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Biology 22 GENETIC CROSSES IN THE FRUIT FLY Drosophila melanogaster

Introduction

Since the turn of the 20th century, Drosophila melanogaster, the common fruit fly, has been a useful organism for the study of genetics. Its relatively short generation time (approximately 10 days at 25oC) yields a large amount of breeding data in a short period of time. Because of its simple food requirements and easy handling in the laboratory, large and varied stocks of Drosophila can be maintained with minimal cost and effort.

The general anatomical features of a male and female wild type fruit fly and the organism’s life cycle are shown in Figure 1. Recognizing various structures of the adult organism, especially sexual differences, will be important in carrying out the experiments to follow. For both male and female flies, locate the head, thorax, and abdomen, the mouth parts (particularly the extendable proboscis), the aristae (feather-like antennae), and the compound eye made up of many individual facets called ommatidia (singular, ommatidium).

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The major sexual differences in Drosophila are apparent from an examination of the adults shown in Figure 1.

1. The last abdominal segment of the male is much larger and more rounded than that in the female.

2. The abdomen of the male contains only five segments while that of the female contains seven.

3. The male's body size is generally somewhat smaller than that of the female.

4. The male has a small, densely packed tuft of bristles known as a sex comb located on one of the outer joints of both forelegs. (The female lacks these). THIS IS THE MOST RELIABLE INDICATOR OF GENDER. BE SURE YOU CAN SEE SEX COMBS ON ANY FLY YOU COUNT AS A MALE!

The complete life cycle of Drosophila is summarized in Figure 1. The eggs are about 0.5 millimeters (mm) in length and bear a pair of filaments at one end that keep them from sinking into the soft food on which they are usually laid. The larvae are small maggots that burrow in the food and spend most of their time eating. Drosophila larvae pass through two molting stages called instars, where they shed their outer protective cuticle, and finally reach a size up to 4.5 mm in length. After their second molt, the larvae crawl off the food onto a dry spot on the container wall where they pupate into small dark cocoons. At 25oC, metamorphosis takes about four days after which the adult fly emerges. At first the adult is somewhat pale in color, its wings are crumpled, and its sex is difficult to distinguish. But within a few hours the wings expand to full size and the adult coloration is visible.

Female fruit flies remain virgins for about six hours after hatching but will mate freely thereafter. Females that are allowed to stay in containers with males when they are older than six hours cannot be considered to be virgins as they have probably mated with one of the males in the container. For this reason flies are separated before or immediately after birth so that only planned matings will occur during experiments.

Procedures

Fruit flies are handled while anaesthetized with triethylamine (Fly NapR) so they won't fly away. Wands soaked in Fly Nap are inserted past the foam stopper into a vial containing flies. The vial is then placed on its side, wand up, until all flies are unconscious. The unconscious flies can be poured out of the vial onto an index card and manipulated with a paintbrush. Your dissecting microscope can be used to help you identify sex, body color, and other body characteristics. New crosses are made by transferring a virgin female and a male to a culture vial containing fresh food. Any fly not needed for further experimentation is to be returned to its vial, placed in the morgue, or crushed so that the environment will not become contaminated with new fly populations!

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Experiments

A. The Inheritance of Body Color

Five stocks of adult male and virgin female Drosophila will be available. Some have the wild type body color that is beige. Wild type body color will be designated by the symbol “Y+”. Others have a naturally occurring mutation causing yellow body color that will be designated by the symbol “y”.

Parent Gender Body ColorA Female BeigeB Female BeigeC Male BeigeD Female YellowE Male Yellow

The question of the experiments to be conducted here is: how is this variation in body color controlled genetically? Only phenotypes of parents and offspring can be observed directly and can be considered results of experiments performed. However an experimenter can infer genotypes for all observed flies, propose a mode or modes of inheritance governing the inheritance of these traits, and test the proposal against the data obtained in the genetic crosses performed. (Please note that the yellow-body trait is controlled by a separate gene from the black-body trait that will be studied in the three-point text cross experiment to be described in Part B.)

