Carolina Labsheets™

In introductory genetics students commonly study the influence of one gene on one phenotype. This is logical since students must understand the simplest cases before tackling the more complicated cases. However, students can easily jump to the conclusion that one gene = one phenotype. In reality, there are few, if any, instances in which this is true.

Eye color in Drosophila is a classic case. A common introductory activity is to have students cross wild-type flies with flies having sepia eyes and study the F₁ and F₂ of that cross. The students then conclude that sepia is recessive to the wild-type allele. This is true but there is no single gene for eye color in Drosophila. The brick-red eye color of wildtype flies is produced by a mixture of pigments, each pigment is the end product of a series of reactions, each reaction is mediated by an enzyme, and each enzyme is a protein that results from the transcription of a DNA gene. A large number of genes, perhaps 100, are involved in producing eye color in Drosophila. This activity introduces students to the concept of interacting genes through a dihybrid cross involving the brown locus (bw) on chromosome 2 and scarlet locus (st) on chromosome 3. Brown pigments are produced through the ommochrome pathway, and bright-red pigments are produced through the drosopterin pathway. These pigments bind to granules within the eyes to produce the characteristic red eye color of wild-type flies.

Homozygous brown is epistatic to all the genes involved in the drosopterin pathway and homozygous scarlet is epistatic to all the genes involved in the ommochrome pathway.

ommochrome pathway

drosopterin pathway

Homozygous brown blocks the binding of red pigments to eye granules, resulting in brown eyes.

ommochrome pathway

drosopterin pathway

Homozygous scarlet blocks the binding of brown pigments to eye granules, resulting in bright-red eyes

ommochrome pathway

drosopterin pathway

If both mutations are present in the homozygous condition, both pathways are blocked, and there is no pigment in the eyes.

ommochrome pathway

drosopterin pathway

Student Lab Sheet

Needed Materials*

171925 Drosophila Gene Interaction Set (2 cultures: brown and scarlet)

empty culture vials and plugs (173076)

vial labels or wax pencils

Formula 4-24® Instant Drosophila Medium (173200)

FlyNap® Kit (173010)

alcohol

Drosophila sorting brushes (173094)

index cards to use as sorting trays

Carolina™ Drosophila Manual (452620)

stereomicroscopes

Optional Materials

Carolina Drosophila Stand (173030)

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

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 2–4.

When your cultures arrive, open the package immediately and inspect the cultures to verify that they have arrived in good condition. The date that the culture was set up is stamped on the vial label. We ship cultures so the flies are beginning to emerge when you receive them. However, cold weather can slow emergence. Each culture will produce approximately 100 flies over a 10-day period and will serve 6–8 groups. Either order enough cultures for the class or plan to expand the parent cultures you receive by subculturing.

Observing and Crossing Parent Flies
Students need virgin females for setting up crosses to obtain their F₁. Clear (remove) all adult flies from the cultures 8–12 hours before students are to set up their crosses. This allows enough time for adults to emerge but not enough time for the females to mate. See the Carolina Drosophila Manual for more details.

The following materials are needed:

vial with medium and plug
vial label
FlyNap® Kit (one for every 6–8 groups of students)
sorting brush
2 index cards
stereomicroscope

Either prepare a vial with medium for each student group or provide materials and instructions to the students so they can prepare their own vials. Materials needed are culture vials with plugs and Formula 4-24® Instant Drosophila Medium. Open the bag of medium and locate the small measuring cup and packet of yeast. Add a level cup of medium to each vial. When all the vials have dry medium, add a level cup of cool water to each vial. Open the yeast packet and sprinkle 6–8 grains of yeast onto the surface of the medium in each vial. Do not add too much yeast. The yeast produces CO2, which in large amounts can cause sterility or even death of the flies. If you want students to check for sex linkage, either provide two vials with medium for each group or have groups do reciprocal crosses (e.g., group 1 does brown-eye females × scarlet-eye males; group 2 does brown-eye males × scarlet-eye females).

Either prepare the fly morgues with alcohol (one morgue is included in each FlyNap® Kit) or provide materials and instructions so the students can do this.

Seven to 10 days after the F₁ vials are prepared, remove the parent flies from the cultures. This is done to prevent mating of the F₁ flies with the parents.

The following materials are needed:

FlyNap® Kit (one for every 6–8 groups of students)
sorting brush
index card

Observing F₁ Flies and Setting Up F₂ Cultures

F₁ flies will begin emerging about 12–14 days after the cultures are set up. Since the F₁ flies can mate only among themselves, it is not necessary to collect virgins to set up cultures for the F₂.

The following materials are needed:

vial with medium and plug
vial label
FlyNap® Kit (one for every 6–8 groups of students)
sorting brush
index card
stereomicroscope

Seven to 10 days after the F₂ vials are set up, the F₁ flies need to be removed from the F₂ cultures.

