Genetics: Testing Hypotheses about Inheritance Introduction Fruit Fly Background and Life Cycle

26 Jun Genetics: Testing Hypotheses about Inheritance Introduction Fruit Fly Background and Life Cycle

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Genetics: Testing Hypotheses about Inheritance
Introduction
Fruit Fly Background and Life Cycle
In this exercise, you will perform reciprocal crosses to determine the inheritance pattern of various traits in Drosophila
melanogaster. This organism has been used in genetic experiments since 1910. Wild type or “normal” traits are red
eyes, wings longer than the body and smooth, and tan or brown body color. An enormous number of mutants,
spontaneous and induced, have been discovered in Drosophila, including mutations that affect external anatomy (eye
color, body size, body color, and wing shape). The most interesting mutations are those that affect behavior. Mutants
have been found that go into shock at loud noises, forget recently learned things and show excessive libido.
Drosophila are useful for laboratory experiments, because they have a short generation time, produce many offspring,
and require no special care or equipment. The life cycle of Drosophila (see Figure 1) is usually completed in 14 days if
incubated at 21°C, or in 10 days at 24°C. The life cycle includes four stages: egg, larva, pupa and adult. The eggs are 2
mm long, sausage-shaped, and white, bearing a pair of filaments at one end, which keep the eggs from sinking into the
soft food on which they are always laid. One day after being laid, the eggs hatch into first instar larvae (little white
maggots), which feed voraciously. After several days, the larvae molt and begin the second instar developing tracheae.
One more molt occurs to produce third instar larvae, which crawl onto a hard dry surface and transform into pupae in
small dark cocoons. Within the pupae, the third instar larvae go through metamorphosis and eventually emerge as
adult flies. A few hours after emergence, the adult is at first light in color with crumpled wings. Within a few hours, the
flies’ wings expand and harden, enabling them to fly.

Figure 1. Diagram of Drosophila life cycle.

How we talk about genetic information
Each cell in living organisms contains DNA, which is made of nucleotide subunits arranged in very long strands. By
winding around structural proteins, the strands become condensed into compact units called chromosomes. Regions
in the DNA, known as genes, carry specific instructions for making proteins. Genes represent unique combinations of
nucleotides that provide information for the genetically-based traits that organisms display.
While all humans have the same genes (that is, each human has a gene that codes for eye color, etc), humans are not
genetically identical. Think of all the variation in the human population. We have different colors of hair, eyes,
different heights, different tendencies towards disease, etc. One reason for the variation we see is that organisms, like
humans, might have different forms of genes, which we term alleles. These alleles code for different proteins, and
therefore result in the expression of different phenotypes.
A dominant allele is expressed phenotypically when present on either a single chromosome or on both homologous
chromosomes (that is, when it’s present on either the chromosome inherited from the mother, or the father, or both).
A recessive allele is masked by a dominant allele and is expressed only when paired with another recessive allele on the
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Genetics: Testing Hypotheses about Inheritance
homologous chromosome—that is, it is only expressed when the chromosomes inherited from both parents have the
recessive allele. A pair of identical alleles (AA or aa) at a genes locus represents the homozygous condition. Two
different alleles (Aa) at a genes locus represent the heterozygous condition. For example, eye color in humans is
determined by a single gene locus (although other loci can modify its effects). If the alleles at that locus are
homozygous dominant (AA) or heterozygous (Aa), the eyes will be brown. If the alleles are homozygous recessive (aa),
the eyes will be blue. Alleles for brown eyes are said to be dominant over alleles for blue eyes.

How is genetic material passed on from generation to generation?
Alleles are passed on from parents to their offspring through reproduction, specifically through gametes (via meiosis)
and fertilization. Meiosis is the process of cell division that happens only in sex cells and is responsible for creating
gametes (sperm and egg). Sperm and egg cells are unique from other body (somatic) cells in that each sperm or egg cell
contains exactly half of the genetic material from each parent. Why do you think this is the case?
To learn more about meiosis, please visit the “Cells Alive” site (http://www.cellsalive.com/meiosis.htm). Watch the
animations for the cell and as you do, track the locations of the chromosomes as they move through the phases of
meiosis. In addition, compare the beginning cells to the end products of meiosis. How many cells does meiosis begin
with? How many cells does meiosis end with? Are the numbers and types of chromosomes the same in both cases?
Drosophila are eukaryotic and diploid, having corresponding sets of genes on paired chromosomes. During each cross,
the chromosomes containing the genes are shuffled by meiosis and combined at fertilization. Different mechanisms of
inheriting a trait involve different patterns of chromosomal movement during reproduction. The key to studying
genetics is to be able to predict the chromosomal movements that would result from different models for how a trait
might be inherited.
It is important to know that female Drosophila can store and utilize sperm from one insemination for a large part of
their reproductive lives. Therefore, only virgin females should be used in making the initial parental crosses. Females of
this species can mate six hours after they have emerged from the pupal case. If all adult flies are emptied from the
culture vial and the vial is left for six hours, all females removed the second time should be virgin. Males of any age may
be used in the crosses.

