Project Summary/Abstract Selfish meiotic drive systems (SMDs) that cheat Mendel’s law of segregation during gametogenesis are ubiquitous in nature. Paradoxically, the wildtype activities of such selfish genes, which permit their biased transmission, have negative fitness costs, such as distortion of sex-ratio (SR) or sterility. Therefore, SMDs are catalysts for intragenomic conflicts and place strong pressure to innovate host suppression mechanisms. Despite their ubiquity and powerful effects on normal transmission, the molecular mechanisms by which SMDs emerge, operate, and are silenced, generally remain poorly understood. My prior works on the evolutionary genomics of simulans clade introduced me to the recently-emerged “cryptic” SMDs in D. simulans (Dsim) and motivated me to study the molecular basis of meiotic drive for postdoctoral work. A significant contribution from my postdoctoral work revealed a crucial role for hairpin RNA (hpRNA)-class siRNA loci in suppressing the SMDs, Distorter on the X (Dox) and Mother of Dox (MDox) in Dsim. Furthermore, transcriptome profiling from RNAi mutants (Ago2 and Dcr2) revealed an array of de novo hpRNAs that were born in the simulans clade and preferentially target X-chromosome genes, suggesting sex chromosomes as a breeding ground for intragenomic conflicts. I hypothesize that the surprising role(s) of RNAi in resolving intragenomic conflicts may be widespread across taxa, and untamed meiotic drive can propel speciation. The proposed work expands on my discoveries utilizing a multidisciplinary approach. First, in Specific Aim 1, using clues to the origin, and their provocative similarity to sperm packaging proteins, I will study how Dox/MDox impair spermatogenesis, employing cutting-edge molecular biology and genomics. The training component of SA1 includes learning the state-of-the-art CRISPR-Cas9 gene editing techniques, and testis cytology. Based on conserved themes in genetic conflicts, I suspect a role for RNAi in resolving intragenomic conflict in mice, analogous to flies, and in Specific Aim 2, I will test this hypothesis with the proprietary Ago-2 catalytic dead (Ago2-CD) mutant mice generated in Dr. Eric Lai’s lab. The proposed work in SA2 will be performed in collaboration with Dr. Scott Keeney, an expert in mouse meiosis and spermatogenesis at Sloan-Kettering Institute. The training component of SA2 includes learning microscopy, image analysis, and morphological characterization of Ago2-CD mutant mice. Finally, in Specific Aim 3, I will build upon the exciting discovery in Dsim that several cryptic SMDs can be uncovered by ablating the RNAi pathway. I propose to employ RNAi mutants as a versatile “tool” to unmask cryptic SMDs in non-model insects, which otherwise take years of laborious genetics to identify and characterize SMDs. The training component described in the proposed work will complement my previous experience, and position me for a productive career as an independent investigator...