AP Biology: Unit 3: Heredity: AP Lab #7: Genetics of Drosphilia
Objectives:
 Use fruit flies to do genetic crosses
 Learn to determine the sex of fruit flies and recognize contrasting phenotypes
 Collect data from Fl and F2 generations and analyze the results of a monohybrid, dihybrid, or sex-linked cross
Background:
Imagine that you have decided to devote your career to genetic research. You want to find mutants with interesting
characteristics and study their inheritance. What organism should you study? It would be best to choose something that
is small, easy to keep, has a short generation time, and produces many offspring. Scientists have adopted certain
organisms as good choices for studying basic processes common to many life forms. These are often called "model
organisms." The fruit fly Drosophila melanogaster is an excellent model organism for genetics research. A scientist can
keep hundreds or thousands of them in a jar and millions in a single room. They are hardy and have simple food
requirements. Drosophila completes its life cycle in about two weeks at room temperature and produces large numbers of
offspring. It is easy to immobilize them for examination and sorting, and they cannot hurt you. Fruit flies have only four
pairs of chromosomes, which can be readily observed in cells of the salivary glands. One pair of these chromosomes is not
completely homologous; these two are designated X and Y. These are the sex chromosomes: females have two X
chromosomes and males have an X and a Y. The other three pair of chromosomes are autosomes. Genes located on
the portion of the X chromosome that has no counterpart on the Y are said to be sex-linked or X-linked.
Research with Drosophila helped to establish facts we now take for granted, for example, that genes are located on
chromosomes and that genes on the same chromosome are inherited together unless they are separated by crossing over. The
phenotypes that are most frequently found in natural populations are designated as wild type. Phenotypes that differ from
the wild type are mutants. Over the years, scientists working with Drosophila have published their results and saved stocks
of mutants they have isolated. As a result, there is a wealth of information in the scientific literature about Drosophila genes
and genetics. Furthermore, stock cultures of hundreds of different genetically pure strains bearing various mutations are
available.
Drosophila develops by complete metamorphosis. Figure 1 provides a summation of the Drosophila life cycle.
Introduction:
This genetics investigation will run for several weeks. You will begin by observing the phenotypes of wild-type flies and
by learning to distinguish male and female flies. After that, you will observe flies of three different mutant strains. Each
mutant strain has a single mutation, and thus differs from the wild- type strain in one trait only. All other traits of the
mutant will be identical to those of the wild-type flies. The mutation most likely will be in eye color or shape, bristle
number or shape, wing size or shape, or antennae size or shape. Make up your own name for the mutations that you
identify in the flies.
Your teacher will have you study the offspring (the First Filial generation, or F 1) produced by crossing two strains of flies.
You will cross these F1 flies to obtain F2 flies. You are responsible for making close observations and keeping accurate records
concerning what happens as these traits are passed from one generation to the next.
The cross you study can be a cross of the wild-type strain with one of the mutants or it can be a cross of two of the mutant
strains. One of the mutant traits is sex-linked. You must determine how each mutant differs from the wild type, whether
the mutations are dominant or recessive, and autosomal or sex-linked.
To successfully complete these activities, you must have a good background knowledge of genetics. In your notebook, write
the things you know about genetics that you think will help you complete these activities. Include at least six facts and
explain how this knowledge will help you. Following are some questions to help you get started, but do not feel limited by
these suggestions. Use your fact sheet as a reference for the duration of this lab; add to and amend your list as needed.
Answer these in your notebook prior to beginning the lab.
1. How will I know which allele is dominant and which allele is recessive?
2. How will I know if I am dealing with a monohybrid or a dihybrid cross? How will this affect my results?
3. Is this a sex-linked gene? How will I know?
4. How will I know what the F 2 flies should look like? What phenotype ratio will I expect in the F 2?
5. How will I determine phenotypic ratios from my raw data?
6. How will I know whether or not my results are valid?
Activity A: Sexing Drosophila and Examining Wild and Mutant Phenotypes
Materials:
Sorting brushes, sorting cards, fly morgue, stereomicroscope, petri dish of wild-type flies, petri dish of mutant flies.