A logical starting place in an experiment such as this is to perform as many crosses among beige-bodied and yellow-bodied male and female flies as possible. The data resulting from these crosses can then be examined to see if patterns of inheritance are exhibited. For instance, are these traits inherited in a dominant/recessive pattern? If so, which trait appears to be dominant? What evidence (phenotypic ratios of offspring) supports that deduction? Does the sex of the flies have any influence on the pattern of inheritance? Which crosses exhibit this and how? After patterns of inheritance have been observed, the experimenters can propose genotypes for all the flies involved, figure out the expected ratios of offspring, and compare the expected ratios with the observed results to test their deductions. A statistical method for testing such comparisons (the Chi-Square Test) is given in this manual.

If completed crosses have not been provided by your instructor, you and your lab partners should set up crosses among the five fly stocks made available to you in order to collect data that will help you to determine the mode(s) of inheritance involved in the inheritance of these body-color traits in Drosophila. Combine flies in fresh culture vials available in lab making sure each cross contains at least one male and one female. If sufficient numbers of flies are available, use three females and two to three males per vial for each cross. Make sure you label all your culture

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vials with the stock letters and phenotypes of the flies used in the cross as well as with your lab group's identification. The vials will be stored by the instructors in the lab prep room. After seven days, the cultures will be returned to you so that you can remove the parents. They will have mated and laid eggs by this time, but none of the progeny will have yet emerged. This step avoids confusing parents with offspring when examining the results of the cross later. (Parents should be deposited in the morgue). Finally fourteen days after starting the crosses, offspring will be collected and counted by phenotype and sex. Data should be recorded in a table like the one included in this manual (see Table A at the end of this section).

If you are asked to collect flies for making the crosses, follow the directions shown below. Depending on the maturation rate of the flies in the culture vials, you may be performing these steps during the laboratory period or at a time of your choosing in the student projects room (SCI 130).

Collecting females

1. Remove all adult flies from the vial at least six hours before you intend to collect virgin female flies.

a. Shake the adult flies into an empty vial and close with a foam plug.b. Soak a wand in Fly-Nap and insert it into the vial to anesthetize the adults.c. Transfer anesthetized adults to the morgue. d. Observe the inside of the culture vial to be sure no adults remain. If so,

repeat steps a through c.

2. Return within six hours to collect virgin females.

a. Shake the adult flies into an empty vial and close with a foam plug.b. Soak a wand in Fly-Nap and insert it into the vial to anesthetize the adults.c. Observe adults under a dissecting microscope to identify females for

crosses. BE SURE NONE OF THE FLIES HAS SEX COMBS! d. Transfer females to a holding vial with food at the bottom or directly to vials

set up for genetic crosses. To keep the flies from adhering to the food, place them against the side of the vial and keep the vial on its side until the flies have recovered from the anesthesia. As long as food and moisture are available, and no males have been accidentally included, females can be kept in holding vials indefinitely. Your group may be asked to collect flies of a specific phenotype to be shared with other laboratory groups. Be sure to accurately label the contents of your holding vials or vials for crosses.

e. If you wish to collect more females after another six hours, observe the inside of the culture vial to be sure no adults remain.

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Collecting males

1. Remove adult flies from the vial at any time before you plan to make the genetic cross.

a. Shake the adult flies into an empty vial and close with a foam plug.b. Soak a wand in Fly-Nap and insert it into the vial to anesthetize the adults. c. Observe adults under a dissecting microscope to identify males for crosses. BE SURE EVERY ONE OF THESE FLIES HAS SEX COMBS! d. Transfer males to a holding vial with food at the bottom or directly to vials set

up for genetic crosses. To keep the flies from adhering to the food, place them against the side of the vial and keep the vial on its side until the flies have recovered from the anesthesia. As long as food and moisture are available, males can be kept in holding vials indefinitely. Your group may be asked to collect flies of a specific phenotype to be shared with other laboratory groups. Be sure to accurately label the contents of your holding vials or vials for crosses.

