Abstract
The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.
Highlights
The study of mutants to elucidate gene functions has a long and successful history; to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation
A set of genetic and molecular tools has been established for this fungus (Nowrousian et al 2005; Pöggeler and Kück 2006; Pöggeler et al 1997, 2003; Walz and Kück 1995), and more than 100 developmental mutants were generated through ultraviolet or ethyl methanesulfonate (EMS) mutagenesis (Kück et al 2009), seven of which have already been characterized using classical complementation analysis, leading to the identification of novel developmental genes (Bloemendal et al 2010; Engh et al 2007b; Kück 2005; Masloff et al 1999; Nowrousian et al 1999, 2007; Pöggeler and Kück 2004)
We describe the identification of the causative mutations in three developmental mutants via a strategy involving wholegenome sequencing of pooled DNA from 40 single spore isolates per strain of three mutants and the wild type, in addition to subsequent bioinformatics analyses
Summary
The study of mutants to elucidate gene functions has a long and successful history; to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information. The analysis of mutants with a distinct phenotype that were generated, for example, by random mutagenesis, is a powerful approach to identify factors essential for biological processes This so-called forward genetics approach is important both in basic research as well as for medical applications, e.g. for the identification of mutations that underlie diseases. The data presented here demonstrate that this strategy allows rapid identification of the developmental genes affected in the mutant strains, is feasible for an organism that can be genetically crossed and for which a reference genome is available, and does not require prior mapping information
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