title: Positional RNA-Seq identifies candidate genes for phenotypic engineering of sexual traits.
abstract: BACKGROUND
  RNA interference (RNAi) of trait-specific genes permits the manipulation of specific phenotypic traits ("phenotypic engineering") and thus represents a powerful tool to test trait function in evolutionary studies. The identification of suitable candidate genes, however, often relies on existing functional gene annotation, which is usually limited in emerging model organisms, especially when they are only distantly related to traditional genetic model organisms. A case in point is the free-living flatworm Macrostomum lignano (Lophotrochozoa: Platyhelminthes: Rhabditophora), an increasingly powerful model organism for evolutionary studies of sex in simultaneous hermaphrodites. To overcome the limitation of sparse functional annotation, we have performed a positional RNA-Seq analysis on different body fragments in order to identify organ-specific candidate transcripts. We then performed gene expression (in situ hybridization) and gene function (RNAi) analyses on 23 candidate transcripts, both to evaluate the predictive potential of this approach and to obtain preliminary functional characterizations of these candidate genes.
  
  
  RESULTS
  We identified over 4000 transcripts that could be expected to show specific expression in different reproductive organs (including testis, ovary and the male and female genital systems). The predictive potential of the method could then be verified by confirming organ-specific expression for several candidate transcripts, some of which yielded interesting trait-specific knock-down phenotypes that can now be followed up in future phenotypic engineering studies.
  
  
  CONCLUSIONS
  Our positional RNA-Seq analysis represents a highly useful resource for the identification of candidate transcripts for functional and phenotypic engineering studies in M. lignano, and it has already been used successfully in several studies. Moreover, this approach can overcome some inherent limitations of homology-based candidate selection and thus should be applicable to a broad range of emerging model organisms.
authors.affiliation: Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland.
authors.forename: Roberto
authors.initials: R
authors.lastname: Arbore
authors.affiliation: Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland.
authors.forename: Kiyono
authors.initials: K
authors.lastname: Sekii
authors.affiliation: D-BSSE, ETH Zürich, Basel, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Beisel
authors.affiliation: Institute of Zoology and CMBI, University of Innsbruck, Innsbruck, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Ladurner
authors.affiliation: ERIBA, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Eugene
authors.initials: E
authors.lastname: Berezikov
authors.affiliation: Evolutionary Biology, Zoological Institute, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland.
authors.forename: Lukas
authors.initials: L
authors.lastname: Schärer
keywords: Macrostomum lignano; Phenotypic engineering; RNA interference; RNA-Seq; simultaneous hermaphrodite