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This experiment focused on the genetic passing of mutations among the Drosophila Melanogaster species. The purpose of this lab was to determine the unknown F1 generation’s genotype from observing the F2 generation’s phenotypical ratio. The hypothesis that was tested stated that based on the 9:3:3:1 phenotypic ratio of the F2 generation, the F1 generation was determined to be heterozygous for both vestigial and ebony mutation traits (VvEe). Based on the class data, a high chi-square value was produced, meaning that the sample data did not fit the 9:3:3:1 model of the Mendelian dihybrid cross. This value was then converted into a p-value which was deemed lower than 0.001, causing the null hypothesis to be rejected. This meant that there was a difference between the observed and expected ratios from the dihybrid F2 generation. Since that data did not fit the hypothesized ratio, there was no evidence to confirm that the F1 generation was in fact heterozygous for each mutation trait; it is only known that the F1 generation was phenotypically wildtype.

Introduction

Heredity and inheritance were first discovered and studied by Gregor Mendel in 1865. His studies made him idealize that trait inheritance is represented by a ratio for each generation of offspring (Schacherer, 2016). In genetics, a dihybrid cross is between two organisms that are identically heterozygous for two separate traits. Heterozygous individuals carry two different alleles for a trait. According to Mendel, a dihybrid cross of two heterozygous individuals has a phenotypic ratio of 9:3:3:1 (Merriam, 2001).

The Drosophila melanogaster species used in this experiment offer several advantages to being studied. The species are known to have a very short generation time which allows separate generations to be studied without having to wait an excessive amount of time. They also have simple dietary requirements and an already well understood genetic background for future experimentation (Partidge & Piper, 2016). A high reproduction rate also allows for more flies or samples to be observed.

In this experiment, Drosophila were bred in a dihybrid cross and then observed to study the passing of genetic mutations. The mutations observed included ebony body color and vestigial wings; the crossing can be seen in Table 1. Both mutations served as recessive traits which were carriers on the F1 generation. The purpose of this lab was to determine the unknown F1 generation genotype from observing the F2 generation’s phenotypes. It was hypothesized that the flies in the F1 generation were heterozygous for both traits: vestigial and ebony, which allows for the F2 generation to follow a 9:3:3:1 ratio. The null hypothesis states that the observed ratios do not deviate from the expected ratios. A chi-square test is used to see how well the observed ratio of mutations fit the expected ratio. The p-value then determines the statistical significance and either rejects or accepts the null hypothesis based on the value.

Unknown Cross F2 Punnett Square

Gametes VE Ve vE ve

VE VVEE VVEe VvEE VvEe

Ve VVEe VVee VvEe Vvee

vE VvEE VvEe vvEE vvEe

ve VvEe Vvee vvEe vvee

Table 1: A Punnett square of the unknown dihybrid cross of the F2 generation with vestigial and ebony mutations.

Materials and Methods

To begin the experiment, a test tube was labeled for the “unknown cross.” Then, one spoon of food (dry medium) was added to the tube followed by 10 ml of DI water which was contained in a beaker. The tube was allowed to sit for two to five minutes until the food solidified. Once the food was checked, two to three grains of yeast were added to the tube by using a brush. Then Drosophila that were already put to sleep were distributed to each group. Out of the Drosophila given, each group choose three female flies and two male flies to place in the tube. The flies were placed into the tube in a way that they would not get trapped by the food. A cotton ball was used to close the tube and keep the flies in. This tube was set aside for two weeks, allowing the flies to reproduce in order to create a F2 generation.

After the 2-week period, the test tubes were returned to the groups and the analysis began. A brush was dipped into the FlyNap solution and then inserted into the tube without removing the cotton ball. After the flies were asleep once again, the cotton ball was removed, and the flies were placed onto a notecard. In order to separate the flies according to their characteristics, a dissecting microscope was used. The characteristics observed included wing type and body color. Wing type was separated into wildtype and vestigial while body color was separated into wildtype and ebony. Once the flies were separated and counted based on their characteristics, the data was added to a class data table. The flies were then moved from the notecard and into a jar filled with ethanol to kill them and prevent from a fly infestation. The test tubes were washed out into the sink and then placed to dry. After all the groups added their data to the table, a chi-square test was run. The test was used to determine if the number of observed traits differed from the number of expected traits. The chi-square value was then used to determine a p-value based on the conversion chart provided. The p-value determines if the data was statistically significant and whether or not to accept or reject the null hypothesis.

