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Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila.

Najarro MA, Hackett JL, Smith BR, Highfill CA, King EG, Long AD, Macdonald SJ - PLoS Genet. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States of America.

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.

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Effects of single gene RNAi knockdown experiments.Gal4-UAS-RNAi female progeny of several genotypes were tested in our caffeine resistance assay against control strains ("Con"). The genes tested were Cyp12d1-d and Cyp12d1-d under QTL Q2, Cyp301a1 under Q3, and Cyp6d5 and under Q9. Each bar represents the mean lifespan (± 1-SD) in the assay across a number of genetically-identical individuals (sample size is in the bottom right corner of each bar), and asterisks represent the significance of Welch's t-test comparing each RNAi genotype to its respective control (ns = not significant, *p < 0.01, **p < 0.001, ***p < 10−10). (A) Driving Gal4 using a ubiquitous promoter with KK-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60100, 109248, 109256, 109771, and 107641. (B) Driving Gal4 using a ubiquitous promoter with GD-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60000, 50507, 21235, and 49269. (C and D) Driving Gal4 ubiquitously in adults using an RU486-inducible promoter in two independent trials, the first (C) with flies on RU486 for 24 hours prior to the assay and throughout, and the second (D) with flies on RU486 for 48 hours prior to the assay and throughout. Genotypes tested are the same as those depicted in (A).
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pgen.1005663.g003: Effects of single gene RNAi knockdown experiments.Gal4-UAS-RNAi female progeny of several genotypes were tested in our caffeine resistance assay against control strains ("Con"). The genes tested were Cyp12d1-d and Cyp12d1-d under QTL Q2, Cyp301a1 under Q3, and Cyp6d5 and under Q9. Each bar represents the mean lifespan (± 1-SD) in the assay across a number of genetically-identical individuals (sample size is in the bottom right corner of each bar), and asterisks represent the significance of Welch's t-test comparing each RNAi genotype to its respective control (ns = not significant, *p < 0.01, **p < 0.001, ***p < 10−10). (A) Driving Gal4 using a ubiquitous promoter with KK-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60100, 109248, 109256, 109771, and 107641. (B) Driving Gal4 using a ubiquitous promoter with GD-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60000, 50507, 21235, and 49269. (C and D) Driving Gal4 ubiquitously in adults using an RU486-inducible promoter in two independent trials, the first (C) with flies on RU486 for 24 hours prior to the assay and throughout, and the second (D) with flies on RU486 for 48 hours prior to the assay and throughout. Genotypes tested are the same as those depicted in (A).

Mentions: To confirm effects of a subset of the P450 genes uncovered by our mapping and expression studies (Cyp12d1-d and Cyp12d1-p, Cyp301a1, and Cyp6d5 under Q2, Q3, and Q9, respectively) we carried out knock-down experiments using the Gal4-UAS RNAi system. When knocked down ubiquitously (RNAi in all cells at all timepoints), all but one of the Gal4-UAS showed a significant reduction in caffeine resistance compared to controls (Fig 3A and 3B), with the remaining genotype showing a minor reduction in resistance (Fig 3B; transformant ID 21235, Cyp12d1-p, Welch's t-test, p = 0.066). In two cases the RNAi appears to interfere with some essential developmental process, since we struggled to generate individuals of the appropriate genotypes for assay (Fig 3A; genes Cyp12d1-d, transformant ID 109248, and Cyp301a1). Since Cyp301a1 appears to be involved in cuticle development, and reduced expression of the gene results in cuticle malformation [48], this may have played a role in our failure to generate large numbers of progeny. We also cannot rule out any minor developmental abnormalities in the other RNAi genotypes that may have led to reduced adult fitness, and a nonspecific reduction in caffeine resistance. To control for any such pleiotropic effects of the test genes during development, we subsequently used RU486-inducible Gal4 to drive RNAi in all cells in young adults (Fig 3C and 3D). Over two trials (differing only in the amount of time the test animals were fed RU486 prior to caffeine+RU486 exposure), the only RNAi knockdown to show a consistent, highly-significant reduction in caffeine resistance was for Cyp12d1-d (Fig 3C and 3D). Since the effect of RU486 on expression knockdown is dose-dependent [49], it is possible higher concentrations of RU486 could have replicated our findings using the ubiquitous Gal4 driver (Fig 3A), and Cyp301a1 and Cyp6d5 remain excellent candidates to underlie QTL Q3 and Q9.


Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila.

