Parental strains and design of genetic crosses.

(A) Experimental design. Two crosses were made between a derivative of CBS7435 (CBS_BGL9) and derivatives of Pp2 (Pp2_BGL5) or Pp4 (Pp4_BGL3). These parental strains each contained a BGL expression cassette and KanR or ZeoR marker, integrated at the GAPDH locus. The resulting diploid interstrain hybrids were sporulated and 1,000 haploid meiotic segregants were isolated from each cross by random spore isolation. BGL secretion in each segregant was assayed and used to choose pools of 30 superior segregants and 30 inferior segregants from each cross. Genomes of the segregants in each pool were then sequenced individually, after which SNP allele frequency plots were constructed for each pool, enabling identification of genomic regions (QTLs) segregating preferentially with contrasting BGL secretion phenotypes in each cross. (B) Quantitative assessment of BGL secretion in the parental strains and controls (CBS_PGAP is a derivative of CBS7435 containing an integrated empty pGAPZα vector; Pp2 and Pp4 are wild-type strains). Bars represent mean data from 3 independent cultures of each strain (open circles). Error bars show standard deviation. Significant differences in BGL secretion between CBS_BGL9 and the other parental strains were tested with unpaired t test and are indicated by asterisks (**, P = 0.0035; and ***, P = 0.0005). Numerical data are listed in S1 Data. (C) Formation of diploids by crossing. Diploid colonies formed by mating grow at the intersections between streaks of the haploid parental strains, when replica plated onto selective media containing zeocin plus geneticin. BGL, β-glucosidase; QTL, quantitative trait locus; SNP, single-nucleotide polymorphism.