Download In this multi-part lab, you will be given two mutant traits to study in

TRANSMISSION GENETICS OF DROSOPHILA
OBJECTIVE: In this multi-part lab, you will be given two mutant traits to study in the fruit fly, Drosophila
melanogaster. You will:
1. demonstrate whether these traits are LINKED
2. MAP the distance between the linked traits.
BACKGROUND
The common fruit fly, Drosophila melanogaster ('black-bellied dew lover' for you Latin buffs), became a
favorite experimental organism of geneticists in the early 1900s. This species was the focus of breeding
experiments in the laboratory of Thomas Hunt Morgan (1866-1945), first at Columbia University, and later at
CalTech. Morgan discovered the first of many morphological mutations, the white-eye mutant, in Drosophila in
1908. The findings of Morgan's research group, which included Alfred Sturtevant (1891-1970), Hermann Muller
(1890-1967), and Calvin Bridges (1889-1938), had a significant impact on the field of genetics. Their publication,
The Mechanism of Mendelian Heredity, in 1915 established Drosophila as an important model system in
experimental genetics. The "Fly Lab" group ultimately provided proof of the chromosomal theory of inheritance: a
cornerstone of modern genetics. In 1933, Thomas Hunt Morgan became the first geneticist to win the Nobel Prize.
Hermann Muller became the second geneticist to win the Nobel Prize in 1946.
The studies of these flies have played an integral role in every aspect of genetic discovery; from transmission
genetics and molecular genetics to behavior and population genetics. Flies have advanced our knowledge about
everything from sex-linkage analyses, giant chromosomes and banding patterns to jumping genes, clock mutants
and evolutionary rates. In fact, we know more about the genetics of this organism than any other species,
including our own.
Why has this organism been the animal of choice in many genetics laboratories? Obviously, the anomalous
giant chromosomes make it useful for banding analyses. In addition, their small size, ease of culture, rapid
generation time and prodigious fecundity make them ideal organisms for any genetics study requiring large
numbers of offspring for analysis. Bacteria and mice have been useful genetic tools for the same reasons.
Here is an amazing example of how flies can generate the incredible numbers of offspring under ideal
conditions is taken from Borrer, Tripple, Horn, and Johnson: An Introduction to the Study of Insects (1989, p. 2).
These authors considered how many flies could be produced by a single pair of flies in one year. They estimate
that 25 generations could be produced in that one-year period. They also assume that a single female lays 100
eggs during her lifetime with a 50:50 sex ratio. If there were no checks on their increase, under optimum
conditions there would be 100 flies in the second generation, 5000 flies in the third generation, and about 1.192 x
1041 flies in the 25th generation. Impressive? They go on to explain that the flies, if packed tightly together, would
form a ball of flies 96,372,988 miles in diameter. This ball would extend from the earth to the sun. Now that is a
lot of flies (and an impressive party fact)!!!!
The fly genome has been recently sequenced and consists of three different autosomes, the X chromosome
and the Y chromosome. Normal flies have eight chromosomes: two copies of each autosome and two sex
chromosomes. Sex in flies is determined chromosomally by the X to autosome ratio. Flies with two copies of the X
chromosome are females. Flies with one copy of the X chromosome and one copy of the Y chromosome are
males.
In this laboratory, you will examine the simultaneous inheritance pattern of two mutant traits in the
Drosophila: you will determine the nature of inheritance of, for example, white eye mutation and ebony body
color mutation by testcross the heterozygous F1 and analyze the F2 offspring. Here is how the heterozygous F1
population came to be: the true-breeding P1 of white eye and ebony body color (double mutations) is crossed
with the true-breeding P1 of normal eye and normal body color. The reciprocal cross (double mutant male x wild
type female, double mutant female x wild type males) was done and both cultures gave rise to phenotypically
identical F1. What does this result tell you about these two mutant traits?
Genetics
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Lab
Once the cross of F1 flies (F1 female flies x homozygous double mutant male flies) has been made, it will take
about one week for the eggs to hatch and the larvae to appear. In the beginning of the second week (Day 7-9),
you need to remove all the F1 parents. During the second week the larva will crawl up the side of the culture vials
and pupate. Toward the end of the second week the F2 flies will emerge from the puparium. At this time you will
need to collect, count, and record data on the F2
flies. The data that will be collected include the
number of male and female flies along with the
expressed traits of each fly.
