Patent ID: 7220893

Claim:
A method of constructing a non-redundant, indexed, saturation, gene-disruption plant library comprising: providing a plasmid having: two identical clusters of unique enzyme-cutting sites and a dissociation element having two ends; transforming a plurality of plants with the plasmid to produce a plurality of transformed plants with the plasmid integrated at different locations within the genome of the plants; mapping the locations of the integrated plasmid in the transgenic plants to identify anchor transgenic plant lines each having the integrated plasmid within its genome at a location from about 200 to about 600 kilobases away from the location of an integrated plasmid in any other anchor transgenic line; obtaining homozygous transgenic anchor plant lines; crossing each of the homozygous anchor transgenic plant lines with a plant having an activator element to form progeny plants, wherein said crossing activates transposition of a portion of the plasmid bounded by the two ends of the dissociation element to form a plurality of progeny plants having different genes disrupted; selecting progeny plants having the activator element segregated out; digesting the plant genome at different unique enzyme-cutting sites to release a DNA fragment produced by digestion of the unique enzyme-cutting sites from each of the transgenic progeny plants, wherein said digesting is carried out by serial, separate use of a plurality of restriction enzymes, specific to one of the unique enzyme-cutting sites; measuring the size of each of the released DNA fragments to determine transposition distances in each of the transgenic progeny plants; and selecting the progeny transgenic plants with the transposition distances which are different from the transposition distances of the other progeny transgenic plants by a pre-determined amount to prepare a non-redundant, indexed, saturation, gene-disruption plant library.