Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Summary

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Abstract

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Introduction

Since the dawn of the field, it has been a prime goal in genetics to understand the mechanistic basis of variation across wild individuals. As we map loci underlying a trait of interest, the emergent genes can be of immediate use as targets for diagnostics and drugs, and can shed light on the principles of evolution. The industry standard toward this end is to test for a relationship between genotype and phenotype across a population via linkage or association¹. Powerful as these approaches are, they have one key limitation—they rely on large panels of recombinant progeny from crosses between interfertile individuals. They are of no use i....

Protocol

1. Preparation of the piggyBac-containing plasmid for transformation

1. Streak out to single colonies the E. coli strain harboring plasmid pJR487 onto an LB + carbenicillin agar plate. Incubate for 1 night at 37 °C or until single colonies appear.
   NOTE: A description of how plasmid pJR487 was cloned can be found in our previous work³.
2. Inoculate 1 L of LB + carbenicillin at 100 μg/mL with a single colony of E. coli containing pJR487 in a 2 L glass flask. Grow overnight at 37 °C with shaking at 200 rpm until saturated (OD₆₀₀ ≥ 1.0).
3. Purify plasmid DNA from the culture using a large-sc....

Results

We mated S. cerevisiae and S. paradoxus to form a sterile hybrid, which we subjected to transposon mutagenesis. Each mutagenized clone was a hemizygote, a diploid hybrid in which one allele of one gene is disrupted (Figure 1A, Figure 2). We competed the hemizygotes against one another by growth at 39 °C and, in a separate experiment as a control, at 28 °C (Figure 1B), and we isolated DNA from each .......

Discussion

The advantages of RH-seq over previous statistical-genetic methods are several-fold. In contrast to linkage and association analysis, RH-seq affords single-gene mapping resolution; as such, it will likely be of significant utility even in studies of trait variation across individuals of a given species, as well as interspecific differences. Also, previous attempts at genome-wide reciprocal hemizygosity analysis used collections of gene deletion mutants, some of which harbor secondary mutations that can lead to false posi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-2 plasmid Gigaprep kits	Zymo Research	D4204	The number of kits required depends on how efficient your preps are in each kit. This kit comes with 5 individual plasmid prep columns. Run 1 L of saturated E. coli culture through each prep column, as using more than 1 L per column can cause clogging of the prep filter, leading to low yield and poor quality DNA.
10X Tris-EDTA (TE) buffer (100 mM Tris-HCl and 10 mM EDTA)	Any	N/A	Filter sterilize through a 0.22 μm filter before use.
1M LiOAc	Any	N/A	Filter sterilize through a 0.22 μm filter before use.
300 mg/mL Geneticin (G418)	Gibco	11811023
52% polyethylene glycol (PEG) 3350	Sigma	1546547	Dissolve in water and filter sterilize through a 0.22 μm filter before use. 1X trafo mix: 228 uL 52% PEG, 36 uL 1M LiOAc, 36 uL 10X TE buffer
Autoclaved LB liquid broth	BD Difco	244620	Make LB liquid broth using your powder from any brand, and milliQ water. Autoclave it before use.
Carbenicillin stock in water (100 mg/mL)	Any	N/A	Filter sterilize through a 0.22 μm filter before use.
Complete synthetic agar plates (24.1cm x 24.1cm) with 5-fluoroorotic acid (5-FOA) [0.2% drop-out amino acid mix without uracil or yeast nitrogen base (YNB), 0.005% uracil , 2% D-glucose, 0.67% YNB without amino acids, 0.075% 5-FOA]	5-FOA: Zymo Research, Drop-out mix: US Biological, Uracil: Sigma, D-glucose: Sigm), YNB: Difco	5-FOA: F9001-5, Drop-out mix: D9535, Uracil: U0750, D-glucose: G8270, YNB: DF0919
DMSO	Any	N/A
E. coli strain carrying pJR487 (CEN-/ARS+ piggyBac-containing plasmid)	N/A	N/A	Request from Brem lab.
Hybrid yeast strain JR507 (S. cerevisiae DBVPG1373 x S. paradoxus Z1, URA-/URA-)	N/A	N/A	Request from Brem lab.
Illumina Hiseq 2500			used for SE-150 reads
Large shaking incubators with variable temperature settings	Any	N/A
LB + carbenicillin agar plates (100 μg/mL)	Agar: BD Difco	Agar: 214010	Make LB agar plates as normal and add carbenicillin to 100 μg/mL before drying.
Nanodrop spectrophotometer	Thermo Scientific	ND-2000
Qubit Fluorimeter	Thermo Scientific	Q33240
Salmon sperm DNA	Invitrogen	15632011
Water bath at 39°C	Any	N/A
Yeast fungal gDNA prep kit	Zymo Research	D6005
Yeast peptone dextrose (YPD) liquid media	BD Difco	Peptone: 211677, Yeast Extract: 212750	Add filter-sterilized D-glucose to 2% after autoclaving.
YPD + G418 agar plates (300 μg/mL)	Agar: BD Difco	Agar: 214010	Make YPD agar plates as normal and add G418 to 300 μg/mL before drying.
YPD agar plates	Agar: BD Difco	Agar: 214010