QTL analysis of high thermotolerance with superior and downgraded parental yeast strains reveals new minor QTLs and converges on novel causative alleles involved in RNA processing

Revealing QTLs with a minor effect in complex traits remains difficult. Initial strategies had limited success because of interference by major QTLs and epistasis. New strategies focused on eliminating major QTLs in subsequent mapping experiments. Since genetic analysis of superior segregants from natural diploid strains usually also reveals QTLs linked to the inferior parent, we have extended this strategy for minor QTL identification by eliminating QTLs in both parent strains and repeating the QTL mapping with pooled-segregant whole-genome sequence analysis. We first mapped multiple QTLs responsible for high thermotolerance in a natural yeast strain, MUCL28177, compared to the laboratory strain, BY4742. Using single and bulk reciprocal hemizygosity analysis we identified MKT1 and PRP42 as causative genes in QTLs linked to the superior and inferior parent, respectively. We subsequently downgraded both parents by replacing their superior allele with the inferior allele of the other parent. QTL mapping using pooled-segregant whole-genome sequence analysis with the segregants from the cross of the downgraded parents, revealed several new QTLs. We validated the two most-strongly linked new QTLs by identifying NCS2 and SMD2 as causative genes linked to the superior downgraded parent and we found an allele-specific epistatic interaction between PRP42 and SMD2. Interestingly, the related function of PRP42 and SMD2 suggests an important role for RNA processing in high thermotolerance and underscores the relevance of analyzing minor QTLs. Our results show that identification of minor QTLs involved in complex traits can be successfully accomplished by crossing parent strains that have both been downgraded for a single QTL. This novel approach has the advantage of maintaining all relevant genetic diversity as well as enough phenotypic difference between the parent strains for the trait-of-interest and thus maximizes the chances of successfully identifying additional minor QTLs that are relevant for the phenotypic difference between the original parents

Genetic mapping of QTLs involved in thermotolerance by pooled-segregant whole-genome sequence analysis.

<p>Genomic DNA samples were extracted from an unselected pool (pool 0) and two pools of thermotolerant segregants able to grow at 41°C (Pool 1) and at 40.7°C (Pool 2), respectively. The DNA for each pool originates from 58 segregants. Pool 1 consists of segregants from the cross between parents 21A and BY4742 and Pool 0 and Pool 2 from the cross between the downgraded parents 21A^DG and BY4742^DG. The top-panel represents the SNP variant frequency of pool 0 (small gray circles), pool 1 (small pink triangles) and pool 2 (small blue diamonds) along with the smoothed SNP frequency profile (black line: pool 0, red line: pool 1 and blue line: pool 2) using a generalized additive model. In the middle panel the log odds ratio (contrast) between the SNP variant frequency of a selected pool and pool 0 is plotted along with simultaneous 95% confidence bands (red region: pool 1 and blue region: pool 2). Horizontal dash lines indicate the threshold (δ = 0.4088). The bottom panel shows 2-sided p-values along the chromosome that are corrected for multiple testing, with horizontal dash lines indicating a cut-off of 0.05. Confirmed QTLs are indicated at corresponding positions, with broken lines indicating QTLs from the original parents, and stippled lines QTLs from the downgraded parents.</p

List of QTLs identified in the mapping with the downgraded parents.

<p>List of QTLs identified in the mapping with the downgraded parents.</p

Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis

High ethanol tolerance is an exquisite characteristic of the yeast Saccharomyces cereyisiae, which enables this microorganism to dominate in natural and industrial fermentations. Up to now, ethanol tolerance has only been analyzed in laboratory yeast strains with moderate ethanol tolerance. The genetic basis of the much higher ethanol tolerance in natural and industrial yeast strains is unknown. We have applied pooled-segregant whole-genome sequence analysis to map all quantitative trait loci (QTL) determining high ethanol tolerance. We crossed a highly ethanol-tolerant segregant of a Brazilian bioethanol production strain with a laboratory strain with moderate ethanol tolerance. Out of 5974 segregants, we pooled 136 segregants tolerant to at least 16% ethanol and 31 segregants tolerant to at least 17%. Scoring of SNPs using whole-genome sequence analysis of DNA from the two pools and parents revealed three major loci and additional minor loci. The latter were more pronounced or only present in the 17% pool compared to the 16% pool. In the locus with the strongest linkage, we identified three closely located genes affecting ethanol tolerance: MKT1, SWS2, and APJ1, with SWS2 being a negative allele located in between two positive alleles. SWS2 and APJ1 probably contained significant polymorphisms only outside the ORF, and lower expression of APJ1 may be linked to higher ethanol tolerance. This work has identified the first causative genes involved in high ethanol tolerance of yeast. It also reveals the strong potential of pooled-segregant sequence analysis using relatively small numbers of selected segregants for identifying QTL on a genome-wide scale

List of QTLs identified in the mapping with the original parents.

<p>List of QTLs identified in the mapping with the original parents.</p

Dissection of QTL1 to identify the causative gene.

<p>(A) Fine-mapping of QTL1 by scoring selected SNPs in the individual thermotolerant segregants. Eight SNPs spanning between 400,000 bp and 550,000 bp on chromosome XIV were scored by PCR in 46 thermotolerant segregants and both SNP variant frequency and FDR p-value were calculated. A 60,000 bp region between SNP2 and 5 showed the strongest linkage. It contained 33 genes and putative ORFs as indicated using the annotations in SGD. The genes containing at least one non-synonymous mutation within the ORF are indicated with an asterisk. (B) Identification of the causative gene <i>MKT1</i> in QTL1. RHA results for <i>MKT1</i>, <i>RHO2</i> and <i>END3</i> in the central region of QTL1 are shown. The strain pairs for the same genes were always spotted on the same plate. The results for the original hybrid diploid 21A/BY4742 and the <i>MKT1</i> reciprocal deletion strains were also from the same plate.</p