Cloning the Arabidopsis GA1 Locus by Genomic Subtraction

Abstract

Arabidopsis thaliana ga1 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 ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.