Procedure:
Your instructor will anesthetize the wild-type and mutant Drosophila strains and then give you petri dishes containing some
of each. Watch the anesthetization process; you will eventually have to do it yourself.
1.
Observe the wild-type flies. Examine them carefully under a stereomicroscope, using a brush to move them around.
Be sure to note eye color and wing shape.
2.
Observe the mutant types and compare them to the wild-type flies. Fill in Table 1 for the phenotype of each
mutant and the contrasting wild-type phenotype in your notebook. Restrict your descriptions to no more than two
words. In your notebook, take good observation notes; you will need to refer to them when you analyze the
offspring of the crosses.
Table 1: Phenotypes of the parental strains of Drosophila
Phenotype
Phenotype
Wild Type:
Mutant A:
Wild Type:
Mutant B:
Wild Type:
Mutant C:
3.
Practice sexing Drosophila. Distinguish male flies from female flies by looking for the following characteristics.
Males are usually smaller than females. Males have dark, blunt posteriors, whereas the females have lighter;
pointed posteriors. Male flies have sex combs (groups of black bristles) on the uppermost joint of the forelegs;
female flies do not. You may wish to make drawings to contrast the wild-type flies with the mutants, and male
flies with female flies.
4.
When you have finished, return the flies to your teacher.
Activity B: Scoring F1 Phenotypes and Setting Up F1 Crosses
Materials:
Sorting brushes, sorting cards, fly morgue, stereomicroscope, Petri dish of F1 flies, culture vial with medium,
foam plug.
Introduction:
You will be assigned to study the offspring (F1) of a cross between two of the four Drosophila strains you observed in Activity
A. Because one of the mutants is sex-linked, you will be told which strain provided the males and which provided the
females for the cross.
We will study the cross of (use the phenotype descriptions from Table 1) – record in your notebook:
x
Female Parent
Male Parent
Procedure
1. Copy Table 2 into your notebook.
Table 2: F1 Data
Date
Phenotype
# females
# males
Total
2.
Examine the F1 flies in the petri dish. In Table 2, record the sex and phenotype of each fly. This data collection is
called "scoring" the flies.
3.
Set up a culture vial for your F1 cross. Place one cup of Instant Drosophila Medium and one cup of water in the vial.
When it solidifies, sprinkle 4-7 grains of yeast on top of the media.
4.
Place five or six male/female pairs of the anesthetized F1 flies in the fresh culture vial. Label the new vial "F1 cross."
Also include on the label the parental strains, the date, and your name.
5.
After you have scored all the Ft flies, recorded the data, and set up the F 1 cross, return the rest of the F1 flies to the
teacher.
Analysis of Results, Activity B: Scoring F1 Phenotypes and Setting Up F1 Crosses
1.
From the data recorded in Table 2 and your observations in Activity A, state a hypothesis about how the
phenotypes are inherited. This should include whether the phenotypes are inherited through one or two gene
loci, whether the alleles are dominant or recessive, and autosomal or sex-linked.
2.
Decide on the genetic symbols you will use for each allele involved in the cross you are studying. Record them in
your notebook and identify them by phenotype.
3.
In your notebook, construct two Punnett squares to show the results of both the parental and F1 crosses.
4.
Predict the expected ratios for the phenotypes of the F2 flies you will obtain from your F, cross.
Activity C: Removing F1 Adults From Vials
Materials:
Sorting brushes, sorting cards, fly morgue, stereomicroscope, F 1 cross vial, Fly Nap° Kit.
Procedure:
1.
Remove the F1 adult flies from the vials and place them into the morgue. To do this, have an empty culture vial and
foam plug ready. Rap the F1 cross vial sharply on your lab bench to knock the adult flies to the bottom. Quickly
remove the plug and put the mouth of the empty vial over the mouth of the cross vial. Invert the pair of vials so
that the empty vial is on the bottom. Rap the empty vial on the table to knock the adult flies into it. Quickly plug
the vial that now contains the flies. Dip a wand into the Fly Nap anesthetic. Rap the vial of flies on the counter
again and quickly insert the wand into the vial, beside the plug. When the flies are asleep, put them in the
morgue.
