Name ______Class ____

AP Biology

Lab 7 Simulation and Lab Report

GENETICS OF ORGANISMS

Directions:

You will be running an online simulation of AP Biology Lab 7: Genetics of Organisms using Drosophila melanogaster (fruit flies). The benefit of this online simulation is that you will be able to perform genetic crosses in a relatively short period of time that would take weeks in a live lab setting. You will then compare your observed experimental results with your predicted expected values to determine if your data has statistical significance using the Chi-Square test for statistical analysis. You will begin this assignment by working through a Chi-Square tutorial lesson. ALL work submissions must be handwritten. Typed reports will not be accepted.

Requirements:

  • Read and complete the Chi-Square tutorial;
  • Read the introduction to lab;
  • Make a prediction of your expected genetic results using Punnett squares;
  • Run the simulation and collect data;
  • Calculate and analyze your data using the Chi-Square test for statistical analysis; and
  • Complete the “Topics for Discussion.”

To be submitted for test grade (all components below are required):

  1. Chi-Square tutorial and practice question
  2. Results & Analysis Section of Lab 7 with conclusion statement
  3. Topics for Discussion

WARNING:

This is an independent assignment. Each student will submit his/her own original work on Wednesday, March 19. This simulation will give unique data to each user; therefore, no two students can receive identical data. Based on the data collected during the lab, each student will also have unique statistical calculations and subsequent analysis answers. Any students submitting identical data or plagiarizing from any other source will receive automatic zeros for this test grade.

Chi-Square Tutorial

Using the Chi-Square Test for Statistical Analysis of Experimental Data

Example 1

Statistics can be used to determine if differences among groups are significant, or simply the result of predictable error. The statistical test most frequently use to determine whether data obtained experimentally provide a good fit or approximation to the expected or theoretical data is the chi-square test. This test can be used to determine if deviations from the expected values are to chance alone, or to some other circumstance. For example, consider corn seedlings resulting from an F1cross between parents that are heterozygous for color.

A Punnett square of the F1 cross Gg X Gg would predict that the expected proportion of green:albino seedlings would be 3:1. Use this information to fill in the Expected (e) column and the (o-e) column in the table below. If there are 84 total seedlings, using the 3:1 ratio, how many would you expect to be green? Albino?

Phenotype / Genotype / # Observed (o) / # Expected (e) / (o-e)
Green / GG or Gg / 72
Albino / gg / 12
Total: / 84

There is a small difference between the observed and expected results, but are these data close enough that the difference can be explained by random chance or was there a confounding factor that caused variation in the sample?

To determine if the observed data fall within acceptable limits, a chi-square analysis needs to be performed to test the validity of the null hypothesis (that there is no statistically significant difference between the observed and expected data). If the chi-square analysis indicates that the data vary too much from the expected 3:1, an alternative hypothesis is accepted.

The formula for chi-square is:

Χ2 = Σ (o-e)2

e

Whereo = observed number of individuals

e = expected number of individuals

= the sum of values (in this case, the differences, squared, divided by the number

expected)

  1. This statistical test will examine the null hypothesis, which predicts that the data from the experimental cross above will be expected to fit the 3:1 ratio.
  2. Use the data from the table above to complete the chi-square table below.

Phenotype / # Observed (o) / # expected (e) / (o-e) / (o-e)2 /
Green / 72
Albino / 12
  1. Your calculations should give you a value for x2 = 5.14. This value is then compared to the Critical Values Table below.

Critical Values of the Chi-Square Distribution

Probability (p) / Degrees of Freedom (df)
1 / 2 / 3 / 4 / 5
0.05 / 3.84 / 5.99 / 7.82 / 9.49 / 11.1
0.01 / 6.64 / 9.21 / 11.3 / 13.2 / 15.1
0.001 / 10.8 / 13.8 / 16.3 / 18.5 / 20.5

How to Use the Critical Values Table

  1. Determine the degrees of freedom (df) for your experiment. It is the number of phenotypic outcomes minus 1. Since there are two possible phenotypes, for this experiment df=1 (2 samples – 1). If the experiment had gathered data for a dihybrid cross, there would be four possible phenotypes and therefore 3 degrees of freedom.
  1. Find the probability (p) value. Under the 1df column, find the critical value in the probability (p) = 0.05 row: it is 3.84. What does this mean? If the calculated chi-square value is greater than or equal to the critical value from the table, then the null hypothesis is rejected. Since for our example X2 = 5.14 and 5.14 > 3.84, we reject our null hypothesis that there is no statistically significant difference between the observed and expected data. In other words, chance alone cannot explain the deviations we observed and there is, therefore, reason to doubt our original hypothesis (or to question our data collection accuracy). The minimum probability for rejecting a null hypothesis in the sciences is generally 0.05, so this is the row you will use in the chi-square analysis for your lab simulation data.
  1. These results are said to be significant art a probability of p = 0.05. This means that only 5% of the time would you expect to see similar data if the null hypothesis was correct; thus, you are 95% sure that the data do not fit a 3:1 ratio.
  2. Since these data do not fit the expected 3:1 ratio, you must consider reasons for this variation. Additional experimentation would be necessary. Perhaps the sample size is too small, or errors were made in data collection. In this example, perhaps the albino seedlings are underrepresented because they died before the counting was performed.

