Drosophila has long been a paradigm for studies in molecular evolution, providing not only important data but serving as a stimulus for theoretical advances and analytical methods. Already it is among the pioneer organisms for studies in evolutionary genomics. The goal of the proposed research is to move beyond the descriptive level of gene-expression microarrays to begin a systematic study of the genetic and molecular basis of expression variation in natural populations. We will use a combination of formal genetic analysis and real-time quantitative reverse transcriptase PCR (rt-qPCR) to identify putative cis-acting regulatory elements that are polymorphic among homozygous lines and their reciprocal hybrids of chromosome 2 and 3 substitution lines. For a subset of these genes, we will sequence coding and flanking regions to try to identify putative regulatory differences, and will carry out analyses of polymorphism and divergence to look for evidence of selection at the amino acid level. The research focuses specifically on genes expressed in testes because as a group these genes have been shown in the preliminary data to be more polymorphic in expression within species and divergent between species than other classes of genes. For reasons of speed, efficiency and economy, initial expression screening will be carried out with young males using microarrays with 20,515 verified PCR products (Eurogentec) querying 96% of all open reading frames. Expression variation will be confirmed with rt-qPCR using testes dissected from third instar wandering larvae. Putative cis-acting regulatory variation will be identified by segregation analysis and by allele-specific PCR genotyping of segregants. Candidate genes that pass these tests will be assayed by rt-qPCR in a wider set of strains, and high and low expression alleles will be sequenced and analyzed along with orthologs from other species to look for polymorphisms in putative cis-acting regulatory elements. The coding sequences will be analyzed for polymorphism and divergence to ascertain whether testes-expressed genes that evolve rapidly at the expression level also evolve rapidly at the amino acid level, and to carry out tests for positive selection. Haplotype analysis will also be carried out to determine whether there is evidence for recent selective sweeps. We will initiate the genetic analysis immediately using rt-qPCR assays of a small but not ideal set of testes-expressed genes that we have identified as polymorphic in laboratory strains using incomplete and female-biased cDNA microarrays.