X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 1

The Study of X Linked Traits in the First and Second Generation of White Eyed Male and Wild

Type Female Cross in Drosophila

Christopher Ellis, Brad Brown, Tim S., Jenna McClure

March 4 2011 – May 6 2011

Honors Biology 10 Period 1

X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 2

Abstract
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 3

Introduction

Genetics is a topic that has been studied for hundreds of years. One of the most notable

geneticists was Gregor Mendel. Mendel studied basic inheritance patterns and gene expression

using pea plants. Mendel determined that the offspring of two parents contains one gene from

each parent (McClean 2000). However, since Mendel’s discoveries in genetics, other scientists

have found exceptions to Mendel’s rules, and have termed these exceptions Non-Mendelian

Genetics. One type of Non-Mendelian genetic inheritance pattern is X-Linked traits.

Drosophila, or fruit flies, were chosen for the study of X-Linked traits for multiple

reasons. Fruit flies are small insects approximately 3mm long, and thus use minimal space when

compared to other species. The diet of fruit flies is simple and does not require extraneous foods

or materials (Manning 2008). Additionally, the life cycle of fruit flies is quite short, lasting

approximately 26 days for a female and approximately 33 days for a male (Arnini 1996). Life

cycles of flies may be altered based on environment or certain genetic mutations. From egg to

adult takes approximately 10 days at room temperature (25°C) (Arnini 1996). The short life span

of fruit flies makes them an ideal subject for the study of genetics, as multiple generations can be

studied in a short period of time.

The life cycle of a fruit fly starts when an impregnated female fruit fly lays an egg. Fruit

flies lay only one egg at a time. The egg hatches in 22 hours, and the larvae eats and grows for

four days. The larvae goes through three larva stages before entering the pupa stage. In the pupa

stage, the pupal case forms, darkens and hardens for 4-6 days. Finally, the pupa ecloses into the

adult stage. Males fruit flies have a smaller body with a rounded black tip at the end of their

body. Females, on the other hand, have a pointed abdomen and are often lighter than males
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 4

(Arnani 1996). The purpose of the experiment was to determine the type of inheritance of the

white eye genetic mutation within fruit flies.

Hypothesis

If white eyed male fruit flies were crossed with wild type female fruit flies and the white

eyed mutation is an X Linked Recessive trait, then the phenotypic ratio of the first generation

will be 4:0, wild type to white eyed, with half of them being male and half of the being female,

and the phenotypic ratio of the second generation will be 3:1, wild type to white eyed, with half

of them being wild type female, one fourth of them being wild type male, and one fourth of them

being white eyed male.

Null Hypothesis

If white eyed male fruit flies were crossed with wild type female fruit flies and the white

eyed mutation is an X Linked Recessive trait, then the phenotypic ratio of the first generation

will not be 4:0, wild type to white eyed, with half of them being male and half of the being

female, and the phenotypic ratio of the second generation will not be 3:1, wild type to white

eyed, with half of them being wild type female, one fourth of them being wild type male, and

one fourth of them being white eyed male.

Rationale

Previous research has shown that the white eye mutation is an X Linked recessive trait,

meaning that it is carried on the X chromosome, which determines sex, and is recessive to wild

type. By performing a Punnett Square for the first generation, the expected genotypic and

phenotypic ratios can be found. The Punnett Square for the first generation can be found in

figure one.
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XWt XWt = Wild Type Male

XW Y = White Eyed Female

Figure 1: Punnett Square for first generation White Eyed Male and Wild Type Female

Cross

Parent Generation: XWt XWt x XW Y

Wild Type Wild Type

XWt XWt

White Eye
XW XWt XW XWt XW

White Eye
Y XWt Y XWt Y

This Punnett Square demonstrates that the expected phenotypic ratio for the first

generation would be all wild type, half male, half female. Using this information a second

Punnett Square for the second generation can be generated. Because all of the females will have

the same genotype, and all of the males will have the same genotype, only one Punnett Square

needs to be created because there is no variance within the flies. The Punnett Square for the

second generation can be found in figure two.

