Genetics of Drosophila melanogaster
Objectives
The purpose of this lab is to use genetic crosses to illustrate independent assortment and
sex-linkage in the fruit fly, Drosophila melanogaster. You will be given monohybrid,
dihybrid, and/or sex-linked crosses with well-defined mutant traits. Over the course of
four weeks, you will observe and record what happens to the mutant traits as they are
passed from one generation to the next. Additionally, you will learn the life cycle of the
fruit fly and learn how to recognize the sex of fruit flies, and several types of classic
mutations. Lastly, you will compare the predicted results with the actual results using a
chi-square analysis.
Drosophila Life Cycle
Fruit flies are an ideal organism for conducting genetic studies. They have a short
generation time and are easily cultured in the lab because they have simple food
requirements and occupy little space. In addition, they produce large numbers of
offspring that can be easily manipulated and examined under a low-power microscope.
The fruit fly life cycle is approximately two weeks at room temperature (see Figure 1).
Several days after a breeding pair has mated, the female lays an ellipsoid egg with two
filaments at one end. They are generally laid on the surface of the medium. The eggs
develop into larvae after about a day. The larvae develop into three progressively larger
larval stages called instars. The larvae appear worm-like and eat almost continuously.
They channel through the culture medium while eating. As the larvae mature, they climb
up the side of the culture tube where the larval cuticle darkens and hardens into a
puparium. Within the puparium, the eyes, wings, and legs become visible as the fly
metamorphoses into an adult. The adult emerges by forcing its way through the anterior
end of the puparium. The newly emerged adult is light in color and its wings are not yet
fully expanded. The female fruit fly does not mate until 10-12 hours following
emergence but once she has mated she can store sperm from multiple partners in
receptacles to fertilize the her eggs as she lays them. Therefore, it is important to use only
unmated females in controlled crosses.
Experimental Design
In the following experiment, you will be given fruit fly crosses with one or two
mutations. The mutations will be in the form of eye color or shape, bristle number or
shape, wing size or shape, or antennae size or shape. You will not be given the any
information about the type of crosses nor will you be given the name of the mutations in
the crosses. You will identify the types of mutations in both the male and female flies by
comparing them to the normal or wild-type individuals (Be sure to draw and describe in
detail each mutation). Once you are sure you have identified a mutant trait, you should
make up a name for that trait.
Procedure
Lab 1
1. Your teacher will provide you with a vial of wild-type fruit flies.
2. Place the culture tube on its side. Use a small brush to soak up FLYNAP (anesthetic)
and insert the brush into the culture tube along the sponge stopper. Be careful not to
get any of the FLYNAP on the sponge stopper. DO NOT OVERANESTHETIZE
THE FRUIT FLIES! The flies should be out for 15-20 minutes.
3. When the flies are anesthetized, place them on an index card to view under a
dissecting microscope.
4. Learn to distinguish male from female flies with the following three characteristics
(see Figure 2). First, males are generally smaller than females. The abdominal tip of
males is dark and blunt whereas females are lighter and pointed abdominal ends.
Also, females have transverse stripes on the abdomen. Third, males have sex combs
that are groups of black bristles on the uppermost joint of the forelegs. Females lack
these combs.
5. Familiarize yourself with the features of the wild-type individuals.
a.
What is the eye color? ____________________
b.
What is the shape of the eye? ____________________
c.
What is the wing size? ____________________
d.
What is the wing shape? ____________________
e.
What is the body color? ____________________
f.
How many bristles are present? ____________________
g.
What is the shape of the bristles? ____________________
1. When you are confident that you can identify the different traits of your fruit flies,
obtain an experimental vial from your teacher.
2. Record the cross number of the vial. These adult flies are the parental generation and
have already mated. The eggs or larvae should be on the surface of the culture
medium. These eggs, larvae, and any pupae represent the F1 (i.e. filial) generation.
3. Anesthetize the adults with the procedure from step 2.
4. Remove the anesthetized parents and observe them carefully. Be sure to separate the
males from the females and note the mutations (if any) present in each sex. If the trait
is not normal (wild-type) you have to describe and name it.
5. Record your data in the table below. What is the parental cross?
__________________
Trait/Mutation
Eye color
Eye shape
Wing size
Wing shape
Body color
Bristle number
Bristle shape
Female
Male
1. Label the experimental vial with the parental cross. This should be based on the data
collected in step 10.
Lab 2
2. Anesthetize the newly emerging F1 fruit flies. Examine the flies carefully. Record
and tally the number of mutations (if any) in males and females separately in the table
below.
Trait/Mutation
Female
Male
1. Place five to six pairs of fruit flies into a new culture vial. For this mating the females
do not need to be virgins.
