We tested the hypothesis that Mutator activity was regulated by several genes, as originally proposed by Robertson (Mutat. Res. 51:21-28, 1978). The difficulty in genetically identifying each of these units may have arisen from the possibilities that [1] more than one gene is required for activity and [2] the number of these genes varies as a result of segregation or of amplification (as was shown for the receptor elements). Variation in copy number of each unit may affect the degree of activity of a given progeny or family.
To test that hypothesis, and to partially overcome the difficulties described above, we looked for co-segregation of Mutator activity and known genetic markers.
a) An active Mutator line was selected (GG65) which contained a Mu1.4 insertion in the Bronze-2 locus (bz2-mu1 allele). In active progenies of this line, a characteristic fine spotting can be easily observed on the kernels; this reflects the excision of Mu1.4 from the indicator allele. GG65 is derived from a Robertson purple Mutator line which was backcrossed four times to a bz2 W23 background.
b) The Mangelsdorf tester (GG63) contains ten morphological traits differing from those of the Mutator line. Each trait is encoded by a single gene, corresponding to one of the ten maize chromosomes.
winter 88: F1 kernels 100% purple
spring 88:
winter 89:
In this material no progeny kernel had become completely inactive, therefore, the degree of spotting was scored visually for each kernel, as an indicator of Mutator activity. Note that all kernels have three doses of bz2-mu1 in their aleurones, therefore, differences of intensity of spotting do not depend on the dosage of the reporter allele. Three classes were established:
2% = 47 K had very few (5-30) spots per kernel (LS = Low spotting)
20% = medium intensity of spotting (MS)
80% = high (HS) or very high (VHS) intensity of spotting.
The Mangelsdorf markers were scored as follows: sugary, red aleurone, and waxy were scored on the F2 kernels; glossy was scored on 2 week old seedlings; liguleless was scored on two month old plants; the other markers could not be scored. All 47 LS K were used from the population of 2500 spotted K. Kernels from the other groups (MS, HS, VHS) were sampled randomly.
If there is no association between a marker and Mutator activity, then the proportion of the recessive phenotype should be similar in all activity groups (LS->VHS). We used the G-test for heterogeneity to compare between proportions. No significant differences were found for sugary, waxy, and glossy, indicating that these markers are
probably not linked to a locus affecting Mutator activity. Significant differences were found for red aleurone and liguleless (P(GH)=0.04 for both markers). For these markers, the frequency of the recessive allele was greater in the medium (0.34 for pr) and low spotted groups (0.425 for lg) than in the High + Very High spotting groups. Although the level of significance of pr association with spotting intensity is low, we found a similar trend of association in a small population of F3 plants from the same cross, as well as in a small population from another cross (data not shown). These observations support the proposition that Pr is linked to a factor which affects Mutator activity. Interestingly, Cy has been mapped distal to Pr (Peterson, MNL 62:3, 1988). The locus we mapped from Robertson's stock may be homologous to Cy.
The five markers used in this study
covered only a small portion of the genome (~10%), and did not explain
all the variation in Mutator activity. Yet, two markers seem to
be linked to loci affecting Mutator activity. This suggests that additional
Mutator activity-related factors exist in the standard Robertson's stocks.
We are currently mapping more precisely the factors which we identified,
and we are looking for additional markers in the genome using RFLP analysis.
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