Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning

To accelerate gene isolation from plants by positional cloning, vector syst ems suitable for both chromosome walking and genetic complementation are hi ghly desirable. Therefore, we developed a transformation-competent artifici al chromosome (TAC) vector, pYLTAC7, that can accept and maintain large gen omic DNA fragments stably in both Escherichia coli and Agrobacterium tumefa ciens. Furthermore, it has the cis sequences required for Agrobacterium-med iated gene transfer into plants. We cloned large genomic DNA fragments of A rabidopsis thaliana into the vector and showed that most of the DNA fragmen ts were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we dem onstrated the practical utility of this vector system for positional clonin g in Arabidopsis. A TAC contig was constructed in the region of the SGR1 lo cus, and individual clones with ca. 80-kb inserts were tested for their abi lity to complement the gravitropic defects of a homozygous mutant line. Suc cessful complementation enabled the physical location of SGR1 to be delimit ed with high precision and confidence.