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Example of a Functional Genomics Study Molecular Ecology 2006 15, 4635-4643 Drosophila • Most species are very poor ecological model organisms. • D. mojavensis is cactophilic: it uses 4 different kinds of cactus host in the Sonoran Desert. • Oviposits in necrotic tissues, exposing larvae to varied toxic chemicals. Objective Identify gene expression differences of 3rd instar larvae reared between two chemically distinct cactus hosts: •Agria (Stenocereus gummosus), native host •Organpipe (Stenocereus thurberi), alternative host Used a custom microarray (6520 anonymous cDNA fragments that were pinned robotically to glass slides) Organpipe vs Agria Cacti • Differ in lipids, triterpenes, and glycosides. •Differ in alcohol content. •Adh is duplicated in D. mojavensis and The paralogs are known to play different roles in host adaptation. Mixed Model Anova Approach 1) Relative hybridization Intensity = Random Technical and Residual Variation Yij = µ + ARRAYi + DYEj + ARRAY × DYEij + Residualij 2) Residual Variation Per Gene = Random Technical and Fixed Technical and Biological Variation Residualijkl = µ + ARRAYi + DYEj + CACTUS + ARRAY x Spotil + Errorijkl Correcting for Multiple Tests Bonferroni correction: More conservative test where the significance threshold is divided by the total number of tests. False Discovery Rate (FDR): Less conservative test that calculates the number of false positives within a set of significant values (P<0.05) and then calculates a new significance threshold , q. Identifying Differentially Expressed Genes Greater Expression Organpipe Greater Expression Agria P value for Each Gene Specific Anova -log(P) Bonferroni (173) False Discovery Rate (1034) Fold Difference Log2 Representation of Up-regulated Genes Among Gene Ontology Categories. Conclusions (i) Cactus host usage affects patterns of gene transcription. (ii) Loci whose function involve detoxification were differentially regulated in response to a cactus host shift. (iii) A subset of the differentially expressed loci may have arisen de novo in the D. mojavensis lineage.