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Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in genes of interest using high-throughput resequencing. A clonal library of 6144 EMS-mutagenized worms was established and screened, resulting in the identification of 1044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation of our results indicates that nonsense mutations for >90% of all C. elegans genes are present in the library. To identify all of these mutations, one only needs to inspect those positions that--given the known specificity of the mutagen--can result in the introduction of a stop codon. We define these positions as nonsense introducing mutations (NIMs). The genome-wide collection of possible NIMs can be calculated for any organism with a sequenced genome and reduces the screening complexity by 200- to 2000-fold, depending on the organism and mutagen. For EMS-mutagenized C. elegans, there are only approximately 500,000 NIMs. We show that a NIM genotyping approach employing high-density microarrays can, in principle, be used for the genome-wide identification of C. elegans knockouts.

Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature-size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature-size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature-size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 x CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature-size rule, which has puzzled biologists for decades., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Recent genetical genomics studies have provided intimate views on gene regulatory networks. Gene expression variations between genetically different individuals have been mapped to the causal regulatory regions, termed expression quantitative trait loci. Whether the environment-induced plastic response of gene expression also shows heritable difference has not yet been studied. Here we show that differential expression induced by temperatures of 16 degrees C and 24 degrees C has a strong genetic component in Caenorhabditis elegans recombinant inbred strains derived from a cross between strains CB4856 (Hawaii) and N2 (Bristol). No less than 59% of 308 trans-acting genes showed a significant eQTL-by-environment interaction, here termed plasticity quantitative trait loci. In contrast, only 8% of an estimated 188 cis-acting genes showed such interaction. This indicates that heritable differences in plastic responses of gene expression are largely regulated in trans. This regulation is spread over many different regulators. However, for one group of trans-genes we found prominent evidence for a common master regulator: a transband of 66 coregulated genes appeared at 24 degrees C. Our results suggest widespread genetic variation of differential expression responses to environmental impacts and demonstrate the potential of genetical genomics for mapping the molecular determinants of phenotypic plasticity., Competing Interests: Competing interests. The authors have declared that no competing interests exist.