In a very productive previous award, the PI developed a medium resolution map (scale of approximately 20 cm) using QTLs to identify genetic factors affecting the shape and size of the male posterior lobe. The proposed research will further resolve this map to a scale of 5-10 cm in regions already known to contain a factor. A second goal is to use mutations within D. melanogaster that affect the morphology of the male posterior lobe and create hybrids to determine whether the wild- type alleles of those mutant genes contribute to species differences. Enhancer-detector P-element insertions that exhibit male-limited lacZ expression in the genital disc will be studied as candidate genes. A collection of homozygous viable inserts with sex-dimorphic expression is in hand. These will be screened for sexually dimorphic lethal inserts in those chromosomal regions already identified as containing QTLs in the interspecific analysis. The lab will conduct a mosaic screen for X-ray induced mutations on the third chromosome and affecting posterior lobe morphology. This chromosome exhibits the greatest density of genes affecting male genital morphology in interspecific crosses. Once a candidate gene has been identified within the species, it will be assessed in a hybrid test using a recessive loss-of- function mutation, crossing the mutant in mel to each of the 3 sister taxa and comparing the resultant male phenotypes with those produced by similar crosses with wild-type mel. Once a candidate gene has been clearly implicated in normal development of the posterior lobe and in hybrid male differences, it will be cloned and characterized in terms of its transcriptional organization, protein sequence, and pattern of expression. P- element mediated transfer of the gene from other species into D. melanogaster will provide a conclusive test of the role of the gene in male genital development. Chimeric gene constructs will be used to further partition the gene into functional regions responsible for its effects to address exactly what aspects of the sequence are causally involved in the morphological differences between species. The pleotrpic effects of the gene and functional regions on other aspects of hybrid morphologies will also be investigated. Eventually, this should provide information regarding the evolution of genes vis-a-vis the selective forces operating in nature.