Example 2

In a study of incomplete dominance in tobacco seedlings, the counts in the table below were made from a cross between two heterozygous (Gg) plants:

Phenotype / Genotype / # Observed (o)
Green / GG / 22
Yellow-Green / Gg / 50
Albino / Gg / 12
Total: / 84

Perform a Punnett square to determine the expected outcome ratio of offspring:

If your solution is correct, your Punnett square for this cross indicates that the expected counts should be in a 1 green:2 yellow-green:1 albino ratio. The expected values for a total count of 84 organisms are therefore:

1 green = / ¼ x 84 = / 21
2 yellow-green = / ½ x 84 = / 42
1 albino = / ¼ x 84 = / 21

Total: / 84

Now, plug your numbers into a chi-square table:

Phenotype / # Observed (o) / # expected (e) / (o-e) / (o-e)2 /
Green / 22 / 21
Yellow-Green / 50 / 42
Albino / 12 / 21

If you worked the table above correctly, you should have calculated X2 = 5.43. If not, go back and try to find you mistake.

Go to the chi-square table, this time for two degrees of freedom (there are three phenotypes: 3-1=2 df). If the X2 value were greater than or equal to the critical value of 5.99, we would reject our hypothesis. Since 5.43 is less than the critical value at p = 0.05, we accept the null hypothesis (this second data set does fit the expected 1:2:1 ratio.)

Chi-Square Practice Problem

Complete the following chi-square analysis problem and submit it with your assignment. These will be assessed as part of your overall test grade.

An investigator observes that when pure-breeding, long-winged Drosophilaare mated with pure-breeding, short-wing flies, the F1 offspring have an intermediate wing length.

When several intermediate-wing-length flies are allowed to interbreed, the following results are obtained:

Observed

230 long wings

510 intermediate-length wings

260 short wings

  1. What is the genotype of the F1 intermediate-wing-length flies? ______
  2. Perform a Punnet Square to determine your expected outcome for the F2 generation.

Write a hypothesis describing the mode of inheritance of the wing length in Drosophila (this is your null hypothesis).

______

  1. Complete the table below:

Phenotype / # Observed (o) / # expected (e) / (o-e) / (o-e)2 /
  1. Calculate the chi-square value for these data.
  2. How many degrees of freedom (df) are there? ______
  1. X2 = ______
  1. Referring to the critical values chart, what is the probability value for these data? ______
  1. According to the critical value of X2, can you accept or reject the null hypothesis? Explain why.

______

Lab 7: Genetics of Organisms Simulation

Overview

In this lab, you will use simulated living organisms to do genetic crosses. You will learn how to collect data from F1 and F2 generations and analyze the results from a monohybrid, dihybrid, or sex-linked cross. The procedures that follow apply to fruit flies.

Objectives

Before doing this lab, you should understand:

  • chi-square analysis of data, and
  • the life cycle of diploid organisms useful in genetics studies.

After doing this lab, you should be able to:

  • investigate the independent assortment of two genes and determine whether the two genes are autosomal or sex-linked using a multigeneration experiment, and
  • analyze the data from your genetic crosses using chi-square analysis techniques.

Introduction

Drosophila melanogaster, the fruit fly, is an excellent organism for genetics studies because it has simple food requirements, occupies little space, is hardy, completes its life cycle in about 12 days at room temperature, produces large numbers of offspring, can be immobilized readily for examination and sorting, and has many types of hereditary variations that can be observed with low-power magnification. Drosophila has a small number of chromosomes (four pairs). These chromosomes are easily located in the large salivary gland cells. Drosophila exists in stock cultures that can be readily obtained from several sources. Much research about the genetics of Drosophila during the last 50 years has resulted in a wealth of reference literature and a knowledge about hundreds of its genes.

The Life Cycle of Drosophila

The Eggs. The eggs are small, oval shaped, and have to filaments at one end. They are usually laid on the surface of the culture medium and, with practice, can be seen with the naked eye. The eggs hatch into larvae after about a day.

The Larval Stage. The wormlike larva eats almost continuously, and its black mouth parts can easily be seen moving back and forth even when the larva itself is less distinct. Larvae tunnel through the culture medium while eating; thus, channels are a good indication of the successful growth of a culture. The larva sheds its skin twice as it increases in size. In the last of the three larval stages, the cells of the salivary glands contain giant chromosomes, which may be seen readily under low-power magnification after proper staining.

The Pupal Stage. When a mature larva in a lab culture is about to become a pupa, it usually climbs up the side of the culture bottle or on to the strip provided in the culture bottle. The last larval covering then becomes harder and darker, forming the pupal case. Through this case the later stages of metamorphosis to an adult fly can be observed. In particular, the eyes, the wings, and the legs become readily visible.

