Advances in quantitative trait analysis in yeast

Abstract

Understanding the genetic mechanisms underlying complex traits is one of the next frontiers in biology. The budding yeast Saccharomyces cerevisiae has become an important model for elucidating the mechanisms that govern natural genetic and phenotypic variation. This success is partially due to its intrinsic biological features, such as the short sexual generation time, high meiotic recombination rate, and small genome size. Precise reverse genetics technologies allow the high throughput manipulation of genetic information with exquisite precision, offering the unique opportunity to experimentally measure the phenotypic effect of genetic variants. Population genomic and phenomic studies have revealed widespread variation between diverged populations, characteristic of man-made environments, as well as geographic clusters of wild strains along with naturally occurring recombinant strains (mosaics). Here, we review these recent studies and provide a perspective on how these previously unappreciated levels of variation can help to bridge our understanding of the genotype-phenotype gap, keeping budding yeast at the forefront of genetic studies. Not only are quantitative trait loci (QTL) being mapped with high resolution down to the nucleotide, for the first time QTLs of modest effect and complex interactions between these QTLs and between QTLs and the environment are being determined experimentally at unprecedented levels using next generation techniques of deep sequencing selected pools of individuals as well as multi-generational crosses.

Figure 1. Experimental measures of natural variation.

Yeasts offer a unique opportunity to engineer changes to measure the impact of phenotypic variants on traits. (A) Reciprocal hemizygosity has high throughput and can be used to test a large number of candidates. Hybrids that differ only in which of two alleles is present/deleted are compared. Deletion collections of multiple strains will soon be available allowing genome-wide systematic studies using hybrids to test all candidates easily or even for discovery of phenotypic effects directly. (B) Allele swapping is less high throughput but allows testing phenotypic effects of specific alleles in different genetic backgrounds. This is more precise than reciprocal hemizygosity. (C) Site-directed mutagenesis is a rapid and precise way of testing known and novel base changes for phenotypic effects. (D) Synthetic biology has the potential of simultaneously testing multiple variants, both natural or artificial, in a single gene or scattered through the genome .

Figure 2. Mapping QTLs and modifiers.

(A) QTL mapping has evolved from the classical approach of individual segregant analysis to the X-QTLs and iQTLs approaches with higher mapping sensitivity and resolution. Analysis of time series data in iQTLs allows dynamic monitoring of allele frequency values . (B) A possible approach to map genetic modifiers using iQTLs. A conditional essential gene, y, is deleted from its original chromosomal location and maintained on a plasmid. This hybrid is intercrossed multiple times to allow reshuffling of parental genomes. Upon loss of gene y, viability relies on the presence of genetic modifier/s, and allelic combinations that result in lethality (dashed cells) will decrease in allele frequency. These modifiers can be detected by comparing allele frequencies of the pool before and after the plasmid loss. When many modifiers are involved, the lethal combinations will be present in low frequency, making them difficult to detect. Further rounds of intercrosses, after loss of gene y, will allow reshuffling of alleles and the generation of more cells with unviable combinations.

Figure 3. Linked quantitative trait loci (QTLs) can arise through normal population genetic processes.

For any given phenotype there are many loci where mutations can have an effect. Different populations will experience mutations in different loci affecting the same phenotype. These mutations can affect a phenotype in a positive (+) or negative (−) way and if nearly neutral ( = ) will remain segregating within a population for awhile. As other mutations occur, advantageous combinations can result with better fitness than either mutation alone or the original parental alleles. Multiple mutations with effects upon a trait will be broken up by recombination if not linked and one or more can therefore be lost. Linked mutations can become fixed as blocks of larger collections of QTLs if the combination of alleles is beneficial. Different populations may evolve different “super”-QTLs, which are revealed when the populations interbreed. Offspring will express a range of phenotypes depending on which QTLs are inherited and how much recombination breaks up the linked groups. Multiple rounds of interbreeding can further break up the linked QTLs revealing individual loci, as illustrated in Figure 2.