Multiple Independent Defective Suppressor-mutator Transposon Insertions in Arabidopsis: A Tool for Functional Genomics

Abstract

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.