Using experiments to test patterns of inheritance
Modern genetics began with the experiments of Gregor Mendel, an Austrian monk with an inquisitive mind. Mendel
performed crosses between garden pea plants and discovered that certain alleles can mask one another (i.e. dominant
alleles mask recessive ones described earlier). A monohybrid cross is one in which the pattern of inheritance of a single
trait (e.g., pea shape) with a pair of alleles (e.g., round or wrinkled peas) is studied. In contrast, a dihybrid cross
involves parents that are identical except for two independent traits (e.g., pea color and shape).
Morgan built upon Mendel’s findings through his discovery that traits may be sex-linked, meaning the gene is carried
on a sex-determining chromosome (X or Y). In Drosophila, the X-chromosome carries sex-linked genes. So in flies,
females have two copies of the X chromosome, and therefore carry two alleles of sex-linked genes, one on each X
chromosome. Male Drosophila, however, only have one X chromosome and therefore only carry one allele of a sexlinked gene. This means that whatever allele is passed on to males through their single X chromosome will be
expressed regardless of whether it is dominant or recessive. Since female flies inherit two alleles for sex-linked genes,
expression of the alleles follows the dominant/recessive pattern described above. Any genes that are present on the
other chromosomes—that is, not on X or Y chromosomes—are said to be autosomal. For example, all of Mendel’s pea
plant traits are inherited on autosomal chromosomes.
Alleles that are naturally common in the wild (e.g., red eyes in fruit flies) are known as wild-type alleles. Less common
alleles (e.g., white eyes in fruit flies) are known as mutant alleles. These are derived from characteristics that are
expressed naturally in the wild. It’s important to note that wild-type alleles are not always dominant, nor are mutant
alleles always recessive.
Reciprocal crosses were important to Morgan’s discovery about sex-linked genes. A reciprocal set of crosses is
composed of a forward cross (where the male parent has the mutant allele and the female parent has the wild-type
allele) and a reverse cross (where the female parent has the mutant allele and the male parent has the wild-type allele).

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For example, Morgan’s forward cross was a red-eyed (wild-type) female with a white-eyed (mutant) male. For the
reverse cross, Morgan used different flies with opposite alleles, in this case, a white-eyed (mutant) female and a redeyed (wild-type) male. The patterns of alleles in the offspring of these reciprocal crosses were markedly different and
as a result, Morgan was able to determine that the white eye-color allele in fruit flies was sex-linked.
In both Mendel’s and Morgan’s (who did experiments with fruit flies) experiments, it was important to begin with
parents that are said to be true breeding. This means that the parents are homozygous for the alleles of interest for
the study. Because the genetic makeup is known for the starting organisms, it enables scientists to track the genotypes
(or the genetic makeup of the organisms) through generations

Genetics with Computer Flies
All concepts about genetic information, the process of meiosis and experiments used to test for different patterns of
inheritance are identical to what was presented in the Drosophila. It is important to remember that all parental virtual
flies are true breeding and that there is no co-dominance or epistasis for any alleles being studied.
There are several key differences between living Drosophila and what we will be doing with the computer simulation.
The first difference is using virtual flies instead of live ones. These virtual flies are a new species we are naming,
Drosophila spartaniensis. This species was created at MSU. The vast majority of the wild type individuals in the
population are small ~3 mm long, with red eyes; two straight wings that extend beyond the abdomen; a tan body; and
non-feathery antennae (just like the live wild type flies we have previously studied). Male flies are readily distinguished
from females in D. spartaniensis by having sex combs on their forelegs and having short, blunt abdomens with three
bands; whereas females have long, pointed abdomens with four bands.
The second difference is the mutant traits being studied. Since we have created these flies then any traits we will be
studying will be inherited in a variety of ways which may be different than a counterpart mutant trait in Drosophila
melanogaster.
The third difference is the instant life cycle. Whenever you mate the virtual flies, their offspring come up immediately
on the screen; there is no waiting for 2-weeks to see them. Don’t forget that you still need to keep track of which
generation you are observing (e.g., parental or F1 or F2).
The fourth difference is the simultaneous viewing of both the forward and reverse crosses. This makes it a lot faster to
use your logic trees to deductively select the mode of inheritance for each mutant trait being studied. The disadvantage
is that you have a lot of flies on the screen at one time, and you have to make sure you are not mixing up the two
crosses nor missing any offspring which may be further down on the problem page.
The fifth difference is the method used to view the virtual flies (no need for the ether or tile or dissecting microscope).
You still have to carefully sex and describe the fly phenotype and to record all the counts.

Hypotheses about Modes of Inheritance
What we’ve learned about meiosis and genetics allows us to examine various alternative hypotheses (modes) about
how a particular allele might be inherited, and we can test their associated predictions by making crosses and
examining their resulting offspring. If the crosses are designed correctly, then each mode of inheritance will lead to a
distinctive phenotypic ratio in the offspring. We can choose among the alternative hypotheses by making statistical
comparisons between the observed phenotypic ratios (the evidence) with the expected ratios that are predicted by
each mode.
Here are four common modes that explain the inheritance of a single allele:
1. Autosomal dominant
2. Autosomal recessive
3. Sex-linked dominant, and
4. Sex-linked recessive.
Below are some common modes that explain the inheritance of two alleles that are assorting independent of one
another in a dihybrid cross analysis:
1. Both alleles are autosomal dominant
2. Both alleles are autosomal recessive
3. First allele is autosomal dominant while the second allele is autosomal recessive
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There are also other modes that can be used to explain the inheritance of two alleles which are different than the ones
listed above:
1. Both alleles are autosomal and located on the same chromosome (linked) [you still have to determine whether
each allele is dominant or recessive]
2. One allele is autosomal while the other allele is sex-linked [you still have to determine whether each allele is
dominant or recessive]
There are several ways to generate predictions from hypothesized modes of inheritance. We will use Punnett
rectangles because they are simple method and easy to remember. For the alleles we will study in lab, it is necessary to
predict the results both of parental crosses—producing the first (F1) generation—and of F1 crosses—producing the
second (F2) generation. The predicted phenotypic ratios are converted to expected frequencies and will be used to
compare with the frequencies of real (or simulated) crosses to test your hypotheses.
Each mode of inheritance (i.e., alternative hypothesis) has a unique F2 generation prediction, and therefore the results
of experiments can be compared to the predicted value to determine how alleles are passed on from parents to
offspring. The best model will be the one whose predictions do not differ statistically from the actual cross values (do
not result in the null hypothesis being rejected). We will use the Chi-Square Goodness-of-Fit test to determine
whether or not our cross data actually matches our predictions.

What you will do
In today’s lab, you will collect data to determine the mode of inheritance of traits in the virtual flies. You will observe
and record the parental flies phenotypes, then you will mate these flies to produce F1 offspring. Once these offspring
are phenotypically described and counted, then you will mate the F1s to generate the F2 offspring. As before, you will
record counts and phenotypes off the offspring (F2s). You will use the logic trees and Punnett rectangles to help you
select one mode of inheritance for the one trait being studied or for the two traits being studied. You will use the Chisquare goodness-of-fit statistical test to analyze the data and to arrive at the final conclusion for the inheritance pattern
for the trait(s) being studied.

Laboratory Objectives
As a result of participating in this exercise, you will:
1. Make predictions for each mode of inheritance.
2. Practice using Punnett rectangles as predictive tools. Develop predictions for F2 offspring using Punnett
rectangles.
3. Mate, sex and describe virtual flies in LON-CAPA.
4. Perform reciprocal crosses using true breeding parents to generate F2 offspring.
5. Evaluate competing hypotheses about modes of inheritance using both virtual and real fly cross data.
6. Use the Chi-Square goodness-of fit test to evaluate support of the selected hypothesis(es).
7. Draw conclusions about modes of inheritance and hypothesis testing.

