Gene expression networks in the Drosophila Genetic Reference Panel

Abstract

A major challenge in modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal traits through networks of intermediate molecular phenotypes. This question is best addressed in a genetic mapping population in which all molecular polymorphisms are known and for which molecular endophenotypes and complex traits are assessed on the same genotypes. Here, we performed deep RNA sequencing of 200 Drosophila Genetic Reference Panel inbred lines with complete genome sequences and for which phenotypes of many quantitative traits have been evaluated. We mapped expression quantitative trait loci for annotated genes, novel transcribed regions, transposable elements, and microbial species. We identified host variants that affect expression of transposable elements, independent of their copy number, as well as microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination, as well as genes regulating transposable element expression. This study provides new insights regarding the role of natural genetic variation in regulating gene expression and generates testable hypotheses for future functional analyses.

Figure 1.

Genetic variation of gene expression in the DGRP. (A) Sexual dimorphism of gene expression. Red indicates significant up-regulation in females; blue, in males. (B) Distribution of H² estimates for annotated genes and NTRs in females. (C) Distribution of H² estimates for annotated genes and NTRs in males. (D) WGCNA modules for annotated genes and NTRs in females. (E) WGCNA modules for annotated genes and NTRs in males. Heatmaps show the pairwise correlation of all genes in each module, sorted by average connectivity, with the most tightly connected module at the top left.

Figure 2.

Genomic location of eQTLs for gene expression and genes they regulate. eQTL chromosome positions (bp) are given on the x-axis, and the genes with which they are associated on the y-axis. Red points denote female-specific eQTLs, blue indicates male-specific eQTLs, and black shows eQTLs shared by males and females.

Figure 3.

Large cis-trans-eQTL genetic network in females and males. Node interior colors indicate genomic location of genes: yellow, euchromatic regions with normal recombination; gray, euchromatic regions with reduced recombination; and blue, heterochomatin. Node border colors denote annotated gene (gray) or NTR (red). Node shape indicates whether a gene is a regulator and/or target: triangles, regulator only; squares, target only; and circles, both regulator and target. The node size indicates the number of node connections. Arrows on the edges point to the target. Edges are color-coded to show female-specific regulation (red), male-specific regulation (blue), and regulation common to both sexes (black).

Figure 4.

Genetic variation of TE expression in the DGRP. (A) Total signal for each TE family, summed over all individual transposon sequences and averaged across all DGRP lines, sex, and replicates. (B) Distribution of copy number independent H² estimates for TE sequences in females. (C) Distribution of copy number independent H² estimates for TE sequences in males. (D) WGCNA modules of TEs for females. (E) WGCNA modules of TEs for males. Heatmaps are depicted as in Figure 1. TE sequences not assigned to any module are included at the bottom right.

Figure 5.

TE genetic regulatory network. Symbols and color-coding are as for Figure 3. Black squares denote TE sequences.

Figure 6.

Genetic variation of microbiome composition. (A) The proportion of microbiome signal in RNA-seq libraries aligned to species in each genus or viral group. (B) Line means of total microbial signal (excluding Wolbachia). (C) Distribution of H² estimates for individual microbe species. (D) WGCNA modules for microbial species. Heatmaps are depicted as in Figure 1. Species not assigned to any module are included at the bottom right.