Two Synthetic 18-Way Outcrossed Populations of Diploid Budding Yeast with Utility for Complex Trait Dissection

Abstract

Advanced-generation multiparent populations (MPPs) are a valuable tool for dissecting complex traits, having more power than genome-wide association studies to detect rare variants and higher resolution than F₂ linkage mapping. To extend the advantages of MPPs in budding yeast, we describe the creation and characterization of two outbred MPPs derived from 18 genetically diverse founding strains. We carried out de novo assemblies of the genomes of the 18 founder strains, such that virtually all variation segregating between these strains is known, and represented those assemblies as Santa Cruz Genome Browser tracks. We discovered complex patterns of structural variation segregating among the founders, including a large deletion within the vacuolar ATPase VMA1, several different deletions within the osmosensor MSB2, a series of deletions and insertions at PRM7 and the adjacent BSC1, as well as copy number variation at the dehydrogenase ALD2 Resequenced haploid recombinant clones from the two MPPs have a median unrecombined block size of 66 kb, demonstrating that the population is highly recombined. We pool-sequenced the two MPPs to 3270× and 2226× coverage and demonstrated that we can accurately estimate local haplotype frequencies using pooled data. We further downsampled the pool-sequenced data to ∼20-40× and showed that local haplotype frequency estimates remained accurate, with median error rates 0.8 and 0.6% at 20× and 40×, respectively. Haplotypes frequencies are estimated much more accurately than SNP frequencies obtained directly from the same data. Deep sequencing of the two populations revealed that 10 or more founders are present at a detectable frequency for > 98% of the genome, validating the utility of this resource for the exploration of the role of standing variation in the architecture of complex traits.

Keywords: MPP; Multiparent Advanced Generation Inter-Cross (MAGIC); budding yeast; de novo assembly; haplotype inference; multiparental populations.

Figure 1

Schematic of the outcrossing process used to make the two 18F12 diploid populations. Both populations were established by a full diallele cross of all 22 isogenic haploid founder strains. A1/B1, A2/B2, A3/B3, and A4/B4 are different mating types of the same strains and are the same strains used in Cubillos et al. (2013). In (A), all pairwise crosses were mixed before the first round of sporulation. This is in contrast to (B), in which mixing did not occur until after an initial sporulation event. In both cases, mixed populations were taken through additional rounds of sporulation and random mating for a total of 12 meiotic generations.

Figure 2

Many alleles of the highly pleiotropic MKT1 gene are segregating among the founder strains, highlighting the potential of uncovering complex allelic series using populations derived from these strains. Seven of these alleles are differentiated by nonsynonymous SNPs, of which six are predicted to be segregating in 18F12v2. Vertical red lines are synonymous SNP differences from the reference S288C strain and black bars are nonsynonymous SNPs.

Figure 3

Combining contiguous long-read sequencing with accurate short-read data enables the detection of structural variants such as those depicted to the left. In (A), a large (> 1 kb) deletion within a vacuolar ATPase (VMA1) is present in one-half of the strains used in this study. This deletion directly overlaps the self-splicing intein PI-SceI. Copy number variants of ALD2, an aldehyde dehydrogenase, were detected (B) and include a duplication of this gene in founder A5 (represented as ALD2-A and ALD2-B), as well as its deletion in founders B5 and B8. In (C), multiple deletions of different lengths in the osmosensor MSB2 were detected in multiple founder strains. Dotplots of a structurally complex region on chr IV are shown for founders AB4 (D) and B7 (E). These plots show alignments of regions from the founder strains (depicted on the y-axis) with the corresponding region from the S288C reference strain (depicted on the x-axis). The red boxes present above the genes in the reference strain map duplications (solid boxes) and deletions (empty boxes) detected in each founder strain to the corresponding reference sequence. In all panels, the Ref is used to highlight the various arrangements of structural variants present in the founder strains. chr, chromosome; Ref, reference strain S288C.

Figure 4

The frequency of SNPs private to a single founder are highly correlated with the estimated haplotype frequencies at these SNPs in 18F12v2. As the frequency of a private SNP should be equal to the corresponding haplotype frequency, this measure provides a benchmark with which the accuracy of our haplotype caller can be measured. Cyan points represent founders that were pooled when estimating haplotype frequencies (“grouped founders”) due to the high degree of sequence similarity between their genomes. Triangles represent mitochondrial SNPs, which, together with SNPs private to pooled founders, represent the bulk of the major outliers. The coefficient of determination was calculated by regressing haplotype frequency onto SNP frequency, excluding SNPs from grouped founders and mitochondrial SNPs.

Figure 5

Genome-wide haplotype frequencies for 18F12v1.

Figure 6

Genome-wide haplotype frequencies for 18F12v2.

Figure 7

Haploids derived from 18F12v1 (A) and 18F12v2 (B) were isolated and sequenced, providing a glimpse into the recombinogenic landscape and haplotype diversity present within these populations.