Methods:
Engagement in Recitation
Part 1. Modeling Meiosis with Foam Cutouts of Chromosomes
Use the foam cutouts provided to model the action of chromosomes during the cell cycle of:
1. Meiotic cells from Prophase 1 through Telophase 2
2. Filling in Punnett squares
Part 2. Developing a Logic Tree for Testing Hypotheses Involving One and Two Traits
Fill in before recitation, the sections in the appended section for “Building Logic Tree a Single Trait”. In groups, you
would work to create the logic tree for the single trait. At recitation, you will receive paperwork for “Building a Logic
Tree for Determining Linkage”.
Be sure to also find the appendix on-line which has more inheritance patterns Punnett rectangles to be used to derive
the predicted (expected) F2 phenotypic ratios which are needed for the Chi-square Goodness-of-Ft statistical testing.
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Exploration during Lab
Part 3. Virtual Fly Problems in LON-CAPA
There will be one single practice problem page for each reciprocal cross (or experiment). In lab, you will be
expected to work in pairs. If you need to re-do the work at another time, you still have access to these pages, and
you can generate new data by clicking on the ‘New Problem Variation’ button.
1.

Observe phenotypes, record data, perform each cross as indicated in the instructions that follow.
a. Set up one table for each cross (forward and reverse) to collect the data from each generation
(parents, F1 and F2). Make sure you have room to collect data for counts and all phenotypes (sex,
eye color, wing characteristic, body color or antennae [if this is visible])
b. Recall that the wild type phenotype is red eyes, normal straight wings (longer than the abdomen),
tan body color and non-feathery antennae (when present). This is just like D. melanogaster.
c. Select an experiment (practice problem).
d. You will see the parents for the forward cross and reverse cross. Collect all the data for the forward
cross in its own table. Next, carefully collect the data for the reverse cross in its own table.
e. To create parent cross, scroll down the page and type the word mate into the box provided and
click on ‘Submit Answer’.
f. To see the F1 offspring for each cross, scroll down the page. Collect the appropriate data in each
appropriate table. Be very careful not to mix up the male and females nor to mix up progeny
between each cross.
g. To create the F1 cross, scroll down the page and type the word mate into the box provided and click
on ‘Submit Answer’.
h. To view the F2 offspring for each cross, be sure to scroll down the page again. Collect the
appropriate data in each appropriate table. Be very careful not to mix up the male and females nor
to mix up progeny between each cross.
i. You do not have to type in the word done in the last box.
Table 1. Typical phenotypes of Drosophila:
Character
Sex:
Eye Color:

Description
Male (short body with sex combs)
Female (long body without sex combs)
Red (Wild Type), Purple, Dwarf Red Eye,
Sepia, or White

Body Color:

Brown (Wild Type), Yellow, or Blackish Grey

Wings:

See Figure 2

Figure 2. Drosophila wing phenotypes.
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Genetics: Testing Hypotheses about Inheritance
2.

Keeping all generation of the virtual flies on the screen, use your data and the logic tree to eliminate
hypotheses. Choose the hypothesis for the mode of inheritance that best predicts your data. Also, use the
following questions to help you eliminate hypotheses.
a. Looking at the parental generation, which specific mutant trait(s) is(are) being studied? Is this a
monohybrid or dihybrid cross analysis? Are there any hypotheses that can be eliminated now?
b. Looking at the F1 offspring in both the forward and reverse crosses, is the mutant trait autosomal or
sex-linked? Which additional hypotheses can be eliminated now?
c. Looking at the F1 offspring, is the mutant trait dominant or recessive? Which hypotheses can be
eliminated?

3.

Test your hypothesis using the Logic Trees and Chi-Square Goodness-of-Fit when there are more than 2
types of F2 offspring.

Assuming the hypothesis you put forth is supported, what is the expected value (ratio) of the F 2 offspring resulting
from this hypothesis? Explain your answers by using the Punnett square(s) that would produce this F2 offspring
phenotypes and ratios. Use the pages which are appended in this handout to come up with the expected ratios and
values for the F2 offspring for each mode of inheritance.

Using the Chi-Square Goodness-of-Fit Test for F2 Offspring
Do Chi-Square Goodness-of Fit analyses for both F2 offspring (forward or reverse) from each reciprocal cross. To
accomplish this, use tables similar to Table 1 for each cross and the expected ratios determined from the
appropriate Punnett rectangles.
Table 1. Example for how you might organize
Phenotype

Observed (O)
Count

Expected (E)
Count

2
Stat

calculations in your lab notebook or in a spreadsheet
(O-E)

2

(O-E)

2

(O-E) /E

Sum (Σ)

4.

Draw conclusions from the Logic Trees and Chi-Square test based on not rejecting the null hypothesis for
both the forward and reverse crosses. This means that if you have to reject the null hypothesis for either
cross then you have selected the wrong hypothesis and need to re-think your logic to choose another
hypothesis.