2. Label the new vial F1. Also, label with the symbols of the cross, the date, and the
your name.
Lab 3
3. Remove the F1 adults from the vial and place them in ethanol. The eggs, larvae, and
pupae represent the F2 generation.
Lab 4
4. Anesthetize the F2 flies. Examine the flies carefully. Record and tally the number of
mutations (if any) in males and females separately in the table below.
Trait/Mutation
Female
Analysis of Results
1. What is the name of the observed mutation(s)?
Male
2. Draw two Punnett squares below to predict the results of the parental and F1 crosses.
You may need to review this information.
3. According to the squares, what are the expected ratios for the genotypes and
phenotypes of the F1 and F2 generation in the experiment?
Expected genotype ratio
Expected phenotype ratio
F1
F2
1.
2.
3.
4.
From the results, define your cross.
Sex-linked or autosomal? ____________________
Monohybrid or dihybrid? ____________________
Dominant mutation or recessive mutation? ____________________
Overview: The Chi-square Test
The chi-square test is used to compare expected values with observed values. For
example, you may be interested in determining whether the phenotypic ratio of a
monhybrid cross with independent assortment is 3:1.
Parental generation (P):
Filial generation (F1):
Female
FF (purple flowers)
Ff (purple flowers)
Male
ff (white flowers)
Ff (purple flowers)
X
X
What is the phenotypic ratio of the F2 generation?
The phenotypic ratio would be 3 purple flowers (FF and Ff) to 1 white flower (ff).
F1 males
F1 females
F
f
F
FF
Ff
f
Ff
ff
Question: Is the observed flower color close enough to the expected 3:1 ratio of purple
to white flowers expected for an independently assorting monohybrid cross?
Your null and alternative hypotheses are as follows:
Ho: The sample comes from a population having a 3:1 phenotypic ratio of purple to white
flowers
Ha: The sample does not come from a population having a 3:1 phenotypic ratio of purple
to white flowers
Your level of significance is set at an alpha () of 0.05 and you will use a two-tailed test.
The chi-square statistic is given by the following equation:
n
2
 = ∑ (o-e)2/e
i =1
o = the observed value in each category
e = the expected value in each category
n = number of categories
∑ means that the expression (i.e. (o-e)2/e) is summed from category i = 1 to n
To calculate 2:
1. For each category (in this case, pink and white flowers), calculate the expected value.
For our example, suppose that you sampled 100 flowers to see how many purple and
white flowers you have. Of those 100 flowers, 75 should be purple and 25 should be
white if you have a monohybrid cross that assorts independently.
2. For each category, subtract the observed value (o) from the expected value (e) to find
the difference (o-e) between the two.
3. Square the difference (o-e)2
4. Divide the value from step 3 by the expected value to find (o-e)2/e
5. Repeat this for all categories.
6. Add up (∑) all the (o-e)2/e, the sum is 2.
Phenotype
Purple
White
Observed
(o)
65
35
Expected
(e)
75
25
(o-e)
(o-e)2
(o-e)2/e
-10
10
100
100
2calculated:
100/75
100/25
5.3333
Summing the last column yielded a value of 5.3333 for 2. This is called the calculated
2 statistic. Next you will have to calculate the degrees of freedom () by subtracting the
number of categories (n) from one. In this example, there are two categories (i.e. purple
and white flowers) so  is equal to 1. Once you have the 2calculated and , you can
compare your calculated value to what is known as the critical chi-square (2critical) value
from Table A-2.
At an  = 0.05 and  of 1, the 2critical is 3.841.
Use the following decision rule:
If the calculated value (2calculated) ≥ the critical value (2critical), then reject the null
hypothesis (Ho) and conclude that the difference found between the observed and
expected values is significantly different and could not occur by chance alone.
2. Given the expected phenotypic ratios of the F2 generation, did your expected ratio
differ from the observed ratio? This question can be answered by performing a chisquare ( analysis. Read the description above for the analysis and then fill in the
following table.
Phenotype
Observed
(o)
Expected
(e)
(o-e)
(o-e)2
(o-e)2/e
Total*:
*Only calculate a total for the first, second, and fifth column.
State clearly the null and alternative hypotheses:
Ho:
Ha:
What is the calculated chi-square statistic (calculated)? __________
What is the critical chi-square statistic ((critical)? __________
What is the significance level? P = __________
2. Why was it necessary for the females of the parental generation to be virgins?
3. Why was it not important for the females of the F1 generation to be virgins when they
were mated?
4. Why were the adult flies removed from the vials at Lab 2 and Lab 4?