Genetic and molecular analysis of mutations in Drosophila melanogaster that lead to differences in expression of microRNA mir-92a and underlie phenotypic evolution

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It is a goal of evolutionary biology to link the variation of morphological traits to the underlying genetic variation. A trait that differs between different Drosophila species but is usually constant within each species is the size of the naked valley, a naked cuticle patch on the second leg. However, several populations of Drosophila melanogaster show size differences of the naked valley. The underlying genetic variation has been mapped to a locus containing a microRNA, mir-92a, which is located in an intron of the protein-coding gene jigr1. mir-92a post-transcriptionally regulates shavenoid (sha), a gene involved in naked valley development. I will first examine the possibility of jigr1 or a second protein-coding gene, Npl4, being involved in the development of the naked valley, as well. Differences in expression of mir-92a in the different Drosophila strains are thought to underlie the naked valley size variation by subsequent differential regulation of sha. Differential expression of mir-92a is likely to be facilitated by mutations in an enhancer. I will analyse whether expression of mir-92a and jigr1 is correlated and thus controlled by the same cis-regulatory elements. In order to identify the size-determining element, I will use the GAL4-UAS system to express GFP as well as mir-92a under control of candidate enhancers from strains with large and small naked valleys, respectively, in the genetic background of a small naked valley. I will identify the specific mutations responsible for the size variation by stepwise altering the enhancer for development of a large naked valley towards the identity of a strain with a small naked valley by PCR-based mutagenesis. The results of these experiments will facilitate the identification of the mutations underlying phenotypic variation of the naked valley within Drosophila melanogaster.