The molecular nature of interspecific barriers between pollen and pistils in higher plants is not currently understood. We plan to study interspecific barriers in tomato, an important crop species with significant genetic and molecular advantages. However, before we embark on the molecular analysis of these barriers, there are several tools we need to have in hand. The objective of this proposal is the development of these tools. First, we will develop a system for the visualization of growing pollen tubes within pistils. Second, we will develop a Fluorescent In Situ Hybridization or "FISH-cassette" for the marking of chromosome arms and identifying linked genes important in reproductive barriers. This cassette will contain a Ds-based engineered transposable element that can be used for both mutagenesis and enhancer trapping. Third, we will take an emerging technology ("secretome" analysis) and test its applicability to the detection of proteins secreted from pollen and pistils. These cellular and molecular systems will have a wide utility in plant biology research beyond this project.
Broader Impacts The work will have a broader impact at several levels. First, undergraduate students at Colorado State University will be involved with many aspects of the work throughout the duration of the project. As noted above, the work will impact the plant research community, since the tools to be developed will have wide application. In addition, the work will permit the expansion of genomic approaches to biodiversity studies with the comparison of domesticated and wild species. Finally, research in plant reproduction represents an area of significant economic importance to society at large, and efforts toward understanding of reproduction at the genomic level have been limited to date. The understanding reproductive barriers is directly relevant to horizontal gene transfer; both in terms of transferring desirable traits from wild species into domesticated ones, and in terms of limiting transfer of transgenes from crops into wild species.
Project outcomes 1. Arabidopsis and tomato pollen promoter-GFP fusions will be available to the scientific community after initial publications upon request from the Bedinger lab. These constructs will be of use to numerous researchers with interests in pollen tube growth and pollen-pistil interactions. 2. The initial transgenic tomato lines with a mapped FISH cassette generated during the funding period will be bulked up and the curation process initiated in preparation for generating a complete series of transgenic lines in the future. Once a complete set of transgenic lines (with a transgene cassette mapped to each chromosome arm) has been generated, these will be announced on the Solanaceae Genomics Network www.sgn.cornell.edu/ and will be made available to the tomato research community. 3. Data from the secretome studies will be deposited in Dr. Jocelyn Rose's developing secretome database, available on his website http://labs.plantbio.cornell.edu/rose/ and sequences will additionally be deposited into GenBank.
