Genetic analysis of su1-R2412, an allele of su1 with an intermediate phenotype
--James, M

Su1 codes for a starch debranching enzyme that is active during starch biosynthesis (James et al., Plant Cell 7:417-429, 1995). Mutant su1-Ref kernels accumulate sugars and the water-soluble polysaccharide phytoglycogen during development, and have a shrunken and overall translucent appearance in the mature dried state. Many su1 alleles have been identified, including some that appear near-normal, or that have a phenotype intermediate to wild type and su1-Ref. I am investigating one of these intermediate alleles, su1-R2412, which arose in a Mutator background. When homozygous, su1-R2412 results in a mildly wrinkled and translucent kernel crown, while the base of the kernel appears normal. This phenotype was observed in less than the expected Mendelian ratios following the self-pollinations of heterozygotes. When su1-R2412 is combined with su1-Ref, an intermediate phenotype results, in which the kernel is slightly shrunken and translucent on the periphery, especially in the crown. This suggests that su1-R2412 has a modulating effect on su1-Ref.

Because su1-R2412 was generated in a Mutator background, the mutation is likely due to the insertion of a Mu element at the su1 locus. To test for the reversion of su1-R2412 to wild type due to Mu element excision, approximately 700 su1-R2412/su1-Ref plants were pollinated by su1-Ref testers (Silver Queen) in an isolation plot in the 1994 summer nursery. Silver Queen also is homozygous for y1, which was used in subsequent analyses as a contamination marker. The resulting ears segregated for the standard sugary and intermediate sugary phenotypes, but also had many starchy kernels, suggesting a high rate of reversion of su1-R2412 to wild type. The frequency of this putative reversion was calculated to be approximately 2.4%. This reversion frequency is higher than that calculated for another allele of su1, su1-R4582::Mu1, by approximately 104.

To test whether the starchy kernels represented stable reversion events, 36 starchy kernels were planted and self-pollinated in the 1995 summer nursery; in addition, 10 kernels that had an intermediate sugary phenotype also were planted. 25 plants derived from the starchy kernels produced ears that segregated for both su1 and y1; however, these ears contained both the standard sugary and intermediate sugary phenotypes, indicating that su1-R2412 was still present. This suggests that these 25 "revertant" kernels may have been starchy as a result of suppression of the mutant phenotype, as described by Barkan and Martienssen (PNAS 88:3502-3506, 1991), rather than as a result of the excision of a Mu element from the su1 locus. The remaining 11 plants from starchy kernels produced ears that segregated for su1, but not y1, indicating that these starchy kernels most likely resulted from contaminating wild type pollen. All 10 plants derived from the intermediate sugary kernels (presumably su1-R2412/su1-Ref) produced ears that contained from 25% to 50% starchy kernels, in addition to both sugary phenotypes; as expected, each ear contained approximately 25% kernels with the standard su1 phenotype (presumably su1-Ref/su1-Ref). Thus, one or two doses of su1-R2412 were able to confer either an intermediate sugary or a normal kernel phenotype.

DNA gel blot analyses with EcoRI and BamHI of populations segregating 1:1 for su1-R2412 showed a RFLP of approximately 2 kb that cosegregated with the su1-R2412 mutant allele. PCR and nucleotide sequence analyses localized this polymorphism to the 5' leader region of su1. Experiments are in progress to investigate 1) the presence and identity of a possible Mu element insertion in this 5' leader region; and 2) the molecular basis for the starchy "revertant" kernels, including possible correlations of the methylation status of this region or element with the mutant or wild type phenotype. 

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