Revealing the genetic structure of a trait by sequencing a population under selection

Abstract

One approach to understanding the genetic basis of traits is to study their pattern of inheritance among offspring of phenotypically different parents. Previously, such analysis has been limited by low mapping resolution, high labor costs, and large sample size requirements for detecting modest effects. Here, we present a novel approach to map trait loci using artificial selection. First, we generated populations of 10-100 million haploid and diploid segregants by crossing two budding yeast strains of different heat tolerance for up to 12 generations. We then subjected these large segregant pools to heat stress for up to 12 d, enriching for beneficial alleles. Finally, we sequenced total DNA from the pools before and during selection to measure the changes in parental allele frequency. We mapped 21 intervals with significant changes in genetic background in response to selection, which is several times more than found with traditional linkage methods. Nine of these regions contained two or fewer genes, yielding much higher resolution than previous genomic linkage studies. Multiple members of the RAS/cAMP signaling pathway were implicated, along with genes previously not annotated with heat stress response function. Surprisingly, at most selected loci, allele frequencies stopped changing before the end of the selection experiment, but alleles did not become fixed. Furthermore, we were able to detect the same set of trait loci in a population of diploid individuals with similar power and resolution, and observed primarily additive effects, similar to what is seen for complex trait genetics in other diploid organisms such as humans.

Figure 1.

Overall strategy. A three-step QTL mapping strategy by crossing two phenotypically different strains for many generations to create a large segregating pool of individuals of various fitness, and growing the pool in a restrictive condition that enriches for beneficial alleles that can be detected via sequencing total DNA from the pool.

Figure 2.

Recombination landscape after multiple rounds of intercrosses. (A) Expansion of the genetic map, measured in recombination units (ru) of 100 times the average number of recombination events from first to 12th generation (bottom) of a 200-kb chromosome XIII locus genotyped at nine markers (top). (B) Genetic background of two segregants from a first (F1) and sixth (F6) generation cross shows a sharp increase in recombination events.

Figure 3.

Changes in allele frequencies pinpoint QTLs. (A–C) WA allele frequency of whole genome (A), chromosome II (B), and IRA1 region (C) of the F12 pool before (blue) and after (green) selection. Lines in gene regions in C denote segregating sites (black) and nonsynonymous segregating sites (red). The sites with intolerable mutations determined by SIFT analysis (Supplemental Material SI; Data set S3) are highlighted with arrows and designated with the amino acid change. (D) Individual examples of mapped QTLs that show differences in QTL strength, beneficial allele, effect of intercross rounds, and ploidy. Each window spans 80 kb and is centered on the locus with the largest allele frequency change in F12 T2 across two replicas. Shaded regions indicate 90% and 95% confidence intervals of the allele frequencies (Supplemental Material SI).

Figure 4.

IRA1 and IRA2 are high temperature growth QTLs. (A) Reciprocal hemizygosity confirms that IRA1 and IRA2 are high temperature growth QTLs. WA/NA hybrids were individually deleted for the IRA alleles and used to assess their contribution to high temperature growth. Plate spotting assay using 10-fold serial dilution demonstrates better growth of the hybrid when the NA allele is present. (B) Competition experiment on hybrids with IRA1/IRA2 reciprocal hemizygous deletions (such as A) that resembles the selective step applied to the pool. Hybrids carrying the NA allele outcompete ones with WA allele after 192 h (T2) of growth at 40°C. (C) Internal level of cAMP is reduced at 40°C, but unchanged at 30°C for WA/NA hybrids with WA alleles deleted at both IRA1 and IRA2 loci, compared to NA alleles deleted. (D) RAS/cAMP signaling contributes to natural variation in heat sensitivity. Defective function of the WA alleles of IRA1 and IRA2 at high temperature results in hyperactive RAS, leading to high level of cAMP and high PKA activity inhibiting the heat transcription induction. As a response to heat stress, the majority of the QTLs selected in the pool are from the NA genetic background (red: NA; blue: WA). Dashed arrow indicates unknown mechanism. Figure adapted from Figure 2 of Santangelo (2006) and reprinted with permission from the American Society for Microbiology.