Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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<.......

Representative Results

Quantification of the mating efficiency of the two mating types
The protocol described here was used to quantify the mating efficiency between two yeast strains-between SK1AMa and SK1AMα and between ScAMa and ScAMα (Figure 3A). In these experiments, the mating between the two haploids was repeated at least 12 times. In each of the repeats of the experiment, at least 100 colonies were streaked.......

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 speciation14,36,37,38. The presence of two mating type.......

Acknowledgements

This work was funded by a DBT/Wellcome Trust (India Alliance) grant (IA/S/19/2/504632) to S.S. P.N. is a Research Fellow supported by a DBT/Wellcome Trust (India Alliance) grant (IA/S/19/2/504632). A.M. is supported by the Council of Scientific and Industrial Research (CSIR), Government of India, as a Senior Research Fellow (09/087(0873)/2017-EMR-I). The authors thank Paike Jayadeva Bhat for discussions.

....

Materials

NameCompanyCatalog NumberComments
AdenineSigma Life ScienceA8626
Agar Powder regular grade for bacteriologySRL19661 (0140186)
Ammonium Sulphate, Hi-ARHiMediaGRM1273
D-(+)-glucoseSigma Life ScienceG8270
Glass Petri platesHiMediaPW008 90 mm x 15 mm dimension
L-ArginineSigma Life ScienceA8094
L-Aspartic acidSigma Life ScienceA7219
L-Histidine monochloride monohydrateSigma Life ScienceH5659
L-IsoleucineSigma AldrichI2752
L-LeucineSigma Life ScienceL8912
L-LysineAldrich62840
L-MethionineSigma Life ScienceM5308
L-PhenylalanineSigma Life ScienceP5482
L-ThreonineSigma AldrichT8625
L-TyrosineSigma Life ScienceT8566
L-ValineSigma Life ScienceV0513
Mating efficiency grid1 cm x 1.5 cm rectangular grid drawn on the Petri plate
Microcentrifuge tubesTarsons500010
PeptoneHiMediaRM001
UracilSigma Life ScienceU0750
Yeast Extract PowderHiMediaRM027
Yeast Nitrogen Base w/o Amino acids and Ammonium SulphateBD Difco233520

References

  1. Neiman, A. M. Sporulation in the budding yeast Saccharomyces cerevisiae. Genetics. 189 (3), 737-765 (2011).
  2. Duina, A. A., Miller, M. E., Keeney, J. B. Budding yeast for budding geneticists: A primer on ....

Explore More Articles

Yeast MatingHaploid Mating EfficiencySaccharomyces CerevisiaeMating AssayMating TypeCell GrowthOptical DensityCell SuspensionIncubationMating Grid

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved