Genomic DNA was extracted using the Qiagen Genomic-tip kit. DNA of the pooled segregants was sequenced using Illumina Hiseq 2000. Detailed procedures of DNA preparation were described previously46 (link). We used paired-end libraries, 101 bp per read, the coverage was ∼50 × . Reads were aligned to the S288c genome using BWA with ‘-n 5 -o 2' options. Single-nucleotide polymorphism (SNP) calling was done using SAMtools47 (link). The allele frequency of S288c was scored at each polymorphic position. As indicated by the segregation pattern, two genomic regions enriched for each parental strain are expected. We defined the region involved in the YJM421 parent as the genomic region comprising most of the mapped SNPs with allele frequency of S288c <0.1, as the S288c alleles were supposed to be absent (chromosome V). As for the region involved in the S288c genome, the YJM421 alleles should have been absent, which would result in a region with no mapped SNP markers (chromosome X). Inconsecutive SNPs that showed high allelic enrichment are considered to be sequencing noise and were not considered in the analysis.
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