Scoring F₂ Phenotypes

F₂ flies will begin emerging about 12–14 days after students have set up their vials. Begin scoring phenotypes of the F₂ on the day after they first begin emerging. More females than males emerge on the first day, but the ratio evens out on successive days. Scored flies should not be returned to the vial—dispose of them in a morgue to prevent counting them twice. Phenotypes can be scored every other day for up to 10 days. For scoring the phenotypes of the F₂ flies, students will need their F₂ vials and the following materials:

FlyNap® Kit (one for every 6–8 groups of students)
sorting brush
index card
stereomicroscope

Optional

As an option you can provide cultures of wild-type flies to compare with the parent flies of the cross. We recommend that you continue to maintain the brown and scarlet parents by subculturing, so that students can make comparisons with the F₁ and F₂ flies. It is easier to distinguish between wild-type red eyes and the bright-red eyes of scarlet flies if you have these stocks for comparison.

The flies with white eyes that show up in the F₂ may confuse students who have done crosses involving sex-linked white. Have students design a set of crosses that would distinguish the genetic makeup of the white-eyed F ₂ flies from that of sex-linked white. A somewhat related question is, “What will result from a backcross of the white-eyed F₂ flies with the brown- and scarlet-eyed parental stocks?”

The pteridine pigments of drosopterin can be separated by paper chromatography. For more information see our 171955 Chromatography of Drosophila Eye Pigments kit.

Answer Key to Questions Asked on the Student LabSheet

1. Using this information, give the genotypes and phenotypes of the parent flies of your cross.
   
   

   Parent Fly	Genotype	Phenotype
   Female	bw/bw, st+/st+	Brown eyes
   Male	bw+/bw+, st/st	Scarlet eyes

   
   or
   
   

   Parent Fly	Genotype	Phenotype
   Female	bw+/bw+, st/st	Scarlet eyes
   Male	bw/bw, st+/st+	Brown eyes

   
   
   
2. Give the expected genotype of the F₁ flies.
   bw+/ bw, st+/ st
3. What additional information would you need in order to predict the phenotype of the F₁ flies?
   Which alleles are dominant. (Some students may add that they would need to know if bw and st are linked and if either is sex linked.)
4. Why is it necessary to remove the parent flies?
   So that they will not mate with their offspring and so they will not be included in the data for the F₁.
   
   Anesthetize the F₁ flies and examine them under a stereomicroscope. Record your observations below.
   All the F₁ flies, both male and female, have red (wild-type) eyes.
5. What does this tell you about the alleles bw and st?
   Both are recessive to their wild-type alleles.
6. Why is it not necessary to select virgin female flies from the F₁ to make this cross?
   All the flies in the F₁ have the same genotype.
7. In the space below, diagram the F₁ cross. Assuming that brown and scarlet are not linked, create a Punnett square and complete it with the expected F₂ phenotypes and their ratios.
   bw/bw+, st+/st × bw+/bw, st+/ st
   
   F₂ Punnett Square
   
   

   Male \ Female	bw+, st+	bw+, st	bw, st+	bw, st
   bw+, st+	bw+/bw+, st+/st+	bw+/bw+, st/st+	bw/bw+, st+/st+	bw/bw+, st/st+
   bw+, st	bw+/bw+, st+/st	bw+/bw+, st/st	bw/bw+, st+/st	bw/bw+, st/st
   bw, st+	bw+/bw, st+/st+	bw+/bw, st/st+	bw/bw, st+/st+	bw/bw, st/st+
   bw, st	bw+/bw, st+/st	bw+/bw, st/st	bw/bw, st+/st	bw/bw, st/st

   
   
   F₂ Ratio        Phenotype
   9                    Red eyes          
   3                    Brown eyes
   3                    Scarlet eyes     
   1                    White eyes
   
   Note: Some students may be very confused at this point because this looks like (and is) a dihybrid cross, but only one characteristic—eye color—is influenced. Class discussion can bring out and challenge student misconceptions of how genes operate.
   
   Sample Data for brown × scarlet F₂
   
   F₂ Phenotypes and Numbers of Flies
   
   

   Date Counted	Phenotype 1	Phenotype 2	Phenotype 3	Phenotype 4
   Total	579	206	178	61
   Expected Ratio	9	3	3	1

8. Are your results compatible with your expected ratios? Explain.
   Answers will vary depending on collected data.
9. In humans, the ability to taste the chemical PTC is inherited. This trait is often presented as a case of inheriting a dominant allele for tasting or a pair of recessive alleles for nontasting of PTC. Based on your experience with eye color in Drosophila, would you be skeptical of this explanation? If so, why?
   I would be skeptical. Taste, like other senses, is a complex trait. It would require many genes to specify the development of the various kinds of taste buds and their sensitivities to different chemical stimuli.
10. Is this a monohybrid or dihybrid cross? Explain your answer.
    It is a dihybrid cross because two gene loci are involved.

Student Lab Sheet