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

For the bleomycin-response variation scantwo, the significant interaction threshold was a LOD 248 score of 4.08. The significant interaction threshold for the scb-1 expression variation scantwo 249 was 4.05. The bleomycin-response variation scantwo summary is available as File S8, and the 250 scantwo summary for scb-1 expression variation is available as File S17. 251 252 Identification of genes with non-synonymous variants: The get_db function within the cegwas 253 R package was used to query WormBase build WS245 for genes in the QTL confidence interval 254 (V:11,042,745-11,189,364). Our initial linkage mappings used the 1,454 marker set (Rockman 255 and Kruglyak 2009) and had a QTL confidence interval larger than the interval found using the 256 whole-genome marker set (described above). Because this confidence interval was larger and 257 more conservative, we kept it for subsequent testing of candidate genes. This expanded interval 258 contained an additional 20 kb on either side of the whole-genome marker set confidence interval 259 (File S9). The snpeff function within the cegwas R package was used to identify variants within 260 the region of interest (File S11 . We performed linkage mapping as described above on these expression data and 315 identified significant peaks for 3,298 probes (File S13). For the H19N07.3 probe 316 (A_12_P104350), the full annotated-LOD data frame is provided (File S14). 317 318 Hemizygosity high-throughput assay: Hemizygosity assays were used to test the effect of zero 319 (two deletion alleles), one (one deletion allele and one wild-type allele), or two (two wild-type 320 alleles) functional copies of a gene product on bleomycin responses. If the phenotype is affected 321 by gene function, one would expect to see bleomycin responses scale with the number of 322 functional alleles of the gene present in each strain. For each heterozygous/hemizygous 323 genotype, 30 hermaphrodites and 60 males were placed onto each of four 6 cm plates and 324 allowed to mate for 48 hours. The same process was completed for homozygous strains to 325 remove biases introduced by the presence of male progeny in the assay. Mated hermaphrodites 326 were transferred to a clean 6 cm plate and allowed to lay embryos for eight hours. After the egg 327 lay period, adults were manually removed from egg lay plates, and embryos were washed into 15 328 mL conicals using M9 and a combination of pasteur pipette rinsing and scraping with plastic 329 inoculation loops. Embryos were rinsed and centrifuged four times with M9 before being 330 resuspended in K medium and 50 µM kanamycin. Embryos hatched and arrested in the L1 larval 331 stage in conicals overnight at 20º with shaking. The next morning, 50 L1 larvae were sorted into 332 . 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 each well of a 96-well plate containing K medium, 10 mg/mL bacterial lysate, 50 µM 333 kanamycin, and either 1% distilled water or 1% distilled water plus bleomycin using the 334 BIOSORT (dose-response assay, Figure S10 , File S1) or by titering (hemizygosity assays, 335 for bleomycin responses in the RIAILs and reports the maximum interaction LOD score for each 404 chromosome pair. File S9 contains information on all genes in the QTL confidence interval plus 405 20 kb on either side. File S10 contains loc