Chromosomal inversions are often detected in humans when individuals present symptoms of genetic diseases. Individuals with two different inversions can have lower fertility because genetic exchange between inverted chromosomes can lead to the formation of sperm or eggs with less or more genetic information. Inversions, however, can also be present in populations at high frequencies with no apparent negative effects on viability or reproduction. This proposal will test hypotheses about how inversions arise, spread, and are maintained in populations. Drosophila pseudoobscura is a model system for the study of inversions because its third chromosome is polymorphic for over 30 different gene arrangements in populations. These arrangements were generated through a series of overlapping paracentric inversions. The inversion frequencies vary among populations forming clines or gradients whose major frequency shifts occur with major climatic zones in the southwestern United States. The gene arrangement frequencies cycle with the seasons and show altitudinal clines. Population cage experiments in the laboratory have shown that different gene arrangements are maintained at stable equilibrium frequencies. This study will use next generation technologies to re-sequence 50 strains of D. pseudoobscura that carry one of six major chromosomal arrangements. Next generation sequencing analysis of mRNA will be used to quantify expression differences among the six different inversion backgrounds for two different Drosophila life stages, larvae and adults. The re-sequencing data will identify inversion breakpoint sequences and improve the D. pseudoobscura genome assembly. The transcriptome data will be used to improve the annotation of gene models in D. pseudoobscura. Molecular population genetic analyses of nucleotide polymorphism and gene expression data will test four classes of hypotheses about how a new inversion spreads through a population: (1) indirect effects due to reduced recombination leading to chromosomes free of deleterious mutations or enhancing the linkage of adaptive genes; (2) genetic drift that fixes underdominant arrangements; (3) direct effects of the chromosomal lesion such as alteration of gene structure or expression; or (4) genetic hitchhiking with an adaptive allele. These analyses will test two hypotheses about how inversions are maintained in populations: (1) by overdominance; or (2) as a protected polymorphism. Genes that show differential expression or protein variation may be manipulated in future experiments designed to understand the molecular genetic basis that underpins inversion evolution.