Making Crosses

1. If the flies you are using have recovered from anesthesia, briefly re-anesthetize them to facilitate transfer for making the crosses.

a. Shake the adult flies into an empty vial and close with a foam plug.b. Soak a wand in Fly-Nap and insert it into the vial to anesthetize the adults.c. Observe adults under a dissecting microscope to identify females or males

for crosses.

2. Add three females of the specified type to an appropriately labeled vial for the cross. To keep the flies from adhering to the food, place them against the side of the vial and keep the vial on its side until the flies have recovered from the anesthesia.

3. Add two or three males of the specified type to the vial. To keep the flies from adhering to the food, place them against the side of the vial and keep the vial on its side until the flies have recovered from the anesthesia.

For example, if the cross is to be A x C, you would add three females of the A group and two to three males of the C group to the SAME vial.

4. Store the vials in the area designated by your instructor.

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Removing parents

Approximately one week after making the crosses, parents must be removed from the vials to avoid confusion with the resulting offspring. Depending on the maturation rate of the flies in the culture vials, you may be performing these steps during the laboratory period or at a time of your choosing in the student projects room (SCI 130).

Make sure you can see larvae and/or pupae in your cultures before removing the parents. Larvae have white wormlike shapes and can be detected by their dark mouthpieces that look like dots moving through the food or by their later emergence from the food to climb the side of the vial. Pupae are dark rigid structures adhering to the side of the vial.

To remove the parents:

a. Shake the adult flies into an empty vial and close with a foam plug.b. Soak a wand in Fly-Nap and insert it into the vial to anesthetize the adults. c. Transfer anesthetized adults to the morgue.d. Observe the inside of the vial to be sure no adults remain. If so, repeat steps

a through c.

B. Construction of a chromosome map in Drosophila: a three-point testcross.

Chromosome maps can be constructed from breeding results using testcrosses involving flies heterozygous at more than one locus, and measuring the frequency of crossing over between the loci. Three loci on the second chromosome, an autosome, have been chosen for this experiment that affect body color, wing size, and eye color. Each locus has a wild type and mutant allele whose traits are described in Table 2. You need to learn to identify each of these.

TABLE 2: Wild type and mutant traits for three loci three loci on the second chromosome (an autosome) in the fruit fly Drosophila melanogaster.

Characteristic Chromosome Mutant phenotype Wild Type Phenotype

Body Color 2Black (bl)Black color on body, tarsi and wing veins, darkens with age

Beige (Bl+)

Wing shape 2Vestigial (vg)Wing size is reduced, wings look shriveled

Long (Vg+)

Eye Color 2Brown (bw)Eye color is red-brown and darkens with age

Brick red (Bw+)

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The cross used for this experiment is set up by first constructing a female heterozygous in all three loci, and then mating her to a male who is homozygous recessive for all three loci. Offspring of this cross will receive three recessive genes from their father, which provide the appropriate genetic background for observing the genetic contribution from their heterozygous mother. The frequency of each phenotype will show how often crossovers occurred between the mother's two copies of chromosome 2. Calculations from the frequencies of observed phenotypes can tell us the map distances (crossover frequencies) between the three loci under study.

The original cross for this experiment was performed by the lab assistants two weeks before you received the flies. That cross was:

Bl+ Vg+ Bw+ bl vg bw Parentals: X

Bl+ Vg+ Bw+ bl vg bw

All of the female offspring from this cross should have the following genotype and show the wild type phenotype:

Bl+ Vg+ Bw+

bl vg bw With no crossing over, the gametes of these females would be either bl+ vg+ bw+ or bl vg bw. However crossovers are extremely frequent between the two copies of the second chromosome so the females can produce a total of eight different gametes with respect to the two alleles at each of these three loci. (See Table C at the end of this section.)

These female virgin flies will be given to you with the stock label of F1. They are to be used in the following cross that you will construct.

