Opaque mutations from Mutator self populations
--Wenpeng Yang, P. SanMiguel, P. Stinard, D. Robertson and J. Bennetzen

Over the course of many years of testing Mutator populations for mutagenic activity, many opaque mutations have been found to segregate on selfcross ears. We have begun the genetic analysis of fifty such ears, segregating for presumably independent opaque mutations. Mutations in opaque1 (o1), opaque2 (o2) and opaque6 (o6) have been found by allelism tests with standard tester alleles. Candidate opaque5 (o5) and opaque7 (o7) mutations have also been conditionally identified.

Eleven of the fifty mutant lines produced opaque seeds that either did not germination (six lines) or germinated and died shortly thereafter (five lines). In this latter class were two of the o6 mutations. We observed in agreement with previous reports (Gavazzi et al., Theor. Appl. Gen. 46:339-345, 1975; Ma et al., Cereal Chem. 52:412-419, 1975; Manzocchi et al., Theor. Appl. Gen. 72:778-781, 1986) that the homozygous seedlings dies shortly after the emergence of the first or second leaf. One of the o6 mutant lines (#3142) was exceptional, however; opaque seed yielded viable seedlings that matured to produce adult plants. In our summer nursery at Purdue, the field-grown o6/o6 seedlings from mutant #3142 first produced yellowish leaves that slowly greened from the veins out as the leaf aged.

The three o6 mutations identified in this study were subjected to further analysis. Opaque seeds from the homozygous-viable o6 mutant were planted in small pots and grown in the greenhouse. After 27 days of growth, the o6/o6 seedlings from line #3142 had produced six leaves; the lower three leaves were yellowish but green near the veins, while the upper three leaves were more uniformly yellow. After 53 days, nine leaves had been produced; the upper four leaves were yellowish and the bottom five were withered and dead. After 80 days, all of the o6/o6 plants had produced thirteen leaves, of which all but the top four had withered and died. Two of these plants were sacrificed for DNA preparation, and the third yielded a reasonable quantity of viable pollen. The viability of the #3142 o6 mutation suggests that it may be due to a partial inactivation of the locus, or to instability (e.g., somatic reversion) of the mutation.

Two of the o6 mutations were backcrossed two or more generations to a B73/Mo17 hybrid and were subsequently tested for cosegregation of the mutant phenotype with a Mutator transposable element. At the same time, the putative parental and mutant lines were scored for Southern hybridization pattern with three restriction fragment length polymorphism (RFLP) probes linked to o6: bnl17.08, bnl19.08 and bnl19.44. The Southern hybridization results indicated that mutations #3142 and 5117 were induced on chromatids derived from the Q67 parent. A Mu element that cosegregated with the opaque phenotype was not found in the two tested families, #3142 and #5117, using several different restriction enzymes and Mu1, Mu2, Mu3, Mu4, Mu5, Mu7, Mu8 and MuA (MuDR) probes. Hence, either these mutations were not induced by Mu element insertion into o6 or the responsible element was not detected in the Southern analysis. This latter possibility could have two origins: either the Mu element responsible is of a subfamily that has not yet been identified or the causative Mu band was obscured by other bands in the Southern blots analyzed. 

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