Discovering Zinc Resistance Loci via Extreme QTL Mapping
Authors: Katherine Hanson 1; Anthony D. Long 2; Stuart J. Macdonald 1,3
Affiliations: 1) Department of Molecular Biosciences, University of Kansas, Lawrence, KS; 2) Department of Ecology and Evolutionary Biology, University of California at Irvine, Irvine, CA; 3) Center for Computational Biology, University of Kansas, Lawrence, KS
Keywords: e. quantitative traits; a. stress responses
Many heavy metals such as zinc, copper and manganese are essential for cellular function and maintaining metal homeostasis is critical. When exposed to toxic levels of heavy metals, including essential metals, organisms can suffer deleterious consequences, including increased risk for cancer and organ failure. Since zinc is involved in many cellular functions, zinc toxicity can have widespread effects, leading to necrosis, inhibition of mitochondria, and impacting the homeostasis of other heavy metals. Zinc toxicity has been primarily studied via functional genetics, employing single-gene mutations and expression knockdowns in a small number of inbred lines. Because the response to zinc toxicity is a complex, polygenic, trait, our goal is to identify those genes segregating for allelic variation for zinc resistance using an unbiased genome wide mapping approach. Drosophila melanogaster is an ideal model to study zinc resistance since it has orthologs of critical genes involved in zinc homeostasis, such as MTF-1 (a transcription factor involved in metal response), zinc transporter (ZnT) proteins and has proved to be a successful model to understand other heavy metal response traits. To identify zinc resistance loci we employed extreme QTL, or XQTL, mapping, a powerful technique that identifies QTL via extreme phenotypic selection of a population. We established a large, outbred population by mixing hundreds of DSPR RILs (Drosophila Synthetic Population Resource Recombinant Inbred Lines), a set of advanced intercross RILs derived from 8 inbred founder lines. We raised animals from this population on media supplemented with toxic levels of zinc, sequencing the pool of surviving, zinc resistant females and a matching control population, replicating the experiment 12 times. We estimate that >4000 animals were tested per replicate, and in each case we selected the top ~7% of animals. At each position in the genome we estimated the founder composition from each pooled sample, identifying QTL as significant frequency shifts between control and selected populations. We succeeded in identifying seven QTL, including one that overlaps with a QTL previously identified for copper developmental viability. Our QTL encompass physical intervals between 320 kb and 880 kb and collectively contain 451 protein coding genes. We implicate MTF-1, Mekk1, a gene involved in cadmium toxicity, present within the shared copper/zinc QTL, and 5 genes found to be associated with zinc resistance in a Drosophila cell line study. For a majority of our QTL only 1 or 2 founder alleles show a substantial frequency change between the control and selected populations, implying that resistant and susceptible alleles are often rare. We identified a series of candidate genes using existing functional data, including previous metal toxicity studies, and gene ontology, and will further test these via gut-specific RNAi knockdowns.