Abstract
The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci. These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. To this end, we inserted a gene encoding red fluorescent protein into the MATa locus. Red fluorescent protein expression caused MATa and a/α diploid vegetative cells and MATa ascospores to fluoresce; MATα cells without the gene did not fluoresce. Heterozygous diploids segregated fluorescent and nonfluorescent ascospores 2:2 in tetrads and bulk populations. The two populations of spores were separable by fluorescence-activated cell sorting with little cross contamination or contamination with diploid vegetative cells. This approach, which we call Fluorescent Ascospore Technique for Efficient Recovery of Mating Type (FASTER MT), should be applicable to laboratory, industrial, and undomesticated, strains.
Highlights
The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance
The approach we describe here is based on the integration of a red fluorescent protein (RFP) gene at the MATa locus, with selection provided by a hygromycin-resistance gene so that the cassette can be introduced into any transformable, haploid or diploid, hygromycin-sensitive strain
Transformation of haploid MATa strains with the StuI fragment of pBC58 (Figure 1A) resulted in the formation of hygromycin-resistant, pink colonies
Summary
The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. Rapid separation of haploids and diploids has been accomplished by incorporation of genetic markers that allow for selection by (1) insertion of a gene-promoter construct expressed only in haploids of one mating type and (2) the use of a recessive resistance marker [e.g., canavanine resistance (Whelan et al 1979)] to select against diploids (Tong and Boone 2007; Ehrenreich et al 2010) Effective, these approaches require the introduction of engineered cassettes via multiple manipulations and entail selections that could bias some analyses. MATa vegetative cells and ascospores tagged contain a visible marker useful for separation of cells by hand or fluorescenceactivated cell sorting (FACS)
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