A catalog of neutral and deleterious polymorphism in yeast

Abstract

The abundance and identity of functional variation segregating in natural populations is paramount to dissecting the molecular basis of quantitative traits as well as human genetic diseases. Genome sequencing of multiple organisms of the same species provides an efficient means of cataloging rearrangements, insertion, or deletion polymorphisms (InDels) and single-nucleotide polymorphisms (SNPs). While inbreeding depression and heterosis imply that a substantial amount of polymorphism is deleterious, distinguishing deleterious from neutral polymorphism remains a significant challenge. To identify deleterious and neutral DNA sequence variation within Saccharomyces cerevisiae, we sequenced the genome of a vineyard and oak tree strain and compared them to a reference genome. Among these three strains, 6% of the genome is variable, mostly attributable to variation in genome content that results from large InDels. Out of the 88,000 polymorphisms identified, 93% are SNPs and a small but significant fraction can be attributed to recent interspecific introgression and ectopic gene conversion. In comparison to the reference genome, there is substantial evidence for functional variation in gene content and structure that results from large InDels, frame-shifts, and polymorphic start and stop codons. Comparison of polymorphism to divergence reveals scant evidence for positive selection but an abundance of evidence for deleterious SNPs. We estimate that 12% of coding and 7% of noncoding SNPs are deleterious. Based on divergence among 11 yeast species, we identified 1,666 nonsynonymous SNPs that disrupt conserved amino acids and 1,863 noncoding SNPs that disrupt conserved noncoding motifs. The deleterious coding SNPs include those known to affect quantitative traits, and a subset of the deleterious noncoding SNPs occurs in the promoters of genes that show allele-specific expression, implying that some cis-regulatory SNPs are deleterious. Our results show that the genome sequences of both closely and distantly related species provide a means of identifying deleterious polymorphisms that disrupt functionally conserved coding and noncoding sequences.

Figure 1. Variation in levels of heterozygosity across chromosome IV.

Sliding windows of heterozygosity were obtained using 1000 synonymous sites and a step size of 500 sites for comparison of S288C and M22 (red), S288C and YPS163 (green), and M22 and YPS163 (blue). The highly polymorphic ENA locus is labeled by a black bar.

Figure 2. Maximum likelihood phylogeny of EHD3 shows introgression of S. paradoxus into some strains of S. cerevisiae.

EHD3 homologs are from S. paradoxus (Spar), S. mikatae (Smik), S. bayanus (Sbay), and two other strains of S. cerevisiae, RM11 and YJM789. All nodes above S. paradoxus show 100% bootstrap support.

Figure 3. Conservation of SNPs containing coding and noncoding sequences in different yeast species.

The fraction of S. cerevisiae SNPs in sequences conserved to S. paradoxus (Spar), S. mikatae (Smik), S. kudriavzevii (Skud), and S. bayanus (Sbay), C. glabrata (Cgla) S. castelli (Scas), S. kluyveri (Sklu), K. lactis (Klac), A. gossypii (Agos), and C. albicans (Calb). Homologous coding and noncoding sequences were identified by TBLASTX and BLASTN, respectively.

Figure 4. Identifying deleterious SNPs using Phylonet.

Using a SNP in the promoter of GPB2 as an example, a profile of conservation was generated from sequences adjacent to a SNP (A) and compared to all other noncoding profiles in the genome, ignoring the SNP containing column. Sixteen of the 178 profile alignments are shown with upper case letters indicating conservation across all species and instances of the derived SNP allele highlighted in red (B). The motif generated from these alignments is shown by a motif logo (C). Extracting the SNP containing positions from each profile alignment (D), a likelihood ratio test was used to determine whether the two SNP alleles were selectively equivalent to one another, measured by comparing the likelihood under equal base frequencies to the likelihood under a model where one allele is preferred over the other (E). Allele-specific expression of GPB2 (F) shows that the S288C allele has reduced expression relative to M22 and YPS163 (P = 0.0027). The difference is found in YPD, YPG, amino acid (AA) starvation, nitrogen (N₂) starvation and heat shock, which shows the maximum difference, 1.85-fold and 2.08-fold in comparison to M22 and YPS163, respectively.

Figure 5. Comparison of deleterious SNPs predicted by different methods.

The overlap between nonsynonymous SNPs predicted by the likelihood ratio test (LRT) and SIFT (A). The numbers in parentheses are for the subset of predictions within perfectly conserved amino acid positions. The overlap between noncoding SNPs predicted by the binding site model and the Phylonet motif model (B). The numbers in parentheses are for the subset of SNPs that occur in both conserved binding sites and conserved Phylonet motifs.