Figure 2: Punnett Square for second generation White Eyed Male and Wild Type Female

Cross

Parent Generation: XWt XW x XWt Y

Wild Type Wild Type

XWt XW

Wild Type
XWt XWt XWt XW XWt

Wild Type
Y XWt Y XW Y
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 6

Figure two shows that the expected outcome for the second generation was half

wild type male, one fourth wild type female, and one fourth white eyed male.

Materials

Wild type fruit fly stock tubes White eyed fruit fly stock tubes

Media Yeast

Water Foam Toppers

Cryaliser Alka Seltzer

Tape Tube Labels

Plastic tubes for f1 and f2 generations

Procedure

1. Stock tubes for both wild type and white eyed flies were allowed to reproduce to provide

an ample amount of flies for experimentation.

2. The tube for F1 generation was made by putting one scoop of yeast, one scoop of water,

and three grains of yeast into the tube.

3. The tube was labeled using a piece of paper to indicate the type of cross contained within

the tube.

4. The stock tube for white eyed flies was anesthetized using a chryalizer. To do this, a

plastic tube was half filled with water. Half of an alka seltzer tablet was then dropped into

the tube, and a cork with tubing was placed over the tube. The tubing was then placed

into the white eyed tube. The CO2 from the alka seltzer and the water successfully put the

flies to sleep. This method of anesthesia was used because it is inexpensive, but also

successful at putting the flies to sleep but not killing them.

 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 7

5. After the flies were asleep, they were poured into a petri dish and five males were found.

These five male white eyed flies were placed into the f1 stock tube to become the males

in the parental generation of the f1 tube.

6. Next, the stock for the wild type fruit flies was emptied, leaving only the larvae left.

7. Approximately 6 hours after the tube was emptied, the newly hatched flies were

anesthetized using the same method as described in step 4. 3 of these females were

retrieved and placed into the f1 stock tube to become the white eyed females for the f1

generation. The flies were retrieved within 6 hours to ensure their virginity. After

hatching from an egg, a fly cannot become fertile for 8 hours. Retrieving them within 6

hours ensures they could not have mated yet, and thus would be virgins. This is important

to ensure that no male wild type flies get into the f1 cross, as this would alter the

accuracy of the experiment.

8. The flies were then given time to reproduce until 100 flies had been created.

9. After 100 flies had been created, the f2 stock tube was created using the methods

described in steps 2-3.

10. The f1 generation was anesthetized using the same procedure described in step 4, and the

flies were placed into a petri dish.

11. Of the flies in the petri dish, 5 males and 5 females were taken and placed into the f2

tube. These offspring from the f1 would be the parent generation for the f2 tube.

12. The remaining flies were placed in the freezer for approximately 6 hours to ensure their

death.

13. After 6 hours, the dead flies were taken out of the freezer, and their eye color as well as

sex were recorded in a data sheet, and then the flies were disposed of.
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14. The f2 generation was given enough time to reproduce and produce 100 flies.

15. After 100 flies were produced in the f2 generation, they were anesthetized as described in

step 4, and then placed in a petri dish and into the freezer for 6 hours.

16. After 6 hours, the dead flies were removed and counted. Their eye color as well as sex

was recorded in a data sheet.

Reciprocal crosses were set up to ensure that the sex of the flies did in fact make a

difference. Because the white eye genetic mutation is a sex linked trait, the sex of the parental

generation makes a difference in the expected outcome for the f1 and f2 generations. Setting up

reciprocal crosses ensured that conclusive data could be attained.

The f1 tubes contained male white eyed flies, and virgin female wild type flies. The wild

type flies had to be virgin to ensure that they did not give birth to a male wild type fly. This

would alter the results as white eyed mutation is a sex linked trait, and therefore the sex of the

parental generation is important and must be controlled. Additionally, only one type of fly was

placed in the tube besides wild type to ensure only one type of breeding was occurring.

This type of setup was necessary so that outside factors which could affect the accuracy

of the results could be eliminated. By crossing only one type of mutation and one type of sex in

the same tube, the type of mating was controlled. This setup ensured that crossbreeding between

different sexes and different mutations did not occur, and ensured that the final results could not

be affected by different types of mutations. If three types of flies were in the tube, the offspring

could not be predicted because the rate and manner of the mating between the three types of flies

could not be controlled. Additionally, if both males and females were put into a tube, the

offspring could not be traced to a specific parent, and thus final results would not be conclusive.
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 9

The setup in this experiment eliminated these possible variables, and thus ensured that only the

hypothesis was being tested.

