title: Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait.
abstract: Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identifiedPMA1andCEM1as causative mutant alleles andTPS1as a causative genetic background allele. The case ofCEM1revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to identify causative alleles underlying many non-selectable, polygenic traits in small collections of haploid strains with multiple induced mutations.
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven. ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Tom Den
authors.initials: TD
authors.lastname: Abt
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven. ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Ben
authors.initials: B
authors.lastname: Souffriau
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven. ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Maria R
authors.initials: MR
authors.lastname: Foulquié-Moreno
authors.affiliation: Agrobiodiversity Research Area, International Center for Tropical Agriculture (CIAT), Cali, Colombia.
authors.forename: Jorge
authors.initials: J
authors.lastname: Duitama
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven. ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
keywords: Mutant screen; QTL mapping; ethyl acetate production; flavor production; genetic basis; genomic mutagenesis; non-selectable trait; polygenic trait