Allele Shuffling of Single-Gene Traits in Drosophila
Martina Pansze
Introduction:
Purpose:
•Use fruit flies to do
genetic crosses
•Learn to determine the sex
of fruit flies and recognize contrasting phenotypes
•Collect data from F1 and F2
generations and analyze the results of a monohybrid, dihybrid, or sex-linked cross
Background
Info: Animas High School chose to use fruit flies (Drosophila) in their study of genetics because they are small,
relatively easy to keep and immobilize, safe, and produce many offspring. For
these reasons, Drosophila have been
used for research with genetics many times in the past and much information is
available, as well as hundreds of genetically pure strains possessing different
mutations. Additionally, Drosophila develops by complete metamorphosis quickly,
fully-grown in only about 9 to 12 days.
Null
Hypothesis: If the p-value is greater than 5%, then
allele shuffling can be said to be random.
Materials
and Equipment:
Materials included in the
completion of this lab include sorting brushes, sorting cards, fly morgue, FlyNap
anesthetizer, stereomicroscope, petri dish of wild-type flies, vials and plugs,
and a petri dish of mutant flies.
Methods:
Sexting
Drosophila and Examining Wild and
Mutant Phenotypes:
1) You are given petri dishes
containing anesthetized wild and mutant Drosophila.
Observe and record the eye color and wing shape phenotype differences of both.
2) Practice sexing the Drosophila into male and female
categories.
3) Deposit the flies in the alcohol
morgue.
Scoring
F1 Phenotypes and Setting Up F1 Crosses:
4) Study the assigned offspring of a
cross between two of the Drosophila
types observed before. This is the F1 generation.
5) Observe and record (score) the
sex and phenotype of each fly.
6) Set up a culture vial for the F1
cross. To do this, place water, instant Drosophila
medium, and yeast or mashed potatoes into the vial.
7) Place approximately twelve (half
male, half female) anesthetized flies of your cross into the culture vial and
label it with your parental strains and name.
8) After you have scored and
recorded the data of all your flies and set up the F1 cross, deposit the
remainder of the flies into the morgue.
Removing
F1 Adults from Vials:
9) Remove the adult F1 flies from
the vials by rapping the vial sharply on a desk and knocking the adult flies to
a second vial. Make sure that the second, clean vial is aligned with the first
and that your mashed potatoes containing F2 larva do not fall into the second
vial. Stick a FlyNap’ed wand into the second vial to anesthetize them, and then
dump the anesthetized adults in the second vial into the morgue.
10) Observe the larvae and eggs in
the vial. You should see channels in the culture medium and possibly the black
mouthparts of the larvae.
Scoring
Phenotypes of F2 Flies:
11) As they emerge, score the F2
adults as to sex (if your cross requires) and the the presence or absence of
the phenotypes assigned to you. To achieve this, transfer the flies to an empty
vial and anesthetize them. When they are asleep, move them to a petri dish and
score them, recording your data. When you have finished scoring the flies,
place them in the morgue.
12) Obtain class total results for
the F2 flies and their phenotypes and record the information.
