Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

Summary

In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies.

Abstract

Saccharomyces cerevisiae is a widely used model organism in genetics, evolution, and molecular biology. In recent years, it has also become a popular model organism to study problems related to speciation. The life cycle of yeast involves both asexual and sexual reproductive phases. The ease of performing evolution experiments and the short generation time of the organism allow for the study of the evolution of reproductive barriers. The efficiency with which the two mating types (a and α) mate to form the a/α diploid is referred to as the mating efficiency. Any decrease in the mating efficiency between haploids indicates a pre-zygotic barrier. Thus, to quantify the extent of reproductive isolation between two haploids, a robust method to quantify the mating efficiency is required. To this end, a simple and highly reproducible protocol is presented here. The protocol involves four main steps, which include patching the haploids on a YPD plate, mixing the haploids in equal numbers, diluting and plating for single colonies, and finally, calculating the efficiency based on the number of colonies on a drop-out plate. Auxotrophic markers are employed to clearly make the distinction between haploids and diploids.

Introduction

Saccharomyces cerevisiae, commonly called budding yeast, is a unicellular eukaryote. It has two mating types, a and α, and exhibits both asexual and sexual reproductive cycles. The a and α mating types are haploids and can divide mitotically in the absence of the other mating type in the surrounding environment, which represents the asexual cycle of yeast. When the two mating types are in close proximity, they stop dividing mitotically and fuse to form a diploid cell. The diploid yeast can either divide mitotically when nutrients are present or undergo meiosis under the conditions o....

Protocol

NOTE: The protocol broadly involves the following steps: (1) patching the haploids in the mating efficiency grids on a YPD plate, (2) mixing the haploids in equal numbers after 24 h incubation and giving the mixed haploids a few hours to mate (7 h in this study), (3) plating the mixed cells on YPD for isolating single colonies after 7 h at 30 °C, and finally, (4) determining the number of diploids formed using the auxotrophic markers. These steps are discussed in detail below (also see Figure 2<.......

Discussion

The quantification of the mating efficiency in S. cerevisiae is essential for carrying out studies related to the genes involved in mating pathways or studying the influence of the external environment on mating behavior. In the past two decades, S. cerevisiae has also become a popular model to address questions related to speciation^14,36,37,38. The presence of two mating type.......

Materials

Name	Company	Catalog Number	Comments
Adenine	Sigma Life Science	A8626
Agar Powder regular grade for bacteriology	SRL	19661 (0140186)
Ammonium Sulphate, Hi-AR	HiMedia	GRM1273
D-(+)-glucose	Sigma Life Science	G8270
Glass Petri plates	HiMedia	PW008	90 mm x 15 mm dimension
L-Arginine	Sigma Life Science	A8094
L-Aspartic acid	Sigma Life Science	A7219
L-Histidine monochloride monohydrate	Sigma Life Science	H5659
L-Isoleucine	Sigma Aldrich	I2752
L-Leucine	Sigma Life Science	L8912
L-Lysine	Aldrich	62840
L-Methionine	Sigma Life Science	M5308
L-Phenylalanine	Sigma Life Science	P5482
L-Threonine	Sigma Aldrich	T8625
L-Tyrosine	Sigma Life Science	T8566
L-Valine	Sigma Life Science	V0513
Mating efficiency grid			1 cm x 1.5 cm rectangular grid drawn on the Petri plate
Microcentrifuge tubes	Tarsons	500010
Peptone	HiMedia	RM001
Uracil	Sigma Life Science	U0750
Yeast Extract Powder	HiMedia	RM027
Yeast Nitrogen Base w/o Amino acids and Ammonium Sulphate	BD Difco	233520