Breakthrough Technologies Efficient Screening of Arabidopsis T-DNA Insertion Lines Using Degenerate Primers1

The sequencing of the Arabidopsis plant genome is providing a fuller understanding of the number and types of plant genes. However, in most cases we do not know which genes are responsible for specific metabolic and signal transduction pathways. Analysis of gene function is also often confounded by the presence of multiple isoforms of the gene of interest. Recent advances in PCR-based reverse genetic techniques have allowed the search for plants carrying T-DNA insertions in any gene of interest. Here we report preliminary screening results from an ordered population of nearly 60,470 indepen-dently derived T-DNA lines. Degenerate PCR primers were used on large DNA pools (n 5 2,025 T-DNA lines) to screen for more than one gene family member at a time. Methods are presented that facilitated the identification and isolation of isoform-specific mutants in almost all members of the Arabidopsis H1-proton ATPase gene family. Multiple mutant alleles were found for several isoforms. The small genome of Arabidopsis simplifies in many ways the study of plant genes required for growth and development. Although the entire genomic sequence of Arabidopsis will be available in the near future, the in planta function of the vast majority of genes within that genome is unknown. I