CROSSING DROSOPHILA MELANOGASTER 1

Crossing ​Drosophila melanogaster t​ o Determine Genotypic and Phenotypic Ratios

Kyle Agudo, David Gelinas, Alexis Jankovich,

Ethan Kerr, Molly Lada,​ & Sam Morabito-Pip​her

MATES OCVTS
CROSSING DROSOPHILA MELANOGASTER 2

Introduction

Drosophila melanogaster ​is the species of fruit flies that we used for the experiment. It is

commonly used in research because it is both simple and complex on the genetic level. In this

experiment, we crossed red-eyed flies with white-eyed flies and we investigated whether or not

the actual phenotypes matched up with the expected phenotypes using punnett squares. In

addition we are using our crosses to confirm the genotype of the flies that is predicted in the

punnett squares.

In species with genetic sex determination, there is often a visible difference in the

chromosomes of males and females as in humans. Most of the chromosomes are identical in both

males and females (though they may carry different alleles) . In fruit flies, females have two X

chromosomes, while males have an X chromosome paired with a Y chromosome, just like

humans (“Drosophila as a Model System”, 2020).

In order for this project to be successful we must correctly distinguish male flies from

female flies. Using a microscope, you can determine that males have sex combs while females

do not. However, an easier method is to know that male fruit flies are generally smaller and have

a dark, rounded abdomen, while the females are larger and have a pale, pointed abdomen.

Background

Drosophila are most commonly known as fruit flies. They feed on ripe or rotten fruit

(Perveen, 2018). The species of Drosophila used for the experiment was ​Drosophila

melanogaster​. Experimentation with ​Drosophila ​has made connections with the developmental

mechanisms and the basic cellular structure it shares with humans and other animals. Research

with Drosophila has led to the discovery of the basis of heredity (O’Kane, 2001). The DNA
CROSSING DROSOPHILA MELANOGASTER 3

genetic code of the species is 50 times greater than ​Escherichia coli​ and 30 times smaller than

the genetic code of humans. Thus, it has the perfect balance of complexity for experimentation

(Echalier, 2018).

White eye color in ​Drosophila​ is a mutant phenotype, caused by a mutation in a gene in

the pigment pathway, discovered in 1910 by Thomas Hunt Morgan. The “white” mutation is

found on the X chromosome and results in lack of pigment and poor eyesight (Heil et al., 2012).

When a homozygous red-eyed wild female was crossed with the white-eyed mutant male, all the

flies in the F​1 generation

​ were found to be red-eyed. This is because the red-eyed color is

dominant over the white-eyed color (Kul Techno Lab, 2019).

Methodology

Before crossing each generation, it was important to keep track of the genotype of the

current flies and also what the genotypes would be of the expected offspring. This information

could be easily managed in Punnett squares where white eye male flies are x’y, wildtype males

are xy, white eye females are x’x’, carrier wildtype females are x’x, and non-carrier wildtype

females are xx (“Drosophila as a Model System”, 2020). As new generations were crossed, old

flies from the previous generation were let go so as they would not mate with their offspring.

Vials containing these old generations were cleaned out and new vials were set up with media

and a stoppers for incoming generations. The medium in the vials was made with a 1:1

solid-to-water ratio, along with about a dozen specks of yeast. After making the medium, the

vials were dried out using paper towels. Even a small amount of water could drown the flies.

To start, the F₁ generation flies (white eye males and non-carrier wildtype females) were

crossed to create a more diverse F₂ generation, which included wildtype males and wildtype
CROSSING DROSOPHILA MELANOGASTER 4

carrier females. After this cross we were left with wildtype carrier males and wildtype females.

