Multiparent recombinant inbred lines crossed to a tester provide novel insights into sources of cis and trans regulation of gene expression.

To understand the relative importance of cis and trans effects on regulation, we crossed multi-parent recombinant-inbred lines (RILs) to a common tester and measured allele-specific gene expression in the offspring. Testing the difference of allelic imbalance between two RIL × Tester crosses is a test of cis or trans, depending on the RIL alleles compared. The study design also enables to separation of two sources of trans variation, genetic and environmental, detected via interactions with cis effects. We demonstrate the effectiveness of this approach in a long-read RNA-seq experiment in female abdominal tissue at two time points in Drosophila melanogaster. Among the 40% of all loci that show evidence of genetic variation in cis, trans effects due to environment are detectable in 31% of loci and trans effects due to genetic background in 19%, with little overlap in sources of trans variation. The genes identified in this study are associated with genes previously reported to exhibit genetic variation in gene expression. Eleven genes in a QTL for thermotolerance, previously shown to differ in expression based on temperature, have evidence for regulation of gene expression regardless of the environment, including the cuticular protein Cpr67B, suggesting a functional role for standing variation in gene expression. This study provides a blueprint for identifying regulatory variation in gene expression, as the tester design maximizes cis variation and enables the efficient assessment of all pairs of RIL alleles relative to the tester, a much smaller study compared to the pairwise direct assessment.