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Getting the Point—Mutations in Maize

Dept. of Agronomy, Purdue University, 1150 Lilly Hall, 915 W. State St., West Lafayette, IN 47906

View larger version (39K): [in this window] [in a new window]   Figure 1. Schematic diagram of TILLING with differentially labeled primers. Amplicons from pooled templates are denatured and allowed to reanneal. The presence of a mutation results in mismatched bases in some templates and these are cleaved by CEL1 into differentially labeled shorter fragments.

View larger version (104K): [in this window] [in a new window]   Figure 2. (A) 2D pooling strategy. Rows and columns of primary plates condensed into individual wells in two adjacent columns of a 2D pool plate. (B) portion of a TILLING gel image (IRD800 channel) showing successive "row" and "column" pairs for two primary plates condensed to the same 2D pool plate, the first has no mutations and the second has two. Heavy boxes, bands corresponding to mutations; light boxes, position of bands for these mutations in the IRD700 channel. These mutations were then sequence verified.

View larger version (26K): [in this window] [in a new window]   Figure 3. Throughput of the Maize TILLING Project. Graph shows fraction of orders in various stages of the TILLING pipeline, as indicated in the legend.

View larger version (99K): [in this window] [in a new window]   Figure 4. EcoTILLING in maize. Portion of a gel image showing the IRD700 channel of a comparison of 24 inbreds to the B73 genome. Gene model for this target is given at top and diagrammed at left of gel with black boxes for exons. Open boxes indicate bands resulting from polymorphisms within exons that are present in duplicate samples (not shown) and have complementary sized products labeled with IRD800 dye. Asterisks indicate 200-bp size and lane marker.