How environmentally-triggered, non-genetic effects are molecularly transmitted over multiple generations and whether regulation of such epigenetic inheritance shows genetic variation in nature represent key questions in current biological research. Here we address these questions by characterizing natural genetic variation in epigenetic inheritance in the nematode Caenorhabditis elegans. We have established experimental paradigms to study multigenerational transmission of fertility defects induced by environmental stressors: (1) chronic exposure to high temperature (25°C), inducing a progressive sterility phenotype across multiple generations; (2) a single, early-life starvation stress inducing fertility defects, such as reduced brood size, across multiple generations. Our preliminary data show for both systems that (a) such transmission likely involves epigenetic inheritance and (b) that C. elegans shows naturally genetic variation in the presence and degree of such multigenerational transmission. The occurrence of this intraspecific variation will allow genetic characterization of mechanisms underlying epigenetic inheritance of fertility effects over multiple generations. We will complement this genetic mapping with the analysis of candidate molecular mechanisms involved in epigenetic inheritance systems: small RNAs and chromatin marks. The project will involve three research groups with complementary expertise in the fields of C. elegans developmental genetics, evolutionary and quantitative genetics, and epigenetic inheritance through small RNA and chromatin pathways. The four principal objectives are: (1) Characterization of natural genetic variation in multigenerational inheritance systems: We will obtain quantitative measures of natural genetic variation in multigenerational transmission of progressive sterility (a) at 25°C and (b) after early-life starvation through analysis of a reference panel of 97 genetically distinct isogenic C. elegans wild isolates. Our preliminary data indicate that variation is found among these wild isolates in both assays. (2) Natural genetic variation and multigenerational changes in small RNA content, histone modification, genome and transcriptome: We will compare piRNA and endo-siRNA content a) among C. elegans wild isolates and b) across generations in control and stress (high temperature and L1 starvation) conditions. Whole-genome sequences will provide information on the genomic piRNA content. Sequencing of genome and transcriptome at later time points will probe the possible accumulation of molecular genetic lesions and gene expression changes. (3) Genetic mapping of natural variation in multigenerational inheritance: We will establish F2 Recombinant Inbred Lines derived from parental isolate pairs with strong differences in transgenerational transmission of sterility defects to map the genes underlying these differences. Quantitative Trait Locus mapping will be complemented by fine-mapping using Nearly Isogenic Lines, allowing the identification of candidate loci. (4) Candidate molecular polymorphisms will be tested by complementation analysis, RNAi, transgenesis and targeted genome editing using the CRISPR-Cas9 technique. The molecular mechanisms of the phenotypic variation will then further studied using isogenic backgrounds differing solely in the identified polymorphisms. Significance: We have established unique and novel C. elegans systems allowing the molecular genetic study of transgenerational inheritance triggered by environmental stressors based on existing natural genetic variation in the species. This project will characterize, for the first time, how natural genetic variation modulates molecular mechanisms of epigenetic inheritance systems. This project therefore addresses a fundamental question in current biological research with wide-ranging implications for multiple disciplines, including epigenetics and gene regulation, developmental and evolutionary biology.