Cloning of the arabidopsis RSF1 gene by using a mapping strategy based on high-density DNA arrays and denaturing high-performance liquid chromatography

Mapping genes by chromosome walking is a widely used technique applicable t o cloning virtually any gene that is identifiable by mutagenesis. We isolat ed the gene responsible for the recessive mutation rsf1 (for reduced sensit ivity to farred light) in the Arabidopsis Columbia accession by using class ical genetic analysis and two recently developed technologies: genotyping h igh-density oligonucleotide DNA array and denaturing high-performance liqui d chromatography (DHPLC). The Arabidopsis AT412 genotyping array and 32 F-2 plants were used to map the rsf1 mutation close to the top of chromosome 1 to an interval of similar to 500 kb. Using DHPLC, we found and genotyped a dditional markers for fine mapping, shortening the interval to similar to 5 0 kb. The mutant gene was directly identified by DHPLC by comparing amplico ns generated separately from the rsf1 mutant and the parent strain Columbia . DHPLC analysis yielded polymorphic profiles in two overlapping polymorphi c amplicons attributable to a 13-bp deletion in the third of five exons of a gene encoding a 292-amino acid protein with a basic helix-loop-helix (bHL H) domain. The mutation in rsf1 results in a truncated protein consisting o f the first 129 amino acids but lacking the bHLH domain. Cloning the RSF1 g ene strongly suggests that numerous phytochrome A-mediated responses requir e a bHLH class transcription factor.