Carolina Labsheets™

In this lab students are introduced to the testcross and its use in revealing whether an organism is homozygous or heterozygous for a trait. However, the term “testcross” is not used, in order that students will be more likely to think through the problem rather than copy a textbook solution. The lab assumes that students have experience anesthetizing flies, sexing flies, and setting up crosses. If this is not the case, plan on spending extra class time covering these procedures, or use our “Introductory Genetics with Drosophila” LabSheet as a lead-in to this lab.

Student Lab Sheet

Needed Materials*

Drosophila cultures:

172100 wild type
172575 sepia or 172320 apterous
172010 F₁ sepia × wild or 172000 F₁ apterous × wild

empty culture vials, plugs, and caps (173076)

vial labels or wax pencils

Formula 4-24® Instant Drosophila Medium (173200)

FlyNap® Anesthetic Kit (173010)

alcohol

Drosophila Sorting Brushes (173094)

index cards to use as sorting trays

Carolina™ Drosophila Manual (452620)

dissecting microscopes

Optional Materials
Our 173050 Drosophila Culture Kit includes 36 vials and plugs, labels, a FlyNap® Kit, Formula 4-24®, sorting brushes, sorting cards, and a Carolina™ Drosophila Manual.

Using Carolina Drosophila Stands (173030) makes anesthetizing flies simple and convenient.

Safety

Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Use personal protective equipment such as safety glasses or goggles, gloves, and aprons when appropriate. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.

Procedures

Students can work individually or in groups of 2 to 4.

When your cultures arrive, open the package immediately and inspect the cultures to verify that they are in good condition. Examine the label on each vial. The label gives the name of the culture and, for mutants, the genetic symbol and chromosome on which the gene is located, for example, “sepia se (3).” The label is stamped with the date that the parent flies were placed in the vial. For F₁ crosses the label is divided into 4 parts. The upper left-hand quadrant gives information on the female flies used for the cross. The upper right gives information on the male flies. Flies should begin emerging 12–14 days after the date stamped on the label. Each culture will produce approximately 100 flies over a 10-day period, and will serve 6 to 8 groups of students.

Observing Parent Flies and Offspring
Students observe the parent flies and their offspring (F₁). Then, they diagram the cross made to produce the F₁ . If students have previously worked with these crosses, this activity can be abbreviated and students can progress to setting up their testcross vials.

Prepare the morgues (wide-mouth bottles) in the FlyNap Kits by half-filling each with alcohol or a mixture of water and detergent.

Students will need the following materials for this activity:

vials of parent flies
vial of F₁ flies
FlyNap Kit (for every 6–8 groups of students)
sorting brush
3 index cards
dissecting microscope

Making a Cross to Reveal a Genotype
Important: This activity should be done within 12 days of receipt of the cultures; otherwise, F₂ flies may begin emerging in the F₁ culture vials, which will invalidate the testcross.

In this activity, students set up a cross to identify the genotype of their unknown. Students will need virgin female flies for their crosses. Clear all flies from the vials 8–12 hours before the activity. This ensures that virgin females will be available for the crosses. (Female flies mate around 15 hours after emerging from pupa and can store sperm.) To make the unknowns, remove the labels from wild type and F₁ vials and replace them with labels marked “Unknown 1” and “Unknown 2.” Students will need an empty vial with medium for their cross; alternatively, provide materials and instructions so they can prepare their own vial.

Students will need the following materials for this activity:

vials of parent flies
vial of unknown flies
FlyNap Kit (for every 6–8 groups of students)
sorting brush
2 index cards
dissecting microscope

Observing the Results of Your Cross
In this activity, students observe the results of their testcross and draw conclusions about the genotype of their unknown flies. It requires about 2 weeks from the time that the cross is set up until testcross flies begin to emerge in the vials. It can take 2 to 3 days longer for mutant flies to emerge than those with the wild-type phenotype, so delay this part of the lab until a few days after the first flies emerge.

Students will need the following materials for this activity:

vials of testcross flies
FlyNap Kit (for every 6–8 groups of students)
sorting brush
index card
dissecting microscope

Optional
Students can make their own F₁ crosses using the parental strains, eliminating the need to order the F₁ cultures. See our “Introductory Genetics with Drosophila” LabSheet for instructions on setting up F₁ crosses. Athough written for a monohybrid testcross, the activity can be done for a dihybrid testcross, using 172100 wild type and 172753 apterous(2); sepia(3) for the parent flies and 172030 F₁ apterous × sepia. (Since the double mutant 172753 is a weak stock, if you are doing your own F₁ cross, use 172320 apterous and cross it with 172575 sepia.) The testcross would involve crossing the 172030 with the 172753 double recessive. The expected phenotype ratio of the testcross flies is 1:1:1:1. Another use of the testcross is to study gene linkage and crossing-over. See our “Three-Point Linkage with Drosophila” LabSheet for details.

Answer Key to Questions Asked on the Student LabSheet

Observing Parent Flies and Offspring

Phenotype of wild type:       red eyes (or normal wings)
Phenotype of mutant:       brown eyes (or no wings)

The F₁ flies are the offspring of a cross between these two parental types. Examine the F₁ flies and record their phenotype here.

Phenotype of F₁:      red eyes (or normal wings)

Which phenotype is due to the action of a dominant allele?
red eyes (or normal wings)

Using the appropriate allele symbols, diagram the cross that produced the F₁ flies.

What is the genotype of the F₁ flies?
Heterozygous; Se/se or Ap/ap

Making a Cross to Reveal a Genotype
You will be given vials of parent flies and a vial of flies of unknown genotype. Using these materials and your knowledge of genetics, what is the best cross you can make to reveal the genotype of the unknown flies? Diagram below the expected results for both heterozygous and homozygous genotypes of the unknown.

Students may default to diagramming a standard cross of the F₁ flies to produce an F₂ . Although this will work, ask them to diagram other crosses that they could make. They should discover that back-crossing to the recessive parent will give more offspring showing the recessive phenotype in the case of the heterozygote, and thus give results that are more reliable.

If the unknown is heterozygous:

Half the offspring show the dominant phenotype and half show the recessive.

If the unknown is homozygous:

All offspring show the dominant phenotype.

Observing the Results of Your Cross
Anesthetize your flies and examine them under magnification. Do the results indicate that your unknown flies are homozygous or heterozygous? How do you know?
If any of the offspring show a recessive phenotype, the genotype of the unknown is heterozygous. If none show a recessive phenotype, the genotype of the unknown is homozygous.

A plant breeder is trying to establish an ornamental corn that produces red seeds; however, when she selfpollinates the plants, the resulting ears have some white seeds. She knows that the allele for white seed is recessive and so some of the red seeds must be heterozygous. What can she do the next planting season to identify the heterozygous plants and keep only seed that will be homozygous red?
She should plant a row of white corn next to the row of red. Each red corn plant is self-pollinated and also crossed onto a white corn plant. If an ear on a white corn plant develops any white seed, the red plant that donated the pollen must be heterozygous, and its seed not saved.

Student Lab Sheet