Two Types of Meiotic Crossovers in Higher Plants

智力优势：在有性生殖过程中，每对同源染色体通常需要至少一次（专性）交叉，以确保染色体的适当分离和产生基因平衡的单倍体配子。在出芽酵母、哺乳动物和植物中，至少有两种途径导致交叉：有干扰的MLH1途径（其中一个交叉的存在降低了附近另一个交叉的可能性）和被认为很少或没有干扰的MUS81途径。如何控制交叉频率和分布以及不同交叉途径如何相互作用是遗传学中的两个基本问题，对基因组进化、物种形成和动植物育种具有理论和实践意义。在全基因组水平上评估这两种交叉途径是困难的，因为这两种类型的交叉不能用连锁图谱来区分。相比之下，细胞遗传学免疫标记方法是理想的，因为它可以明确地识别和定位每种类型的个体交叉。本项目将利用光（LM）和电镜（EM）对番茄粗线染色体上MLH1和MUS81蛋白的免疫定位，直接评估每种途径对高等植物杂交和干扰的贡献。MLH1是大多数晚期重组结节（LNs）的组成部分。l标记交叉位点，在番茄中被广泛研究。MUS81被认为是mlh1阴性LNs的一个组成部分，EM免疫定位将用于验证这一假设。后续工作将利用MLH1和MUS81的同时LM免疫定位，将这些焦点定位到野生型番茄植株和MLH1和MUS81基因沉默系的粗线染色体传播上。这些关于两类杂交在不同遗传情况下的作用的数据将在一个综合的杂交数学模型中加以利用。这项工作将是首次在任何物种中进行此类研究，并将在开花植物全基因组水平上为干扰型和非干扰型杂交的贡献和相互作用提供新的见解。更广泛的影响：这项工作将定义正常和突变开花植物中两种不同交叉途径的贡献和相互作用。这一信息将有助于设计针对遗传重组特别感兴趣的目标区域的育种方法，以改善农业品系。研究中产生的有用工具（如抗体和转基因植物）将与同事分享。人力资源开发是本研究的重要内容，将有一名研究生和一名博士后密切参与研究。每个人都将得到首席研究员的个人关注和定期指导，两人都将在内布拉斯加大学专门研究植物基因沉默方法的实验室接受培训。此外，本科生将参与许多方面的研究。在最初的培训期之后，每位本科生将被鼓励从事一个支持研究总体目标的独立项目，并在科罗拉多州立大学（CSU）的本科生年度论坛上展示他们的研究成果，该论坛名为“庆祝本科生研究和创造力”。通过与科罗拉多州立大学的麦克奈尔项目合作，将专门招募代表性不足和少数民族的学生。

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.