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.
Most have heard the truism that all humans are unique. Most of our genetic uniqueness comes about through a special cell division called meiosis that eventually produces reproductive cells (gametes). An important part of meiosis is shuffling of genetic information between chromosomes in a process called crossing over. In crossing over, comparable parental (homologous) chromosomes are broken at the same site, and the corresponding genetic segments are traded between the homologous chromosomes. Although crossing over is a highly conserved process that is integral to the successful completion of meiosis in nearly all organisms, we do not understand how crossing over is controlled, although we know several "rules" that characterize crossovers. One rule is that every pair of homologous chromosomes has to have at least one (obligate) crossover so that each gamete receives a complete set of chromosomes (containing all necessary genes) at the end of meiosis. Failure to follow this rule can lead to gamete death and/or genetic defects. Recent research has shown that both animals and plants have two different pathways of crossing over. Pathway I is highly regulated, accounts for about 80-95% of all crossovers, and is responsible for the obligate crossover between each pair of homologous chromosomes. The protein MLH1 is critical for Pathway I crossovers. Less is known about Pathway II, but it accounts for only 5 – 20% of all crossovers and is apparently less regulated. In most genetic studies, it is not possible to distinguish crossovers formed through Pathway I (Class I crossovers) from those formed through Pathway II (Class II crossovers). In this research, we developed a new approach to distinguish Class I crossovers from Class II crossovers. We used MLH1 antibody labeling together with light and electron microscopy to examine recombination nodules (RNs) on meiotic chromosomes. RNs are protein complexes that mark all crossover sites, and RNs that contain MLH1 protein mark Class I crossovers. Similarly, RNs without MLH1 protein mark Class II crossovers. This innovative approach allowed us to map nearly 3000 crossovers to examine the characteristics of each pathway and to determine how the two pathways interact in a normal (non-mutant) organism, in this case, cherry tomatoes. Such a study on the two crossover pathways had not been done previously in any organism. The significance of this research is twofold: One is training the next generation of scientists. During the course of this project, four undergraduate students were taught good research practices including hands-on training in wet lab procedures. Two of the students contributed significantly to the project and were co-authors of a published paper. We anticipate publication of another paper on a separate aspect of the project, which will include the other two students as co-authors. Of the four students, one is currently in medical school, another is training to be a physician’s assistant, a third is planning to attend graduate school, and the youngest student will soon apply to medical school. In addition, a post-doc and research associate were crucially involved in the project. Both received specialized cytological training from the principal investigator, and both developed mentoring skills by training and supervising the undergraduate students. The second significant aspect of this research is its contribution to society. In this regard, one of our most interesting discoveries is that Class I and Class II crossovers have different distributions along chromosomes. In particular, Class II crossovers are disproportionately found in regions of the genome that do not often crossover. Because these regions do not crossover much, it is difficult to utilize the many genes in these regions for improving plants by breeding. Research by others has shown that it is possible to specifically increase the number of Class II crossovers in plants. Combining this result with our findings, we think it is likely that increasing the number of Class II crossovers would likely mobilize this relatively inaccessible genetic material and increase the speed and effectiveness of plant improvement programs for tomatoes and other crops. For example, such an approach could facilitate integration of beneficial genes from wild species. The genomes of wild plants often carry important genes that can be used to increase yield and/or improve disease and drought resistance in crops. However, this resource is often unexploited because the genes cannot be transferred to cultivated species without adding genes that reduce the agronomic value of crops. Increasing crossing over using Pathway II could provide a way to exploit these genetic resources more effectively and thereby lead to a more secure food supply.
