Dihybrid Cross: Sepia x Vestigial
A dihybrid cross is a cross in which individuals that differ in two traits are crossed. The two traits in this cross arethe wing mutant vestigial and an eye color mutation sepia. In vestigial flies, the wings are small and irregular in shape. In sepia flies, the eyes are a dark brownish color. You will begin with the F1 progeny of a mating between homozygous sepia flies with homozygous vestigial flies. The sepia and vestigial genes are on different chromosomes: sepia on chromosome 3 and vestigial on chromosome 2. Further information on these genes can be seen at FlyBase.
The experiment has three parts:
Compare wild, sepia and vestigial flies:
Work in groups of 2-3 students. On your bench you will have three vials of flies: vestigial, sepia and wild type (Oregon R). Using the procedures described in your handout for the Monohybrid Cross, anaesthetize the flies for examination. Begin with the wild type flies and then examine each of the mutants. After the flies have been transferred to the methylene chloride chamber, and the last fly has stopped moving, leave the flies in the chamber for an additional 2 minutes. Tap the anaesthetized flies out onto an indiex card and divide the flies among the members of your group. Examine the eyes, body color, wing morphology and eye color of each type of fly and describe them for your notebook. After you have examined the flies, return them to the vial.
Prepare culture tubes for the F2 generation:
You will be given a vial of F1 flies from a cross between vestigial and sepia flies. You will prepare a new food vial and transfer the F1 flies. Your instructor will demonstrate the food preparation procedure. After you have prepared the new food vials, let them rest for about 10 minutes before you transfer the F1 flies.
Examine and transfer the F1 flies:
Examine the F1 flies in the vial. What do their wings and eye color look like? Which eye color and wing morphology are the dominant traits? What is the genotype of the F1 flies? The symbols for the ebony and vestigial alleles are e and v. To transfer the F1 flies, take the vial, knock it against the bench bad to bring the flies briefly to the bottom of the vial, and then invert the food vial over the vial with the flies. Invert the pair of vials, and gently knock the flies into the food vial. Quickly put the plug in the vial. Your instructor will demonstrate the transfer procedure. Label the culture, including the date and your initials. Place the new cultures into the basket on the front bench.
Collect and count the F2 progeny.
After one week, you will check the cultures to be sure that larvae are plentiful, and remove the parent generation. Then the flies will be returned to incubation for an additional week. What stages of the life cycle can you see in the culture vials after a week?
After two weeks, you will collect the hatched F2 flies and examine them. To count the flies, transfer them in small batches to a dry empty vial, and then anaesthetize them. Never anaesthetize flies in their food vials. Count and record the number of flies with normal wings, vestigial wings, normal eye color and sepia eye color. How many different conbinations of these traits might you see? What is the predicted fraction of the progeny of each type?
You may need to return to the laboratory after the lab period to continue to count your flies. The necessary materials will be left in the lab for you. When you have counted as many flies as you can remove from the culture, perform a Chi Square analysis to see if your progeny counts conform to the predicted F2 ratio. See the Monohybrid Cross handout for a reminder on how to perform the Chi Square analysis.
Laboratory Material for each group of students (2-3 students per group):
Laboratory Materials on the front prep bench: