Yeast - A Dihybrid Cross

Kerry F. Chesser
1994 Woodrow Wilson Biology Institute

Introduction

Mating in yeast, as in most organisms, is especially dramatic. Organisms which reproduce sexually have haploid and diploid stages. In flowering plants and animals it is only the gametes that are haploid and the phenotype can be observed only in this diploid stage. When Mendel made dihybrid crosses to study the inheritance of two different traits, such as seed color and seed shape, he could only observe these traits in the diploid cells of parents and offspring. Mendel had to use statistical probability in order to calculate what the genotype of the gametes was likely to be.

The phenotypes of some traits can be seen in the haploid-cell colonies of Saccharomyces cerevisiae. Yeast cells may occur as either haploid or diploid. Haploid cells occur as either mating-type a or mating-type a (alpha). When cells of these opposite type come in contact, they secrete hormonelike substances called mating pheromones, which causes these cells to develop into gametes. These gametes then fuse and form a diploid zygote. This is similar to fertilization in animals, but here both parents contribute cytoplasm and nuclei. The resulting yeast zygotes reproduce asexually by budding. Diploid yeast cells do not mate, but in times of stress, such as a lack of certain nutrients, may sporulate and form spores. These spores may be found together looking like ball-bearings in a transparent sac called an ascus.

Objectives

In this investigation, you will make a yeast dihybrid cross and follow two forms of each of two traits: red growth versus cream color, and tryptophan-dependent (must have this amino acid supplied in order to grow) versus tryptophan-independent (does not require supplying of the amino acid). These mutants were originally isolated by yeast geneticists using the methods originally devised by Beadle and Tatum for studying mutations. In this method they isolate mutants that cannot make some essential substance. The ability of the normal strain to make this substance and the inability of the mutant strain to do so illustrate two forms of a trait.

In a dihybrid cross of these yeasts, if the red strain of one mating-type is crossed with a cream-colored strain of the other, then the resulting diploid strain is cream-colored. If this trait is determined by a single gene, then there must be a single allele that determines cream color in the haploid strain and another that determines red color in the diploid one. By observing the color of the growth of the haploid strain, you can accurately determine which allele it carries. However, with the cream-colored diploid, you would be uncertain of the genotype. This diploid could be carrying either one allele for red and one for cream color or two alleles for cream color. Further, the trait for dependence or independence toward tryptophan operates in much the same way. A haploid which is tryptophan-dependent must carry the allele for this dependence (a defective form of this gene) and an independent haploid must carry the allele for tryptophan-independence (a functional form of this gene). Finally, a tryptophan-independent diploid could be either homozygous or heterozygous for this allele.

If symbols are used for these traits, the allele for the dominant cream form is R and the recessive red form is r, the tryptophan-independent (normal form of that gene is T and the dependent form is t. The trait for color is visible but determining tryptophan dependence or independence will require a growth test.

The table below shows how the possible combinations of these two traits can be determined by testing the yeast for growth on two kinds of media: one which is nutritionally complete (COMP) and another lacking in tryptophan (MIN).

Materials (per team):

clean flat toothpicks

glass marker pen

2 paper templates for strain streaking

agar plate of complete growth medium (COMP)

agar plate of minimal growth medium minus adenine (MIN)

COMP plate with 24-hr. cultures of yeast strains

a1, a2, a3, a4,
a1, a2, a3, a4

Procedure

Part A : Predicting the Gametes

The alleles of the diploid parents of this dihybrid cross would have been r/r T/T and (rT).

1. Complete the diagram shown below in Figure 1b for a cream, tryptophan-dependent parent (R/R t/t).
Probabilities of gametes that could be formed by yeast organisms that are homozygous for two traits
2. Using the appropriate symbols, describe for each parent strain the genotypes and phenotypes of all the possible gametes as well as the probabilities of their occurrence.
3. These two pure-breeding parents produce haploid gametes which mate and form diploid zygotes of the F2 generation. Using symbols, describe the genotype and phenotype of the diploid zygotes that could be formed from the fusion of these gametes. At this point your diagrams should show that they will all have the dihybrid genotype R/r T/t.

