Name: genetic mapping of thermotolerance differences between species of saccharomyces yeast via genome-wide reciprocal hemizygosity analysis
Uploaded: 2019-08-12T14:30:00
Description: Watch this Scientific Journal Video about Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis at JoVE.com

We thank J. Roop, R. Hackley, I. Grigoriev, A. Arkin and J. Skerker for their contributions to the original study, F. AlZaben, A. Flury, G. Geiselman, J. Hong, J. Kim, M. Maurer, and L. Oltrogge for technical assistance, D. Savage for his generosity with microscopy resources, and B. Blackman, S. Coradetti, A. Flamholz, V. Guacci, D. Koshland, C. Nelson, and A. Sasikumar for discussions; we also thank J. Dueber (Department of Bioengineering, UC Berkeley) for the PiggyBac plasmid. This work was supported by R01 GM120430-A1 and by Community Sequencing Project 1460 to RBB at the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility. The work conducted by the latter was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

1. Preparation of the piggyBac-containing plasmid for transformation
<ol>
	<li>Streak out to single colonies the E. coli strain harboring plasmid pJR487 onto an LB + carbenicillin agar plate. Incubate for 1 night at 37 &#176;C or until single colonies appear. 
	NOTE: A description of how plasmid pJR487 was cloned can be found in our previous work3.</li>
	<li>Inoculate 1 L of LB + carbenicillin at 100 &#956;g/mL with a single colony of E. coli containing pJR487 in a 2 L gla...

<ol>
	<li>Flint, J., Mott, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+molecular+basis+of+quantitative+traits:+successes+and+pitfalls.">Finding the molecular basis of quantitative traits: successes and pitfalls.</a> Nature Reviews Genetics. 2, 437-445 (2001).</li><li>Allen Orr, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+species+differences.">The genetics of species differences.</a> Trends in Ecology and Evolution. 16, 343-350 (2001).</li><li>Weiss, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+interspecific+differences+in+yeast+thermotolerance.">Genetic dissection of interspecific differences in yeast thermotolerance.</a> Nature Genetics. 50, 1501-1504 (2018).</li><li>Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+loci+that+cause+phenotypic+variation+in+diverse+species+with+the+reciprocal+hemizygosity+test.">Identification of loci that cause phenotypic variation in diverse species with the reciprocal hemizygosity test.</a> Trends in Genetics. 30, 547-554 (2014).</li><li>Steinmetz, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+architecture+of+a+quantitative+trait+locus+in+yeast.">Dissecting the architecture of a quantitative trait locus in yeast.</a> Nature. 416, 326-330 (2002).</li><li>Scannell, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Awesome+Power+of+Yeast+Evolutionary+Genetics:+New+Genome+Sequences+and+Strain+Resources+for+the+Saccharomyces+sensu+stricto+Genus.">The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus.</a> G3 (Bethesda). 1, 11-25 (2011).</li><li>Wetmore, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+mutant+fitness+in+diverse+bacteria+by+sequencing+randomly+bar-coded+transposons.">Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons.</a> MBio. 6, e00306-e00315 (2015).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15, 550 (2014).</li><li>Wilkening, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+high-throughput+approaches+to+QTL+mapping+in+Saccharomyces+cerevisiae.">An evaluation of high-throughput approaches to QTL mapping in Saccharomyces cerevisiae.</a> Genetics. 196, 853-865 (2014).</li><li>Kim, H. S., Huh, J., Riles, L., Reyes, A., Fay, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noncomplementation+screen+for+quantitative+trait+alleles+in+saccharomyces+cerevisiae.">A noncomplementation screen for quantitative trait alleles in saccharomyces cerevisiae.</a> G3 (Bethesda). 2, 753-760 (2012).</li></ol>

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...

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&mdash;they rely on large panels of recombinant progeny from crosses between interfertile individuals. They are of no use in the study of species that cannot mate to form progeny in the first place. As such, the field has had little capacity for unbiased dissection of trait differences between reproductively isolated species2.
In this work we report the technical underpinnings of a new method, RH-seq3, for genome-scale surveys of the genetic basis of trait variation between species. This approach is a massively parallel version of the reciprocal hemizygote test4,5, which was first conceived as a way to evaluate the phenotypic effects of allelic differences between two genetically distinct backgrounds at a particular locus (Figure 1A). In this scheme, the two divergent individuals are first mated to form a hybrid, half of whose genome comes from each of the respective parents. In this background, multiple strains are generated, each containing an interrupted or deleted copy of each parent&rsquo;s allele of the locus. These strains are hemizygous since they remain diploid everywhere in the genome except at the locus of interest, where they are considered haploid, and are referred to as reciprocal since each lacks only one parent&rsquo;s allele, with its remaining allele derived from the other parent. By comparing the phenotypes of these reciprocal hemizygote strains, one can conclude whether DNA sequence variants at the manipulated locus contribute to the trait of interest, since variants at the locus are the only genetic difference between the reciprocal hemizygote strains. In this way, it is possible to link genetic differences between species to a phenotypic difference between them in a well-controlled experimental setup. To date the applications of this test have been in a candidate-gene framework&mdash;that is, cases in which the hypothesis is already in hand that natural variation at a candidate locus might impact a trait.
In what follows, we lay out the protocol for a genome-scale reciprocal hemizygosity screen, using yeast as a model system. Our method creates a genomic complement of hemizygote mutants, by generating viable, sterile F1 hybrids between species and subjecting them to transposon mutagenesis. We pool the hemizygotes, measure their phenotypes in sequencing-based assays, and test for differences in frequency between clones of the pool bearing the two parents&rsquo; alleles of a given gene. The result is a catalog of loci at which variants between species influence the trait of interest. We implement the RH-seq workflow to elucidate the genetic basis of thermotolerance differences between two budding yeast species, Saccharomyces cerevisiae and S. paradoxus, which diverged ~5 million years ago6.

