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G3 (Bethesda). 2012 Apr;2(4):449-52. doi: 10.1534/g3.111.001826. Epub 2012 Apr 1.

FASTER MT: Isolation of Pure Populations of a and α Ascospores from Saccharomycescerevisiae.

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1
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.

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.

KEYWORDS:

BUD5-TAF2; MATa; budding yeast; fluorescence-activated cell sorting; hygromycin resistance; red fluorescent protein

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