Why is it necessary to remove the F1 flies?
2.
Observe the F2 generation, which is currently in the form of eggs and/or larvae in the vial. You should be able
to see channels in the culture medium and may be able to see the black mouthparts of larvae moving under the
surface of the medium.
Activity D: Scoring Phenotypes of F2 Flies
Materials
Sorting brushes, sorting cards, fly morgue, stereomicroscope, Fl cross vial, Fly Nape Kit.
Procedure
1. Every other day or so after the F2 adults begin to emerge, score them as to sex and the presence or absence of
the phenotypes observed in the parental flies. To do this, transfer the adults to an empty culture vial and
anesthetize them as described in Step 1 of Activity C. When the flies are asleep, sprinkle them onto a card or
into a petri dish and score them. Record the data in Table 3 in your notebook. (It is only necessary to record
data on males and females if you are analyzing the sex-linked cross.) The more F2 flies collected, the more
reliable the data will be. When you have finished scoring the flies, put them in the morgue. Do not return
them to the cross vial.
Table 3: F2 Group Data
F2 Group Data for ___________________ x ____________________
Phenotype
(If sex-linked, record male and female as phenotypes.)
Total
Total Scored: _______
2. Obtain class totals for the F2 flies and their phenotypes and record the information in Table 4 in your notebook.
Table 4: F2 Class Data
F2 Group Data for
___________________ x ____________________
Phenotype
Total
(If sex-linked, record male and female as phenotypes.)
F2 Group Data for
___________________ x ____________________
Phenotype
Total
(If sex-linked, record male and female as phenotypes.)
F2 Group Data for
___________________ x ____________________
Phenotype
Total
(If sex-linked, record male and female as phenotypes.)
Analysis of Results, Activity D: Scoring Phenotypes of F2 Flies
1. Using the class data from Table 4, calculate actual phenotype ratios for each of the F2 flies and record this
information in your notebook.
2. Refer to the prediction you made in 4 of Analysis of Results, Activity B. Do your actual results support your
prediction? Explain.
3. Look back at your hypothesis in 1 of Analysis of Results, Activity B. Give below any needed modifications or additions
to your hypothesis about how the phenotypes are inherited. Explain why the change(s) is/are necessary. If no
changes are needed, write "none."
4. Refer to the background knowledge fact sheet that you produced at the beginning of this lab. Make any
necessary additions, corrections, or clarifications to it that you feel are necessary. Has your knowledge of
genetics changed as a result of doing this lab? If so, summarize how.
Activity E: Data Analysis Using the Chi-Square Test
Introduction:
Does your data actually support your hypothesis about how the trait is inherited? Genetics, like gambling, deals
with probabilities. When you flip a coin, you have the same chance of getting a head as a tail: a one-to-one ratio. That
does not mean that if you flip a coin 100 times you will always get 50 heads and 50 tails. You might get 53 heads and 47
tails. That is probably close enough to a one-to-one ratio that you would accept it. But what if you get 61 heads and
39 tails? At what point do you begin to suspect that something other than chance is at work in determining the fall of
the coin?
Review your data and hypothesis. You have assumed that chance (and chance only) has been operating in the
assortment and recombination of alleles that gave rise to the Fl and F2 you investigated. Thus, any variation of your
observed results as recorded above from the expected results is due to chance. This is known as the "null hypothesis."
Does your data actually support the null hypothesis?
The chi-square test (x2) is a statistical test used to determine how well observed ratios fit expected ratios. The
difference between the number observed and the number expected for a phenotype is squared and then divided by
the number expected. This is repeated for each phenotype class. The x2 value is the sum of these values for all cases.
