Multiple independent defective Suppressor-mutator transposon insertions inArabidopsis: A tool for functional genomics

A new system for insertional mutagenesis based on the maize Enhancer/Soppre ssor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-D NA construct carried a nonautonomous defective Spm (dSpm) element with a ph osphinothricin herbicide resistance (BAR) gene, a transposase expression ca ssette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negativ e selection markers were applicable to soil-grown plants, allowing the reco very of new transpositions on a large scale. To date, a total of 48,000 lin es in pools of 50 have been recovered, of which similar to 80% result from independent insertion events. DNA extracted from these pools was used in re verse genetic screens, either by polymerase chain reaction (PCR) using prim ers from the transposon and the targeted gene or by the display of insertio ns whereby inverse PCR products of insertions from the DNA pools are spotte d on a membrane that is then hybridized with the probe of interest. By sequ encing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permit ted a comparison of the chromosomal distribution of transpositions from var ious T-DNA locations.