Part B: Basic Genetics Experiments

A Dihybrid Cross

The dihybrid cross, made between two strains that carry different traits, is most commonly associated with Punnet's Square. This is a diagram used to display the combinations of traits that are found in the children produced by the cross. In yeast, because you can observe the phenotypes of the haploid cells, you can directly see much more of what is going on and why.

In most plants and animals phenotypes can only be observed in the diploid stage because the only haploid cells are single celled gametes. Mendel observed diploid phenotypes of peas and then calculated the most likely genotypes of the gametes. Baker's yeast, Saccharomyces cerevisiae, has the ability to grow as stable haploid colonies and the phenotypes of the colonies can be observed. Because there is only one set of genes in haploid cells (compared to two sets in diploids), the haploid phenotype is a direct reflection of the cell genotype. It is this characteristic of yeast that makes it a powerful tool for understanding inheritance patterns.

Experiment:


In this experiment you will use a set of haploid strains to produce the diploid strains that would be produced in the second generation of the traditional experiment. Your text may call this the F2 generation. This set of haploid strains represents the eight different haploid genotypes (spores or gametes) a dihybrid cross where the genese for the two traits are inherited independently. We will use a simple shorthand description of the strains that is similar to the way many textbooks describe Mendel's crosses with peas.
In the following table, A and B refer to the two different mating types (sexes). The symbols R and r stand for the different forms (alleles) of a gene controlling the color trait. This is the same trait studied in the experiment A Simple Cross. If you did that experiment you know that R is dominant and cream colored and r is recessive and red. The symbols T and t stand for alleles of another gene that controls the cell's ability to grow on an agar medium that we call MV. As you might guess, T is dominant and can grow on MV while t is recessive and cannot. (Of course, all the strains can grow on YED medium).

The eight haploid strains you will study can be described as follows: 


Genotype    Phenotype on:      MV
            YED               
 ART,BRT     cream-colored     grows
 ARt,BRt     cream-colored     doesn't grow
 ArT,BrT     red               grows 
 Art,Brt     red               doesn't grow

Time Line:

1st Day: 50 min. Make mating grid and subculture haploid strains
2nd Day: 20 min. Make mating mixtures
3rd Day: 10 min. Replica plate to MVA
4th Day: 50 min. Record and analyze results

Materials:

Making a Prediction:

Use the information in the mating grid to figure out the genotype of each cross. Use the genotypes to predict the phenotype of each cross. Do you expect to get a 9:3:3:1 phenotype ratio?
Testing your Prediction:
1. 1st Day: Tape the mating grid to the bottom of a YED plate.
Using a fresh sterile toothpick for each strain make spots of the haploid strains (gametes) across the top and down the side of the plate. Use the grid as a guide. Incubate the plate overnight.
2. 2nd Day: Prepare mating mixtures. Using sterile toothpicks transfer and mix the haploid strains in all possible combinations at the intersections of the grid. Remember to use a new toothpick for each different strain and mixture.
Incubate the plate overnight.
3. 3rd Day: Use toothpicks or replica plating equipment to replica plate the YED plate onto MVA (MV + adenine).
Incubate the plate overnight.
4. 4th Day: Read the colors of the mixtures from the YED plate and the growth pattern from the MVA plate.
Record your results in a table and then compare you results with your predictions.

5. How did your results compare with your predictions?( Teacher Tips )


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Last updated Friday August 19 2005