Two Types of Meiotic Crossovers in Higher Plants

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Intellectual merit: During sexual reproduction, each pair of homologous chromosomes typically requires a minimum of one (obligate) crossover to assure proper segregation of chromosomes and production of genetically balanced haploid gametes. At least two pathways lead to crossovers in budding yeast, mammals, and plants: the MLH1 pathway with interference (in which the presence of one crossover reduces the likelihood of another crossover nearby) and the MUS81 pathway that is thought to show little or no interference. How crossover frequency and distribution are controlled and how different crossover pathways interact are two fundamental questions in genetics that have both theoretical and practical implications in genome evolution, speciation, and plant and animal breeding. Evaluating the two crossover pathways at a genome-wide level is difficult because the two types of crossovers cannot be distinguished using linkage mapping. In contrast, a cytogenetic immunolabeling approach is ideal because it allows individual crossovers of each type to be unequivocally identified and localized. This project will directly evaluate the contribution of each pathway to crossing over and interference in higher plants using light (LM) and electron microscopic (EM) immuno¬localization of MLH1 and MUS81 proteins on pachytene chromosomes of tomato (Solanum lycopersicum). MLH1 is a component of most late recombination nodules (LNs). LNs mark crossover sites and have been extensively studied in tomato. MUS81 is thought to be a component of MLH1-negative LNs, and EM immunolocalization will be used to test that hypothesis. Subsequent work will use simultaneous LM immunolocalisation of MLH1 and MUS81 to map these foci on pachytene chromosome spreads from wild-type tomato plants and from MLH1 and MUS81 gene-silenced lines. These data on the roles of the two types of crossovers under different genetic situations will be exploited within a comprehensive mathematical model of crossing over. This work will be the first to perform such investigations in any species and will provide novel insights into the contributions and interactions of both interference and non-interference types of crossovers at a genome-wide level in flowering plants. Broader Impacts: This work will define the contribution and interaction of two different crossover pathways in both normal and mutant flowering plants. This information will be useful in designing breeding approaches to target regions of special interest for genetic recombination in order to improve agricultural lines. Useful tools generated in the study (such as antibodies and transgenic plants) will be shared with colleagues. The development of human resources is an important part of the work, and one graduate student and one post-doctoral fellow will be closely involved in the research. Each will receive individual attention and regular mentoring by the principal investigator, and both will receive training in a laboratory that specializes in plant gene-silencing approaches at the University of Nebraska. In addition undergraduate students will be involved in many aspects of the research. After an initial training period, each undergraduate will be encouraged to work on an independent project that supports the overall goals of the research and to present their research results at a yearly forum for undergraduates at Colorado State University (CSU) called Celebrate Undergraduate Research and Creativity. Underrepresented and minority students will be specifically recruited by working in partnership with the McNair Program at CSU.