A nematode-specific gene underlies bleomycin-response variation in Caenorhabditis elegans

Shannon C. Brady; Stefan Zdraljevic; Karol W. Bisaga; Robyn E. Tanny; Daniel E. Cook; Daehan Lee; Ye Wang; Erik C. Andersen

Here, we used a high-throughput fitness assay to measure bleomycin responses across a panel of 104 249 RIAILs (Andersen et al. 2015) and then performed linkage mapping to identify quantitative 105 trait loci (QTL) that underlie bleomycin-response variation. We used near-isogenic lines (NILs) 106 to validate the largest effect QTL on chromosome V. Our results from the NIL assays suggested 107 that epistatic loci underlie bleomycin responses, but a two-factor genome scan was unable to 108 detect significant genetic interactions. Next, we created and tested CRISPR-Cas9 mediated 109 deletion alleles to investigate all six candidate genes in the QTL region. We identified a 110 nematode-specific gene, H19N07.3, that underlies this QTL. Although this gene does not contain 111 a protein-coding variant between the N2 and CB4856 strains, its gene expression varies across 112 the RIAIL panel. Interestingly, a genome-wide association (GWA) approach identifies different 113 QTL than the linkage mapping approach, suggesting that both common and rare variants underlie 114 bleomycin response variation. Given the genetic complexity underlying the bleomycin response 115 phenotype, this study highlights the power of the C. elegans model system to identify elusive 116 causal genes. 117 118 MATERIALS AND METHODS 119 120 Strains: Animals were grown at 20º on 6 cm plates of modified nematode growth medium 121 (NGMA), which contained 1% agar and 0.7% agarose, spotted with OP50 bacteria (Ghosh et al. 122 2012). The two parental strains used in this study were N2 and CB4856. N2 is the canonical 123 laboratory strain of C. elegans that has been extensively studied (Brenner 1974) . CB4856 is a 124 well studied Hawaiian wild isolate that is genetically divergent from N2 and has a characterized 125 genome (Wicks et al. 2001; Swan et al. 2002 ; Thompson et al. 2015) . The N2 and CB4856 126 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/565218 doi: bioRxiv preprint 8 of 48 by the BIOSORT for several fitness parameters. Because bleomycin exposure can affect animal 150 proliferation (brood size), animal growth (length), and animal development (optical density), the 151 fitness parameters we measured with the BIOSORT included brood size (n), animal length (time 152 of flight, TOF), and optical density (extinction time, EXT). 153 154 Bleomycin-response trait measurements and processing: Phenotypic measurements collected 155 by the BIOSORT were processed using the R package easysorter (Shimko and Andersen 2014) . 156