Revised Fall 2018 Studying Mechanisms of Inheritance using Drosophila melanogaster Learning Objectives: 1. Describe the life cycle of Drospohilia. 2. Explain why the fruit fly is an ideal model organism for inheritance studies. 3. Be able to describe the features that differentiate male flies from female. 4. Describe sex-linked inheritance and use correct terminology and letters to represent the genotypes of female and male flies for sex-linked traits. 5. Perform crosses and use Punnett squares to determine genotype and/or phenotype percentages or ratios in offspring and to predict genotype and phenotype of offspring for sex-linked traits. This lab will continue over several weeks. Part 1: Setting up the F1 cross Part 2: Remove F1 generation flies Part 3: Collecting data for the F2 generation Introduction Gregor Mendel is known as the father of modern genetics. In the 1860 s, he published the results of his hybridization studies with Pisum sativum (garden pea) in which he proposed a mechanism for the inheritance of traits from parent to offspring. In the early 1900 s, following the rediscovery of Mendel s work, T.H. Morgan began a series of studies of inheritance in Drosophila melanogaster, the common fruit fly. This organism has now been used for over 100 years to study the mechanisms of inheritance in animals. Why is Drosophila used as a model organism for genetic studies? When choosing a model organism for biological studies, there are a certain characteristics which make a model organism ideal: (1) Drosophila are small; you can house 1000 s of flies in a small area; (2) they have a short life cycle (on the order of days); (3) they are easy to maintain for multiple generations; (4) they produce many, many offspring in a short period of time; and (5) they have a number of different heritable characteristics that are easily distinguishable. The purpose of this experiment is to determine if the inheritance pattern of the alleles that determine the eye color trait are autosomal dominant, autosomal recessive, sexlinked dominant or sex-linked recessive. This can be determined by setting up a series of crosses between male and female flies which are red eyed (normal) or white eyed (mutant). Looking at the traits of the offspring will allow us to determine the pattern of inheritance of the eye color trait. We will need to look at the offspring from the crosses described in Figure 1. Figure 1: P, F1 and F2 crosses used to determine the inheritance pattern of eye color trait 1
Part 1: Setting up our F1 cross: Revised Fall 2018 The parental cross (white eyed female X red eye male) was completed several weeks ago. All male offspring were white eyed and all female offspring were red eyed. You will need to know this information to help you predict the inheritance pattern of eye color. Today you will set up the F1 cross. The data from this cross will help you to confirm your prediction. 1. Obtain a culture vial containing flies of this F 1 generation. 2. Anesthetize the flies. Because Drosophila can fly, they must be immobilized in order to set up your crosses and analyze progeny. If anesthetizing flies in their original culture vial, use the following procedure (your instructor may do this for you): a) Obtain an anesthetizing wand. Dip the absorbent end of the wand into the Fly Nap and remove excess liquid by running the wand across the rim of the bottle. b) Tap the vial of flies against a hard surface so that the flies are gently knocked to the bottom of the vial. c) Use one finger to push the vial plug slightly to the side and quickly stick the anesthetic end of the wand into the vial. (Do this quickly or the flies could escape!). d) Lay the culture vessel on its side while the flies are anesthetized. (DO NOT leave the vial upright while the flies are unconscious or you risk drowning them in the media!) e) Wait until the flies have stopped moving, then empty the flies out onto an index card or piece of white paper. 3. You need to be able to select both male and female flies in order to have success with your crosses. Being able to determine the sex of the offspring will also allow you to see if any of the traits you are observing are sexlinked. Physical characteristics that are useful for distinguishing males and females are as follows: Body size males tend to be slightly smaller than females Tip of abdomen dark in males; lighter, striped & more pointed in females Sex combs males have sex combs on the uppermost joint of their forelegs; females do not sex combs Using these markers separate the males from the females using the brush provided. Check with your instructor to see if you have done this correctly. Make sure you can easily identify the eye color as this is the trait you will be observing. It can be difficult to make these observations using the naked eye, to aid in viewing specimens in more detail we can use a dissecting microscope. You should be familiar with the parts and use of a dissecting microscope after completing your Pre-Lab activity. Why are you NOT using a compound light microscope for this? 4. Transfer 5 female flies and 5 male flies to a newly prepared culture vial (vial plus food). Leave the vial on its side until the flies wake up. Label your vial with your group name, date and lab section. 2
