellow seeds from these reciprocal crosses in an isolation plot and score for ears with 50% yellow seeds expected on a y1 y1 plant in a field with predominantly Y1 y1 plants. If mutations are being induced in the female gametophyte as well as the male gametophyte, they would be recognized as y1 y1 plants produced by yellow seeds. If it is assumed that discordant seeds occur in the same frequency for the population summarized in Table 1 as it did in the 1985 reciprocal crosses, 46 of the 135 white seeds from the crosses of Mu2 as a male would be discordant. If these 46 seeds are removed from the total of the white seeds, the frequency of y1 mutants from the male Mu2 crosses (i.e., 3.22 x 10-4) now is closer to the frequency of y1 mutants in the female Mu2 crosses (i.e., 1.99 x 10-4). This frequency, however, is still higher than that observed when Mu2 plants are used as females and the difference is still significant at the 1 percent level. Thus the discordant seeds cannot account for all of the difference observed between the male and female crosses of Mu2 plants. It appears that there are more germinal mutants induced when Mu plants are crossed as males than when they are crossed as females. This confirms previous observations on reciprocal crosses involving Mu plants (D.S. Robertson, Mol. Gen. Genetics 200:9-13, 1985).

In summary, there has been presented additional evidence that Mu can induce mutants in the male gametophyte. Additional tests will be necessary to determine if Mu is also active in the female gametophyte. Also, evidence is presented confirming earlier observation that more Mu-induced mutants are observed in the male progeny of a given plant than in the female progeny.

Table 1. Reciprocal crosses of Mu2 Y1 Y1 Wx Wx X y1 y1 wx wx gl1 gl1--Total of 1985-1986 experiments.

Donald S. Robertson
 
 


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