The formula for X 2 is:
x2 = total of (observed - expected)2 for all cases
expected
The calculated value for x2 is then compared to the values given in a statistical table, such as the one shown here.
Chi-Square Values and Probabilities
15% or less is considered significant}
Degrees of Freedom
1
2
3
4
5
6
7
8
9
10
p = 99%
0.000157
0.020
0.115
0.297
0.554
0.872
1239
1,646
2.088
2.558
95%
0.00393
0.103
0.352
0.711
1.145
1.635
2.167
2.733
3.325
3.940
80%
0.0642
0.446
1.005
1.649
2.343
3.070
3,822
4.594
5.380
6.179
50%
0.455
1.386
2.366
3.357
4.351
5.348
6.346
7.344
8.343
9.342
20%
1.642
3,219
4.642
5.989
7.289
8.558
9.803
11.030
12.242
13.442
5%
3.841
5.991
7.815
9.488
11.070
12.592
14.067
15.507
16.919
18.307
1%
6.635
9.210
11.345
13.277
15.086
16.812
18.475
20.090
21.666
23.209
In this table, note the column "Degrees of Freedom." The number of degrees of freedom is always one less than the
number of different phenotypes possible. For a monohybrid F2 there are two possible phenotypes (the wild and the
mutant) so the number of degrees of freedom is determined by the following calculation: 2 -1 = 1. For a dihybrid F2,
there are four possible phenotype combinations and 3 degrees of freedom. The numbers to the right of the "Degrees of
Freedom" column in the table are X 2 values. The percentages given at the top of each column represent the probability
that the variation of the observed results from the expected results is due to chance. If the probability value is greater
than 5%, we accept the null hypothesis; that is, our data fits the expected ratios. Following are two examples, one for a
monohybrid cross and one for a dihybrid cross.
In an F2 population of 100 grains of corn, there are 60 purple grains and 40 yellow grains (expected ratio would be 3:1 or
75 purple:25 yellow). Then:
X2 = (60 – 75)2 + (40 – 25)2 = 3 + 9 = 12.0
75
25
According to the chi-square table for x 2 = 12 with 1 degree of freedom, the probability = <0.01; therefore, these
results do not support the expectation (or null hypothesis) of a 3:1 ratio because the probability is less than 5% that
deviation from the expected ratio is due to random chance.
List at least three possible sources of error that could explain why the data in this example does not fit the
expected 3:1 ratio.
Now, consider the following data for F2 corn grains of a dihybrid cross that began with a corn that had purple and
smooth (starchy) grains with a corn that had yellow and wrinkled (sweet) grains. The alleles for purple and smooth are
dominant. The expected phenotype ratio is 9 purple, smooth:3 purple, wrinkled: 3 yellow, smooth:1 yellow, wrinkled.
The expected numbers are calculated from the 9:3:3:1 ratio.
1
2
3
4
Total
Phenotype
purple, smooth
purple, wrinkled
yellow, smooth
yellow, wrinkled
4
Phenotype
count
577
204
176
59
1016
Expected
numbers
571.5
190.5
190.5
63.5
1016
X2 = (577 – 571.5)2 + (204 – 190.5)2 + (176 – 190.5)2 + (59 – 63.5)2 = 2.43
571.5
190.5
190.5
63.5
The probability of 2.43 from the table for three degrees of freedom is greater than 30% but less than 50%. This means that a
deviation as large or larger would be expected to occur by chance more than 30 percent of the time. Such a deviation is
not significant, so we accept the null hypothesis in favor of the 9:3:3:1 ratio. Note that this acceptance is provisional.
Additional data could always cause us to reject the null hypothesis.
Procedure:
Calculate the chi-square statistic for the class data. What does this tell you about the data collected on these crosses?
List some possible areas of error. Record in your notebook.
Conclusion:





What is your conclusion? What did you learn?
Was your hypothesis supported or not? Why
What went well? Why?
What didn’t go well? Why?
What were your sources of error? What would you improve if you were able to conduct this experiment again?