Locus by Genomic Subtraction

Arabidopsis thaliana ga l mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive g a l deletion mutant, gal-3. The cloned sequences correspond to a 5.0-kb deletion in the gal-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the gal-3 mutant is located at the GAl locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GAl locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in gal-3 complemented the dwarf phenotype when integrated into the gal-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga l alleles within the 5.0-kb region deleted in mutant gal-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GAl locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GAl locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GAl region of the Arabidopsis genome.