Drosophila II Lab Report

Abstract: Throughout the species of Drosophila, there are two-thousand distinct species. Within the lab, Drosophila is used due to its short life span, and its ability to reveal information regarding both genetic variation and general genetics to those participating in research. The experiment was conducted in two separate parts, with each part requiring its own individual lab time. The purpose of the first part was to breed the flies and observe the offspring produced from this process, and the second part was conducted in order to observe any changed that occurred in the F2 generation. After observing these changes in the F2 generation, the class was able to determine the genotype and phenotype of the F1 generation. The hypothesis stated that the phenotype of the F2 generation would interpret the F1 generation, however the class focused more on the Null hypothesis. The Null hypothesis states that the observed ratios would not deviate from the expected ratios. To begin the experiment, a vial filled with water, a dry media, yeast was created. After the vial was completed, the class put two males and three females within the vile in order to initiate breeding. After three weeks of breeding, the F2 generation was observed and recorded. The data presented by the TA yielded a 9:3:3:1 ratio. This ratio consisted of 306 wild type flies, 71 mutated eye/wild type winged flies, 54 wild type/ mutated winged flies, and 15 mutated eye/winged flies. The expected values for the flies were 250.875: 83.625: 83.625: 27.875. From the chi-squared analysis completed through an excel graph, the chisquared value was 30.46038862. The p-value derived from this chi-square test was < .00001, meaning there is a statistical significance between the observed and expected values. In this case, the high value of the chi-squared test supports the fact that the sample does not fit the model, thus leading to a rejection of the Null hypothesis. Introduction: Drosophila consists of two-thousand species, and each of these individual species feed on yeast, mold, and/or bacteria (Markow & O’Grady, 2008). In the lab conducted in class, Drosophila melanogaster was the specific species chosen to research, and this species is popularly observed and analyzed among other researchers in the field of genetics. Drosophila melanogaster is more commonly known as the fruit fly, and the fruit fly has been used in countless genetic studies across research, due to the similarities to mammals and the large genome sequence (Kornberg & Krasnow, 2000). The fruit fly also has a short generation time, which allows researcher to analyze the quick mutations occurring in offspring. These mutations include specifications such as white/sepia eye mutations, vestigial/apterous wing mutations, or the ebony body color mutation. Wildtype eye color is red, however a possible mutation in the cross could be eye colors such as white or sepia. The wildtype wings are normal and healthy, however a possible mutation in the cross may be vestigial or apterous. Vestigial wings are shown as shriveled, and apterous wings are shown as no wings present on an offspring. Lastly, the wildtype body color is yellow or tan, and the fly has black stripes across its abdomen. A possible mutation in the cross could be an ebony body color. (Mckay, 2010). Efficient understandings of chromosomal mechanics, genetic variation, genetic linkages, and gender calculations can be achieved through studying Drosophila melanogaster (Kornberg & Krasnow, 2000). The frequency in alleles yielded created from dihybrid crosses yielded a 9:3:3:1 ratio. Dominant wild type occurred most frequently in the offspring, yet the appearance of the eye and wing mutations prove that the recessive traits are still seen (DeBenedictis, 1978). The purpose of this experiment was to cross organisms of the same Drosophila species to produce an F2 generation, which would ultimately reveal the genotype of the F1 generation. The initial Null hypothesis stated that the observed ratios would not deviate from the expected values. After the experiment was conducted, a chi-squared test was conducted from the observed and expected values, and a Punnett square was used to display the phenotype ratio of the dihybrid cross. Ultimately, both the chi-squared test and the Punnett square was used to determine if the hypothesis was supported. F1 Generation F1: AaBb X AaBb Results from cross: 9 WT/WT, 3 WT/mut, 3 mut/WT, 1 mut/mut Gametes AB Ab aB ab AB AABB AABb AaBB AaBb . Materials and Methods: Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb As mentioned previously, the experiment was conducted in two individual parts. The first part of the experiment was conducted by preparing a culture vial to host the breeding of the fruit flies. To begin the first part, formula 4-24, a jumbo-sized cotton ball, and dry yeast were used. One spoonful of the formula (dry media) was added to the vial, followed by between 10-12 mL of water, in order to completely soak the media. An important step in the process was to place the vial on its side in order to prevent any media from becoming loose. Next, two to three grains of yeast were added to the vial. Now, the vial was set aside, still on its side, and the flies were prepared for insertion into the vial. Adult flies were anesthetized and placed on a white note card. They were observed under a microscope, specifically through their differences in eye color, wing shape, and body color. Next, the flies were separated by sex, with two males placed on the left side of the note card, and three female flies placed on the right side of the notecard. After the males and females were distinguished, the two males and three females were placed into the vial, and