Project Summary Multicellular organisms face constant mutational pressures due to DNA damage and errors in cell division. New mutations continually accumulate throughout development and can result in cancer, aging, and infertility. With the increased sensitivity to detect mutations by deep sequencing, it has become clear that even healthy individuals are genetically heterogeneous and carry numerous post- zygotic mutations in both their somatic and germinal cells. This proposal aims to understand how organisms cope with ongoing mutation during development, focusing on maize and Arabidopsis. First, we will investigate the accumulation of mutations in different tissues throughout the life cycle. To follow new mutations, we focus on transposons as it is possible to quantify rare, recent mutations with high sensitivity. We hypothesize that development is organized in a way that maintains the diversity of the meristem stem cell niche, avoiding population bottlenecks in critical reservoir cells and thus preventing any single somatic mutation from reaching fixation across the organism. We will test whether plants with altered growth patterns and meristem defects accumulate new mutations more frequently because they are unable to maintain stem cell diversity. Second, we will investigate chromosomal regulation in the soma and germline during maize pollen development. Each grain of pollen is a simple 3-celled organism, with a single somatic cell and two germ cells. We will study the activation of the haploid pollen genome and then determine how epigenetic marks that differentiate soma and germline are established and maintained. Genome changes will be followed using single-cell RNA-seq, chromatin profiling, and microscopy. We will identify genes that regulate this process using a novel strategy to perform genetic screens directly in haploid pollen, allowing millions of mutations to be assayed per plant. Ultimately, this work will shed insight into how organisms regulate multicellular development and chromosomal structure to reduce the impact of new mutations.