A set of genetically diverged Saccharomyces cerevisiae strains with markerless deletions of multiple auxotrophic genes. 
Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S. cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species.  The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European and Sake) and represent clean, non-mosaic lineages of S. cerevisiae, meaning that their genomes differ essentially by monomorphic and private SNPs.  In this study, one representative strain for each of the five S. cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions, so that all dominant markers remained available for further genetic manipulations.  A set of 50 strains was assembled, including eight haploid and two diploid strains for each lineage.  These strains carry different combinations of leu2∆0, lys2∆0, met15∆0, ura3∆0 and/or ura3∆::KanMX-barcoded deletions with marker configurations resembling that of the BY series, which will allow large-scale crossing with existing deletion collections.  This new set of genetically tractable strains provides a powerful tool kit to explore the impact of natural variation on complex biological processes.