<table>
	<tbody>
		<tr>
			<td>1-2 plasmid Gigaprep kits</td>
			<td>Zymo Research</td>
			<td>D4204</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>10X Tris-EDTA (TE) buffer (100 mM Tris-HCl and 10 mM EDTA)</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Filter sterilize through a 0.22 &#956;m filter before use.</td>
		</tr>
		<tr>
			<td>1M LiOAc</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Filter sterilize through a 0.22 &#956;m filter before use.</td>
		</tr>
		<tr>
			<td>300 mg/mL Geneticin (G418)</td>
			<td>Gibco</td>
			<td>11811023</td>
			<td></td>
		</tr>
		<tr>
			<td>52% polyethylene glycol (PEG) 3350</td>
			<td>Sigma</td>
			<td>1546547</td>
			<td>Dissolve in water and filter sterilize through a 0.22 &#956;m filter before use. 1X trafo mix: 228 uL 52% PEG, 36 uL 1M LiOAc, 36 uL 10X TE buffer</td>
		</tr>
		<tr>
			<td>Autoclaved LB liquid broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>Make LB liquid broth using your powder from any brand, and milliQ water. Autoclave it before use.</td>
		</tr>
		<tr>
			<td>Carbenicillin stock in water (100 mg/mL)</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Filter sterilize through a 0.22 &#956;m filter before use.</td>
		</tr>
		<tr>
			<td>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]</td>
			<td>5-FOA: Zymo Research, Drop-out mix: US Biological, Uracil: Sigma, D-glucose: Sigm), YNB: Difco</td>
			<td>5-FOA: F9001-5, Drop-out mix: D9535, Uracil: U0750, D-glucose: G8270, YNB: DF0919</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain carrying pJR487 (CEN-/ARS+ piggyBac-containing plasmid)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Request from Brem lab.</td>
		</tr>
		<tr>
			<td>Hybrid yeast strain JR507 (S. cerevisiae DBVPG1373 x S. paradoxus Z1, URA-/URA-)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Request from Brem lab.</td>
		</tr>
		<tr>
			<td>Illumina Hiseq 2500</td>
			<td></td>
			<td></td>
			<td>used for SE-150 reads</td>
		</tr>
		<tr>
			<td>Large shaking incubators with variable temperature settings</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LB + carbenicillin agar plates (100 &#956;g/mL)</td>
			<td>Agar: BD Difco</td>
			<td>Agar: 214010</td>
			<td>Make LB agar plates as normal and add carbenicillin to 100 &#956;g/mL before drying.</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorimeter</td>
			<td>Thermo Scientific</td>
			<td>Q33240</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath at 39&#176;C</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast fungal gDNA prep kit</td>
			<td>Zymo Research</td>
			<td>D6005</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast peptone dextrose (YPD) liquid media</td>
			<td>BD Difco</td>
			<td>Peptone: 211677, Yeast Extract: 212750</td>
			<td>Add filter-sterilized D-glucose to 2% after autoclaving.</td>
		</tr>
		<tr>
			<td>YPD + G418 agar plates (300 &#956;g/mL)</td>
			<td>Agar: BD Difco</td>
			<td>Agar: 214010</td>
			<td>Make YPD agar plates as normal and add G418 to 300 &#956;g/mL before drying.</td>
		</tr>
		<tr>
			<td>YPD agar plates</td>
			<td>Agar: BD Difco</td>
			<td>Agar: 214010</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic mapping of thermotolerance differences between species of saccharomyces yeast via genome-wide reciprocal hemizygosity analysis

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.

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 &#176;C and, in a separate experiment as a control, at 28 &#176;C (Figure 1B), and we isolated DNA ...

Watch this Scientific Journal Video about Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis at JoVE.com

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

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.

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

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 ...

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