Techniques: Drosophila Genetics
Background
Modern genetics began with the work of Gregor Mendel. Through Mendel's studies of
pea plants, he developed the laws of independent assortment and segregation. These two
laws state that when organisms are crossed and offspring are produced, the offspring
receive one gene from each parent for a particular trait and all traits are inherited
independently of one another (modern genetics has found that in some cases genes are
linked). Mendel saw these laws at work in his pea plants, as he observed patterns in the
number of each phenotype (appearance) in different offspring. These laws are utilized
today to explain heredity (what traits are passed from one generation to the next) and
variation (the differences between parents and their offspring).
Through his experiments, Mendel determined that there can be multiple forms of the
same gene. These alternate forms of genes, called alleles, code for slightly different
expressions of a genetic trait. He saw that some alleles exhibited dominance and others
exhibited recessive tendencies. When a dominant allele was combined with a recessive
allele, only the dominant allele was expressed in the phenotype. In modern genetics, as
in Mendel's experiments, a dominant allele is represented with a capital letter and a
recessive allele is represented with a lowercase letter. Of note, genetic dominance or
recession does not directly correlate with a wild-type (normal) or mutant phenotype, but
in this exercise, wild-type eye color and wing size correlates with dominance.
Drosophila melanogaster (fruit fly) has become a model organism for many reasons,
including ease of use. Fruit flies are inexpensive to support, and millions fit in a single
room. Drosophila has simple food and storage requirements, completes its life cycle in
approximately 12 days, and has a genome packaged in only four chromosomes.
Furthermore, Drosophila is safe to work with, large enough to be examined with the
naked eye, and easily anesthetized for observation and sorting. There is also a great deal
of genomic homology between Drosophila and human beings, making the fruit fly an
invaluable tool for disease research. Drosophila exhibits complete metamorphosis, going
through egg, larval, pupal, and adult stages of life.
In the early 20th century, the geneticist Thomas Hunt Morgan used Drosophila to provide
the first evidence supporting the chromosome theory of inheritance, which claims that
chromosomes carry genes and that all genes on a chromosome tend to be inherited
together. Since Morgan's time, Drosophila has been used in thousands of experiments;
hundreds of mutations have been characterized.
Sexing Flies
Wild-type fruit flies have brick red eyes, are yellow-brown in color and have transverse
black rings across their abdomen. They exhibit sexual dimorphism: females are about 2.5
millimeters (0.098 in) long; males are slightly smaller and the back of their bodies is
darker. Males are easily distinguished from females based on color differences, with a
distinct black patch at the abdomen, less noticeable in recently emerged flies, and the
sexcombs (a row of dark bristles on the tarsus of the first leg). Furthermore, males have a
cluster of spiky hairs (claspers) surrounding the reproducing parts used to attach to the
female during mating. For a visual representation, refer to the figure below.
Using Drosophila, you will investigate and learn the basic principles of monohybrid and
dihybrid inheritance. During your observations, you will keep accurate records of the
numbers of flies with two different mutant traits; this is called "scoring" the flies. To
repeat, wild-type flies have red colored eyes. Sepia flies have dark-brown colored eyes.
Wild-type flies also have full wings (oh, really, tell us something we don’t know Doc).
Apterous flies do not possess wings. The figures below show the description of wild-type
and mutant flies; you can see (viewing clockwise from top left) fly wings vary from wildtype (top left) to miniature, vestigial and finally apterous (bottom right). In this exercise,
you will observe the way in which these two mutant traits are passed from one generation
to the next and then construct a hypothesis describing the mode of inheritance for each
mutation.
When setting up crosses, the first two lines of flies that are crossed are called the parental
(P) generation. The offspring of the parental generation are called the First Filial (F1)
Generation. The offspring of the F1 generation are the Second Filial (F2) Generation,
and so on. When labeling crosses, the female description always comes first. For
example, if we were crossing wild-type females with sepia eyed males, we would label
the culture vial "wt x sepia." This standardized notation eliminates the need to
distinguish between males and females when labeling crosses.
Purpose
The purpose of this exercise is to familiarize you with the application of the fruit fly in
monohybrid and dihybrid trait inheritance through experimental mating.