F1 females x black, vestigial, brown males:

Bl+ Vg+ Bw+ bl vg bw x

bl vg bw bl vg bw

Both male and female progeny from this cross, labeled F2, constitute the experimental results. Anesthetize the flies and then proceed to classify them according to their phenotypes with respect to these three loci. Record your results in a table similar to the one included in this lab manual.

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Data Treatment and Discussion

A. Data treatment for determining body color mode of inheritance.

Use a table such as that included in this manual to collect the results of the crosses you performed. Then start by examining the phenotypic ratios of offspring obtained to see if any patterns of inheritance are apparent. If any crosses seem to indicate that patterns exist, propose tentative hypotheses about the mode(s) of inheritance of body color. Use these to deduce genotypes for all of the parent flies and to calculate expected ratios of offspring. Then use the Chi-Square test, the description of which follows, to test whether the observed experimental results might represent the pattern you hypothesized in preparing your expected results. Remember that all the crosses used together must support your proposed mode of inheritance in order for you to accept it. Don't forget to look for evidence of both systematic and intrinsic error as you analyze the data.

The Chi-Square Test (X2)

When data fall into discrete categories, they can be tested for "goodness of fit" by a statistical test called the Chi-Square test. This test assumes that in many trials of an experiment, the number of samples in any category will vary by a normal distribution. That is, statistically the values for a category will fall within +1 s (where s = the standard deviation due to statistical variation only) of the mean or EXPECTED value about 66% of the time. The Chi-Square test measures how far from the expected value a specific experimental value is, and then expresses the probability that a value could fall there by chance and still represent the hypothesis. The farther from the expected value an experimental value is, the less likely it is to represent support of that hypothesis.

The Chi-Square test actually tests an hypothesis relative to statistical variation ONLY. If an experiment contains biological error or systematic errors, they must be considered in addition to the statistical error in order to completely analyze the data.

The test involves first determining a predicted ratio of results for the experiment. This prediction is derived from an hypothesis mode of inheritance that you deduce from the observed data. It is shown on your work sheet as the "Hypothesis: Expected Ratios". Then the observed data is examined to see how closely it fits this ratio. The formula used for Chi-Square is related to the formula for the standard deviation except that no square root is taken. The level of significance, or the probability level, that will be accepted as supporting the hypothesis must be selected. For most biological applications, a level of P>.05 (within +2s) is used as a standard for determining whether data fit a specific hypothesis. There is a 5% probability of accepting data that fit the hypothesis by chance, but that is an acceptable level of uncertainty in most situations.

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Each genetic cross is treated as a separate experiment although they collectively represent one hypothesis. At the end, you will need to ask whether collectively all six experiments support your hypothesis. For instance, if one of the six crosses has a low probability, you will have to decide whether this directs you to a new overall hypothesis or to greater error analysis.

X2 = (observed number –expected number) 2

expected number

where this sum is found for all the categories making up one experiment (one genetic cross). The number of categories is n and the degrees of freedom (df) are (n-1). A table is used to find, for any degree of freedom, what the statistical probability (P) is that this X2 represents support of the hypothesis. A sample calculation of X2 is shown below, and a work sheet and X2 probability table are provided for you to use.

Table 2: A Sample Calculation using X2 to calculate the probability that observed data matches a predicted ratio of offspring. Predicted Ratio = 9:3:3:1

Phenotype ObservedNumber

ExpectedRatio

Expected Number(ratio x total)

(O-E) (O-E)2 (O-E)2/E

Wild Type 2834 9/16 2808 26 676 0.24

Vestigial 920 3/16 936 16 256 0.27

Ebony 951 3/16 936 15 225 0.24

Vestigial Ebony

287 1/16 312 25 625 2.00

Total offspring = 4992 Total =2.75 = X2

df = n-1= 3; P > 0.3. Since P>0.05, a match between expected and observed data is accepted.

