SUCCESSFUL PCR-BASED REVERSE GENETIC SCREENS USING AN EN-1-MUTAGENIZED ARABIDOPSIS-THALIANA POPULATION GENERATED VIA SINGLE-SEED DESCENT

The development of an Arabidopsis population via single-seed descent i s described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-l. A PCR st rategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thal iana. The method employs PCR reactions on pooled DNA. Positive amplifi cation using a target-specific primer and an En-l-specific primer on r ow, column and single-tray pools identifies the putative insertion mut ant. In a control experiment two insertion mutants of the PIN gene wer e successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generatio n and showed cosegregation with the pi,il phenotype. These examples de monstrate that the inheritance of inserts of the autonomous element En -1 is stable enough to make a proper genetic analysis feasible in a ge nomic background with multiple En-1 inserts.