Assignments for majors

To begin an experiment, you must first design the phenotypes for the flies that will be mated. In addition to wild-type flies, 29 different mutations of the common fruit fly, Drosophila melanogaster, are included in FlyLab. The 29 mutations are actual known mutations in Drosophila. These mutations create phenotypic changes in bristle shape, body color, antennae shape, eye color, eye shape, wing size, wing shape, wing vein structure, and wing angle. For the purposes of the simulation, genetic inheritance in FlyLab follows Mendelian principles of complete dominance. Examples of incomplete dominance are not demonstrated with this simulation. A table of the mutant phenotypes available in FlyLab can be viewed by clicking on the Genetic Abbreviations tab which appears at the top of the FlyLab homepage. When you select a particular phenotype, you are not provided with any information about the dominance or recessiveness of each mutation. FlyLab will select a fly that is homozygous for the particular mutation that you choose, unless a mutation is lethal in the homozygous condition in which case the fly chosen will be heterozygous. Two of your challenges will be to determine the zygosity of each fly in your cross and to determine the effects of each allele by analyzing the offspring from your crosses.

One advantage of FlyLab is that you will have the opportunity to study inheritance in large numbers of offspring. FlyLab will also introduce random experimental deviation to the data as would occur in an actual experiment! As a result, the statistical analysis that you will apply to your data when performing chi-square analysis will provide you with a very accurate and realistic analysis of your data to confirm or refute your hypotheses.

For your ease in completing each assignment, the background text relevant to the experiment that you will perform is italicized, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.

Assignment 1:
Getting to Know FlyLab: Performing Monohybrid, Dihybrid, and Trihybrid Crosses

  1. To begin a cross, you must first select the phenotypes of the flies that you want to mate. Follow the directions below to create a monohybrid cross between a wild-type female fly and a male fly with sepia eyes.

    1. To design a wild-type female fly, click on the Design button below the gray image of the female fly. Click on the button for the Eye Color trait on the left side of the Design view. The small button next to the words "Wild Type" should already be selected (bolded). To choose this phenotype, click the Select button below the image of the fly at the bottom of the design screen. Remember that this fly represents a true-breeding parent that is homozygous for wild type alleles. The selected female fly now appears on the screen with a "+" symbol indicating the wild-type phenotype.

    2. To design a male fly with sepia eyes, click on the Design button below the gray image of the male fly. Click on the button for the Eye Color trait on the left side of the Design view. Click on the small button next to the word "Sepia." Note how eye color in this fly compares with the wild-type eye color. Choose this fly by clicking on the Select button below the image of the fly at the bottom of the Design screen. The male fly now appears on the screen with the abbreviation "SE" indicating the sepia eye mutation. This fly is homozygous for the sepia eye allele. These two flies represent the parental generation (P generation) for your cross.

    3. Based on what you know about the principles of Mendelian genetics, predict the phenotypic ratio that you would expect to see for the F1 offspring of this cross and describe the phenotype of each fly.

    4. To select the number of offspring to create by this mating, click on the popup menu on the left side of the screen and select 10,000 flies. To mate the two flies, click on the Mate button between the two flies. Note the fly images that appear in the box at the bottom of the screen. Scroll up to see the parent flies and down to see the wild type offspring. These offspring are the F1 generation. Are the phenotypes of the F1 offspring what you would have predicted for this cross? Why or why not? Note: The actual number of F1 offspring created by FlyLab does not exactly equal the 10,000 offspring that you selected. This difference represents the experimental error introduced by FlyLab.

    5. To save the results of this cross to your lab notes, click on the Analyze Results button on the lower left side of the screen. The view will change to a summary of the results for this cross. Note the number of offspring, proportion of each phenotype and observed ratios for each observed phenotype. Click the Add Data to Notebook button at the top right side of the window. The Notebook will appear in a new window. To comment on these results in your notebook, click to move the cursor to the space above the dashed line and type a comment such as, "These are the results of the F1 generation for my first monohybrid cross." Close the Notebook window and click the Return to Lab button in the upper left side of the FlyLab window to return to the Mate screen.

    6. To set up a cross between two F1 offspring to produce an F2 generation, be sure that you are looking at the two wild-type offspring flies in the box at the bottom of the screen. If not, scroll to the bottom of this box until the word "Offspring" appears in the center of the box. Click the Select button below the female wild-type fly image, then click the Select button below the male wild-type fly image. Note that the two F1 offspring that you just selected appear at the top of the screen as the flies chosen for your new mating. Click on the Mate button between the two flies. The F2 generation of flies now appears in the box at the bottom of the screen. Use the scroll buttons to view the phenotypes of the F2 offspring.

