1) The experiment aimed to determine if mating Drosophila melanogaster in a dihybrid cross would yield results similar to Mendel's 9:3:3:1 ratio by observing inheritance of eye color (red vs white) and body color (ebony vs brown). 2) The F1 generation showed more dominant traits than recessive, but not all were dominant as in Mendel's experiments. The F2 results using a chi-square test did not match the expected 9:3:3:1 ratio. 3) Errors were made in transferring flies between generations that may have impacted the results, which were ultimately deemed inconclusive. Proper fly handling and food preparation are needed to obtain clear results.

1. Dihybrid Cross Mating of Drosophila Melanogaster
Joniqua Christopher, Danielle Coco, Brianna Nicolas and Pume Chikowi
The Abstract
The organism that will be experimented on is a fruit fly, the scientific name of it is Drosophila
melanogaster. Drosophila can live in small spaces, produce a large amount of offspring, have a
short life span, and have many varieties of different characteristics. Drosophila melanogaster can

2. 1
survive when the area is room temperature (about 70°F), and has a source of food like culture
medium where they eat and lay their eggs. In this experiment Drosophila melanogaster are used
to find out how recessive and dominant alleles of eye color and body color are inherited through
parental crosses of F1 and F2 generations. These should follow Gregor Mendel’s principles. In
F1, when crossing red eyed flies with the white eyed flies it is expected to be a 3:1 ratio. In F2, it
is expected to be a 9:3:3:1 ratio but our results were different since the flies in one of our vials
died. Our key findings were that our results were inconclusive since not all of our F1 generation
were red eyed with ebony bodies like Mendel would assume. In the F2 generation we had more
red eyed ebony body flies to white eyed brown body flies
The Introduction
The hypothesis of this experiment is to determine if mating the Drosophila melanogaster through
a dihybrid cross will yield similar results to Mendel’s Law of Independent assortment of 9:3:3:1.
Phenotypes are physical characteristics like red eyes or brown bodies. Genotypes which are the
genetic makeup or DNA of organisms, determines what they will look like. Alleles are one of the
possible forms of genes, and most genes have two alleles. An allele can be dominant or
recessive. If an organism has one of each allele or is heterozygous for a trait, then the dominant
trait is shown (Dd). If an organism is homozygous for a trait, a recessive allele is only shown
when there are two of them (dd).
Gregor Mendel known as the “Father of Genetics”, first defined alleles, and he is known for his
carefully designed plant breeding experiments. These experiments helped him develop the
concept of a gene and toward the end of his experiments on pea plants, he discovered the
principle of inheritance (322 Brooker). Inheritance is when traits from the parents are passed
down to the offspring. Mendel specifically studied and crossed hybrid garden pea plants, that
were the same species but had different characteristics. He used pea plants for different reasons
like they have many varieties with different characteristics, are self fertilizing (male and female
gametes), and he could cross fertilize them (322 Brooker). In cross fertilization, Mendel could
remove stamens from a purple flower, and transfer the stamens from a white flower to a purple
flower. In his experiment he cross fertilized the parental generation (P) a tall plant with a dwarf
plant and the offspring (F1) were all tall monohybrids (322 Brooker). Then he let F1 self fertilize
to produce an F2 generation and there was a 3:1 ratio of tall to dwarf plants. To reiterate, the
inheritance pattern of the P generation was true breeding TT x tt, the F1 offspring Tt which were
all tall, and F2 was TT:Tt:tt. This means there was one homozygous dominant tall trait, one
heterozygous tall plant and one homozygous dwarf plant. Mendel concluded that tall trait was
dominant and dwarf trait was recessive. Mendel also discovered the Law of Segregation which
proved that when any individual produces gametes, the copies of a gene separate so that each
gamete receives only one copy. A gamete will receive one allele or the other. This is later proven
in meiosis. In meiosis, the paternal and maternal chromosomes are separated and the alleles with
the traits of a character are segregated into two different gametes. Mendel also discovered the
Law of Independent Assortment states that alleles of different genes assort independently of one
another during gamete formation (326 Brooker). While Mendel's experiments with mixing one