Revised Fall 2018 Part 2: Remove F1 generation flies Fruit Fly Life Cycle: The life cycle of Drosophila can take place in plastic culture vials that contain a source of food. The life cycle has four stages: egg, larva, pupa, and adult. You should be able to see all four stages present in the cultures you will use today. The eggs are small and white (usually found on the surface of the food). The larva hatch within 24 to 36 hours, and burrow into the food at the bottom of the vial; they look like little wriggling grains of rice. After about 5 days, they crawl out of the food onto the sides of the vial and transform into brown, oblong pupae. During the pupation stage, the larvae metamorphose into adult flies. After about 4 days, the adult flies emerge from the pupae and begin the life cycle anew. The entire cycle takes approximately 10 to 12 days to complete. Observe your culture vials for larvae and pupae. Anesthetize and remove the F 1 generation from the vials; discard them in the morgue. Figure 2: Fruit Fly life Cycle (Carolina Biologicals) Part 3: Collecting data about F2 generation Today you will look at the F2 generation for the eye color trait. Collecting this data will allow us to determine the pattern of inheritance of the eye color trait. Before you collect your data write out your hypothesis regarding the inheritance pattern of the alleles that determine the eye color trait (is the pattern autosomal dominant, autosomal recessive, sex-linked dominant or sex-linked recessive?). You should have used the parental cross and outcome (F1 data) along with the Punnett squares completed as part of the pre-lab activity 2 to help you determine your hypothesis. Hypothesis: You can use your prediction of pattern of inheritance to calculate the expected ratio of phenotypes in the F2 generation. To do this you will need to show a Punnett square of the F1 cross and calculate the expected percentages for each phenotype in the F2 generation. What are the genotypes of the F1 generation? Punnett square (be sure to correctly represent the female and male genotypes) Female F1 Male F1 3
Revised Fall 2018 This Punnett square gives the expected ratios of the phenotypes in the F2 generation What % of the F2 generation is expected to be red-eyed male? What % of the F2 generation is expected to be white-eyed male? What % of the F2 generation is expected to be red-eyed female? What % of the F2 generation is expected to be white-eyed female? What % of the F2 generation is expected to be male? female? To determine the actual % or ratios of the different phenotypes in the F2 generation we need to count the flies with each phenotype and complete the table below with class data. 1. Anesthetize the adult flies in the F 2 generation. 2. Identify the sex and eye color of each individual. Record your data (along with the data from the other six lab groups) in the table below. Discard the counted flies in the morgue after determining their phenotypes Table 1: Data for phenotypes of the F 2 generation from the F1 (red eyed female X white eye male) cross: Lab Group Males Females Red-eyed White-eyed Red-eyed White-eyed 1 2 3 4 5 6 7 Class Total % (total # with phenotype/total # of flies) 4
Revised Fall 2018 Analysis of the Results: What are the genotypes of the individuals who were in the P generation? What are the genotypes of the F 1 generation? Compare the ratios that you expected (from the Punnett square) to the actual numbers that were counted: What % of the F2 generation is expected to be red-eyed male? What % of the F2 generation did you count as red-eyed male? What % of the F2 generation is expected to be white-eyed male? What % of the F2 generation did you count as white-eyed male? What % of the F2 generation is expected to be red-eyed female? What % of the F2 generation did you count as red-eyed female? What % of the F2 generation is expected to be white-eyed female? What % of the F2 generation did you count as white-eyed female? What % of the F2 generation is expected to be male? female? What % of the F2 generation was male? female? How do the actual ratios compare to the expected ratios? If there is a difference, propose an explanation for this difference? Does the data you collected support your hypothesis and prediction for the inheritance pattern of the alleles that determine the eye color trait? 5