Najarro MA, Hackett JL, Smith BR, Highfill CA, King EG, Long AD, Macdonald SJ - PLoS Genet. (2015)

Effects of single gene RNAi knockdown experiments.Gal4-UAS-RNAi female progeny of several genotypes were tested in our caffeine resistance assay against control strains ("Con"). The genes tested were Cyp12d1-d and Cyp12d1-d under QTL Q2, Cyp301a1 under Q3, and Cyp6d5 and under Q9. Each bar represents the mean lifespan (± 1-SD) in the assay across a number of genetically-identical individuals (sample size is in the bottom right corner of each bar), and asterisks represent the significance of Welch's t-test comparing each RNAi genotype to its respective control (ns = not significant, *p < 0.01, **p < 0.001, ***p < 10−10). (A) Driving Gal4 using a ubiquitous promoter with KK-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60100, 109248, 109256, 109771, and 107641. (B) Driving Gal4 using a ubiquitous promoter with GD-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60000, 50507, 21235, and 49269. (C and D) Driving Gal4 ubiquitously in adults using an RU486-inducible promoter in two independent trials, the first (C) with flies on RU486 for 24 hours prior to the assay and throughout, and the second (D) with flies on RU486 for 48 hours prior to the assay and throughout. Genotypes tested are the same as those depicted in (A).
© Copyright Policy
Related In: Results  -  Collection

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pgen.1005663.g003: Effects of single gene RNAi knockdown experiments.Gal4-UAS-RNAi female progeny of several genotypes were tested in our caffeine resistance assay against control strains ("Con"). The genes tested were Cyp12d1-d and Cyp12d1-d under QTL Q2, Cyp301a1 under Q3, and Cyp6d5 and under Q9. Each bar represents the mean lifespan (± 1-SD) in the assay across a number of genetically-identical individuals (sample size is in the bottom right corner of each bar), and asterisks represent the significance of Welch's t-test comparing each RNAi genotype to its respective control (ns = not significant, *p < 0.01, **p < 0.001, ***p < 10−10). (A) Driving Gal4 using a ubiquitous promoter with KK-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60100, 109248, 109256, 109771, and 107641. (B) Driving Gal4 using a ubiquitous promoter with GD-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60000, 50507, 21235, and 49269. (C and D) Driving Gal4 ubiquitously in adults using an RU486-inducible promoter in two independent trials, the first (C) with flies on RU486 for 24 hours prior to the assay and throughout, and the second (D) with flies on RU486 for 48 hours prior to the assay and throughout. Genotypes tested are the same as those depicted in (A).
Mentions: To confirm effects of a subset of the P450 genes uncovered by our mapping and expression studies (Cyp12d1-d and Cyp12d1-p, Cyp301a1, and Cyp6d5 under Q2, Q3, and Q9, respectively) we carried out knock-down experiments using the Gal4-UAS RNAi system. When knocked down ubiquitously (RNAi in all cells at all timepoints), all but one of the Gal4-UAS showed a significant reduction in caffeine resistance compared to controls (Fig 3A and 3B), with the remaining genotype showing a minor reduction in resistance (Fig 3B; transformant ID 21235, Cyp12d1-p, Welch's t-test, p = 0.066). In two cases the RNAi appears to interfere with some essential developmental process, since we struggled to generate individuals of the appropriate genotypes for assay (Fig 3A; genes Cyp12d1-d, transformant ID 109248, and Cyp301a1). Since Cyp301a1 appears to be involved in cuticle development, and reduced expression of the gene results in cuticle malformation [48], this may have played a role in our failure to generate large numbers of progeny. We also cannot rule out any minor developmental abnormalities in the other RNAi genotypes that may have led to reduced adult fitness, and a nonspecific reduction in caffeine resistance. To control for any such pleiotropic effects of the test genes during development, we subsequently used RU486-inducible Gal4 to drive RNAi in all cells in young adults (Fig 3C and 3D). Over two trials (differing only in the amount of time the test animals were fed RU486 prior to caffeine+RU486 exposure), the only RNAi knockdown to show a consistent, highly-significant reduction in caffeine resistance was for Cyp12d1-d (Fig 3C and 3D). Since the effect of RU486 on expression knockdown is dose-dependent [49], it is possible higher concentrations of RU486 could have replicated our findings using the ubiquitous Gal4 driver (Fig 3A), and Cyp301a1 and Cyp6d5 remain excellent candidates to underlie QTL Q3 and Q9.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States of America.

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.

Show MeSH
Related in: MedlinePlus