Materials per team
2 parental (P1 & P2) fly stocks
Yeast
Sorting Brushes
Nap kit
Bleach Solution
1 first filial (F1) fly stock
Vials and Plugs
Sorting Cards
Microscope
Drosophila medium
Adhesive Labels
Fly
Tap Water
Preparation before implementation
Peruse through the Fruit Fly Reference Manual (posted online as a .pdf document) and
become familiar with the kit materials and activities before you begin the activities with
your class. A little more than two weeks (18 days) prior to our F1 mating procedure (Fly
Lab day 2), male and female mutants of each parental (P1 and P2) strain were crossed by
Carolina Biosciences, Inc. in a vial of medium (the date that the parental flies were put
together in the vial is written on the label affixed to the vial). The flies were allowed to
interact for 7 days, giving the flies enough time to mate and lay eggs. After this period,
the parental flies were removed, leaving only the eggs in the vial. These eggs will
produce offspring that will emerge from pupae 14 to 18 days after the cross date, yielding
plenty of F1 adult flies available for examination and use in setting up in-bred crosses (F1
males mate with F1 females; in other words, brother mates with sister) producing the F2
generation. Thus, due to the long incubation period (14 to 18 days), we may ‘score’ our
mutant F2 flies over an extended duration of two lab periods (in other words, Fly Lab day
4 could be extended to include another period).
Procedures
(Fly Lab day 1) – Observing, Manipulating, and Sexing Fruit Flies (P generation)
1. There will be a total of 4 groups, consisting of a maximum of 6 students each. Two
groups will be in the ‘odd’ group; the other two groups will be in the ‘even’ group.
Ask your instructor to designate what your group is (odd or even).
2. Your instructor will prepare media filled vials (4 total; 1 vial per group) and will
incapacitate the flies you are about to employ by anesthetization. Watch the
instructor while the procedures are being performed; you will be doing it during the
next lab period. (When you are ready to put the flies to sleep, let your instructor
know and he/she will begin the procedure). If the anesthetized flies start to wake up
during your examination, let your instructor know immediately! We don’t want flies
escaping and flying around the lab during the next couple of weeks (contamination
issues)!
3. Two groups (A and B) will receive a card with several anesthetized mutant flies (P1
strain). Using a microscope (under wide-field scope) and sorting brushes, take turns
observing and sexing the flies. Look at the general features of the fly, but pay special
attention to eye color, wing shape and bristles (sex anatomy indicates). Take note of
these phenotypic observations in the P1 strain (eye color:__________, wing
shape:__________).
4. The other two groups (C and D) will receive a second card with several anesthetized
mutant flies with a different mutation. This is the (P2) strain. Characterize these flies
according to eye color and wing shape (eye color:__________, wing
shape:__________).
5. Compare the two groups of flies. What difference do you notice between P1 and P2
(besides the sexes within the groups)? From your observations:
P1 is which mutant? ___________
P2 is which mutant? ___________
6. Now you will perform a Parental (P) cross to produce a Filial (F1) generation. We
already have an F1 generation (from the breeding company) that we will employ
during Fly Lab day 2. I want you to perform a Parental cross as practice before you
perform the real-deal on Fly Lab day 2. Each group will receive 1 media filled vial
to place mating groups into. Your mating groups should consist of 5 males from P1
and 5 females from P2 (Groups A and B), or 5 males from P2 and 5 females from P1
(Groups C and D). Why do you employ this mating scheme? Thus, you will rely on
each other (groups) to get the appropriate sex and mutant. Just remember: when
returning anesthetized flies to a vial that contains medium, place the vial on its side
so the flies do not become stuck in the medium. Turn the vial upright after the flies
recover.
7. Return the remaining flies to their appropriate, original culture vials. If you are
confused, ask your instructor.
(Fly Lab day 2) – Setting-Up the F2 Vials (Mating the F1 Flies with each other)
1. You will prep your materials for the cross at the instructor’s station. Each group
should have a volunteer step forward. Using the plastic cup provided with the
Drosophila Medium (flaky powder), add one level measure to each vial to be used for
your F2 cultures. Your group will have a total of two vials for this generation.