finally the vial was sealed with the jumbo cotton ball. The vial remained on its side, and was labeled with a sharpie, “Uknown Cross.” After three weeks the vial was retrieved in order to begin the second part of the experiment. Although the class unfortunately did not have the opportunity to conduct the second part of the experiment, there is a guide which accurately describes the process. To conduct the second part, a fly-nap wand was inserted into a vial of fly nap, and excess fly nap was scraped off on the rim of the vial. The flies were then tapped to the bottom of the vial, and the wand was carefully inserted by the side of the stopper, meanwhile avoiding getting fly-nap on the stopper. Next, the vial was placed on its side, so the flies do not get stuck in the media, and the experimenter waited until the flies stopped moving. Next, the wand was removed, and the flies were placed under a dissecting microscope. Each fly was examined, and the eye-color, body color, wings and sex of each fly was recorded. An important step was that if the flies started to wake up, a Petri-dish was placed over them to re-anesthetize them. After these steps were concluded, the results were written, and the flies were placed in an ethanol-kill jar. The media was then washed out. Finally, a bottle brush was used to scrape off any remaining pupal cases. To begin calculations, a chi-square analysis was used, with a p-value but of p = .05. Results: The observed number of F2 generation flies were separated and categorized into four groups. The total number of flies in the wild type/ wild type category was 306, the total number of flies in the mutated eye/wild type category was 71, the total number of flies in the wild type eye/mutated wing category was 54, and the total number of flies in the mutated wing/ mutated eye category was 15 (Table 1). The expected values varied slightly from the observed values. The expected value for the wild type/ wild type category was 250.875, the expected value for the mutated eye/ wild type category was 83.625, the expected value for the wild type eye/ mutated wing category was 83.625, and the expected value for the mutated wing/ mutated eye category was 27.875 (Table 1). Based on this data, a chi-squared value was 30.46038862 was derived. Furthermore, a Punnett square was created to show the phenotypic ratio of 9:3:3:1 (Table 2). The degree of freedom was 3, and the p-value cutoff was 0.05 (Table 3). The p-value derived from the chi-square test was < .00001, meaning there is a statistical significance between the observed and expected values (Table 4). In this case, the high value of the chi-squared test supports the fact that the sample does not fit the model, thus leading to a rejection of the Null hypothesis. Table 1: This table calculates the chi-square value off the observed and expected data from the experiment Table 2: This table shows the Dihybrid Punnett square for the F2 of unknown cross. It yeilds a 9:3:3:1 ratio Gametes AB Ab aB ab AB AABB AABb AaBB AaBb Ab AABb AAbb AaBb Aabb aB AaBB AaBb aaBB aaBb ab AaBb Aabb aaBb aabb Table 3: This table is a stat analysis info table. It summarizes the important F2 Generation statistics Table 4: This is a p-value/chi-squared value table. This table allows researchers to determine whether they should support or reject the Null hypothesis. Discussion: The purpose of this experiment was to use the F2 generation of Drosophila observations in order to determine the F1 generation genotype and phenotype. The Null hypothesis stated that the observed values would not deviate from the expected values. The expected values were 250.875 (WT/WT), 83.625 (Eye/WT), 83.625 (WT/Wing), and 27.875 (Eye/Wing) (Table 1), created from the 9:3:3:1 ratio yielded from the Punnett square (Table 2). Table 1 presents the fact that the observed values all deviated from the expected values. Furthermore, the data does not fit the ratio presented, thus leading to a rejection of the Null hypothesis. This conclusion was then confirmed through the chi-squared test. The p-value cutoff was p = .05, and there were 3 degrees of freedom. From calculations, the p-value was < .00001. This means that our data is statistically significant because the sample value does not fit the model value more than 5% of the time. Subsequently, conclusions can be drawn stating that the parent generation of Drosophila was heterozygous. Possible limitations within the experiment that may have affected the results of the experiment are possibly misplacing females and males in the first part of the experiment. This could have affected the offspring in the F2 generation. Another limitation could be that the mutations were inaccurately categorized. For future experiments, using a larger experimental group, and having maybe three rounds (one round- placement, two-rounds- confirming) of the phenotypic mutations occur to ensure there are no misplacements. The P generation genotypes are predictable. Table 5 summarizes the P generation, which yields a ratio of 1:1, half the population of being red-eyed females and the other half being white-eyed males. Controls were used throughout this experiment to ensure the smallest possible chance of error from occurring. For example, all vials of fruit flies had the same gestation period of three weeks. If this aspect of the experiment was not controlled, there could have been more flies produced, thus altering the outcome of the experiment. This research is significant because it allows for a deeper understanding of chromosomal mechanics, genetic variation, genetic linkages, and gender calculations in not only just Drosophila melanogaster, yet other mammals with similar genetic sequences. Table 5: Punnett square of P generation.