Analysis of natural variation reveals neurogenetic networks for Drosophila olfactory behavior

Abstract

Understanding the relationship between genetic variation and phenotypic variation for quantitative traits is necessary for predicting responses to natural and artificial selection and disease risk in human populations, but is challenging because of large sample sizes required to detect and validate loci with small effects. Here, we used the inbred, sequenced, wild-derived lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) to perform three complementary genome-wide association (GWA) studies for natural variation in olfactory behavior. The first GWA focused on single nucleotide polymorphisms (SNPs) associated with mean differences in olfactory behavior in the DGRP, the second was an extreme quantitative trait locus GWA on an outbred advanced intercross population derived from extreme DGRP lines, and the third was for SNPs affecting the variance among DGRP lines. No individual SNP in any analysis was associated with variation in olfactory behavior by using a strict threshold accounting for multiple tests, and no SNP overlapped among the analyses. However, combining the top SNPs from all three analyses revealed a statistically enriched network of genes involved in cellular signaling and neural development. We used mutational and gene expression analyses to validate both candidate genes and network connectivity at a high rate. The lack of replication between the GWA analyses, small marginal SNP effects, and convergence on common cellular networks were likely attributable to epistasis. These results suggest that fully understanding the genotype-phenotype relationship requires a paradigm shift from a focus on single SNPs to pathway associations.

Fig. 1.

Association between SNPs in the DGRP and variation in olfactory behavior. (A) Line means for olfactory behavioral response to 0.3% (vol/vol) benzaldehyde for DGRP males (blue bars) and females (pink bars). Error bars are SEMs. (B) Genome-wide association analyses. All SNPs from single-marker analyses with P < 10⁻⁵ are shown. (Upper) Associations based on females are indicated by red dots, males by blue dots, and sexes pooled by black dots. (Lower) The triangle depicts LD between SNPs as measured by r², with the five major chromosome arms demarcated by black lines. The heat map indicates the magnitude of LD with red corresponding to complete LD and blue to absence of LD. Upper shows the significance threshold (−log₁₀P), the effect size in phenotypic SD units (a/σ_P), and the minor allele frequency (MAF).

Fig. 2.

Validation of candidate genes using mutants. Candidate genes from the GWA analyses were confirmed by using P{MiET1} mutants. Eighteen mutants were tested for females (A, red bars) and males (B, blue bars) along with their corresponding controls (black). Error bars indicate the SEMs. *0.01 ≤ P ≤ 0.05; **0.001 ≤ P ≤ 0.01; ***0.0001 ≤ P ≤ 0.001.

Fig. 3.

Functional interactions among candidate genes. (A) An enriched (P < 0.01) cellular network (31) among candidate genes with at least one SNP (P < 10⁻⁵) in the GWA analysis. Candidate genes are indicated by rectangles, missing genes (i.e., genes without significant associations) by triangles, and metabolites by circles. Components of the network associated with distinct interconnected cellular processes are highlighted by the colored backgrounds. (B) Validation of the connectivity of the predicted network by qRT-PCR of transcripts in the Pkc53e P{MiET1}-insertion mutant. Error bars indicate the SEMss. *0.01 ≤ P ≤ 0.05; **0.001 ≤ P ≤ 0.01; ***0.0001 ≤ P ≤ 0.001.

Fig. 4.

Cellular network for olfactory behavior identified by combined GWA, vGWA, and AIL analyses. An enriched (P < 0.005) cellular network (31) allowing for one missing gene derived from genes containing at least one SNP within 1 kb in any of the GWA, vGWA, and AIL analyses (P < 10⁻⁵). Significant genes are indicated by rectangles, missing genes by triangles, and metabolites by circles. Components of the network associated with distinct interconnected cellular processes are highlighted by the colored backgrounds.