Introduction:

Drosophila melanogaster are an ideal organism to be used in genetic experiments due to their ability to reproduce quickly and with many offspring. They possess very specific traits for eye color, body color, and wing type. This makes it easy for the traits to be followed from generation to generation. These phenotypic traits are extremely beneficial once the experiments have been conducted, but they also have a rather simplistic genotype. Their genomes have been studied for years and the genes for the phenotypes that will be examined within this experiment have been extensively explored.
Fruit flies are usually very uniform in how they look. This makes experiments involving breeding for different phenotypes very easy. You can clearly see with the naked eye most of the traits. For this experiment we are breeding wild type males with white eye females. The wild type male fruit flies all possess the dominant trait for red eyes. The white eyes of the female are actually an X-linked recessive trait (The National Health Museum 1999-2009). By crossing certain genotypes of flies with others, we can easily predict what the offspring will look like and the proportion of each phenotype that will occur from the cross. Figure 1:

Image from Access Intelligence of the National History Museum.

Figure 1 helps to explain the way that sex-linked traits work and how they occurred within the experiment that was conducted. In the first Punnett square, the male has white eyes and was crossed with a red eyed female. This resulted in all of the offspring having red eyes. The red eyes females have the genotype of (RR) and the males have the genotypes of (XwY) this resulted in all of the daughters to be heterozygous with the genotype of (Rw) and to have red eyes and all of the sons to be (RY) and also have red eyes. For this experiment, we conducted the second cross within this diagram. The females are homozygous for the white eye trait, since it is recessive, have the genotype of (ww). The males in the experiment have the genotype of (RY). This produces daughters that have red eyes and males that have white eyes. Since the male only have one X chromosome and he is inherited from his mother who was homozygous for the recessive trait of white eyes, he will possess white eyes. White the daughters have

two X chromosomes and the dominant trait of red eyes show through. The cross that was done in the second Punnett square is the same cross that was conducted to prepare for this experiment. It will be demonstrated again under the Methods section. For this experiment we are curious about the F2 generation from the cross. This would be the set of offspring that is produces from the red eyed females and the white eyed males we got after crossing the white eyed female with the wild type male. The ratio that we are hoping to produce would be a testcross. It is the simplest cross involving dominant traits being crosses with homozygous recessive (Rutgers Life Science). This would be the dominant trait of red eyes crossed with the recessive white eyed trait. The predicted and hoped for ratio would be a 1:1:1:1. Since the F1 generation consisted of 50% - 50% of both phenotypes, the F2 generation will consist of 25% of each of the four phenotypes. This is the basis for the following experiment. Materials**: Vials Labels Plastic mesh inserts (so adults flies have some place to land) Form or cotton caps for vials Instant medium (food for the flies) Bakers yeast Paint brushes 4 x 6 index card (to sort flies on) Dissecting microscope FlyNap (Triethylamine) Notebook to record data and observations o ** all materials were taken from page 5 of the Genetics Lab Manual Fall 2011

Methods: Before even working with Drosophlia melanogaster, 2 culture vials for the mating and developing of the following generations was prepared. This is a simple procedure. The vials were labeled with the group name, fly phenotypes being crossed, where the parent flies came from and the date. Roughly 10mL of a dry medium was added to a clean vial followed by an equal amount of water. If there was still some dry substance in the vial, more water was added to make sure everything wet. Once this was accomplished, a pinch of yeast was added and a mess netting for the flies to rest upon. This process was then repeated for the second vial. The vials were then capped and the preparation of the fruit flies began (Genetics Lab Manual Fall 2011 pg 5). The phenotypes being crossed have already been determined and the generation in question is the F-2 generation. The F-1 generations will be segregated into the two vials that were prepared for mating. Extensive notes were taken while doing these crosses. Stock vials containing the F-1 generations were available for selection. The white eyed females were being crossed with wild type males for this experiment. Notes on which crosses were recorded and the location of where the flies came from were also noted. In order for the flies to be anesthetized, you must remove them from the stock vial into the clean vials. These are not the vials that were prepared earlier, but rather clean vials that have nothing within them. The cap to the clean vial was removed and one group member held said clean vial while another member prepared to remove the cap from the stock vial still containing the mature Drosophlia.

