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

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

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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 MethodsParental GenerationTo 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 GenerationOnce 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

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F2 vial. Observe the mating process over a week and write how many of each characteristic is found.

ResultsAccording 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

re RrEe Rree rrEe rree

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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.

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Phenotypes Observed Expected Calculation

White Eyed Brown Body Males 1/16

69 69/440 =.156

(.156 - .0625)2 = .140.0625

Red Eyed Ebony Body Males9/16

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.

DiscussionThe 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 flies, this proves Mendel’s Law of Inheritance. Even in our F1 we had significantly more

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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.

ConclusionIn 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)

AppendicesDifferences in Drosophila Body Shape in Gender

Life Cycle of Drosophila melanogaster

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Example of the Different Eye Colors

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