Genomic analyses of new genes and their phenotypic effects reveal rapid evolution of essential functions in Drosophila development

It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the genome-wide experimental testing of gene effects and improvements in the computational identification of new genes (< 40 million years ago, Mya) open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues in techniques and concepts related to phenotypic effect analyses of genes, particularly of those that recently originated. Here we reported our analyses of these issues, including reproducibility and efficiency of knockdown experiment and difference between RNAi libraries in the knockdown efficiency and testing of phenotypic effects. We further analyzed a large data from knockdowns of 11,354 genes (~75% of the Drosophila melanogaster total genes), including 702 new genes (~66% of the species total new genes that aged < 40 Mya), revealing a similarly high proportion (~32.2%) of essential genes that originated in various Sophophora subgenus lineages and distant ancestors beyond the Drosophila genus. The transcriptional compensation effect from CRISPR knockout were detected for highly similar duplicate copies. Knockout of a few young genes detected analogous essentiality in various functions in development. Taken together, our experimental and computational analyses provide valuable data for detection of phenotypic effects of genes in general and further strong evidence for the concept that new genes in Drosophila quickly evolved essential functions in viability during development.

Author summary It is conventionally believed that the genes in control of important biological functions, processes and structures are conserved in evolution. Using recently developed RNAi knockdown libraries in combination of CRISPR knockout analyses, we showed that a high proportion in a large number of evolutionary new genes (~32.2% in 702 new genes, aged < 40 million years ago) in D. melanogaster are essential for the survival in development. We found that the frequency of essentiality in gene functions is stable in the species’ various ancestral stages and unchanged among ancient genes, unveiling a constant gene evolution that has driven evolution of Drosophila species in the Sophophora subgenus. We detected the transcriptional compensation effect from CRISPR knockout for highly similar duplicate copies. We experimentally examined the reproducibility, knockdown efficiency and performance of knockdown libraries, revealing the validness of RNAi knockdown in detection of essential phenotypic effects of genes. Our experimental and computational analyses provided strong evidence for the concept that evolutionary new genes in Drosophila quickly evolved essential functions.