3. 2
trait always resulted in a 3:1 ratio between dominant and recessive phenotypes, his experiments
with mixing two traits (dihybrid cross) showed 9:3:3:1 ratios. Mendel then determined that there
is no relation and that different traits are inherited independently of each other.The goal of this
experiment is to find out if our F2 will be an exact 9:3:3:1. This can be done by interbreeding
two true breeding types of Drosophila melanogaster which are red eyed with ebony bodies and
white eyed with brown bodies.
Materials and Methods
Parental Generation
To begin this experiment there needs to be true breeding stocks of Drosophila melanogaster with
red eyes and ebony bodies and white eyes with brown bodies. First, gather two plastic vials and
two sponge stoppers. Put a 1:1 ratio of cornmeal medium (fruit fly food) to water in the vials.
Wait five minutes for it to absorb. Label one vial “Cross 1” in here is where 3 red eyed ebony
males and 4 white eyed brown female fruit flies will mate. Write REM x WBF F1 Gen. Label the
other vial “Cross 2” in here is where 3 white eyed brown males and 4 red eyed ebony female
fruit flies will mate. Write WBM x REF F1 Gen. Gather four vials of Drosophila melanogaster.
Place the vials of flies into an ice bucket for approximately 5 minutes. Take 2 petri dishes, put
ice on one and be sure to put the dry petri dish over it. After five minutes make sure the flies are
asleep, gently remove them from the tube with a small paintbrush, and put them on the petri dish.
Place the petri dish under a dissecting microscope and analyze the flies closer to sort them out
based on eye and body color. Place 3 red eyed ebony males in the Cross 1 vial with the small
paintbrush. In the same vial, place 4 white eyed brown females using the same technique. In the
Cross 2 vial place 3 white eyed brown males and 4 red eyed ebony females with the small
paintbrush. Close the vials with a sponge stopper and constantly watch the mating progression
within the next 6 days to observe the F1 generation offspring.
F1 Generation
Once the F1 generation offspring can be seen tunneling through the food as larvae, it is time to
remove the parental Drosophila. In order to do this, they need to be asleep. Place the Cross 1 and
Cross 2 vials in an ice bucket for approximately five minutes. When they are asleep, use the
small paintbrush to remove them and dispose of them. After about a week, the larvae will
become pupae and cling to sides of the vials where they will eventually develop into an adult fly.
Observe the mating process over a week and write how many of each characteristic is found
when they are fully developed.
F2 Generation
Once the F1 are now adult flies, begin preparing two new vials with a 1:1 ratio of cornmeal
medium (fruit fly food) to water in the vials. Wait five minutes for it to absorb. Label one vial
Cross 1 again, and write REM x WBF F2 Gen. Label the other vial Cross 2, and write WBM x
REF F2 Gen. Take the original F1 vials and place them in an ice bucket for five minutes to put
them asleep. Then remove the flies from Cross 1 F1 and carefully place them in the new Cross 1
F2 vial with the small paintbrush. Repeat process and transport the Cross 2 F1 into the Cross 2

4. 3
F2 vial. Observe the mating process over a week and write how many of each characteristic is
found.
Results
According to Mendel’s Law of Inheritance, the true breeding P generation monohybrid cross will
yield all red eyed offspring for F1. Since we are looking at 4 different traits, red eyes, white eyes,
ebony bodies, and brown bodies, only the dominant alleles should be shown in the phenotypes.
To test Mendel’s Law of independent assortment, cross two pure breeding strains of Drosophila
melanogaster and observe the inheritance of eye
color which is red and white and body color
which is ebony and brown. Determined which
allele is dominant by crossing them. The capital
‘R’ allele means red eyes and the lower case ‘r’
allele means white eye. The capital ‘E’ allele
means ebony body and lower case ‘e’ is a brown
body. So red eyes and brown bodies are
dominant while white eyes and brown bodies are
recessive.
RR- Red Eyes (Homozygous Dominant) EE- Ebony Body (Homozygous Dominant)
Rr- Red Eyes (Heterozygous Dominant) Ee- Ebony Body (Heterozygous Dominant)
rr- White Eyes (Homozygous Recessive) ee- Brown Body (Homozygous Recessive)
Independent Assortment Punnett Square True Breeding
RE Re rE re
RE RREE RREe RrEE RrEe
Re RREe RRee RrEe Rree
rE RrEE RrEe rrEE rrEe

