Gregor Mendel studied the genetics of inheritance by crossing individuals with variations in specific traits and observing the occurrence of these traits in the offspring. His conclusions led to his first law: the Law of Segregation. This law states that a trait is determined by two factors that separate during meiosis and unite during fertilization. These factors remain as independent units and do not blend in the offspring. You will be studying the inheritance of certain characteristics by performing your own crosses between some specific variations in traits in the common fruit fly, Drosophila melanogaster.
The Life Cycle of Drosophila melanogaster
Drosophila melanogaster, the common fruit fly, normally feeds on sugars and yeast produced by rotting fruit. The fruit fly has been extensively used in genetic experiments primarily because it is grown easily in the lab, reaches maturity in 10-14 days, and one female can produce hundreds of offspring. Also, many different mutations are known and have been studied.
Fruit flies have a fairly complicated life cycle that is completed within 10-14 days. They spend the first day or two of their lives as eggs. These hatch into larvae (instars) that eat and grow for 6 to 8 days. Next the pupa emerges and lives on the walls of the culture vessel for 3-4 days before hatching into the winged, free-flying adult form. These adults mate 6-8 hours after hatching. See Figure 1 for a summary of this life cycle.
Note: This lab will occasionally require you to come in on your own time to work with the flies.
Figure 1. Life Cycle of Drosophila melanogaster.
Where in the culture bottle will you find the following life stages of the fruit fly?
eggs? ______larvae? ______
pupae? ______adults? ______
From your observations, draw each of these stages in Drosophila melanogaster development. (Do not simply copy the above figures.)
Egg Larva Pupa Adult
Genetics of Drosophila melanogaster
Drosophila melanogaster is a diploid organism, having two alleles for each gene located on homologous chromosomes. It has four pairs (2n = 8) of chromosomes, three pairs of autosomes and one pair of sex chromosomes. As in humans, the female is XX and the male is XY. The cultures provided for you are pure breeding, that is, they are homozygous, the two alleles being the same for the characteristic to be studied. Wild type Drosophila melanogaster may be designated as ++ or by capital letters for the traits to be examined. Examples of wild type traits are: red eye color, indicated by “R”; long (normal) wing length, indicated by “L”; and gray body color, indicated by “G”.
Examining flies:
1. The use of ether to anesthetize flies allows them to sleep for 50 minutes without being
killed. Follow directions in Figure 2 for the anesthetizing directions.
2. Mark a white card with a line drawn in the middle. Label the two sides “male” and
“female”. Place the sleeping flies in the center.
3. Closely examine ++ type flies and especially note the following characteristics:
eye color ______
wing length ______
body color ______
4. Using a soft brush separate the males from the females by the following
characteristics. Have your instructor check that the flies have been sexed correctly.
See Figure 1 for males and females.
Size: males: usually smaller than females.
Body shape: males: the tip of the abdomen is black and rounded
females: the tip of the abdomen is pointed and has light and
dark bands.
Forelegs: males: have dark spots called sex combs
females: have no sex combs
5. In preparation for your first cross, place three males into a new culture vial and three
females into another new culture vial. See Figure 3. Label the vial to indicate the sex
strain of flies, the date, and your name and lab session. Remember to leave the vial on
its side until the flies recuperate. Otherwise, they will get stuck in the sticky medium.
Figure 2. Use of ether on Drosophila melanogaster
Figure 3. Transferring anesthetized flies to a vial.
Fill in Table 1.
Table 1. External characteristics observed in male and female fruit flies.Characteristic observed / Male / Female
Overall size
Color of abdomen
Shape of abdomen tip
Are sex combs present?
Crosses
Monohybrid crosses involve following the inheritance pattern of one genetic trait or characteristic through several generations. These crosses will be set up between three wild type female flies and three males from one of the mutant types. Reciprocal crosses involving the same traits will also be made between wild type males and mutant females. We will be working with several mutant types: white-eye = r, vestigial (short) wings = 1, and ebony (black body) = g.
The wild type characteristics are dominant and designated by a capital letter. The phenotypes (observed characteristics) and genotypes (alleles present) for the wild type flies have been filled in on Table 3. Eye color alleles are indicated as superscripts on chromosome X (for example, Xr) because this is a sex-linked trait. The mutant alleles are recessive and designated by a small letter.
Your instructor will assign the crosses for your lab group to perform.Possible crosses:
Monohybrid, autosomal crosses
Wild males x vestigial wing female
Wild females x vestigial wing male (reciprocal cross)
Wild male x ebony/black body female
Wild female x ebony/black body male (reciprocal cross)
Monohybrid, sex-linked
Wild female x white-eye male
Wild male x white-eye female (reciprocal cross)
Procedures
1. Collect and anesthetize mutant flies of the two appropriate types as indicated by the instructor.
2. Note the distinguishing characteristics of mutant flies. How do you distinguish the mutants from the wild type?
3. Separate according to sex.
4. Place three females or three males of the appropriate mutant type in the vial with three wild-type flies of the opposite sex. Remember to keep the vial on its side until the flies have recuperated.