If a single category in an experiment contains a very small number of samples--fewer than five-- the X2 test does not work properly. For that experiment ONLY, the values under "Observed - Expected" should be decreased by 0.5. This result will better approximate the X2 test under these conditions.

There is also an exceptional rule for analyzing unexpected data: If a single category has an expected value of zero but an observed value of l or more, the category (or categories, if there is more than one within a single cross) should be combined and analyzed with the smallest observed category for which data is expected. (Degrees of freedom are calculated from number of categories expected.) However, the unexpected data must be discussed separately. (i.e. its source or cause must be

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explored, and you must consider whether this data is due to error or to a wrong hypothesis.)

Table 3: Chi-Square Probability Table. Chi-Square values are shown for specific probabilities for up to three degrees of freedom (df). Find the Chi-Square value closest to the one you calculated in the correct row. Express the probability of a match between your observed and expected data as being less than, greater than, or equal to the probability shown.

df < 1s P = 0.5

1s P= 0.3

2s P=0.05

3s P=0.01

1 0.455 1.074 3.841 6.635 2 1.386 2.408 5.991 9.210 3 2.366 3.665 7.816 11.345

B. Calculation of map distances from three-point testcross data.

Crossover values are based on a statistical sample of probable events. The greater number of flies you have counted, the more likely that the data will exhibit a statistically reliable sample of the crossover frequencies we are studying. For this reason, it is usually a good idea to combine the flies counted by all lab sections in order to have a large sample for this calculation.

Start by placing the phenotype classes that represent gametes formed by the same cross-over event together, then calculate the percentage, or frequency, with which each pair occurred. These frequencies will by themselves yield crossover distances (map distances) in two point crosses, but a three point cross requires further analysis and calculation.

The pair of phenotype classes with the highest frequency should represent the non-cross-over (parental) classes. You can check this because you know the genotype of the mothers of these flies. The pair of classes with the smallest frequency should represent the double crossover classes since we expect two crossovers to occur with lower frequency than a single crossover. The two remaining pairs should be the single crossover classes.

To determine the gene order, i.e. which gene is in the middle, observe which gene changes places when comparing the parental combinations to the double recombinant classes. The gene that changes places is the middle gene.

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Table 4: Sample data arranged by phenotype pairs with the frequency of each pair calculated below. Parental, double crossover, and single crossover classes are indicated.

Phenotype +++ abc a++ +bc ab+ ++c a+c +b+Observed Totals 436 741 107 122 313 236 390 382Combined Totals 1177 229 549 772Percentage(frequency) 43% 8% 20% 28%Deduced class

Parental Types

Double Crossover

Single Crossover

Single Crossover

FIGURE 2: The recombination results of double crossovers. Two possible results of double crossovers are shown. The top sketch shows the results of double crossing-over if the locus order on the chromosome is abc. The lower sketch correctly illustrates the results obtained in the sample data of Table 4 above.

Table 4 shows some sample data sorted in the manner described above, and Figure 2 shows two possible rearrangements of the chromosomes that might result from a double crossover. The second sketch shows the correct interpretation of the data shown in Table 4.

The double crossover classes immediately indicate the sequence of the loci on the chromosome. Use your data and the sketches in Figure 2 to figure out the order of the loci among the bl, vg and bw loci you are studying.

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Once the order of the genes is determined, the relative distances apart can be calculated. Cross-overs between b and a (in Table 4) are found by combining the frequency of the single cross-over between b and a (the last two columns of Table 4) with the double cross-over frequency (since every double crossover involves both of the single cross-overs). Thus crossovers occurred between the b locus and the a locus in our example a total of 28 % + 8 % = 36% of the time. The two loci are said to be 36 map units apart. Similarly crossovers between the a and c locus occurred 20 % + 8 % = 28 % of the time. We can now label a sketch of the chromosome as shown in Figure 3 below.

Figure 3: A chromosome map showing the location of the three loci whose relative map distances were calculated from data shown in Table 4.

