DEVELOPMENT OF LARGE DNA METHODS FOR ARABIDOPSIS

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This proposal concerns the development of technologies which will facilitate the isolation and analysis of large segments of a plant genome. At present, the cloning of plant genes about which nothing more is known than genetic map position is technically difficult. Current restriction fragments length polymorphism (RFLP) maps of most plat genomes contain large gaps which makes standard chromosome "walking" techniques difficult, if not impossible. Our approach to this problem is to develop methods for the cloning and introduction into plant cells of large segments of plant DNA. These studies will involve the development of "large DNA" methods for chromosome walking, gene isolation and transfer using a model plant system, Arabidopsis. An important aim of this work will be the construction of a low resolution physical map of the Arabidopsis genome using large segments of cloned DNA. Genomic libraries of large Arabidopsis DNA-containing yeast artificial chromosomes (YACs) will prepared and cloned into Saccharomyces cerevisiae. YACs will be analyzed by pulsed-field gel electrophoresis (PFGE) using contour-clamped homogeneous electric field (CHEF) apparatus. Overlapping YACs will be identified and linked using both molecular and yeast genetic methods. Two unrelated but equally important chromosome walking/linking projects will be initiated. We propose to: 1) link up macro-regions of the Arabidopsis genome using YACs and PFGE and 2) clone the entire centromere region of an Arabidopsis chromosome.