Verification of single nucleotide polymorphisms affecting sleep in Drosophila. Yazmin L. Serrano, Susan T. Harbison. Laboratory of Systems Genetics, National Heart Lung and Blood Institute, Bethesda, MD.

   Human genome-wide association studies (GWAS) have been used to identify thousands of Single Nucleotide Polymorphisms (SNPs) associated with complex traits and disease. Demonstrating the causality of these SNPs is difficult in human populations; however, it may be possible to validate candidate polymorphisms and determine their likely mechanism of action in genetically tractable model organisms such as Drosophila. Here we employ an artificial selection procedure to validate candidate SNPs for sleep, a complex trait whose genetic basis is not well understood. In a previous study using the fully sequenced Drosophila Genetic Reference Panel (DGRP), we observed significant genetic variation for night sleep duration. Association of 2,490,165 non-singleton SNPs in the DGRP with night sleep revealed 160 significant SNPs. We have chosen a subset of 96 SNPs from this study for further validation. We designed SNP genotyping assays based on Taqman PCR chemistry and verified their ability to distinguish allelic differences among the five longest- and five shortest-sleeping DGRP lines. We crossed these lines in a full diallel design and measured night sleep in the resulting progeny. Most of the progeny from crosses between long sleepers and short sleepers had intermediate levels of sleep, suggesting that the SNP haplotypes behave additively; however, some haplotypes were dominant. To determine the effect of single SNP alleles on sleep, we created an outbred population by mating the progeny of this cross randomly for 15 generations, allowing long-sleeper and short-sleeper alleles to recombine. From the outbred population, we created duplicate selection lines for long sleep and short sleep, along with an unselected control. We are currently measuring sleep and SNP genotypes in successive generations to verify the key SNPs involved in sleep.