Part B: Predicting the Gametes of the F1 Diploids

The diagram for the segregation of the R, r, T, and t alleles becomes more complicated because there are two equally probable ways that the alleles of the gene can separate at M1. This is illustrated in Figure 2a and 2b. Since the probabilities of these two patterns of segregation are equal, the two diagrams together illustrate the relative numbers of all possible gametes.

4. In Figure 2, fill in the missing symbols on each chromatid.
Gametes that could be formed by yeast organisms heterozygous for two traits
5. What is the total number of different gametes in this diagram?
6. Give the symbols of all of the different genotypes shown.
7. How many times is each genotype represented?
8. What is the probability of each genotype occurring among the total number of gametes shown?
9. Draw a checkerboard diagram for all possible crosses between these gametes. This diagram should illustrate the 16 different combinations that were predicted. Use mating-type a and mating-type a haploid strains instead of male and female gametes. Construct the diploid genotype that would result from corresponding gametes in each square. Does the diagram predict a 9: 3: 3: 1 ratio of phenotypes? What phenotypes are represented by these ratios?

Part C :Testing the Predicted Phenotypes

Day 0

10. Make two templates for setting up crosses by copying Figure 3 so that each diagram fits the bottom of a Petri dish.
11. Tape one of the templates to the bottom of a Petri dish of complete growth medium (COMP) so that you can read it through the agar.

12. Transfer a bit of each strain onto the agar directly over its corresponding label by touching the flat end of a clean toothpick to strain a1 on the dish your teacher provides. Next, gently drag the toothpick on the COMP agar plate and make a streak about 1 cm long. Discard the used toothpick in the waste container provided by your teacher. Use a new toothpick for each transfer and repeat this procedure for all eight strains. Be careful not to touch the end of each toothpick to anything but yeast or sterile agar.
13. Invert the dish and incubate it at room temperature.
14. Be sure to wash your hands before leaving the laboratory.

Day 1

15. On the same COMP plate, make a mating mixture for each of the type a strains with each of the a type strains. This is accomplished by using the flat end of a clean toothpick to transfer a small amount of the a1 strain of freshly grown cells to the agar above the boxes on the template. Repeat this procedure for a2, a3, a4, using a new toothpick for each strain. Through this same procedure, transfer a bit of the freshly grown cells of each a to each of the boxes to the right of the a strain. With a clean toothpick, mix each pair of spots together. Be certain to use a new toothpick each time you mix pairs together.
16. Invert the plate and incubate at room temperature.
17. Wash your hands before you leave the laboratory.

Day 2

18. Also test each mating mixture and parent strain for its ability to grow on MIN agar. Again, to keep track of the tests, tape a copy of the template to the bottom of the MIN plate. Transfer and mix the strains in the same manner as was done on the COMP plate.
19. Invert and incubate the plate at room temperature.
20. Wash hands well before leaving the laboratory.

Day 3

21. Prepare a score sheet and record the color and growth phenotypes of each parent haploid strain and F2 diploid on the score sheet for both plates.
22. Tabulate the different phenotypes observed among the F2 diploids and the number of times each one occurred among the 16 crosses.
23. Compare the F1 phenotypes with the predictions you made in Part A. Explain how your results either support or contradict your predictions.
24. Discard all plates according to your teacher's instructions.
25. Thoroughly wash your hands after completing the laboratory.

Acknowledgment: This article has drawn on "Patterns of Inheritance, Investigation 8.3 A Dihybrid Cross," Biological Science: An Ecological Approach, 7th ed. Kendall/Hunt: Dubuque, Iowa, 1992, pp. 192-96.

Teacher Instruction Page

Preparing Media

YED (Yeast Growth Medium)
Yeast Extract ....................... 5.0 g
Dextrose ........................... 10.0 g
Agar ................................... 15.0 g
Distilled Water ..................... 1.0 L

Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate

To 1.0 L of Yeast Nitrogen Base (purchase ready-made or make from powder) add:

Ammonium Sulfate ........ 0.52 g
Dextrose ........................... 2.0 g

Sporulation Medium (YEKAC)

Potassium Acetate ...... 5.0 g
Yeast Extract ............ 1.25 g
Agar ........................... 15.0 g

Note: All cultures, media, laboratory manuals, videotapes, etc. may be inexpensively ordered from:

Dr. Tom Manney
Department of Physics
Kansas State University
Cardwell Hall
Manhattan, KS 66506-2601
Ph: (913) 532-6789

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