PROJECT SUMMARY Changes in the copy number of large genomic regions, termed copy number variations or CNVs, contribute to phenotypic diversity and facilitate important processes such as host range and drug resistance. Despite decades of CNV research, many questions regarding their formation and dynamics remain unanswered. Due to distinctive genome characteristics, facile in vitro propagation, and the relative simplicity of CNV formation, Plasmodium falciparum is an exceptional model to study many aspects of CNV-based adaptation. Experimental evolution demonstrates how this haploid asexual parasite rapidly acquires CNVs in the form of tandem duplications. By studying the junctions of these CNVs, we have identified the precise genome characteristics that contribute to their formation in this organism; A/T-rich features of the genome both trigger DNA breakage and facilitate subsequent error-prone repair. Based on this finding, we hypothesize that the extreme AT content of the P. falciparum genome (>80%) specifically contributes to its highly adaptive nature. We aim to explore this hypothesis using different human-infective Plasmodium species, which have genomes exhibiting a range of AT- content (differing by >20% overall and ~30% in intergenic regions). To do so, we will generate highly accurate genome assemblies to characterize CNV junctions and the sequences that lead to their formation. We will identify genome features that contribute to DNA breakage and CNV formation in vivo. We will measure the frequency of novel CNV generation, under both basal and stressed conditions. These proposed studies will facilitate our creation of a genomic map of adaptive potential for different malaria species, provide firm evidence for our model of CNV formation, and define constraints influencing CNV evolution. This knowledge, and the novel methods developed during this project, will pave the way to developing approaches to limit CNV-based adaptation in diverse microorganisms, cancers, and other cell types under rapid evolution.