This protocol is a simple method to quantify the efficiency with which different strains and species of yeast mate with each other. The proposed method is simple, robust, and efficient. The method can be extended to any strain of yeast.
The method described in this study can be used to understand how speciation occurs in yeast. Moreover, the strains that we describe can be particularly useful to design experiments with gene flow. The method's strength is that it's really simple.
Some slight adjustments like the time of incubation might be needed depending on the particular strain used. Begin reviving the haploids A and Alpha from frozen stocks by streaking the yeast inoculum on a yeast extract peptone dextrose, or YPD agar plate. Allow them to grow for 48 hours at 30 degrees Celsius to obtain single colonies.
After incubation, inoculate single colonies from the YPD plate and five milliliters of YPD medium, and incubate at 30 degrees Celsius for 48 hours with 250 RPM shaking. The cells are in the stationary phase of growth after this incubation period. On a fresh YPD plate, draw mating efficiency grid as a 1 by 1.5 centimeter rectangle divided into three boxes, each with a dimension of 1 by 0.5 centimeter.
Inoculate five microliters of the YPD culture of the two mating types on the leftmost and rightmost rectangles. This volume corresponds to roughly 5 times 10 to the fifth haploid cells in each section. Incubate the plates for 24 hours at 30 degrees Celsius.
To mix an equal number of yeast cells, remove about 1/3 of the yeast growth developed in the outer boxes using a sterile toothpick and resuspend the removed cells in 20 microliters of water in a sterile 1.5 milliliter vial. Follow this for each haploid. Then, dilute five microliters of this suspension in two milliliters of water and measure the optical density using a spectrophotometer at 600 nanometers.
Based on the optical density value and the number of cells per milliliter at one optical density, add the required volumes of both haploids in a fresh and sterile 1.5-milliliter vial. Mix it well using a pipette. The final volume of this suspension is generally around six to eight microliters.
Next, inoculate this suspension in the center grid. Incubate the plate at 30 degrees Celsius for seven hours, allowing the haploids to mate. After seven hours of incubation, scrape the cells from the center rectangle using a toothpick or a pipette tip and dilute in two milliliters of sterile water.
Carry out further dilutions, and then spread the cell suspension on YPD agar to obtain single colonies. After plating, incubate the YPD plates at 30 degree Celsius for 36 to 48 hours to develop single colonies. Ensure that a few hundred individual colonies are obtained from each mating experiment for screening to ensure the statistical significance of the data.
To identify the diploid colonies on the plate, transfer the single colonies by streaking them individually onto a double drop-out plate, lacking the amino acids that the strains are auxotrophic for. Incubate the plates at 30 degrees Celsius for 48 hours and calculate the mating efficiency, nu. The robustness of the protocol enabled easy differentiation of the mating efficiency between the two strains, SK1AM and ScAM.
While the SK1 strains mated with a very high efficiency, the ScAM strains mated with a relatively lower efficiency. The mating efficiency of the ScAM strains dropped significantly when the environment did not contain glucose as a primary carbon source, indicating that mating is an energetically expensive process and growth on alternate carbon sources qualitatively reduces the efficiency of mating. When allowed to mate for different time periods, no mating was observed for the first four to five hours.
The first diploids appeared only after four to five hours, indicating that this is the time needed for the mating process to be completed in the given environment. After that, the mating efficiency increased rapidly. Beyond seven hours, mating efficiency calculations were influenced by the fact that mitotic growth started on the plate.
Ensure that equal number of both haploids are mixed. Deviations from this would lead to variations in independent repeats of the protocol. How gene flow impacts adaptation and speciation is an open question.
Since the auxotrophic markers are inserted next to the MAT locus in our strains, this helps address this important question.
