1. Introduction Obtaining insights into cellular complexity requires a thorough understanding of small silencing RNA pathways. Small silencing RNAs, characterized by their length of 21 to 35 nucleotides, have been classified into three prominent families: small interfering RNAs (siRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs). The Argonaute family protein is comprised of AGO and PIWI clads proteins. AGO is loaded with miRNAs and siRNA whereas piRNAs are loaded into the PIWI clades protein. piRNA exhibits hallmark characteristics that set them apart from siRNAs and miRNAs: they mostly start with a uridine base and retain 2ʹ-O-methyl modifications at their 3ʹ termini[1]. A unique aspect of piRNAs biology emerges in placental mammals where approximately 100 genes are specifically expressed at the pachytene stage of meiosis I, resulting in the production of pachytene piRNAs. In adult mouse testis, pachytene piRNAs are the most abundant group of small RNAs. During the process of male gametogenesis, pachytene piRNAs regulate the formation of functional sperm by silencing specific mRNAs. Counterintuitively, genes producing pachytene piRNAs and pachytene piRNAs themselves are not conserved among placental mammals[2]. This raises the question of how spermatogenesis relies on RNA molecules whose sequences evolve rapidly. We hypothesize that rapid changes in pachytene piRNAs sequences result in silencing distinct sets of mRNAs in different species, therby probing the reproductive barrier between species, i.e., speciation. To address this question, we will use mouse sub-species (i.e., Mus musculus domesticus, Mus musculus musculus, and Mus musculus castaneus) that are geographically isolated and retain reproductive barriers between each other. The initial step is to map pachytene piRNA producing genes in mouse sub-species which involves sequncing large transcripts and piRNAs to demarcate transcripts producing pachytene piRNA. After defining pachytene piRNA-producing genes in mouse sub-species, we will perform comparative genome analyses to pinpoint species-specific pachytene piRNA genes. To further test our hypothesis that differences in piRNA sequences target different mRNAs, we perform degradome sequencing which enable us to capture piRNA-guided mRNA cleavage products. This comparative investigation, conducted between mouse subspecies, becomes imperative to elucidate the underlying mechanisms of genetic variation in pachytene piRNAs and its possible contribution to speciation and reproductive biology. 2. Research Objective The primary aim of this project is to examine the diverse sequences and expression profiles of pachytene piRNAs in placental mammals and to uncover the functional significance of pachytene piRNAs in germ cell biology by identifying their specific mRNA targets.