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Two-Point Linkage with Drosophila

Carolina Labsheets™

In this lab students explore the genetic consequences of gene linkage using an F1 cross of Drosophila.

172050 Two-Point Linkage:The vial of F1 flies is from a cross between females having yellow bodies and white eyes (172290 yellow white) and wild-type males having gray bodies and red eyes (172100 wild type). Yellow (y) and white (w) are recessive and are located on chromosome 1. Chromosome pair 1 consists of the sex chromosomes. The loci for yellow (linkage position 0.0 ) and white (linkage position 1.5) are on a portion of the X chromosome that has no homolog on the Y; thus, yellow and white are inherited only through the X chromosome, making them sexlinked (or X-linked). Because two sex-linked genes are used, the results of linkage can be seen in the F1. Recombinations resulting from crossovers can be seen in the F2.

The activity assumes that students have previously worked with Drosophila and know how to sex flies and perform other common tasks. If this is not the case, refer students to the pertinent sections of the Carolina Drosophila Manual. Students should also be familiar with dihybrid and sex-linked crosses.

Since Drosophila crosses must be set up before shipment, please give 2-week notice for delivery of the cultures; otherwise, the crosses may not be available when you need them.

Needed Materials

two-point linkage Drosophila cultures

FlyNap® Kit (173010)

empty culture vials and plugs (173076)

vial labels or laboratory markers

Formula 4-24® Instant Drosophila Medium (173200)

alcohol (for fly morgue)

Drosophila sorting brushes (173094)

index cards to use as sorting trays

Carolina™ Drosophila Manual (452620)



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.


Have students work individually or in pairs. Each person or pair needs an empty culture vial with plug, sorting brush, index card, Carolina™ Drosophila Manual, and access to a stereomicroscope. Provide a 172050 culture and a FlyNap® kit for every 3–4 groups.

When your cultures arrive, open the package immediately and inspect the cultures to verify that they have arrived in good condition. Examine the label on each vial. The label is divided into four parts. The upper left-hand quadrant gives information on the female flies used for the cross, and the upper right gives information on the male flies. The lower left-hand quadrant is stamped with the date that the parent flies were placed in the vial. F1 flies should begin emerging 12 to 14 days after the date on the label. Each culture will produce approximately 100 flies over a 10-day period. Cultures should not be used beyond 12 days after receipt because offspring of the F1 flies may begin to emerge.

Either prepare the fly morgues with alcohol (one morgue is included in each FlyNap® Kit) and set up culture vials with Formula 4-24 Medium or provide materials and instructions so the students can prepare the morgues and vials themselves.

Optional: In the interest of time, it is possible to stop this lab after the prediction of the expected F2 results and not set up the F2 cultures. Calculation of linkage can be performed using the supplied sample data.


With green Hydra (132810), students will observe symbiotic green algae within the Hydra’s tissues. Place a Hydra in a drop of water on a slide. Add a coverslip to compress it and observe under low and high power. To release algae cells for better viewing, cut off a portion of a tentacle and place it in a drop of water on a slide. Add a coverslip and apply pressure with the eraser of a pencil to crush the tentacle. This will release the algal cells into the water.

Students can design their own experiments to test the response of Hydra to other stimuli. Possibilities include responses to pH, gravity, salt concentration, and light.

Answer Key to Questions asked on the Student LabSheet

Assume that the alleles for yellow and white are recessive to the wild-type alleles. Choose symbols for the alleles that you will use in your following work. Note: Students can use the standard allele symbols shown below or the class may come up with their own. Either way, ensure that all groups use the same symbols to facilitate communication.

    y       allele for yellow body

   y+      allele for gray body (wild type)

   w       allele for white eye

  w+      allele for red eye (wild type)

Assume that the loci for yellow and white are on separate chromosomes.

P1 Female, yellow body, white eyes     x     P2 Male, gray body, red eyes
                      y/y, w/w                           x                           y+/y+, +w/+w                      

F1 genotype y/y+, w/w+      F1 phenotype gray body, red eyes

Assume that the loci for yellow and white are linked on the same autosome and are inherited together.

P1 Female, yellow body white eyes     x     P2 Male, gray body red eyes
                        y w/y w                         x                          y+w +/y +w +                      

F1 genotype y w/y+w +     F1 phenotype gray body, red eyes

Place the card on the stage of a dissecting microscope and sort the flies by sex and phenotype. Record the results below.

Phenotype Males Females
Gray body, red eyes 0 12
Yellow body, white eyes 8 0

Do your results show linkage or non-linkage/ Explain?
The results show linkage. The combination of yellow body and white eyes was inherited together as was the combination of gray body and red eyes.

What additional assumption(s) must you make to explain the results you observed in the F1?
The traits of body color and eye color are sex-linked. Or: the alleles for body color and eye color are linked on the X chromosome and do not have homologs on the Y chromosome.

Using your new assumptions, diagram and explain this cross:

P1 Female, yellow body white eyes     x     P2 Male, gray body red eyes
                        y w/y w                         x                          y+ w+/Y0                      

  y+w + Y0
y w y w/y+w + y w/ Y0

F1 Phenotypes: 1 gray-bodied red-eyed female to 1 yellow-bodied white-eyed male

What would be the expected F2 of this cross? Show a Punnett square or branching diagram of the cross and give the phenotypes and their ratios of the F2. Note: Essentially this is a testcross.

y w/y+y +     x     y w/ Y0

  y w Y0
y w y w/ y w y w/ Y0
y+ w+ y+ w +/ y w y+ w+/ Y0

F2 Phenotypes: 1 yellow-bodied white-eyed female to 1 gray-bodied red-eyed female to 1 yellow-bodied whiteeyed male to 1 gray-bodied red-eyed male. Note: If the sex of the flies is disregarded, this is a 1:1 ratio of yellowbodied white-eyed flies to gray-bodied red-eyed flies.

If there is crossing-over between the gene loci for body and eye color, what new genotype and phenotype combinations will be produced in the F2? Notice that crossovers will occur only in the female.

Crossing-over in the female flies will produce the new gamete combinations of y+ w and y w+.

  y w Y0
y+ w y+ w/ y w y+ w/ Y0
y w+ y w +/ y w y w+/ Y0

F2 Recombination Phenotypes: gray-bodied white-eyed female, yellow-bodied red-eyed female, gray-bodied white-eyed male, yellow-bodied red-eyed male

The following is sample data for the F2. Your class data will differ but should show the preponderance of noncrossovers but a few crossovers. The tight linkage of 1.5 means that a large number of flies must be scored to obtain even a few examples of crossover.

Non-recombinants Count   Recombinants Count
yellow bodied white-eyed female


  gray-bodied white-eyed female 2
gray bodied red-eyed female 149   yellow-bodied red-eyed female 1
yellow bodied white-eyed male 131   gray-bodied white-eyed male 0
gray bodied red-eyed male 140   yellow-bodied red-eyed male 3

Do your results show that crossing-over has occurred? If so, use your data to calculate the linkage distance as the percentage of recombinants.

Map distance = total recombinants/total flies counted x 100 = 6/554 x 100 = 1.1