Development of a system for efficient chromosome walking in Arabidopsis

The small genome size of Arabidopsis and the low level of repetitive DNA sequences make this crucifer an attractive system for chromosome walking to isolate genes. Mapping of a mutant locus relative to restriction fragment length polymorphism (RFLP) markers provides the first step towards isolating the corresponding gene. The RFLP marker closest to the target gene serves as a starting point. The distance between gene and marker is generally in the range of 50-200 kb. In order to facilitate chromosome walking of this magnitude, we constructed a yeast artificial chromosome (YAC) library of Arabidopsis. Large fragments of Arabidopsis DNA were cloned into a YAC vector and transformed into yeast. The library contains more than 10 equivalents of the Arabidopsis genome. YACs containing sequences of RFLP markers of Arabidopsis revealed an average insert size of 150 kb. Thus, 1-3 (contiguous) YACs should be sufficient to clone genes from Arabidopsis by chromosome walking. In order to use the system to isolate genes involved in the signal transduction of abscisic acid, we fine-mapped the mutant loci abi-1 and abi-2, which confer abscisic acid insensitivity, relative to RFLPs and isolated the corresponding YACs.