Sequential elimination of major-effect contributors identifies additional quantitative trait loci conditioning high-temperature growth in yeast

Abstract

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

Figure 1.—

Growth of the studied strains at high temperature. Spot dilutions showing growth at 30° and 41° of the YJM421/S288c background strains used to map Htg QTL. Tenfold dilutions were spotted on a YPD plate and incubated for 48 hr. All strains are diploids.

Figure 2.—

(A) Targeted backcross mapping strategy to sequentially uncover QTL. Consider a quantitative trait in a (+) strain, defined by multiple QTL alleles (red bars) with different contributions to a phenotype (the larger the type size, the larger the phenotypic effect). F₁ segregants of a cross, between (+) and (−) parental stains, show a range of phenotypes. The best F₁ (+) segregant is backcrossed to the (−) parent. Analysis of these backcross segregants would fine map and identify QTL-1. Similar analysis of the next best F₁ (+) segregant that lacks QTL-1 (shown as blue ×) would then fine map QTL-2 in the next backcross segregants. Iterative fixation of major-effect QTL by a targeted selection of F₁ segregants for further backcrossing would map minor-effect QTL. (B) Genomic linkage scans of backcross segregants using microarrays. Genotypes of 24 and 21 backcross segregants of F₁-14d/S288c and F₁-50b/S288c, respectively, were determined by DNA hybridization. Vertical lines along chromosomes are SFPs; segregants are stacked by chromosome; YJM421-derived regions are in red, S288c-derived regions in blue. Htg QTL are marked with triangles (fixated ones indicated in white and newly detected in black). In F₁-14d/S288, Htg-QTL-1 (chromosome XIV) was fixed and Htg-QTL-2 (chromosome XIV) and Htg-QTL-3 (chromosome XV) were mapped. In F₁-50b/S288c, Htg-QTL-1 and Htg-QTL-2 (chromosome XIV) were fixed and Htg-QTL-3 (chromosome XV) and Htg-QTL-4 (chromosome IV) were mapped.

Figure 3.—

Fine mapping of Htg QTL. Percentage of inheritance of YJM421-derived alleles among 71 and 65 BC₁ backcross segregants of F₁-14d/S288c and F₁-50b/S288c, respectively. For each QTL, boxes and names denote annotated genes.

Figure 4.—

Sequence variation analysis of the Htg-QTL-2 (chromosome XIV; 395,662–410,654 bp) among six Htg⁺ and seven Htg⁻ yeast strains. Each column represents a strain and each row a sequence variant relative to S288c (black). White are synonymous and yellow are nonsynonymous variants. Variants with significant marker-trait association are marked (*). Rows (a–f) show Htg⁺ strains: YJM421 (a), YJM326 (b), YJM320 (c), YJM280 (d), YJM339 (e), and YJM789 (f). Rows (g–m) show Htg⁻ strains: YJM270 (g), YJM269 (h), YJM627 (i), YJM1129 (j), W303 (k), SK1 (l), and S288c (m).

Figure 5.—

RHA analysis of NCS2, MKT1, NCS2-MKT1, and NCS2-288(71L). Solid bar denotes the hybrid strain, XHS740, used as reference and for comparison in each competition. Shaded bars denote hemizygous hybrid strains containing the YJM421-derived allele(s) while open bars denote those that contain the S288c-derived allele(s). The striped bar denotes the hemizygous hybrid strain containing the 71L Htg⁺ SNP allele of YJM421 inside the otherwise S288c-derived allele of NCS2. All assays were carried out in the diploid hybrid background (YJM421/S288c) at 41°. For each gene, three independent measurements were taken and averaged after normalization between replicates and genes using a reference hybrid strain. All NCS2-421/Δ, MKT1-421/Δ, and Δ/NCS2-288(71L) hybrids grow similar to each other but significantly better than the hemizygotes with S288c-derived alleles (Tukey–Kramer HSD test, P < 0.05).

Figure 6.—

Functional analysis of NCS2. Overlap in gene lists between genes differentially expressed (>1.5-fold) in ncs2 deletion strain and genes that when deleted cause a fitness defect at 37°. GO category enrichments are indicated for the genes common between the two data sets.

Figure 7.—

Architecture of the chromosome XIV Htg⁺ complex QTL. MKT1 (Htg-QTL-1) and NCS2 (Htg-QTL-2), shown in red, are two linked Htg genes on chromosome XIV. Boxes denote annotated genes. On top, physical and genetic distance between the QTL alleles is shown, as calculated from random F₁ segregants of the YJM421/S288c background. Along with the QTL names, the percentage of association of each Htg⁺ QTL individually is indicated (see Table 1). The combined inheritance of the linked QTL among Htg⁺ F₁ segregants in the YJM421/S288c background is shown at the bottom.