5. Label the vials.
Complete Table 2 filling in the information about the phenotypes and genotypes of the
wild and mutant flies.
Table 2. Phenotypes and genotypes of wild and mutant flies.Trait / Wild type / White Eye / Vestigial Wing / Ebony Body
Eyes:
Phenotype / Red eyes
Genotype / XRXR, XRY
Wing length:
Phenotype / long wings
Genotype / LL
Body:
Phenotype / Gray (normal) body
Genotype / GG
Do a punnett square for the crosses performed and fill in expected ratio of phenotypes expected in the offspring of the F1, first filial generation.
Punnet square of monohybrid, autosomal cross.
P1
Female Male
Genotype / XPhenotype
Predicted F1 generation:
Female Male
Genotype / andPhenotype
------Punnet square of monohybrid, sex-linked cross.
P1
Female Male
Genotype / xPhenotype
Predicted F1 generation:
Female Male
Genotype / andPhenotype
Week 2
After seven days, remove the adult flies from the culture and place in the morgue. This prevents incestuous breeding between the parent generation and their offspring. (Some flies just have no morals at all!) Return the culture vial to its place for 5-7 days more of incubation.
Week 3
Flies that hatch are your F1, first filial generation. Anesthetize the flies as previously, and place them in the center of the card and separate them according to sex.
Monohybrid, autosomal cross:
Are both sexes alike? ______
Do the offspring look like the wild type parent or the mutant parent? ______
Find the group in the lab that performed the reciprocal cross.
Do their results agree with yours? ______
Place three females or three males of the appropriate mutant type in the vial with three wild-type flies of the opposite sex. Remember to keep the vial on its side until the flies have recuperated. Label the vials. The remaining flies should be placed in the morgue.
Monohybrid, sex-linked cross:
Are both sexes alike? ______
What is the phenotype of the F1 female? ______
What is the phenotype of the F1 male? ______
Find the group in the lab that performed the reciprocal cross.
Do their results agree with yours? ______
If they are not the same, how do they differ? ______
Week 4
After seven days, remove the adult flies from the culture and place in the morgue.
Return the culture vial to its place for 5–7 days more of incubation.
Flies that hatch are your F2, second filial generation.
Monohybrid, autosomal cross:
Anesthetize the flies as previously, and place them in the center of the card and separate them according to phenotype. Count the number of flies of each phenotype and enter in Table 3.
Are both sexes alike? ______
Do the offspring look like the wild type parent or the mutant parent? ______
Find the group in the lab that performed the reciprocal cross.
Do their results agree with yours? ______
Punnet square of F1 monohybrid, autosomal cross.
Actual F1 (Is it the same as you predicted?)
Female Male
Genotype / xPhenotype
Predicted F2 generation:
Female Male
Genotype / andPhenotype
------Table 3. F2 generation results of monohybrid, autosomal cross in Drosophila.
F1 Phenotypes / Number of Flies(Sex-linked only) / (Sex-linked only) / Total
Total
Give the actual phenotype ratio that you have observed ______
Compare the actual phenotype ratio with the expected ratio from your punnett square. Are they the same? ______
Exactly the same? ______Why might the actual results differ from what you predicted? ______
Monohybrid, sex-linked cross:
Anesthetize the flies as previously, and place them in the center of the card and separate them according to sex and phenotype. Count the number of flies of each category and enter in Table 4.
Are both sexes alike? ______
What is the phenotype of the F2 female? ______
What is the phenotype of the F2 male? ______
Find the group in the lab that performed the reciprocal cross.
Do their results agree with yours? ______
If they are not the same, how do they differ? ______
Punnet square of F1 monohybrid, sex linked cross
Actual F1 (Is it the same as you predicted?)
Female Male
Genotype / xPhenotype
Predicted F2 generation:
Female Male
Genotype / andPhenotype
Table 4. F2 generation results of monohybrid, sex-linked cross in Drosophila.
(Sex-linked only) / (Sex-linked only) / Total
Total
Give the actual phenotype ratio that you have observed ______
Compare the actual phenotype ratio with the expected ratio from your punnett square. Are they the same? ______
Exactly the same? ______Why might the actual results differ from what you predicted? ______
Extra Credit:
As demonstrated by your instructor, perform a Chi-squared analysis to evaluate your results.
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Spring 2006