Results

The total number of Drosophila observed in the experiment was 80 individual flies. Of the 80 flies, 62 were wildtype, 15 had a vestigial mutation, 1 had an ebony mutation, and 2 had both mutations. The expected numbers were 45 wildtypes, 15 vestigial mutations, 15 ebony mutations, and 5 flies with both mutations. Table 2 follows the process of converting the observed and expected numbers into chi-square values by first subtracting the expected from the observed in each mutation category. Then, the result was squared for each category and divided by the expected to create chi-square values for each mutation. All of them added together resulted in the chi-square value of 21.2888889.

Unknown Cross Chi-squared Data Table

  WT/WT vestigial/WT WT/ebony vestigial/ebony Total

O 62 15 1 2 80

E 45 15 15 5 80

O-E 17 0 -14 -3 N/A

O-E2 289 0 196 9 N/A

O-E2/E 6.42222222 0 13.0666667 1.8 21.2888889

Table 2: Observed and expected values are calculated to determine the chi-square value.

Then, in order to convert the chi-square value to a p-value, the degree of freedom was determined to be 3 since there were 4 phenotypic possibilities. The chi-square value and degree of freedom (df) were used to find the p-value according to table 3, which results in the p-value being below 0.001. The three statistical values (chi-square, df, and p-value) are shown in table 4.

Chi-square values vs. P-values

Degrees of Freedom P = 0.9 P = 0.5 P = 0.2 P = 0.05 P = 0.01 P = 0.001

1 0.016 0.46 1.64 3.84 6.64 10.83

2 0.21 1.39 3.22 5.99 9.21 13.82

3 0.58 2.37 4.64 7.82 11.35 16.27

Table 3: This table shows how each chi-square value correlates to a p-value. Data table retrieved from Biology 101 Laboratory Manual, Chapter 7.

Statistical Analysis Summary Table

df 3

Chi-square 21.2888889

P-value <0.001

Table 4: This table summarizes the statistical information obtained in this experiment.

Discussion

Based on Table 2, the resulting chi-square value was 21.2888889, which is considered high. This value means that the sample used in the experiment does not fit the Mendelian model of the 9:3:3:1 phenotypical ratio. After converting the chi-square value to a p-value by using Table 3, the resulting p-value was lower than 0.001. This value calls for the null hypothesis to be rejected which means that the observed ratios do in fact deviate from the expected ratios. The experimental data from the F2 generation did not follow the Mendelian ideal of a heterozygous dihybrid cross having a 9:3:3:1 phenotypical ratio. This can be explained by our class data having a very small F2 generation that resulted in only 80 total flies. Since the Drosophila melanogaster is known to have a very high reproduction rate, a larger F2 generation was expected to observe from. Though the amount of food in each tube and yeast grains added were controlled as well as the number of flies in each F1 generation, each group had a drastically different amount of F2 generation flies produced. This was a limitation to our particular experiment.

By using the Punnett square of the F2 generation or Table 1, the F1 generation could be hypothesized to be heterozygous for each trait while being phenotypically wildtype. This would cause the genotype of the F1 generation to be VvEe as the mutations are present on the recessive allele. This is also reversely explained by the idea that if you cross two heterozygous individuals in the F1 generation, the F2 generation will follow a 9:3:3:1 phenotypic ratio. However, due to the rejection of the null hypothesis, the F1 generation was not confirmed to be heterozygous for each mutation trait; the generation was only known to be wildtype.

Drosophila are used in several biological studies since they deliver efficient answers. More specifically, Drosophila is a great model for studies related to genetic diseases. They can be used in developmental biology such as cancer development by allowing the defects present in flies to be patterned in order to better the understanding of human development (Wangler et al., 2015).

References

Merriam J. 2001. Mendelian Ratio. Encyclopedia of Genetics :759–761.

Piper, M. D., & Partridge, L. 2016. Protocols to Study Aging in Drosophila. Methods in molecular biology 1478: 291-302.

Schacherer J. 2016. Beyond the Simplicity of Mendelian Inheritance. Comptes Rendus Biologies 339(7-8): 284–288.

Wangler, M. F., Yamamoto, S., & Bellen, H. J. 2015. Fruit flies in biomedical research. Genetics 199(3): 639-53.

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