Functional Diversification, Redundancy and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster

Large multigene families, such as the insect odorant binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 re-integration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (“transcriptional niches”) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families. Overall design: RNA sequencing on 14 transgenic Drosophila melanogaster lines, separately by sex, with two biological replicates per line (56 samples total). Lines are grouped into eight genotypes which differ in their mutations in the four Obp50a-d genes. Four genotypes ("Obp50a," "Obp50b," "Obp50c," and "Obp50d") have premature termination codon (PTC) mutations in all but one of the four Obp50a-d genes (each of these genotypes consists of two lines, except for the genotype with no PTCs in Obp50d, which consists of a single line). One genotype ("Obp50Neg") has PTCs in all four Obp50a-d genes (two lines). One genotype ("Obp50Pos") has no PTCs in any of the four Obp50a-d genes (one line). One genotype ("Obp50NegBCD") has missense mutations in Obp50b and PTCs in Obp50a, Obp50c, and Obp50d (two lines). One genotype ("Obp50cBCD") has missense mutations in Obp50b and PTCs in Obp50a and Obp50d (two lines).