These male and females were then crossed to obtain the expected 1:2:1 ratio of wildtype males,

wildtype females, and white males respectively. However, looking at their phenotype would not

be sufficient as now half of the wildtype females were expected to be carrying the white eye

gene (and of course all the white males were also carrying this gene). The next step was to allow

this generation to mature, which generally took 14 to 17 days, and then to separate them. To

separate the flies, the flies would first be transferred into an empty vial using a funnel, with the

wide end on the fly-containing vial, and the skinny end in the upside-down empty vial. The

bottom vial containing the flies would be tapped to encourage the flies to climb up the funnel and

into the empty vial. Once the flies were transferred, a piece of paper would be placed over the

funnel in the empty vial to prevent flies from escaping. The vial was then vigorously tapped to

knock the flies to the bottom and allow us to take the funnel off and cap the empty vial. Vials

were then immersed in an ice bath for around three to five minutes or until they were

unconscious. Leaving them in for any longer could result in death. Then, using a paintbrush for

moving them around and the occasional microscope for close-up examination, they were

separated into the three groups of wildtype males, wildtype females, and white males into their

own vials. The lights on the microscope were not used because that woke up the flies faster. The

reverse process of getting the flies into the empty vial would be used to get the separated flies

into the new vial. The wildtype females, while some carried the gene for white eyes and others

did not, were crossed with the white eyed males without actually knowing which were which.

This meant a total of two crosses: x’y with xx and x’y with x’x. This new generation, the F​3
CROSSING DROSOPHILA MELANOGASTER 5

generation, was the expected 3:3:1:1 ratio of the respective wildtype males, wildtype females,

white eyed males, and white eyed females.

Data

The parent generation of wildtype females and white eyed males was crossed to produce

the F₁ generation of carrier females and wildtype males (figure 1). The F₁ generation was

crossed to produce the F₂ generation of wildtype females, wildtype males, carrier females, and

white eyed males (figure 2). Out of the F₂ generation, we crossed only the white eyed males and

all of the females (figure 3). This is so that all of the females produced would be either carriers or

white eyed, so there would be no confusion when counting them. This cross produced our final,

F₃ generation with a ratio of 3 wildtype females to 3 wildtype males to 1 white male to 1 white

female.

Figure 1​: Punnett square of the parent generation cross.

CROSSING DROSOPHILA MELANOGASTER 6

Table 1​: Data from our F₁ generation.

1 2 3 4 Total

Phenotype Wildtype Wildtype White males White 4

class males females females

Actual 21 22 0 0 43

number

Expected 21 21 0 0 42

number

Figure 2​: Punnett square of the F₁ generation cross.

CROSSING DROSOPHILA MELANOGASTER 7

Table 2​: Data from our F₂ cross.

1 2 3 4 Total

Phenotype Wildtype Wildtype White males White 4

class males females females

Actual 26 59 28 0 113

number

Expected 28 56 28 0 112

number

Figure 3​: Punnett square of the F₂ generation cross.

Figure 4​: Additional punnett squares of the F₂ generation cross had we crossed both white males

and wildtype males.

CROSSING DROSOPHILA MELANOGASTER 8

Table 3​: Data for our final F₃ generation.

1 2 3 4 Total

Phenotype class Wildtype Wildtype White males White 4

males females females

Actual number 51 47 14 15 127

Expected 45 45 15 15 120

number

Results

Chi Test for our F₁ generation:

x²= (21-21)² / 21 + (22-21)² / 21

x²= 0.048

df= 2-1= 1

P-value= 0.80

Chi Test for our F₂ generation:

x²= (26-28)² / 28 + (59-56)² / 56 + (28-28)² / 28

x²= 0.304

df= 3-1= 2

P-value= 0.90

Chi Test for our F₃ generation:

CROSSING DROSOPHILA MELANOGASTER 9

x²= (51-45)² / 45 + (47-45)² / 45 + (14-15)² / 15 + (15-15)² / 15

x²= 0.956

df= 4-1= 3

P-value= 0.80

Discussion

Our project was successful. The goal was to cross multiple generations of flies to produce

the same offspring as the expected results. Starting with the parent generation, we ended with the