There are three major questions that you will
be able to answer at the completion of this
experiment:
1) Are the traits inherited independently?
2) Are any of these traits sex-linked?
3) If they are linked, how far apart are they?
Life cycle of Drosophila: (Adapted from
Carolina Biology Supply Company)
There are four distinct stages in the life of the
fruit fly: egg, larva, pupa, and adult. At 21ºC a
fresh culture of D. melanogaster will produce new
adults in two weeks; eight days in the egg and
larval stages, and six days in the pupal stage. The
adult fruit flies may live for several weeks.
The day after the egg is laid, the larva
hatches. The larva molts twice; that is, it sheds
the cuticle, mouth hooks, and spiracles.
Metamorphosis occurs within the puparium. The
pupa begins to darken just prior to the emergence
of an adult fly. About one day before emergence,
the folded wings appear as two dark elliptical
bodies, and the pigment in the eyes is visible
through the puparium.
The figure shows the life cycle and sexual dimorphism of Drosophila
melanogaster. The male (1) is generally smaller than the female (2).
The male also has a more rounded abdomen that is black tipped
rather than striped. The enlarged foreleg of the male reveals special
bristles known as the sex comb, a characteristic lacking in the female.
Metamorphosis is complete, from egg (3), larval stages (4, 5, and 6),
and pupa (7) through adult.
When metamorphosis is complete, the adult emerges by forcing its way through the anterior end of the
puparium. At first the fly is light in color, the wings are unexpanded, and the abdomen is long. In a few hours the
wings expand, the abdomen becomes more rotund, and the color gradually darkens.
Two days after emerging, a female can start laying eggs. After maturity, they are fertile as long as they live.
PROCEDURE:
You will receive two true breeding stocks, one is wild type and the other is homozygous for two mutant traits
listed below.
Gene name
Gene Symbol
Wild type
+
Flies with red eyes and other normal standard characteristics
White
w
Eye problem. Eyes white.
Miniature
m
Wing problem. Wings just slightly longer than abdomen and
proportionately narrower.
Genetics
Description
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Lab
Take some time to examine these flies. Use the figures on the following pages to help you sex the flies,
identify virgin females, and identify the mutant characteristics.
Anesthetizing:
To anesthetize the flies, place several drops of triethylamine on the sponge in the lower anesthetizing
chamber and seal the bottom. Transfer the flies by gently tapping down the culture vials into the collecting
chamber. Use the plastic stopper to trap the flies inside and watch them through the top of the chamber. When
the flies no longer climb to the top of the chamber they are ready to handle. This process will take a few minutes.
Care must be taken to not over expose the flies to the triethylamine, since it can kill the flies.
You can also use carbon dioxide (CO2) to temporarily immobilize the flies. A light stream of CO2 can be
introduced into the culture vials by means of the tank and regulator located in the back of the laboratory. The
instructor will show you how to use the regulator. When using the CO2 method, remember to hold the vial on its
side to avoid drowning the flies in the nutrient medium. Once the flies are no longer active, they are ready to be
studied. The CO2 method is not as effective as the triethylamine. You have to act fast before the flies wake up and
fly away.
Sexing:
Before you start, you must be able to distinguish the sexes, and distinguish the alternative phenotypic
forms of your strains. The later is relatively easy on initial inspection of the flies. Sex determination takes a bit
more practice. Look for the following characteristics:
Virgins:
Female Drosophila have an organ in their reproductive tract (called the spermatheca) that allows them to
store sperm from a mating and use that sperm at a later date to fertilize eggs. Therefore, we must use virgin
females in all of our crosses. It takes approximately 8 hours for a newly emerged female adult to gain sexual
maturity; therefore, all females used in crosses must be less than 8 hours old. These females can be easily
distinguished by the presence of the meconium on the ventral side of their abdomen. Your vials were cleared this
morning so there should be plenty of virgin females present.