Pre-Lab Activity Part A: Prior to the first part of the fruit fly lab (setting up the F1 cross) you should: Revised Fall 2018 1. Read Chapter 14 Mendel and the Gene in your Biological Science textbook (Freeman, 6th edition pages 289-315) 2. Define the terms below: Sex chromosomes Autosomes Genes Alleles Homozygous Heterozygous Hemizygous Dominant Recessive Genotype Phenotype Wild-type Mutant P generation F 1 generation F 2 generation 2. The Dissecting Microscope When a biologist needs to look at a specimen too large or thick to be viewed by a compound binocular microscope and too small to be viewed in sufficient detail with the naked eye they will use a dissecting microscope or stereoscopic microscope. Review the following functions of the parts of the dissection microscope and label the diagram of a dissecting microscope. 1. Adjustment focus knob allows you to focus the objective on the specimen. 2. The image magnified by the objective lens in use is passed up through the body tube into the oculars. Each ocular contains two lenses for a total magnification of 10X. 3. Light control switches: There are two switches that control the light on this scope. One of them turns on either the top light or the bottom light or both. The other knob controls the intensity of the light. 4. Magnification adjustment knob: On this scope instead of individual objectives you can adjust the amount of magnification by turning this knob. 5. Two light sources: On this scope you have an in base light source and an upper light source. You may use either one or both depending on the type of specimen you are observing. 6
Pre-Lab Activity Part B: Revised Fall 2017 Prior to the last part of the fruit fly lab (data collection F2 generation) you should complete the activities below. Using the Parental cross and outcome information given to you in this handout you can predict whether the inheritance the inheritance pattern of the alleles that determine the eye color trait are autosomal dominant, autosomal recessive, sex-linked dominant or sex-linked recessive 1. What are the phenotypes of the individuals who were in the P generation? 2. Complete the Punnett square below for the P generation cross assuming that the inheritance pattern is autosomal dominant for red eye. Notes: Remember that the genotype is the combination of alleles. A single letter is used to represent an allele. The assigned letter usually comes from the dominant trait (R for red eye color). A capital letter is for the dominant trait R red eye and a lower case letter for the recessive trait r for white eye) What are the genotypes of the individuals who were in the P generation? Remember that the P generation is a pure bred line and will be homozygous for their trait (be sure to express the genotypes correctly using the information above). 3. Complete the Punnett square below for the P generation cross assuming that the inheritance pattern is autosomal recessive for red eye (dominant for the white eye phenotype). What are the genotypes of the individuals who were in the P generation? Remember that the P generation is a pure bred line and will be homozygous for their trait (be sure to express the genotypes correctly). 7
Revised Fall 2017 4. Complete the Punnett square below for the P generation cross assuming that the inheritance pattern is x- linked dominant for red eye. Notes: Remember that for sex-linked traits the allele is on the sex chromosome (X or Y). To show this the letter used to represent an allele is superscript to the sex chromosome it is on (X R or X r for alleles that are linked to the X chromosome). What are the genotypes of the individuals who were in the P generation? Remember that the P generation is a pure bred line and will be homozygous for their trait (be sure to express the genotypes correctly using the information above). 5. Complete the Punnett square below for the P generation cross assuming the inheritance pattern is x-linked recessive for red eye (dominant for the white eye phenotype). What are the genotypes of the individuals who were in the P generation? Remember that the P generation is a pure bred line and will be homozygous for their trait (be sure to express the genotypes correctly). 6. Now look at each Punnet square that you completed above. Determine the ratio of phenotypes that you would expect in all four of the Punnett squares (% red eyed, % white eyed etc). In only one cross will the ratio of the offspring phenotypes match the data from the parental cross provided to you (Data: offspring males all white eyed, offspring females all red eyed). Which one matches? This information allows you to predict what the pattern of inheritance might be for the red eye trait in the Fruit Fly. This data suggests that the inheritance pattern of the alleles that determine the eye color trait is ** The F1 cross data that you collect will help you to confirm whether this predicted pattern of inheritance is true. ** Post-Lab Activity: You may be required to submit an assignment based on this lab. You instructor will provide you with the details required for your submitted assignment. 8