Project Summary Parthenogenesis (i.e., reproduction without mating) has evolved from sexual reproduction in nearly all major eukaryotic groups. In parthenogenesis, chromosomally unreduced (e.g., diploid) gametes result from modified forms of meiosis. Understanding the genetic mechanisms underlying the modification of meiosis in parthenogenetic lineages is of significant public health interest because meiosis is central to sexual reproduction. Using parthenogenesis to understand the genetic regulation of meiosis is also a highly innovative approach, with its natural history perspective most likely yielding novel knowledge about meiosis. Using a combination of evolutionary and functional genomic approaches, this project examines the genetic bases of cyclical and obligate parthenogenesis in the freshwater microcrustacean Daphnia, which represents an excellent tractable experimental system with well-understood biology and many genomic tools. Daphnia is well known for its cyclical parthenogenesis (CP) life cycle, i.e., propagating asexually under favorable environmental conditions and switching to sexual reproduction in response to stressful environment. Interestingly, some populations of the species D. pulex (backcrosses of two parental CP species D. pulex and D. pulicaria) reproduce by obligate parthenogenesis (OP) because they lost the capability to engage in sex. This project has two long-term goals. First, considering that a single Daphnia genome can encode the genetic machinery for both reproductive strategies, it is hypothesized that environment-mediated gene expression changes, especially the neofunctionalization of duplicated meiosis genes (e.g. Cdc20), play a role in the origin of CP. To test this, ovary- specific gene expression changes between parthenogenesis and meiosis in CP D. pulex and D. pulicaria will be examined to produce a high quality set of candidate genes. To determine the functional role of these candidates in CP (e.g., master regulator genes), we will perform RNAi knockdown of each candidate in CP isolates and examine the associated phenotypic effects using animal reproduction assay and cytological examination of developing oocytes. Second, concerning the origin of OP, we hypothesize that the genetic incompatibility between the two ecologically divergent species CP D. pulex and D. pulicaria is a key factor. For the previously identified candidate genes for OP, we will test (1) whether they experience differential gene expression in OP isolates compared to CP isolates and (2) whether their expression is mis-regulated (i.e., genetic incompatibility). The former will be achieved by ovary-specific RNA-seq in OP and CP clones at different reproductive stages, whereas the latter will be based on allele-specific RNA-seq analysis of the parental species and OP isolates. For the candidate genes showing differential expression and mis-regulation, RNAi knockdown and phenotype screening will be performed to verify their functional role in OP, whereas CRISPR/Cas9 editing will be used to identify the causal SNPs for each gene. Lastly, we will re-create OP animals by introducing OP-causal SNPs to CP Daphnia pulex using CRISPR/Cas9 editing.