Genetics
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Lab
Setting up Crosses:
As a group, you need to set up two crosses, each should ideally have 3-4 males and 3-4 virgin females:
Cross 1: Wild type males crossed to homozygous double mutant (white, miniature) females
Cross 2: Homozygous double mutant (white, miniature) males crossed to wild type females
What you need to do:
1. Prepare two vials of fly medium.
2. Set up reciprocal crosses between the wildtype and homozygous double mutant stocks:
a. Anesthetize some flies from your wildtype stock. Using a dissecting microscope, learn how to
distinguish males and virgin females.
i. Collect 3-4 males and put into cross#1
ii. Collect 3-4 virgin females and put into cross#2
b. Anesthetize flies from your mutant stock. Using a dissecting microscope, observe the mutant
phenotypes
i. Collect 3-4 virgin females and put into cross#1
ii. Collect 3-4 virgin males and put into cross#2
c. Label your crosses with the following information:
i. Your group initials
ii. Gender and genotype of flies in the cross (use zodiac signs and gene symbols to save space)
iii. Today’s date
3. Lay your vials on their sides until the flies wake up. Once awake, the vial can be stored upright in the
incubator in the project room.
4. About 7-9 days afterward, remove these P1 adults, so they will not be mistaken for F1 adults that are about to
appear. You do so by anesthetizing all the adult P1 flies and discard them in the oil morgue.
5. Expect F1 adults to begin appearing about 11 to 12 days after the P1 flies were placed in the bottles. Examine
F1 animals for sex and phenotype, considering both traits being studied.
6. Set up a test cross between your F1 generation and the original homozygous recessive stock. Be sure you
label your vials as in step 2c above. As above, you will set up reciprocal crosses.
7. About 7-9 days afterward, remove these F1 adults, so they will not be mistaken for F2 adults that are about to
appear. You do so by anesthetizing all the adult F1 flies and discard them in the oil morgue.
8. Expect progeny from your testcrosses to begin appearing about 11 to 12 days after the F1 flies were placed in
the bottles. Examine these “F2” animals for sex and phenotype, considering both traits being studied.
9. Use the data from both crosses (the F1 and the progeny from the testcrosses) to do the following:
a. Validate that your two mutant traits (genes) are in fact linked. Do this by performing a Chi-squared
test.
b. Map the recombination distance between the genes
Data analysis: the Chi-squared test
The purpose of the chi-square (X2) test is to determine whether experimentally obtained data constitute a
good fit to, or a satisfactory approximation of, a theoretical, expected ratio; that is, the X2 test enables one to
determine whether it is reasonable to attribute deviations from a perfect fit to chance. Obviously, if deviations are
small, they can be more reasonably attributed to chance than if they are large. The question we try to answer
with the x2 test is, "How small must the deviations be to be attributed to chance alone?" The formula for X2 is as
follows: X2 =
∑(O - E)2 / E, where O = the observed number of individuals in a particular phenotype, E = the
expected number in that phenotype, and ∑= the summa on of all possible values of (O - E)2 /E for the various
phenotypic categories.
Genetics
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Lab
Table of X2 Values*
df
P = 0.99
0.95
0.80
0.50
0.20
0.05
0.01
1
.000157
.00393
.0642
.455
1.642
3.841
6.635
2
.0201
.103
.446
1.386
3.219
5.991
9.210
3
.115
.352
1.005
2.366
4.642
7.815
11.345
4
.297
.711
1.649
3.357
5.989
9.488
13.277
5
.554
1.145
2.343
4.351
7.289
11.070
15.086
6
.872
1.635
3.070
5.348
8.558
12.592
16.812
7
1.239
2.167
3.822
6.346
9.803
14.067
18.475
8
1.646
2.733
4.594
7.344
11.030
15.507
20.090
9
2.088
3.325
5.380
8.343
12.242
16.919
21.666
10
2.558
3.940
6.179
9.342
13.442
18.307
23.209
15
5.229
7.261
10.307
14.339
19.311
24.996
30.578
20
8.260
10.851
14.578
19.337
25.038
31.410
37.566
25
11.524
14.611
18.940
24.337
30.675
37.652
44.314
30
14.953
18.493
23.364
29.336
36.250
43.773
50.892
*Selected data from R.A. Fisher and F. Yates, Statistical tables for biological, agricultural and medical research
(London: Oliver and Boyd, 1943).
Genetics
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Lab