2. Add one level measure of tap water to each vial.
3. From the packet of yeast provided, sprinkle four to six grains on the surface of the
medium. Take care not to add too much yeast, as an excessive amount can damage
the culture.
4. Label each vial: “F2: Mutation 1 x Mutation 2.” Each group should now have a total
of two labeled F2 vials.
5. Next, I will need another volunteer from each group to help me anesthetize the flies.
Tap the flies to the bottom of the vial without disrupting the media. Quickly insert a
wand dipped in FlyNap into the vial, between the plug and the wall of the vial. Leave
the wand in place until most of the flies have dropped to the bottom of the vial, and
then remove it. When the flies are asleep (there may be some trembling of arms and
legs), put them on sorting cards for scoring. Do not leave the flies in the
anesthetization vial for a long time. If FlyNap got on the plug when you inserted the
wand, it will continue to anesthetize the flies after the wand has been removed and
can kill them. When returning anesthetized flies to a vial that contains medium, place
the vial on its side so the flies do not become stuck in the medium. Turn the vial
upright after the flies recover.
6. The instructor will dispense the anesthetized F1 flies into four equal aliquots (one
aliquot for each group); each aliquot contains both males and females. Each group
will examine the phenotypes of their aliquot of F1 flies by microscope. As you
become familiar with characterization, you will be able to forego the microscope and
view with the naked eye. Be sure to separate the males from females by placing the
flies on two separate (labeled) sorting cards.
7. Score the F1 flies and enter the information in the first row of the F1 Data Table.
Your data should include the date as well as the number of males and the number of
females displaying the wild-type phenotype, the Mutation 1 phenotype and the
Mutation 2 phenotype. Let your instructor know your data, as well. The instructor
will compile the data and put it on the board, for you to enter in the second row of the
F1 Data Table. The F1 Data Table is shown below.
8. Give the instructor your flies. Be sure to label the cards with the sex and the type of
mutation of the flies.
9. Put five male/female pairs into each “F2: Mutation 1 x Mutation 2” vial. Remember
to pair 5 males with one type of mutation with 5 females of the other type of
mutation.
10. Complete the F1 Punnett Squares (in the Worksheet section) for the predicted modes
of inheritance (the genes are autosomal and non-linked, and exhibit a dominant
pattern of expression) for the F1 generation. Remember: during your breeding, the
female parents have one mutation and the male parents have the other. We will
declare that (dark eye color is mutation 1 – sepia, the absence of wings is mutation 2
– apterous). I suggest designating wild-type red eye color as “R”, sepia as “r” and
wild-type wings as “W”, apterous wings as “w”. Of course, upper-case represents
the dominant allele, lower-case represents the recessive allele.
11. Compare the observed ratios of the F1 flies to the predicted ratios stated in the F1
Punnett Squares (in the Worksheet section). Answer questions 1-4 in the worksheet
section.
12. Based on your hypothesis, create dihybrid Punnett squares and branch diagrams for
the F2 generation (Worksheet section, F1 Punnet Squares, #5).
(Fly lab day 3) – Clearing the F2 Vials
1. Anesthetize the adult flies (if you need a reference, look at step 5 in Fly lab day 2).
2. Remove the F1 adults that were transferred into the F2 vials. This is known as
"clearing" the vials. Follow the FlyNap instructions when anesthetizing the flies. lt is
imperative that all adult flies be cleared from the F2 vials at this point. Otherwise,
there will be generational crossover, wherein F1 flies mate with their F2 offspring.
3. Dispose of the anesthetized flies by placing them in a morgue full of bleach. Note:
The F2 generation of flies is in the form of eggs and larvae in the vials. You should
be able to see trails in the medium left by the larvae. You may also be able to see the
black mouthparts of the larvae as they move through the medium.
(Fly Lab day 4) – Scoring the F2 Flies
1. Anesthetize the F2 flies (if you need a reference, look at step 5 in Fly lab day 2).
2. Remove the flies from the vial.
3. Score the flies and record the data in the F2 Data Table (Worksheet section). Your
data should include the date as well as the number of males and the number of
females displaying the wild-type or mutant phenotype(s).
4. Dispose of the scored flies in a bleach filled morgue.
5. Answer questions 5 through 8 (Worksheet section).
WORKSHEET
Drosophila Genetics