Transformation of Brachypodium distachyon to Analyze CNS Enhancer Activity and Genome Fractionation in Maize

Fractionation is the deletion of one or the other but not both of a pair of homeologous genes following an ancient whole genome duplication. In maize these deletion events result from intra-chromosomal recombination. Analyses of expression data (RNAseq) between homeologous gene pairs in maize revealed that genes from maize1 (the least fractionated subgenome) tended to be expressed more than duplicates of the same genes from maize2. Significant differences between maize1 to maize2 expression have been found to occur in specific tissues, such as maize pollen. To determine the polymorphisms responsible for these expression differences, we have compared the noncoding sequences surrounding pairs of maize genes which showed different patterns of expression. Syntenic orthologs from other grass species (sorghum, foxtail millet, rice, Brachypodium) are included in the analysis to identify conserved non-coding sequences (CNSs) which are functionally constrained and may act in the regulation of gene expression. In order to test the enhancer activity of these CNSs, several have been cloned into the binary vector pGPro8 (GenBank JN593327), which is modified to include a minimal 35S promoter (35SMin) ahead of a GUS reporter gene. Here we describe the stable transformation of Brachypodium distachyon (Bd21-3) plants to test the activity of pollen-specific CNS enhancers identified through analysis of fractionation mutagenesis in maize. Stable T₀ plants carrying the pGPro8-35SMin vector with potential CNS enhancers demonstrate pollen and anther-specific GUS expression.