Abstract
Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5–14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes.
Highlights
Living organisms are subjected to a barrage of toxic compounds, or xenobiotics, in their environment and their diet
We identify several short genomic regions that collectively explain a substantial portion of the heritable variation for caffeine resistance, and find that several of these regions harbor members of known detoxification enzyme families
We show that variation in the number of copies of Cyp12d1 is positively associated with resistance
Summary
Living organisms are subjected to a barrage of toxic compounds, or xenobiotics, in their environment and their diet. The Monarch butterfly (Danaus plexippus) is resistant to cardenolides, a class of secondary metabolites toxic to most animals, produced by their milkweed host plant. This resistance is due to at least one amino acid change in the Monarch Na+, K+-ATPase gene that prevents dietary cardenolides binding to the protein, making the protein insensitive to cardenolide inhibition [4, 5]. Cytochrome P450 monooxygenases (P450s) act on the toxic compounds to decrease their toxicity The products of these reactions subsequently become substrates for phase two enzymes, such as glutathione-S-transferases (GSTs) and UDP-glucuronosyltransferases (UGTs), which add large, charged side groups onto substrate molecules making them easier to excrete. As a result of a series of naturally-occurring gene duplication events and transposable element (TE) insertions, overexpression of the P450 Cyp6g1 is primarily responsible for resistance to the insecticide DDT (dichlorodiphenyltrichloroethane) in Drosophila [10,11,12]
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