    7. Examine the phenotypes of the offspring produced and save the results to your lab notes by clicking on the Results Summary button on the lower left side of the Mate view. Note observed phenotypic ratios of the F2 offspring. Click the Add to Lab Notes button at the bottom of the panel. Click the OK button to close the panel.

    8. To validate or reject a hypothesis, perform a chi-square analysis as follows. Click on the Chi-Square Test button on the lower left side of the screen. To ignore the effects of sex on this cross, click on the Ignore Sex button. Enter a predicted ratio for a hypothesis that you want to test. For example, if you want to test a 4:1 ratio, enter a 4 in the first box under the Hypothesis column and enter a 1 in the second box. To evaluate the effects of sex on this cross, simply type a 4 in each of the first two boxes, and type a 1 in each of the last two boxes. Click the Test Hypothesis button at the bottom of the panel. A new panel will appear with the results of the chi-square analysis. Note the level of significance displayed with a recommendation to either reject or not reject your hypothesis. What was the recommendation from the chi-square test? Was your ratio accepted or rejected? Click the Add to Lab Notes button to add the results of this test to your lab notes. Click OK to close this panel.

    9. To examine and edit your lab notes, click on the Lab Notes button in the lower left corner of the screen. Click the cursor below the recommendation line and type the following: "These are my results for the F2 generation of my first monohybrid cross. These data do not seem to follow a 4:1 ratio." To print your lab notes, you can export this data table as an html file by clicking on the Export button. In a few seconds, a new browser window should appear with a copy of your lab notes. You can now save this file to disk and/or print a copy of your lab notes. Click the Close button at the bottom of the panel to close the panel.

    10. Repeat the chi-square analysis with a new ratio until you discover a ratio that will not be rejected. What did you discover to be the correct phenotypic ratio for this experiment? Was this what you expected? Why or why not? What do the results of this experiment tell you about the dominance or recessiveness of the sepia allele for eye color?

  2. Click on the New Mate button in the lower left corner of the screen to clear your previous cross. Following the procedure described above, perform monohybrid crosses for at least three other characters. For each cross, develop a hypothesis to predict the results of the phenotypes in the F1 and F2 generations and perform chi-square analysis to compare your observed ratios with your predicted ratios. For each individual cross, try varying the number of offspring produced.

    What effect, if any, does this have on the results produced and your ability to perform chi-square analysis on these data? If any of your crosses do not follow an expected pattern of inheritance, provide possible reasons to account for your results.

  3. Once you are comfortable with using FlyLab to perform a monohybrid cross, design a dihybrid cross by selecting and crossing an ebony body female fly with a male fly that has the vestigial mutation for wing size.

    Develop a hypothesis to predict the results of this cross and describe each phenotype that you would expect to see in both the F1 and F2 generations of this cross.

    Analyze the results of each cross by Chi-square analysis and save your data to your lab notes as previously described in the assignments for a monohybrid cross.

    Describe the phenotypes that you observed in both the F1 and F2 generations of this cross. How does the observed phenotypic ratio for the F2 generation compare with your predicted phenotypic ratio? Explain your answer.

  4. Use FlyLab to perform a trihybrid cross by designing and crossing a wild-type female fly and a male fly with dumpy wing shape, ebony body color, and shaven bristles.

    Develop a hypothesis to predict the results of this cross and describe each phenotype that you would expect to see in the F2 generation of this cross. Perform your cross and evaluate your hypothesis by Chi-square analysis. What was the trihybrid phenotypic ratio produced for the F2 generation?

Assignment 2:
Testcross

A testcross is a valuable way to use a genetic cross to determine the genotype of an organism that shows a dominant phenotype but unknown genotype. For instance, using Mendel's peas, a pea plant with purple flowers as the dominant phenotype could have either a homozygous or a heterozygous genotype. With a testcross, the organism with an unknown genotype for a dominant phenotype is crossed with an organism that is homozygous recessive for the same trait. In the animal- and plant-breeding industries, testcrosses are one way in which the unknown genotype of an organism with a dominant trait can be determined. Perform the following experiment to help you understand how a testcross can be used to determine the genotype of an organism.

  1. Design a female fly with brown eye (BW) color (keep all other traits as wild-type), and design a male fly with ebony body color (E; keep all other traits as wild-type).  Mate the two flies.  Examine the F1 offspring from this cross and save your data to your lab notes.  Add to your data any comments that you would like.

    To determine the genotype of an F1 wild-type female fly, design a male fly with brown eye color and ebony body color, then cross this fly with an F1 wild-type female fly.  Examine the results of this cross and save the results to your lab notes. 

