Investigation of promoter variation and DNA methylation. (a) Percentages of genes with and without variations in their promoter regions. (b) Percentages of promoter variation of genes with differential expression. (c) An example of a gene with the same promoter sequence but with differential expression abundance between BT×623 and Keller. (d) Differentially methylated cytosine in their promoter regions between BT×623 and Keller. (e) and (f) An example of a gene with differential expression abundance by microarray and qRT-PCR analysis, respectively. (g) Promoter motif analysis of the gene Sb01g001893. (h) and (i) Transient expression analysis of the promoter-GFP constructs. (h) The bright-field image under visible light. (i) The image showing GFP expression abundance under confocal microscopy in bombarded sorghum shoots. Top and bottom panels in (h) and (i) show the images from the promoters without motif SEF4MOTIFGM7S and with the motif, respectively. Bars in (h) and (i) are 250 μM. To test their transformation efficiency, total of 10 microscope views were selected in each cassette each repeat to count transformed cells with detectable GFP activity. On average, 20.2 and 19.9 cells in each view for the motif SEF4MOTIFGM7S-containing cassette and non motif cassette, were detected, respectively. No difference has been detected in their transformation efficiency by t-test (P<0.01).