Results

F1 Cross:

Parental generation: XWt XWt x XW Y

Expected genotypic ratio: 50% XWt XW : 50% XWt Y

Expected phenotypic ratio: 50% Wild Type Males : 50% Wild Type Females

Observed phenotypic ratio: 50% Wild Type Males : 50% Wild Type Females

Table 1: Recorded Fly Characteristics in f1 Generation

Cross White Eyed Male x Wild Type Female

Generation F1
Traits White Eyed Wild Type
Sex Male Female Male Female
Expected 0 0 50% = 50 50% = 50
Observed 0 0 50 50
(n = 100)

Table 2: Chi Square Calculations for f1 Data

Fruit Fly Observed # Expected # Observed – (Observed – (Observed # -

Phenotype Expected Expected) 2 Expected #)2
Expected #
White Eyed 0 0 0 0 0.00
Male
White Eyed 0 0 0 0 0.00
Female
Wild Type 50 50 0 0 0.00
Male
Wild type 50 50 0 0 0.00
Female
Total 100 Df = (n-1) = (2-1) = 1 X2 = 0.00
The Chi Squared value equals 0.00 with 1 degree of freedom.
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F2 Cross:

Parental generation: XWt XW x XWt Y

Expected genotypic ratio: 25% XWt XWt : 25% XW XWt : 25% XWt Y : 25% XW Y

Expected phenotypic ratio: 50% Wild Type Female : 25% Wild Type Male : 25% White Eyed

Male

Observed phenotypic ratio: 45% Wild Type Female: 32% Wild Type Male : 23% White Eyed

male

Table 3: Recorded Fly Characteristics in f2 Generation

Cross White Eyed Male x Wild Type Female

Generation F2
Traits White Eyed Wild Type
Sex Male Female Male Female
Expected 25% = 29 0 25% = 29 50% = 58
Observed 27 0 37 52
(n = 116)

Table 4: Chi Square Calculations for f2 Data

Fruit Fly Observed # Expected # Observed – (Observed – (Observed # -

Phenotype Expected Expected) 2 Expected #)2
Expected #
White Eyed 27 29 -2 4 0.138
Male
White Eyed 0 0 0 0 0.00
Female
Wild Type 37 29 8 64 2.207
Male
Wild type 52 58 -6 36 0.621
Female
Total 100 Df = (n-1) = (3-1) = 2 X2 = 2.966
The Chi Squared value equals 2.966 with 2 degrees of freedom.
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 11

.Figure 3: Chi Squared Value Chart

Conclusion

The f1 data was tested for statistical significance using a Chi Square test (see table 2 and

figure 3). The Chi Square test rejected the null hypothesis (X2 = 0.00 df = 1, P = < 0.5). The f2

data was also tested for statistical significance using a Chi Square test (see table 4 and table 3).

The Chi Square test rejected the null hypothesis (X2 = 2.966 df = 2, P = < 0.25).
 X LINKED TRAITS OF WHITE EYED MALE AND WILD TYPE FEMALE CROSS 12

References

Arnini, C. (1996). Using Drosophila to Teach Genetics. Retrieved May 7, 2011 from:

http://www.yale.edu/ynhti/curriculum/units/1996/5/96.05.01.x.html

Manning, G. (2008). A Quick and Simple Introduction to Drosophila Melanogaster. Drosophila

Virtual Library. Retrieved May 7, 2011 from: http://ceolas.org/VL/fly/intro.html

McClean, P. (2000). Mendel’s First Law of Genetics. Mendelian Genetics. Retrieved May 7,

2011 from: http://www.ndsu.edu/pubweb/~mcclean/plsc431/mendel/mendel1.htm

Wonnacott, R. J., Wonnacott, T. H. (1982). Statistics: Discovering Its Power. Retrieved May 8,

2011 from: http://www.monarchwatch.org/grafx/read/eggs/table.gif