    What was the phenotypic ratio for the offspring resulting from this testcross?  Based on this phenotypic ratio, determine whether the F1 wild-type female male was double homozygous or double heterozygous for the eye color and body color alleles.  Explain your answer.  If your answer was double homozygous, describe an expected phenotypic ratio for the offspring produced from a testcross with a double heterozygous fly.  If your answer was double heterozygous, describe an expected phenotypic ratio for the offspring produced from a testcross with a homozygous fly.

Assignment 3:
Lethal Mutations

Five of the mutations in FlyLab are lethal when homozygous. When you select a lethal mutation from the Design view, the fly is made heterozygous for the mutant allele. If you select two lethal mutations that are on the same chromosome (same linkage group, or the "cis" arrangement), then the mutant alleles will be placed on different homologous chromosomes (the "trans" arrangement). Crosses involving lethal mutations will not show a deficit in the number of offspring. FlyLab removes the lethal genotypes from among the offspring and "rescales" the probabilities among the surviving genotypes. Hence, the total number of offspring will be the same as for crosses involving only nonlethal mutations. Perform the following crosses to demonstrate how Mendelian ratios can be modified by lethal mutations.

  1. Design a cross between two flies with aristapedia mutations for antennae shape. Mate these flies.

    What phenotypic ratio did you observe in the F1 generation? What were the phenotypes? Perform an F1 cross between two flies with the aristapedia phenotype. What phenotypic ratio did you observe in the F2 generation? How do these ratios and phenotypes explain that the aristapedia mutation functions as a lethal mutation?

    To convince yourself that the aristapedia allele is lethal in a homozygote compared with a heterozygote, perform a cross between a wild-type fly and a fly with the aristapedia mutation.

    What results did you obtain with this cross?

  2. Design a cross between two flies with curly wing shape and stubble bristles.

    Develop a hypothesis to predict the phenotypic ratio for the F1 generation. Mate these flies. What phenotypic ratio did you observe in the F1 generation?

    Test your hypothesis by Chi-square analysis. Repeat this procedure for an F1 cross between two flies that express the curly wing and stubble bristle phenotypes.

    Are the phenotypic ratios that you observed in the F2 generation consistent with what you would expect for a lethal mutation? Why or why not? Explain your answers.

Assignment 4:
Epistasis

The genetic phenomenon called epistasis occurs when the expression of one gene depends on or modifies the expression of another gene. In some cases of epistasis, one gene may completely mask or alter the expression of another gene. Perform the following crosses to study examples of epistasis in Drosophila.

  1. Design and perform a cross between a female fly with vestigial wing size and a male fly with an incomplete wing vein mutation. Carefully study the phenotype of this male fly to be sure that you understand the effect of the incomplete allele.

    What did you observe in the F1 generation? Note: It may be helpful to click up and down in this display box to closely compare the phenotypes of the F1 and P generations.

    Was this what you expected? Why or why not? Once you have produced an F1 generation, mate F1 flies to generate an F2 generation.

    Study the results of your F2 generation, then answer the following questions.

    Which mutation is epistatic? Is the vestigial mutation dominant or recessive? Determine the phenotypic ratio that appeared in the dihybrid F2 generation, and use chi-square analysis to accept or reject this ratio.

  2. Perform another experiment by mating a female fly with the apterous wing size mutation with a male fly with the radius incomplete vein structure mutation. Follow this cross to the F2 generation.

    Which mutation is epistatic? Is the apterous wing mutation dominant or recessive?

Assignment 5:
Sex Linkage

For many of the mutations that can be studied using FlyLab, it does not matter which parent carries a mutated allele because these mutations are located on autosomes. Reciprocal crosses produce identical results. When alleles are located on sex chromosomes, however, differences in the sex of the fly carrying a particular allele produce very different results in the phenotypic ratios of the offspring. Sex determination in Drosophila follows an X-Y chromosomal system that is similar to sex determination in humans. Female flies are XX and males are XY. Design and perform the following crosses to examine the inheritance of sex linked alleles in Drosophila.

  1. Cross a female fly with a tan body with a wild-type male.

    What phenotypes and ratios did you observe in the F1 generation?

    Mate two F1 flies and observe the results of the F2 generation.

    Based on what you know about Mendelian genetics, did the F2 generation demonstrate the phenotypic ratio that you expected? If not, what phenotypic ratio was obtained with this cross?

  2. Perform a second experiment by crossing a female fly with the vestigial wing size mutation and a white-eyed male. Describe the phenotypes obtained in the F2 generation. Examine the phenotypes and sexes of each fly.

