Figure 1.
Distribution of Sporulation Efficiencies of Diploid Segregants Obtained from the Crosses S288c × SK1
Generation of diploid segregants is described in Materials and Methods. Sporulation efficiencies and standard errors of parents and hybrids are in italics. Assessment of sporulation was carried out after 7 d on solid sporulation medium. Equal amounts of DNA from 21 segregants at each of the two “tails” were pooled to generate the “high” and “low” DNA pools (gray bars).
Figure 2.
Shown are short sequences with known SNPs. The upper part shows reconstruction of mixtures of DNA of the strains SK1 and S288c, testing the ability to evaluate reliable allele frequencies of SNPs in DNA pools. DNA of strains SK1 and S288c were pooled at various ratios and a short genomic region containing a known SNP in the gene SPS18 was sequenced. The two alleles could clearly be distinguished even in pools with allele ratios of 8:2 and 9:1. The signal height of the DNA pool sequence was found to be a very good estimator for the allele frequency (correlation coefficient r = 0.99, p < 0.0001).
The figure contains sequences of the “high” and “low” DNA pools from the genes RAS2 and YNL100W and from polymorphic DNA segments flanking the candidate region on Chromosome 14. In each sequence, the SNP position is labeled by a black box or arrow (the SNP in the promoter of RAS2 is in position −52).
Figure 3.
Hybridization of DNA from Parents and Pools of Segregants (“Low Tail” and “High Tail”) to Affymetrix S98 Microarrays
For each chromosome, the top horizontal line (green) represents hybridizations of S288c DNA and the second line (red) represents hybridizations of SK1 DNA. The third and the fourth horizontal lines represent the hybridizations of the “low” and the “high” pools, respectively. Each horizontal array (comprised of four lines) represents a given yeast chromosome and the physical genomic positions along the chromosome. The small vertical bars represent probes containing polymorphisms between strains SK1 and S288c (alleles are colored according to their parental colors). The small vertical bars on the third and fourth lines of each chromosome represent the inherited allele in the pools: green is S288c and red is SK1. Inheritance of a mixture of alleles is marked either yellow (composition closer to S288c) or pink (closer to SK1). Three regions show consistent inherited differences in allele frequencies between the low and the high pools (boxes). These regions are located on Chromosome 2 (95–157 kb from the left end), Chromosome 7 (500–612 kb), and Chromosome 14 (400–585 kb).
Figure 4.
Effects of Reciprocal Hemizygosity and of Allele Replacements on Sporulation
(A) Sporulation efficiency of pairs of hybrid strains (S288c × SK1) with single-gene heterozygous deletions. Strains deleted for the S288c allele (only the SK1 allele is present) are presented by black bars and the isogenic strains deleted for the corresponding SK1 allele (only the S288c allele is present) are presented as diagonally hatched bars. The non-deleted hybrid is presented for reference (gray bar).
(B) Sporulation efficiency of double-gene and four-gene deletion mutants. Every pair consisted of two isogenic hybrid strains (S288c × SK1), each with two (or four) hemizygosities: One strain had deletions of the two (or four) sporulation-promoting alleles (empty bars) and the other had deletions of the corresponding sporulation-inhibiting alleles (bars with horizontal lines).
(C) Sporulation of the four-gene deletion mutants. In the hybrid strain containing the four sporulation-promoting alleles (left microscopic image), almost all cells formed asci, whereas in the strain with the sporulation-inhibiting alleles (right image), most of the cells did not form asci. The genotypes of the two “reciprocal” strains are given below each image.
(D) Sporulation efficiencies of a diploid S288c strain and two isogenic allele-replacement strains, one containing the two SWS2 alleles from strain SK1 and the other containing the two RAS2 alleles from SK1. A fourth isogenic strain contains, homozygotically, only a single additional A in the promoter poly-A stretch of RAS2, as found in strain SK1. For each strain, sporulation was assessed four times. The average sporulation efficiencies and their confidence intervals (p = 0.95) are shown.
Figure 5.
Sequence Comparisons of Part of the RAS2 Promoter in Ten Winery Strains
The published [27] assessment of sporulation efficiency are: H, high; M, moderate; L, low. Our assessment of sporulation efficiency (percent) is under “Spo.” The first codon of the open reading frame (ATG) is marked by “Start.” The black arrowhead indicates the deletion of adenine in the poly-A stretch. Based on their DNA sequences, the ten winery strains are closely related to each other and to strain S288c, whereas they differ from SK1 in many SNPs throughout the genome, by approximately 1 in 150 bp [26].