Mapping Novel Traits by Array-Assisted Bulk Segregant Analysis in Saccharomyces cerevisiae

Abstract

We demonstrate a new method, microarray-assisted bulk segregant analysis, for mapping traits in yeast by genotyping pooled segregants. We apply a probabilistic model to the progeny of a single cross and as little as two microarray hybridizations to reliably map an auxotrophic marker, a Mendelian trait, and a major-effect quantitative trait locus. M APPING the genes responsible for traits remains one of the most basic goals of genetics. There are several techniques available for reaching this goal, especially in model organisms such as the yeast Saccharomyces cerevisiae. The dense and well-characterized genetic map of S. cerevisiae supports traditional tetrad analysis, which is still widely used in spite of its requirements for time-and labor-intensive crosses and dissections. More recently developed genomics techniques take advantage of parallel genotyping of single feature polymorphisms (SFPs) and other small differences between two strain backgrounds (Winzeler et al. 1998(Winzeler et al. , 1999(Winzeler et al. , 2003Brem et al. 2002;Steinmetz et al. 2002;Deutschbauer and Davis 2005). Other approaches use the strains created by the yeast deletion project as a dense collection of genetic markers that can be crossed in parallel to a query strain ( Jorgensen et al. 2002). Finally, a new approach looks for mutations directly by hybridizing DNA from a strain of interest to a reference sequence microarray (Gresham et al. 2006).All of these approaches have limitations that reduce their value as routinely applied methods for mapping suppressors or other mutations. Most highly parallel methods are still resource intensive, requiring 10 or more array-typed segregants. Mapping strategies that use the yeast deletion collection are appropriate only for limited classes of phenotypes and are tedious and timeconsuming without substantial robotic automation. Methods that find DNA sequence changes directly are unable to link these changes to particular phenotypes without extensive follow-up work.Here we describe a microarray-based methodmicroarray-assisted bulk segregant analysis-that applies a highly parallel genotyping strategy to mapping genes in pooled populations of yeast. Bulk segregant analysis measures the variation present in pools of segregants that have been sorted according to phenotype and uses the correlation between these measurements and the pool phenotype to assign a likely map location. This is an improvement over methods that require individual genotyping, as it simultaneously measures the average genotype of progeny with a given phenotype. The approach of bulk segregant analysis was first developed (Michelmore et al. 1991) and adapted for microarraybased genotyping (Borevitz et al. 2003;Hazen et al. 2005) in plants. We have adapted the method for use in yeast and have also developed an analytical model that takes advantage of the linkage that is expected to occur between loci at similar map locations. Incorporating this cosegregation into the mapping model greatly improves the accuracy of the method a...

Influence of genotype and nutrition on survival and metabolism of starving yeast

“…Complementation tests revealed that these mutants do not complement each other, implicating a single locus. One of the mutants, DBY11202, was used for both array-assisted bulk segregant analysis (14) and mutation detection by using overlapping tiling arrays of the entire yeast genome (15). The bulk segregant analysis identified one linkage peak on chromosome IV (Fig.…”

“…Linkage analysis was performed by using a strategy described previously (14). In short, strain DBY11202 was backcrossed twice to get a 2:2 segregation (resistant; high viability upon leucine starvation, sensitive; low viability).…”

Mapping Mendelian traits in asexual progeny using changes in marker allele frequency

“…One such method is array-assisted bulk segregant analysis (Brauer et al, 2006), which has been used to map traits in yeast. Here, yeast strains differing in genetic background and trait value are crossed.…”

Back to the Future: Mutant Hunts Are Still the Way To Go

“…Historically, complex traits have been studied among strains or individuals found in nature. The advances in the efficiency of mutagenesis and whole-genome sequencing after pooled linkage analysis (Brauer et al 2006;Ehrenreich et al 2010) will allow unbiased screens in the laboratory not only for single mutations that confer traits but for the de novo creation and analysis of polygenic mutants as well. This approach opens the door for the identification and analysis of phenotypes previously unapproachable in the lab (for example: Koschwanez et al 2013).…”