Aim 1. We created an outbred population of flies from the five longest-sleeping and five shortest-sleeping DGRP lines. We performed a full diallel cross of these lines, and then randomly mated flies from the F1 generation of this cross to create the first generation of outbred flies. We continued the random mating procedure for 22 generations, allowing SNPs from the long and short-sleeping flies to recombine. We chose 96 representative SNPs from the genome-wide association study that segregate in the outbred population and developed Taqman assays for each SNP.
Aim 2. From the outbred population, we created six selection populations. Two populations are being selected for short sleep, two for long sleep, and the remaining two populations are unselected controls. We are measuring sleep in each population every generation. We have chosen a subset of flies to create the subsequent generation as appropriate (for example, the shortest-sleeping flies from the population selected for short sleep become the parents for the next generation). We ascertained the effectiveness of the selection procedure by plotting the means of each selection population and comparing them to the controls. We have used high-throughput genotyping methods to genotype single flies prior to selection and at various generations during the selection procedure. We have determined the SNP allele frequencies in single flies of the outbred population prior to selection, and we will compare these frequencies that of SNPs in subsequent generations. We will associate SNP genotypes of single flies with their corresponding sleep. Significant SNPs denote genomic regions responsive to the selection procedure.
Aim 3. For each generation of selection, we froze groups of flies, separated by each selection population and sex. Genomic DNA will be extracted from these samples and sequenced to determine the frequency of all SNP alleles present in the genome. We will associate these alleles to mean sleep phenotypes for the group. This procedure should pinpoint single SNPs responsive to selection, and these SNPs are expected to fall within the genomic regions localized in Aim 2.
Aim 4. Polymorphic variants may influence gene expression, and the signal of gene expression may be detectable across generations. We froze groups of flies of each sex and selection population for each generation. Total RNA will be extracted from these samples, and messenger RNA sequenced. We will then associate gene expression with sleep phenotypes in each generation.