The mutant Ae-5180 was discovered as a single sugary/glassy kernel in a y1 wx gl8 x Y1 Wx Gl8 Mu isolation plot. Fortuitously, gl8 is located 11 cM from ae on chromosome 5. By seedling testing the plump non-sugary kernels from the self of the plant arising from the original mutant kernel, and scoring for gl8 (71 gl, 16 Gl), we were able to determine that Ae-5180 arose on the homologue of chromosome 5 that came from the Mutator parent. While this is not conclusive evidence that Ae-5180 is Mutator-induced, it increases the likelihood of this being the case.
The best case for Ae-5180 being Mutator-induced comes from molecular evidence. From the third generation male outcross of a heterozygous Ae-5180 plant to standard, we selected 6 sugary kernels (Ae-5180/+) and 6 starchy (+/+) kernels. We grew these kernels in our 1987 summer nursery and harvested immature ears for sibling segregation analysis. We extracted DNAs, digested them with EcoRI (which cleaves outside the Mu1 element), and electrophoresed them on agarose gels. In addition, EcoRI-digested Standard B70, Q60, and y1 wx gl8 tester were electrophoresed on the same gel, as controls. We Southern-blotted onto nylon membranes, hybridized with the central MluI fragment of Mu1 made radioactive with 32P-dCTP by random hexamer priming, and prepared autoradiographs. We observed a segregating 6.6 Kb Mu1-homologous band in heterozygous mutant plants, but not in sibling non-mutant plants and controls. The number of copies of Mu1-homologous elements in the sibling plants based on our EcoRI digests is approximately 12. We will continue outcrossing our Ae-5180 lines in order to reduce the copy number further. If the 6.6 Kb Mu1-homologous band that cosegregates with Ae-5180 is the result of a Mu element insertion within the Ae gene, then this band should persist. We will also rerun these sibling segregation analyses using other restriction enzymes that cleave outside Mu1.
One unexpected result of this study was that our standard control lanes had a few Mu1-homologous bands in them; these probably represent endogenous Mu1-homologous sequences. The last standard to which Ae-5180 was crossed for this analysis was Q60. An 8.6 Kb band common to all 12 sibling plants, both mutant and nonmutant, and to Q60 appeared on the autoradiograph. This demonstrates the inherent consequence of endogenous Mu elements in standard lines, i.e. a Mu1-homologous element carried by a standard could potentially result in a band that might obscure a segregating mutant band and ruin the analysis.
Philip S. Stinard and Brent Buckner
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