Figure 4. QTL mapping identifies a plasmid instability locus on Y9 chromosome V.

(A) We plotted the mean Y9 SNP allele frequency in 20 kb windows for the ‘non-permissive’ (red) and ‘permissive’ (black) pools of meiotic haploid progeny from BY4742/Y9 heterozygous diploid parents. Associations with a plasmid instability locus would show an increased representation of Y9 alleles in the non-permissive pool and a decreased representation of the BY4742 haplotype in the permissive pool (dotted line indicates equal representation). The increased representation of Y9 alleles on chromosome XIV in both pools is a result of a segregating disomy in the Y9 parent that we show does not affect the plasmid instability phenotype (Figure 4—figure supplement 2). (B) Based on the allele frequencies of individual SNPs, we used MULTIPOOL to calculate LOD scores for association with the plasmid instability phenotype. We observe a highly significant LOD score (10.00) on chromosome V. The peak is fairly sharp and reaches maximal LOD score at chrV:122.3–122.7 kb (sacCer3 coordinates). All loci have allele frequencies skewed in the expected direction; the restrictive pool is enriched for Y9 alleles. (C) MULTIPOOL 90% (54 genes, 91.2 kb, chrV:92.4–183.6 kb) and 50% (16 genes, 23.1 kb, chrV:107.3–130.4 kb) credible intervals for the chromosome V QTL. Among the 16 genes in the 50% credible interval is MMS21, which encodes an essential SUMO E3-ligase.

Figure 4—figure supplement 1. Schematic of QTL mapping by bulk segregant analysis (Magwene et al., 2011).

We crossed non-permissive Y9 and permissive BY4742 haploid cells to create heterozygous diploids. We expect any Y9 alleles associated with plasmid instability (yellow star) to be concentrated in pools of meiotic progeny that show plasmid instability with SCAMPR analyses. Therefore, by determining where the Y9 allele frequency is elevated in the non-permissive pool and depleted in the permissive pool, we can identify genetic loci that are likely to significantly contribute to the plasmid instability phenotype. We use allele frequencies as input to the MULTIPOOL algorithm to calculate LOD scores that indicate statistical likelihoods of each genetic locus contributing to the plasmid instability phenotype (Edwards and Gifford, 2012).

Figure 4—figure supplement 2. Aneuploidy of chromosome XIV in Y9 strain.

(A) Whole genome sequencing reveals ~2X coverage of chromosome XIV in the sequenced Y9 haploid strain, indicating that this parent is disomic for this chromosome. This disomy segregates among the meiotic progeny from BY4742/Y9 heterozygous diploids. (B) Y9-derived diploid strains that were euploid or aneuploid for chromosome XIV were identified by qPCR and phenotyped (Pavelka et al., 2010). We find no difference in plasmid instability phenotypes, indicating that disomy of chromosome XIV is not likely to contribute to this trait.

Figure 4—figure supplement 3. Y9 chromosome V is most strongly associated with plasmid instability.

(A) Over-representation of chromosome V Y9 alleles in the non-permissive pool (red) and under-representation in the permissive pool (black). Each datapoint is an individual SNP. (B) Based on the allele frequencies shown in (A), we calculated LOD scores and 50% (chrV:107.3–130.4 kb), 90% (chrV:92.4–183.6 kb) credible intervals for association with the plasmid instability phenotype. The highest LOD score in the region is approximately 10.00 at chrV:122 kb.

Figure 4—figure supplement 4. Deletion of URA3 from Y9 haploid cells does not affect their plasmid instability phenotype.

Independently derived biological replicates (Rep1 through 5) of Δura3 Y9 cells are not different from wildtype Y9 in terms of their plasmid instability phenotypes. ***p<0.0001, Kruskal-Wallis test, n.s. = not significant.