Successful PCR-based reverse genetic screens using an En-1-mutagenised Arabidopsis thaliana population generated via single-seed descent

The development of an Arabidopsis population via single-seed descent is described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-l. A PCR strategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thaliana. The method employs PCR reactions on pooled DNA. Positive amplification using a target-specific primer and an En-l-specific primer on row, column and single-tray pools identifies the putative insertion mutant. In a control experiment two insertion mutants of the PIN gene were successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generation and showed cosegregation with the pi,il phenotype. These examples demonstrate that the inheritance of inserts of the autonomous element En-1 is stable enough to make a proper genetic analysis feasible in a genomic background with multiple En-1 inserts.