5. 4
re RrEe Rree rrEe rree
Number of Drosophila melanogaster Parental Generation
Phenotypes Parental Gen
Red Eyes Ebony Body
Male
3 males
White Eyes Brown
Body Female
4 females
Total: 7 flies
F1 Generation Offspring
Cross 1 Cross 2
Red Eyed Ebony Males - 78 White Eyed Brown Male - 25 + 2*
White Eyed Brown Females - 22 Red Eyed Ebony Female - 6 + 11*
Total = 100 Flies Total = 44* Flies
F2 Generation Offspring
Cross 1 Cross 2
Red Eyed Ebony Males - 134 White Eyed Brown Male - 0 + 42*
White Eyed Brown Females - 99 Red Eyed Ebony Female - 0 + 22*
Total = 233 Flies Total = 64* Flies
This data is shown to be inconclusive since the F2 generation have failed to reproduce and they
all died. As a result, our group had to incorporate another group’s data to perform a Chi Square
Test. The red data with the asterisk represents the additional data we received from another
group.

6. 5
Phenotypes Observed Expected Calculation
White Eyed Brown Body
Males 1/16
69 69/440 =
.156
(.156 - .0625)2
= .140
.0625
Red Eyed Ebony Body Males
9/16
212 212/440 =
.481
(.481 - .5625)2
= .011
.5625
White Eyed Brown Body
Females 3/16
121 121/440 =
.275
(.275 - .1875)2
= .040
.1875
Red Eyed Ebony Body
Females 3/16
38 38/440 =
.086
(.086 - .1875)2
= .054
.1875
Total 440 flies total --------- Sum = .245
The degrees of freedom is 3, so critical chi square value is = 7.82 , our result probability
.245, this is more likely than our significance level so we accept the null hypothesis.
Discussion
The experiment suffered many flaws since our data was not correct. Our hypothesis was rejected since it
did not match up with the critical chi square value, and our result was not in range. In the F1 offspring
there was an error somewhere it could have been made when changing vials, putting the flies to
sleep or removing all of the flies. But it is evident that we were not accurate in the week when
we were observing hatching of the larvae. When removing the adult P generation parents we
accidentally took out some of the newly grown F1 adults. Also going from F1 to F2 we did not
put a correct ratio of water to food since we were rushing. As a result the food was very dry and
instead of staying a vibrant blue color, it later turned to a mixture of yellow and brown color,
thus the flies had no proper food to eat in F2 Cross 2. This is why we had to use data from
another group to makeup for our loss. Even with another group’s data our results were still
inconclusive. To avoid our error next time we must closely watch the flies and very carefully
distinguish the different body and eye colors. We should also add a careful and correct amount of
medium to water so the Drosophila can have proper food The degrees of freedom is 3 and in
order for our findings to be significant it had to be a .05 probability and our critical chi square
value had to be 7.82. Our probability was .245 and that is more likely than our significance level
so we accept the null hypothesis. Our hypothesis was that the dihybrid cross will have the same
results as Mendel’s 9:3:3:1. Mendel’s experiment would show that there are more dominant red
eyed ebony body flies than there are recessive white eyed brown body flies. Surprisingly, in our
F2 cross there were 250 red eyed ebony body flies as opposed to 190 white eyed ebony body

7. 6
flies, this proves Mendel’s Law of Inheritance. Even in our F1 we had significantly more
dominant traits than recessive traits, but all were not dominant like in Mendel’s case. This is
shown in the above tables. However the exact data did not match Mendel.
Conclusion
In conclusion, the phenotype of the F1 and F2 progeny confirmed that the red eyes and ebony
bodies are in fact dominant. It is also true that white eyes and brown bodies are recessive.
Although not all of our F1 were red eyed with ebony bodies like in Mendel’s case, a majority of
them were. This may be due to the errors that were made. In F2 with the chi square test, it
showed that our result probability was more likely than our significance level so we accept the
null hypothesis.
References
Brooker, Robert J. "Chapter 16 Simple Patterns of Inheritance." Biology. 3rd ed. New York, NY:
McGraw-Hill, 2014. N. pag. Print.
Acknowledgements- Ronya Farraj (Other Group Info), Danielle Coco, Brianna Nicolas and
Pume Chikowi (worked together)
Appendices
Differences in Drosophila Body Shape in Gender
Life Cycle of Drosophila melanogaster

8. 7
Example of the Different Eye Colors