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:
- Mating-type a strains
- HA0 (RT), HAR (rT), HAT (Rt),
HART (rt)
- Mating-type à> strains
- HB0 (RT), HBR (rT), HBT (Rt),
HBRT (rt)
- Petri plate of YED
- Petri plate of MV + adenine
- Sterile toothpicks
- Mating grid and tape
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