Are P-locus epiallele methylation status and phenotype set during inflorescence or embryo development by maternal influence?
--Bradeen, J; Timmermans, M and Messing, J

P-pr is an epiallele of the full red P-rr allele characterized by variable patterned pericarp and red cob (Das and Messing, Genetics 136:1121). Also characteristic of P-pr is somatic instability as evidenced by frequent cob sectors. Methylation status of the P-pr epiallele in leaf DNA correlates inversely with kernel pigmentation levels and the P-pr pericarp ranges from virtually fully pigmented (similar to P-rr) to virtually unpigmented (Das and Messing, Genetics 136:1121; personal observations). In the current study, we examine causes of variability of P-pr phenotype and determine when the methylation status of the epiallele is set. Two approaches were used: comparison of within and between plant P-pr methylation levels for plants originating from "sector" and "nonsector" portions of sectored cobs ("sector study") and correlations between P-pr phenotypes of sibling plants and their original cob position ("ear map study").

In the sector study, ten BC1 cobs ((P-pr x 4Co63:P-ww) x 4Co63:P-ww) with distinct large sectors were selected and ten seeds were planted from each sector and nonsector portion of each cob. Three inch leaf tip samples (or whole leaves for small leaves) were collected from every true leaf of every plant. P-pr phenotypes were determined at harvest for all P-pr/P-ww heterozygotes and support previous observations that somatic sectors yield penetrant phenotypic modification. SalI digestions of DNA extractions from each leaf of phenotypically selected plants were hybridized with clone p15 (Das and Messing, Genetics 136:1121), allowing determination of methylation status. Methylation status correlated inversely with pigmentation, as expected. Importantly, methylation status was consistent for each leaf of every plant; by the time the first leaf was harvested, methylation status and consequently P-pr phenotype had been determined. This suggests seedling environmental factors are likely not important in determining methylation status or P-pr phenotype within that plant. Furthermore, these results suggest P-pr methylation status and phenotype are determined prior to or at germination, most likely in either the gametes or embryo.

In the ear map study, ten BC1 cobs were selected with differing but uniform pigmentation levels. Ear maps were prepared and seed order was randomized prior to planting. Following harvest, cobs were superimposed upon corresponding enlarged ear maps, allowing visual analysis of cob position effects on P-pr phenotype. Although original ear map cobs were uniform (i.e. lacking apparent sectors), progeny cob pigmentation was confined to particular ear map regions, with similarly colored cobs arising from seed from a common region. These results are consistent with the possibility that P-pr phenotype is determined during female inflorescence development, with kernels that give rise to similarly colored cobs arising from a common progenitor cell. (Note that determination of P-pr methylation status during embryo development is not precluded by these observations. However, determination during embryo development requires methylation status to be set for each individual embryo, and mechanisms explaining the observed clustering of similarly colored cobs must be more complex.) These results further suggest maternal somatic instability is an important factor in generating variability in progeny cob pigmentation and probably methylation levels (work in progress). The mechanism giving rise to somatic instability that has no apparent phenotype in the individual but affects the phenotype of its progeny (as observed in the ear map study) may be the same as or different from that which gives rise to visible sectors in the affected individual (as observed in the sector study). Ear map experiments designed to determine environmental effects on phenotype suggest these effects are likely minimal. However, environmental effects on future generations have not yet been determined (work in progress). 

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