Nested Deletions: A New Tool for Plant Genomics Research

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Plant genomes are laden with local sequence duplications and clusters of homologous genes. To simplify the analysis of these duplications and gene clusters, this project will develop a new genetic technology to generate chromosomal deletions quickly and efficiently. This approach is based on the finding that transposable elements can participate in alternative transposition pathways that generate novel recombination products, including large deletions and duplications. The system described here utilizes a transgene construct containing maize Ac/Ds transposon ends in tandem orientation within a I/dSpm transposon (Ned1; Nested deletions 1). The Ned1 construct will be transformed into maize; subsequent crosses will introduce the En/Spm transposase to mobilize Ned1 to various genomic locations, and the Ac transposase to activate the deletion process. The action of Ac transposase on the Ac termini within Ned1 generates an unlimited set of nested deletions with one end anchored at the transgene locus. The Ned1 construct contains marker genes for detection of both Ned1 transpositions and Ac--induced deletions, as well as sequences for easy cloning of deletion endpoints via plasmid rescue. The expected outcome of this project is the production of maize plants containing the Ned1 transgene construct. These plants and their progeny will be tested for 1) transposition of Ned1 by En/Spm transposase; 2) induction of deletions by Ac transposase; 3) transmission of deletions to progeny plants. Additionally, the Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful for the research community. This approach could be extended to the production of a set of maize lines containing Ned1 elements at dispersed sites throughout the genome that could be used to isolate deletions and other rearrangements in specific regions of the genome.Deliverables: 1. This project will generate maize plants containing the Ned1 transgene construct. 2. Ned1 transgenic plants and their progeny will be tested for a) transposition of Ned1 induced by En/Spm transposase; b) formation of deletions induced by Ac transposase; c) transmission of deletions to progeny plants. 3. Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful to the research community. Contact Information for Deliverables: Thomas Peterson, 2206 Molecular Biology, Iowa State University, Ames, IA 50011thomasp@iastate.edu