Results
and Analysis:
|
Cross 1: Ss x Ss
|
|
|
|
|
|
|
Phenotype
|
Observed
|
Expected
|
Chi-Square
Value
|
p-Value
|
Significant
(Y/N)
|
|
Sepia-Eyed
|
326
|
299.25
|
2.391186
|
>5%
|
N
|
|
Wild-Eyed
|
871
|
897.75
|
0.797062
|
>5%
|
N
|
|
Total
|
1197
|
1197
|
3.188248
|
>5%
|
N
|
|
|
|
|
|
|
|
|
Cross 2: VvSs x VvSs
|
|
|
|
|
|
|
Phenotype
|
Observed
|
Expected
|
Chi-Square
Value
|
p-Value
|
Significant
(Y/N)
|
|
Vestigial Wild
|
197
|
258
|
14.422481
|
<5%
|
Y
|
|
Vestigial
Sepia
|
231
|
86
|
244.476744
|
<5%
|
Y
|
|
Wild Wild
|
708
|
774
|
5.627907
|
>5%
|
N
|
|
Wild Sepia
|
240
|
258
|
1.255814
|
>5%
|
N
|
|
Total
|
1376
|
1376
|
265.782946
|
<5%
|
Y
|
|
|
|
|
|
||
|
Cross 3: W+ W-
x W+ W-
|
|
|
|
|
|
|
Phenotype
|
Observed
|
Expected
|
Chi-Square
Value
|
p-Value
|
Significant
(Y/N)
|
|
Wild Female
|
214
|
216.5
|
0.288683
|
>5%
|
N
|
|
Wild Male
|
240
|
216.5
|
2.550808
|
>5%
|
N
|
|
White Female
|
184
|
216.5
|
4.878753
|
>5%
|
N
|
|
White Male
|
228
|
216.5
|
0.610854
|
>5%
|
N
|
|
Total
|
866
|
866
|
8.329098
|
<5%
|
Y
|
To test the accuracy of the hypothesis the Chi-Square method was
used. This method is used to determine how well observed ratios fit expected
ratios and is modeled by the equation: xsquared = total of
((observed-expected)squared/expected) for all cases. The value of xsquared is
then compared to the values in a statistical table. The value that comes out of
this depends on the degrees of freedom. The degree of freedom is one value less
than the amount of phenotypes possible. In this lab, if the results are 5% or
less then they are considered significant, meaning our null hypothesis is
accepted. Below is a model equation demonstrating the first cross (Ss x Ss)
Sepia-Eyed results.
Using the Chi-Square equation, xsquared of Sepia Eyes=((326-299.25)squared)/299.25.
By solving this it can be concluded that xsquared=2.39, the Chi-Square value.
Observing the chart and keeping in mind that the degree of freedom is one, it can
also be seen that the p-value of this data is >5%, because 5% is 3.841 on
the chart. So, for this test, our null hypothesis is proved true.
Conclusion
and Discussion:
The purpose of this lab is to use Drosophila to learn about genetics and
determine whether or not the allele shuffling among the flies is random or not.
If the hypothesis were to be correct, then the data would indicate that the p-values
are all deemed significant. To test this we bred the Drosophila twice, studying genetics past on through the F1 and F2 generations,
and sorted the flies to find the results.
Above in the total column of the data result charts it can be noted that the results
in crosses two and three were determined to be significant, while the first
cross was not. Using this information and the fact that this means that the
total p-values were above 5%, it can fairly be concluded that the majority of
the genetic crosses tested fit the hypothesis and therefore the allele shuffling
can indeed be determined to be random. The dependent variable (the flies
breeding) was not influenced severely by any independent variable or else the results
would not show the allele shuffling to be random, but rather the variable would
cause a disruption in the breeding and it would be visible through the
phenotypes.
The results are significant because
they have proved that allele shuffling is random in Drosophila, and to draw a larger hypothesis, all other animals as
well. These concepts connect with what is being studied in class because,
although they are fruit flies, which are thought to be pesky, the concepts seen
through this lab apply to humans, too, as far as general passing on of genetic information.
It also was interesting to breed the crosses and then actually observe the
results of the breed and see the changes in the generations evolve right before
your eyes.
Animas High School is fairly
confident in the results and accuracy of this lab, even though the results were
not quite as consistent as they could have been (differing cross one results)
the majority was still regular. Possible causes for the irregularity of the
lab, or perhaps unnoticed errors, are numerous. This was the first time that
many lab students sexed and scored phenotypes, so they could have made a
mistake on that account. Also, a small amount of flies may have been let loose
accidentally when transferring vials, moving, anesthetizing, or sorting the
flies that were therefore left out of the final results. Also the environment
of the classroom or the food given in the culture vials could have unwittingly
abolished some phenotypes because of related complications. Perhaps in a future
lab more efforts could be focused on not letting loose flies.
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