Now, using the methods explained for the abc example, determine the sequence of genes and their map distances apart for the bl vg bw loci on the second chromosome of Drosophila using the class data.

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Drosophila Genetics Lab Report

Title: The title of the laboratory report should reflect both the purpose(s) of the experiment and the name of the experimental organism.

Introduction: The introduction should include the hypothesis you have formulated for the inheritance of body color in Drosophila. You should provide a brief rationale for your hypothesis.

ResultsInheritance of body color

1. Report your data and calculations in tabular format. You may use the examples provided in these instructions. Be sure to place a meaningful caption above each table.

2. For each cross, determine the expected genetic ratio and apply a chi-square test to determine the goodness of fit.

Three Point Testcross1. Report your data in tabular format. 2. Find the correct gene order for bl, vg, bw and calculate the distances

between these genes.

DiscussionInheritance of body color

There are just two alleles involved in the inheritance of body color in the system you are studying. Write a discussion for the data you collected to show first how you deduced the mode of inheritance of body color in flies and then how you tested your deduction against the observed data to determine if your hypothesis was supported. Be certain to consider the following questions while writing your discussion.

a. Is there evidence for dominance or recessiveness of these alleles? If so, describe the evidence and identify the crosses that demonstrate this relationship. Show how you used the data to decide which allele is dominant and which is recessive. Remember that only phenotypes, which can be observed directly, may be considered as evidence.

b. Is there evidence for sex linkage? If so, describe the evidence and the method of deduction.

c. Discuss the results of the chi-square analysis in determining whether the mode of inheritance you proposed is supported by the data.

d. Consider the sources of error in this experiment. Discuss the direction, size and probable cause(s) of the error. Be sure to suggest only sources of error that could explain discrepancies in the experimental results, especially for crosses where the chi-square p value is less than 0.05.

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DiscussionInheritance of body color (continued)

e. Evaluate the entire collection of data relative to the predicted genetic ratios and the error you observed. Will you accept your deduced mode of inheritance for body color in fruit flies? Be sure to explain your reasoning. You must either accept the results of all six crosses or sufficiently account for probable sources of error for any crosses with low probability values in order to validate the proposed genetic mechanism for body color inheritance.

DiscussionThree Point Testcross

a. Describe how the data was used to determine genetic map distances. b. Consider the sources of error in this experiment. Discuss the direction,

size and probable cause(s) of the error. How have the sources of error affected the calculated values for map distances?

ConclusionInheritance of body color

State your conclusions concerning the mode of inheritance of beige and yellow body color in Drosophila melanogaster.

Three Point Testcross

State your conclusions regarding the map distances between loci for body color, wing size and eye color on the second chromosome of Drosophila melanogaster.

Sample Tables for Drosophila Lab Results

Table A: Results of the Body Color Experiment

Cross Phenotypes of Parents

Beige female

Observed Beigemale

DataYellow female

Yellow male

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Table B: Determination of Parental Genotypes

Parent Gender Body Color Proposed GenotypeA Female BeigeB Female BeigeC Male BeigeD Female YellowE Male Yellow

Table C: Expected Fractions of Offspring for Body Color Experiment

Cross Genotypes of Parents

Beige female

Expected Beigemale

FractionsYellow female

Yellowmale

Table D: Chi-Square Analysis

Phenotype Observed Number

ExpectedRatio

Expected Number(fraction x total)

(O-E) (O-E)2 (O-E)2/E

Total = Chi-square _______

Number of degrees of freedom=Probability of a match=

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Table E: Results of the Three Point Testcross

Phenotype Mother’s contribution to Genotype

Number of Offspring

beige, long, red Bl+ Vg+ Bw+

black, vestigial, brown bl vg bw

beige, vestigial, brown Bl+ vg bw

black, long, red bl Vg+ Bw+

beige, long, brown Bl+ Vg+ bw

black, vestigial, red bl vg Bw+

beige, vestigial, red Bl+ vg Bw+

black, long, brown bl Vg+ bw