    Is there a sex and phenotype combination that is absent or underrepresented? If so, which one? What does this result tell you about the sex chromosome location of the white eye allele?

Assignment 6:
Recombination

Mendel's law of independent assortment applies to unlinked alleles, but linked genes--genes on the same chromosome -- do not assort independently. Yet linked genes are not always inherited together because of crossing over. Crossing over, or homologous recombination, occurs during prophase of meiosis I when segments of DNA are exchanged between homologous chromosomes. Homologous recombination can produce new and different combinations of alleles in offspring. Offspring with different combinations of phenotypes compared with their parents are called recombinants. The frequency of appearance of recombinants in offspring is known as recombination frequency. Recombination frequency represents the frequency of a crossing--over event between the loci for linked alleles. If two alleles for two different traits are located at different positions on the same chromosome (heterozygous loci) and these alleles are far apart on the chromosome, then the probability of a chance exchange, or recombination, of DNA between the two loci is high. Conversely, loci that are closely spaced typically demonstrate a low probability of recombination. Recombination frequencies can be used to develop gene maps, where the relative positions of loci along a chromosome can be established by studying the number of recombinant offspring. For example, if a dihybrid cross for two linked genes yields 15% recombinant offspring, this means that 15% of the offspring were produced by crossing over between the loci for these two genes. A genetic map is displayed as the linear arrangement of genes on a chromosome. Loci are arranged on a map according to map units called centimorgans. One centimorgan is equal to a 1% recombination frequency. In this case, the two loci are separated by approximately 15 centimorgans. In Drosophila, unlike most organisms, it is important to realize that crossing over occurs during gamete formation in female flies only. Because crossing over does not occur in male flies, recombination frequencies will differ when comparing female flies with male flies. Perform the following experiments to help you understand how recombination frequencies can be used to develop genetic maps. In the future, you will have the opportunity to study genetic mapping of chromosomes in more detail using PedigreeLab.

  1. To understand how recombination frequencies can be used to determine an approximate map distance between closely linked genes, cross a female fly with the eyeless mutation for eye shape with a male fly with shaven bristles. Both of these genes are located on chromosome IV in Drosophila. Testcross one of the F1 females to a male with both the eyeless and shaven bristle traits. The testcross progeny with both mutations or neither mutation (wild-type) are produced by crossing over in the double heterozygous F1 female. The percentage of these recombinant phenotypes is an estimate of the map distance between these two genes.

    Draw a map that shows the map distance (in map units or centimorgans) between the locus for the shaven bristle allele and the locus for the eyeless allele.

  2. To understand how recombination frequencies can be used to determine a genetic map for three alleles, mate a female fly with a black body, purple eyes, and vestigial wing size to a wild-type male. These three alleles are located on chromosome II in Drosophila. Testcross one of the F1 females to a male with all three mutations. The flies with the least frequent phenotypes should show the same phenotypes; these complementary flies represent double crossovers.

    What is the phenotype of these flies? What does this tell you about the position of the purple eye allele compared with the black body and vestigial wing alleles? Sketch a genetic map indicating the relative loci for each of these three alleles, and indicate the approximate map distance between each locus.

Assignment 7:
Group Assignment

Work in pairs to complete the following assignment. Each pair of students should randomly design at least two separate dihybrid crosses of flies with mutations for two different characters (ideally choose mutations that you have not looked at in previous assignments) and perform matings of these flies. Before designing your flies, refer to the Genetic Abbreviations chart in FlyLab for a description of each mutated phenotype. Or view the different mutations available by selecting a fly, clicking on each of the different phenotypes, and viewing each mutated phenotype until you select one that you would like to follow. Once you have mated these flies, follow offspring to the F2 generation.

  1. For each dihybrid cross, answer the following questions. Perform additional experiments if necessary to answer these questions.

    1. Which of these traits are dominant and which traits are recessive?
    2. Are any of these mutations lethal in a homozygous fly? Which ones?
    3. Are any of the alleles that you followed sex-linked? How do you know this?
    4. Which alleles appear to be inherited on autosomes?
    5. If any of the genes were linked, what is the map distance between these genes?

  2. For at least one of your crosses, attempt to perform the cross on paper using a Punnett square to confirm the results obtained by FlyLab.

  3. Ask another pair of students to carry out one of the crosses that you designed. Did they get the same results that you did in the F1 and F2 generations? Did they develop the same hypotheses to explain the results of this mating as you did? Explain your answer.

  4. Once you have completed this exercise, discuss your results with your instructor to determine if your observations and predictions were accurate.