The stock vial was lightly tapped to remove the flies from the top of the vial. The cap was quickly removed and the clean vial was placed on top to allow for the flies to travel up into the vial. A few moments passed to allow for the stragglers to make their way up into the clean vial. This process was then repeated for the second clean vial and the second stock vial. Once the flies were removed from the stock vials and safely placed within the clean vials, they were ready to be anesthetized. This was done simply by placing one of two wands dipped in FlyNap (Triethylamine) into the clean vial (Genetics Lab Manual Fall 2011 pg 5). After about 30 seconds, the flied began to fall asleep and dropped to the bottom of the vial. Sleeping Drosophlia were removed from the vial and placed upon an index card. A dissecting microscope was used by some group members, while others could simply look at the flies to determine phenotypes. Paint brushes were used to move the flies around on the note card to separate them without harming them. Since the cross was done on white eyed females and wild type males, there was no need to be concerned with the sex of the fly. Eye color determined whether it was a male or female. 10 males and 10 females were separated from the rest of the anesthetized Drosophlia. The remaining flies were placed within a chemical filled jar to be humanly euthanized. Of the 20 living flies remaining, 5 females and 5 males were placed within one of the earlier prepared vial. The remaining 5 females and 5 males were placed within the other vial. These vials were then placed in a safe location in the laboratory for the flies to wake and proceed to mate and create the F-2 generation (Genetics Lab Manual Fall 2011 pg 6). A sufficient amount of notes were taken of when the flies where checked, when the larva had developed, and then the F-2 generation were developed. Punnett Squares were created to help predict the future generations. When the F-2 generations matured and were ready to be counted, the process was exactly the same as when the segregation of the F-1 generation occurred. The flies were removed from the created vials into clean vials to be anesthetized with the FlyNap. Once asleep, each vial was placed upon a note card and counted. The Punnett Squares that were done before the counting helped to give an idea of what should be expected. The only significant difference was that now there would need to be sexing of the flies. As described within the introduction, there are differences among the males and the females. The dissecting microscope was needed to determine the sex of the flies. Notes were taken of how many of each phenotype. These results were then turned into a Punnett Square (Figure 1 and 2) and analyzed in the results. Parent generation phenotypes: 50% males with red eyes; 50% females with white eyes. Parent generation genotypes: 50% XW+Y ; 50% XwXw Figure 2: X Xw
w

XW+ XW+Xw XW+Xw

Y XwY XwY

Predicted F1 generation phenotypes: 50% females with red eyes; 50% males with white eyes. Predicted F1 generation genotypes: 50% XW+Xw ; 50% XwY

Figure 3: XW+ Xw Xw XW+Xw XwXw Y XW+Y XwY

Predicted F2 generation phenotypes: 25% females with red eyes; 25% males with red eyes; 25% females with white eyes; 25% males with white eyes. Predicted F2 genotypes: 25% XW+Xw ; 25% XW+Y ; 25% XwXw ; 25% XwY

Results: Figure 4:
Probabilities: 0.5 0.05 0.46 3.84 1.39 5.99 2.37 7.82 3.36 9.49 4.35 11.07

df: 1 2 3 4 5

0.9 0.02 0.21 0.58 1.06 1.61

0.01 6.64 9.21 11.35 13.28 15.09

*This data will be used to figure out if the Chi2 data will be rejected or fail to be rejected. (Genetics Lab Manual Fall 2011 pg 25)

Figure 5: Group 1 Drosophlia melanogaster Chi2 of offspring

Phenotype: Red Eye Females White Eye Females Red Eye Males White Eye Males TOTALS: O: 101 85
125 121 432

E: 108 108 108 108

432

Ratio: 1 1
1 1 4

O-E: -7 -23
17 13

(O-E)^2: 49 529
289 169 Chi Value:

(O-E)^2/E: 0.45 4.90

2.68 1.56 9.59

HO: The data collected will fit a 1:1:1:1 ratio. Degrees of Freedom: 4-1=3 Chi2 Critical Value: 7.82 Calculated Value: 9.59 Fail to Reject or Reject The Drosophila offspring data collected will be similar to the predicted ratio of 1 red eye female to 1 white eye female to 1 red eye male to one white eye male. In the above cross there was 3 degree of freedom. The critical value for this degree of freedom with a .05 level of significance was 7.82. After completing the table, I found my critical value to be 9.59; therefore I reject the hypothesis due to the calculated value being greater than the chi2 table value.