F3 generation. For our final cross, breeding the F2 generation, we bred only white eyed males

with wildtype and carrier females. While the generation before had also produced wildtype

males, we released them as using them would have made it impossible to know if our project

worked. Crossing wildtype males with wildtype females would produce wildtype offspring,

however crossing wildtype males with carrier females would produce some offspring that

appeared to be wildtype but were actually carriers. There would be no way to identify if a female

fly was wildtype or a carrier just by looking at them. By only using white eyed males in the

cross, we knew that none of the females produced would be homozygous-wildtype. Therefore,

any female flies with red eyes could be classified as carriers. Since white females were produced,

that confirms that the 75% of the females that were wildtype were carriers, as only a 1:1 ratio of

homozygous-wildtype-to-heterozygous-wildtype (carrier) could produce 75% red-eyed females

and 25% white-eyed females when crossed with white-eyed males, which would also result in all

of the red-eyed females being carriers (figure 3). This allows us to confirm the genotypes of the

flies in the previous generation along with the flies in the F3 generation.
CROSSING DROSOPHILA MELANOGASTER 10

Using punnett squares (figure 3) we predicted what the outcome would be, or the

expected number, for our final cross. After counting the actual number of flies produced by

phenotype (table 3), we ran a chi test and x²= 0.956. The degree of freedom was 3, as we had 4

different phenotype classes minus 1. Using these numbers on the p-value table we found our

p-value to be .80. We also calculated results for the F1 and F2 generation, getting p-values of 0.8

and 0.9, respectively. The null hypothesis is that there is no significant difference between the 2

sets. The higher the p-value is, the stronger correlation there is between our data and the

expected data (Beers, 2019). Conversely, a low p-value (less than 0.05) would reject the null

hypothesis that the differences in data sets are caused purely by random chance, and would rather

more strongly support the alternative hypothesis, which is that the differences in the data is due

to a non-random cause (Statistics Dictionary, n.d.). In other words, the differences in our data

and the expected data would be because of human error, such as breeding the wrong flies or

misidentifying their gender. Because the p-value is greater than 0.05, you fail to reject the null

hypothesis, which means that there is no significant difference between the actual number and

the expected number of flies; therefore, our results of a ratio of 3 wildtype females:3 wildtype

males: 1 white male: 1 white female accurately represented the phenotypic ratio we should have

had based on the punnett squares, with a very high p-value indicating strong correlation between

the expected ratio and the actual ratio.

Had there been a p-value under 0.05 and we rejected the null hypothesis, we would be

able to use the p-values in the other generation’s data to find during what cross the human error

was made. For example, had the p-value been 0.90 for the F2 generation and 0.04 for the F3

generation, we could conclude that a human error was made in the F2 cross. We could then use
CROSSING DROSOPHILA MELANOGASTER 11

the resulting phenotypic ratio and how it differs from what we expected to determine what error

was made in the cross. However, since our p-values were very high throughout the experiment,

that procedure is unnecessary, as there were no human errors made in this experiment.

Conclusion

In conclusion, our project was successful and we produced the expected offspring for our

F​3​ generation. The p-values were well over 0.05, failing to reject the null hypothesis. In addition,

the phenotypic ratios could be successfully used to confirm the genotypes of the flies.

Acknowledgements

On behalf of our group, we would like to give special thanks to Mr. Sprague for his

continued support and acquisition of the flies and materials used in our experiment.
CROSSING DROSOPHILA MELANOGASTER 12

References

Beers, B. (2019, October 27). P-Value Definition. Retrieved January 12, 2020, from

https://www.investopedia.com/terms/p/p-value.asp.

Drosophila as a Model System. (2020). Retrieved January 12, 2020, from

http://www.indiana.edu/~oso/lessons/Genetics/Drosophila.html.

Heil, C. S. S., et al. (2012, September 12). Witnessing Phenotypic and Molecular Evolution in

the Fruit Fly. Retrieved January 12, 2020, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583343/.

O'Kane, C. J. (2001). Drosophila Melanogaster. Retrieved January 12, 2020, from

https://www.sciencedirect.com/topics/neuroscience/drosophila-melanogaster.

Perveen, F. K. (2018, February 28). Introduction to Drosophila. Retrieved January 12, 2020,

from

https://www.intechopen.com/books/drosophila-melanogaster-model-for-recent-advances-i

n-genetics-and-therapeutics/introduction-to-drosophila.