Pl-Bh is an unusual allele of the anthocyanin regulatory gene Pl . Whereas Pl pigments vegetative tissues a uniform purple and does not affect kernel pigmentation, Pl-Bh pigments both vegetative tissues and kernels in a variegated and irregular manner (Cocciolone and Cone, Genetics 135:575-588, 1993). Pl and Pl-Bh have essentially identical nucleotide sequences in almost 7 kb of DNA including flanking and coding regions (O. Hoekenga, MNL this issue). Pl-Bh does differ from Pl by being hypermethylated at specific MspI/HpaII sites 3' of the coding region. The hypermethylation of Pl-Bh is correlated with altered Pl gene expression in husks. The mutation responsible for both the altered expression and the hypermethylation is considered cis-linked because it always co-segregates with the Pl locus. The following model explains how increased methylation may lead to the blotched phenotype. We envision an altered chromosome structure (Center of Modification or CM), located at a cis-linked site some distance from the gene, where a DNA methylation signal initiates. This signal is propagated stochastically along the chromosome toward Pl-Bh. In some cell lineages, the methylating signal moves through Pl-Bh, the gene becomes hypermethylated, and expression is altered. In other cell lineages, the signal does not reach Pl-Bh, the gene is not hypermethylated, and expression is normal.
The model predicts that Pl linked to CM yields the blotchy phenotype. In an effort to genetically define the CM, we attempted to separate it from the Pl-Bh sequence by recombination. Our rationale was that uncoupling the essentially normal Pl sequence from the CM by recombination should result in normal Pl expression. The following cross was set up and both proximal and distal morphological and RFLP markers were used to verify recombination.
A recombination event between the Pl sequence and the CM should result in normal Pl expression. A plant carrying this recombinant chromosome would be recognized as having uniform purple pigmentation as opposed to sun-red (pl) or blotchy (Pl-Bh) pigmentation. Approximately 20,000 plants were screened during the past two summer pollinating seasons. No purple recombinant plants were found. This indicates a genetic distance of < 0.01 cM between the CM and the Pl locus. Alternatively, the CM may act as a suppressor of recombination such that crossovers between the CM and Pl locus may not be recoverable.
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