5.

Now start over at step 1 with a new reciprocal cross (experiment).

Explanation
Discuss in your groups:
1. Why are reciprocal crosses helpful when trying to determine patterns of inheritance?
2. What hypotheses did you pose to test using a Chi-Square?
3. What were your conclusions from your Chi-Square?
4. Why are studying patterns of inheritance important?

Expand
Discuss in your group:
1. How does meiosis link with what you did in lab today?
2. What are the limitations of meiosis? Advantages?
3. How does meiosis differ from mitosis? (link back to mitosis exercise)
4. How does meiosis and what you’ve done today help explain why two siblings are never genetically identical
(unless they are identical twins)?
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Building a Logic Tree for a Single Trait
If the allele is autosomal dominant:

Parental genotypes:

Forward Cross
Male A / A Female + / +

Parental phenotypes:

male mutant A and female wild type

Reverse Cross
Male + / + Female A / A
male wild type and female mutant A

Sperm genotypes:

A

+

Ova genotypes:

+

A

Punnett rectangle to generate F1 offspring:
Ova Genotype

Ova Genotype

+

A

Sperm
Genotype

Sperm
Genotype

A

+

F1 offspring:
F1 genotypes:
F1 phenotypes:

If the allele is autosomal recessive:
Parental genotypes:
Parental phenotypes:

Forward Cross
Male a / a Female + / +
male mutant a and female wild type

Reverse Cross
Male + / + Female a / a
male wild type and female mutant a

Sperm genotypes:

a

+

Ova genotypes:

+

a

Punnett rectangle to generate F1 offspring:
Ova Genotype

Ova Genotype

+

a

Sperm
Genotype

Sperm
Genotype

a

+

F1 offspring:
F1 genotypes:
F1 phenotypes:

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Building a Logic Tree for a Single Trait, continued
If the allele is sex-linked (X-linked) dominant:
Forward Cross
Parental genotype:
Male A / Y Female + / +
Parental phenotypes:
Sperm genotypes:

Reverse Cross
Male + / Y Female A / A

male mutant A and female wild type

male wild type and female mutant A

A and Y

Ova genotypes:

+ and Y

+

A

Punnett rectangle to generate F1 offspring:
Ova
Genotype

Ova
Genotype

+

A

Sperm
Genotypes

Sperm
Genotypes

A

Y

+

Y

F1 offspring:
F1 genotypes:
F1 phenotypes:

If the allele is sex-linked (X-linked) recessive:

Parental genotypes:
Parental phenotypes:
Sperm genotypes:

Forward Cross
Male a / Y Female + / +
male mutant a and female wild type

Reverse Cross
Male + / Y Female a / a
male wild type and female mutant a

a and Y

+ and Y

+

a

Ova genotypes:

Punnett rectangle to generate F1 offspring:

Y

a

Sperm
Genotypes

a

Ova
Genotype

+

Sperm
Genotypes

Ova
Genotype

+

Y

F1 offspring:
F1 genotypes:
F1 phenotypes:

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Building a Logic Tree for a Single Trait, continued
How can you tell from looking at the F1 offspring whether or not a trait is autosomal or sex-linked?

If it’s autosomal, how can you tell from the F1 offspring whether the trait is dominant or recessive?

If it’s sex-linked (X-linked), how can you tell from the F 1 offspring whether the trait is dominant or recessive?

Write an efficient flowchart that shows logic (based on the work you did not theory) for deciding among the possible
modes of inheritance.

9

If both alleles are autosomal recessive and not linked:
Forward Cross
Parental genotypes: Male a / a + / + Female + / + b / b

Reverse Cross
Male + / + b / b Female a / a + / +

Parental phenotypes: male mutant a and wild type

male wild type and mutant b

Female wild type and mutant b
Sperm genotypes:

only a +

Ova genotypes:

female mutant a and wild type
only + b

only + b

only a +

Punnett rectangle to generate F1 offspring:

a

F1 offspring
F1 genotypes:

Ova
Genotype
a +
Sperm
Genotype
s

Sperm
Genotype
s

Ova
Genotype
+ b

+

+ b

F1 male + / a b / +
F1 female + / a b / +

F1 phenotypes:
Punnett rectangle to generate F2 offspring:
Ova Genotype

a

+

+

+

a

b

Ova Genotype

+ b

a

+

+

a

b

a

a

a

b

+ b

b

+

+

+

+

Sperm Genotype

+

+

+ b

a

Sperm Genotype

+ b

+

F2 of…