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.

Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae

“…Strains used this study belong to the Saccharomyces Genome Resequencing Project (SGRP; Liti et al, 2009), a collection of isolates from around the globe that have been tagged with unique genetic barcodes, and made heterothallic so they can be easily crossed (Cubillos et al, 2009). We focus on four of these strains, listed in Table 1, which have been previously phenotypically characterized (Warringer et al, 2011), and extensively genome sequenced (e.g., Bergström et al, 2014;Linder et al, 2020). Linder et al (2020) further modified these haploid strains so that MATa and MATα haploids have specific drug resistance markers linked to the mating-type locus, so that MATa strains can be recovered in media supplemented with 100 µg/mL nourseothricin sulfate ("NTC"), MATα strains can be recovered in media supplemented with 300 µg/mL hygromycin B ("hyg"), and mated a/α diploids can be recovered in media containing both drugs.…”

“…We focus on four of these strains, listed in Table 1, which have been previously phenotypically characterized (Warringer et al, 2011), and extensively genome sequenced (e.g., Bergström et al, 2014;Linder et al, 2020). Linder et al (2020) further modified these haploid strains so that MATa and MATα haploids have specific drug resistance markers linked to the mating-type locus, so that MATa strains can be recovered in media supplemented with 100 µg/mL nourseothricin sulfate ("NTC"), MATα strains can be recovered in media supplemented with 300 µg/mL hygromycin B ("hyg"), and mated a/α diploids can be recovered in media containing both drugs. For the initial phenotypic characterization of each strain following heat shock, a/α diploids were created by streaking cultures of each haploid strain in a cross shape on YPD plates (so that mating types came into contact where the streaks cross), allowing growth at 30 • C for 48 h, and replica plating onto YPD/NTC plates and YPD/hyg plates.…”

“…After this time, ∼200 µL aliquots of this mating mix were spread onto 10 YPD/NTC/hyg plates and allowed to grow for 48 h at 30 • C. These mated diploid cells were then scraped off agar plates with a sterile glass slide, transferred to fresh YPD media, and archived as the "ancestral" 4X population. The 4X population was then taken through 12 cycles of outcrossing, using a protocol implementing various techniques for random spore isolation adapted from methods used in previous work (Burke et al, 2014) as well as by Linder et al (2020). Briefly, newly mated diploid cells were grown overnight in 10 mL YPD media.…”

Data_Sheet_2.xlsx

“…To address this question, we estimated genome-wide haplotype frequencies in the recombinant population following 12 outcrossing cycles, with and without the heat-shock step. Haplotype frequencies were estimated using the sliding window haplotype caller described in Linder et al (2020) and software the authors have made available as a community resource: https://github.com/ tdlong/yeast_SNP-HAP. With the haplotyper.limSolve.code.R script, we estimated haplotype frequencies across 30 kb windows with a 1 kb step size.…”

“…As more diverse natural isolates are cataloged and made amenable to genetic interventions (e.g., Cubillos et al, 2009), multiple investigators have established so-called "multiparent mapping populations" by crossing strains from various geographical origins. Cubillos et al (2013) created a 4-parent population, and Linder et al (2020) created an 18parent population, as community resources for mapping complex traits in yeast. While including more parental strains in a mapping population increases its capacity for trait mapping, it also requires random spore analysis, as tetrad dissection becomes prohibitively laborious.…”