Figure 6: Group 2 Drosophlia melanogaster Chi2 of offspring

Phenotype: Red Eye Females White Eye Females Red Eye Males White Eye Males TOTALS: O: 95 95
107 62 359

E: 89.75 89.75 89.75 89.75

359

Ratio: 1 1
1 1 4

O-E: 5.25 5.25

17.25 -27.75

(O-E)^2: (O-E)^2/E: 27.56 0.31 27.56 0.31

297.56 770.06 Chi Value: 3.32 8.58 12.51

HO: The data collected will fit a 1:1:1:1 ratio. Degrees of Freedom: 4-1=3 Chi2 Critical Value: 7.82 Calculated Value: 12.51 Fail to Reject or Reject The Drosophila offspring data collected will be similar to the predicted ratio of 1 red eye female to 1 white eye female to 1 red eye male to one white eye male. In the above cross there was 3 degree of freedom. The critical value for this degree of freedom with a .05 level of significance was 7.82. After completing the table, I found my critical value to be 12.51; therefore I reject the hypothesis due to the calculated value being greater than the chi2 table value. Figure 7: Combined Group 1 & Group 2 Drosophlia melanogaster Chi2 of offspring
Phenotype: Red Eye Females White Eye Females Red Eye Males White Eye Males TOTALS: O: 196 180
232 183 791

E: 197.75 197.75 197.75 197.75

791

Ratio: 1 1
1 1 4

O-E: -1.75 -17.75

34.25 -14.75

(O-E)^2: 3.06 315.06

1173.06 217.56 Chi Value:

(O-E)^2/E: 0.02 1.59

5.93 1.10 8.64

HO: The data collected will fit a 1:1:1:1 ratio. Degrees of Freedom: 4-1=3 Chi2 Critical Value: 7.82 Calculated Value: 8.64 Fail to Reject or Reject The Drosophila offspring data collected will be similar to the predicted ratio of 1 red eye female to 1 white eye female to 1 red eye male to one white eye male. In the above cross there was 3 degree of freedom. The critical value for this degree of freedom with a .05 level of significance was 7.82. After completing the table, I found my critical value to be 9.59; therefore I reject the hypothesis due to the calculated value being greater than the chi2 table value.

Discussion: Figure 2 and Figure 3 show the Punnett square break downs of the Drosophila given for the experiment. It was found that the offspring for the experiment should result in a 1:1:1:1. This was the expectations that was hoped to be found after conducting the crosses of the F1 generations. Unfortunately, this isnt what was found after conducting three different Chi2 analyses. Figure 5 was the Drosophila cross that was conducted by the group that I was working in. There were 101 adult females with white eyes, 85 females with red eyes, 125 wild type males, and 121 males with white eyes. The Chi2 value for this was 9.59. Since this value was higher that the Chi2 value for our degrees of freedom, we had to reject that it fit our projected ratio. This could be slightly skewed due to flies escaping white trying to move the flies from one vial to another. There may have been a significant amount of one phenotype lost that the other. Looking at Figure 6, the same results are seen. It does not fit the predicted ratio for the desired outcome. This group had 95 red eye females, 95 white eye females, 107 red eye males, and then 62 white eye males. The Chi2 value was 12.51. This value was very far off from the needed value 7.82. Finally, the two groups were added together and the Chi2 value was taken for this in Figure 7. There were 196 red eyed females, 180 white eyed females, 232 red eyed males and 183 white eyed males. The Chi2 value was 8.64. This is closer to the desired value but still called for it to be rejected. One solution to this would be to up the level of significance to .01. This would raise the Chi2 value to 11.35. The results for Figure 5 and Figure 7 would then be able to be accepted and would be considered the correct ratio for the predicted 1:1:1:1. After pondering about what could have caused the results to come out in and undesirable fashion, I have come up with a few theories as to why this may have happened. One such reason for this could be that since the genes are sex linked, the probability of males possessing red eyes may be higher. When looking at the totals in Figure 7, you can see that there is a rather large amount of wild type males. These males possess the red eyes rather than the white eyes. The other three seemed to be relatively close in number, but the dominant wild type males definitely prevails over the other phenotypes. Another problem that may have occurred was that during our experiment the temperature within the room where the Drosophila larva and offspring were kept was below the normal temperature needed for this experiment. This may have skewed the results slightly causing some traits to not develop and others to develop. And finally, my last theory on why the ratios did not come out right would be that the flies that escaped and the larva that did not develop into mature flies could make up for the lack of numbers. This experiment could be improved simply by being extremely careful about the working conditions and the development of the larva into the mature flies.