The Adult Stage. When metamorphosis is complete, the adult flies emerge from the pupal case. They are fragile and light in color and their wings are not fully expanded. These flies darken in a few hours and take on the normal appearance of an adult fly. They live a month or more and then die. A female does not mate for about ten to twelve hours after emerging from the pupa. Once she has mated, she stores a considerable quantity of sperm in receptacles and fertilizes her eggs as she lays them. To ensure a controlled mating, it is necessary to use females that have not mated before (virgins).

It is important to realize that a number of factors determine the length of time of each stage in the life cycle. Of these factors, temperature is the most important. At room temperature (about 25°C), the complete cycle takes ten to twelve days.

Design of the Experiment

You will be assigned to study a certain mode of inheritance using particular genetic crosses of flies having one or two mutations. The modes of inheritance most commonly used are:

1. Monohybrid. In these experiments the mode of inheritance is determined when a single

contrasting pair of characteristics is involved.

2. Dihybrid. In these experiments the mode of inheritance is determined when two pairs of

contrasting characteristics are considered simultaneously.

3. Sex-linked. In these experiments the mode of inheritance is determined when the mutant

characteristic is associated with the X-chromosome.

To make these experiments interesting and challenging, you will not be told the mode of inheritance, nor the name for the particular mutation you are studying. Study the wild type flies (both male and female) until their phonotypic characteristics are familiar. Flies having one or two mutations can then be identified by making comparisons with the wild type flies. The most commonly studied mutations are in the eye color or shape, bristle number or shape, wing size or shape or antenna size or shape.

Procedure

1. Go to

2. Enter as guest. This will take you to the simulated lab you saw in class.

Read the instructions at the bottom of the page on how to operate the system.

3. Click on the computer to begin selecting your traits and ordering your flies. This will take you

to the menu screen. Click on "order files."

4. Select the female fly on the right sketch a picture of the female fly and make observations

about the female body structure. Record your observations in Data Table 1.

5. Repeat step 4 for the male fly body structure and record your data.

6. Select the female fly again. With the female selected, select "eye color" from the tabs on the

right of the page.

7. Click on the wild type (+) eye color. Then, click the green shopping cart beneath the fly to add

them to your cart.

8. Click on the male fly. Select "eye color," and choose the white-eyed trait. Add the males to

your cart.

9. Click on the green shopping cart button at the top right of the page to view your contents.

Make sure you have female wild type-eyed flies and male white-eyed flies.

10. Click checkout and choose "yes" for "Do you really want to order?" This will take you back

to the simulated laboratory.

11. These flies will serve as your Parental generation. Choose a letter to represent your tested

character and record your parental generation geneticcross in your results. Remember, if

you think this trait is sex-linked, you must include the sex chromosomes in your genotype

(example: XQY x XQXQ). If you do not think this is sex-linked trait, you do not need the sex

chromosomes in your genotype (example: QQ x qq).

12. Unpack you flies. The software will automatically create a mating jar for you. Click on the

mating jar to place it in the incubator.

13. Watch your flies reproduce. When the process is complete, click on the incubator to open

the door. Click on the mating jar to remove the offspring flies and prepare them for

viewing.

14. The software will automatically place your F1 flies under the microscope. Click on the red

"sort flies" button on the lower right of the page.

15. Place the cursor over each pile of flies. Record the gender, eye color, and number of

offspring in Data Table 2.

16. Click on the pile of female offspring to zoom in to one offspring and make observations.

Now, click the red "use in new mating" button on the lower right of the page. Click OK.

Zoom out.

17. Repeat step 16 for the pile of male offspring.

18. Return to Lab.

19. Click on the new mating jar to place it into the incubator. This will generate your new F2

generation.

21. Watch your flies reproduce. When the process is complete, click on the incubator to open

the door. Click on the mating jar for Cross #2 to remove the offspring flies and prepare them

from viewing.

22. The software will automatically place your F2 flies under the microscope. Click on the red

"sort flies" button on the lower right of the page.

23. Place the cursor over each pile of flies. Record the gender, eye color, and number of

offspring in Data Table 3.

Results

Data Table 1: Drosophila body structure by gender

Female / Male

Parental Generation Cross: ______x ______

Data Table 2: Observed F1 Generation Data

Phenotype and Symbol / Females / Males

Data Table 3: Observed F2 Generation Data

Phenotype and Symbol / Females / Males

Analysis of Results

1.Describe and name the observed mutation(s).

______

2. Write a hypothesis that describes the mode of inheritance of the trait(s) you studied. This is

your null hypothesis (as described in the Chi-Square tutorial).

______

3. Refer to your textbook and notes and review Punnet squares. In the space below, construct

twoPunnet squares to predict the expected results of both the parental and F1 crosses from

your null hypothesis.

Parental Cross:

F1 Cross:

4. Refer to the Punnet Squares above. In the box below, record the expected ratios for the

genotypes and phenotypes of the F1 and F2 crosses in the experiment.

Expected Genotypic Ratio / Expected Phenotypic Ratio
F1
F2

5. Do the actual (observed) results deviate from what you expected? If so, explain how.