Linkage analysis of QTLs on chr. II, IV and XIII with different groups of segregants. (a) Glycerol and ethanol yield (on glucose) of the parental strains, 26B (■) and ER7A (▲), and the hybrid diploid strain 26B/ER7A (●). Glycerol and ethanol yield of the first isolated F1 segregants from 26B/ER7A (○), of the additional F1 segregants (□) and of the F5 segregants (◊). For screening purposes, one fermentation was carried out in 5 ml YP 10% glucose. Glycerol and ethanol yield of all segregants, ER7A and the diploid 26B/ER7A were related to the yield of 26B, which was set as 100% (b) Segregants were selected for low glycerol (<120% glycerol yield, stippled line) and high ethanol (>99% ethanol yield, stippled line) yield (on glucose) after each round of screening, resulting in the following segregant groups: 22 F1 segregants used for pooled-segregant whole-genome sequence analysis (○), 22 additional selected F1 segregants (□), and 26 F5 segregants (◊). These segregants were reconfirmed in 100 ml YP 10% glucose. Values for glycerol and ethanol yield are the average of three replicates. (c) SNP variant frequency (top) and respective P-value (bottom) were determined by allele-specific PCR in individual segregants of the sequenced selected pool (●), additional F1 selected pool (○), the total F1 selection of 44 (▲), the selection of F5 segregants (△), and the total selection of all 70 segregants (■) to fine-map the QTLs on chr. II, IV and XIII, which were detected with EXPloRA. The statistical confidence line (for P-value ≤ 0.05) is indicated with a stippled line.