Materials:
Vials of pure bred parental flies for one, two, or three crosses (if starting at beginning of mating)
• Vial of experimental mated cross (if starting at collecting of F1 offspring)
• 4 vials containing media for each cross
• 1 bottle EDVOTEK® BugOutTM Solution
• Camel-hair paintbrush to manipulate flies
• Filter paper
• Foam lids
Pasteur pipets
• Petri plate
• Drosophila culture tubes
• Drosophila growth media
• Dissecting microscope
• Microscope
• Vegetable or mineral oil for morgue
• Alcohol for morgue
• Flask with fitted cork (morgue)
• NaCl solution
• Slide and cover
• Acetocarmine Stain
• Glass slide
Part A: Flies getting in Each Other's Jeans (Genes.. ayy lmao)
Hypothesis: If pure breeding parents of two genes are crossed for two generations, then one should be able to determine whether the genes are autosomal or sex-linked and also be able to use use Chi-square analysis to see if the actual results follow Mendel's Law of Independent Assortment.
Procedures:
Obtain a vial of parental flies from your teacher. Anesthetize the flies as instructed by your teacher and practice looking at the fl ies under the dissecting microscope. You should note the sex differences. Also note the type of mutation written on the vial.
1. Obtain a vial of F1 flies from your teacher.
• Note the vial number and parental cross marked on the vial in your notebook.
• Using standard fly handling techniques, collect the F1 flies and observe their phenotypes.
• Record the results below.
2. Place 5-6 male and female F1 flies into a fresh vial with media.
3. After 10 days, remove the F1 parents and discard into the fly morgue.
4. The F2 generation flies will begin to emerge within several days.
• Remove the F2 generation flies as they begin to emerge to an empty vial without media and anesthetized as described under the section titled Using EDVOTEK® BugOutTM solution.
• Observe the flies under the dissecting microscope. Record the F2 results over a 3-4 day period. The more data you accumulate the more significant the results.
5. Analyze the data using the chi-squared statistic as previously described. Obtain the class results for the other crosses in this experiment and analyze the results using chi-squared as well.
Monohybrid Cross:
The monohybrid cross (vials numbered 1 to 4) should yield all red eyes in the F1 generation and a 3:1 ratio of red to sepia in the F2. Record red eye and sepia eye results for the F1 and F2 generation in your notes. Determine the X2 values.
The null hypothesis for the F2 generation would be a 3:1 ratio meaning that red eyes would be 75% and sepia would be 25%. The chi square value is 0.2156 and the degrees of freedom is 1. The maximum p value for 0.05 at 1 degree of freedom is 3.84. 0.2156 is less than 3.84 meaning we accept the null hypothesis.
Sex Linked Cross
The gene for eye color is carried on the X chromosome. As with humans, Drosophila males are XY, and females are XX. The possible F1 and F2 phenotypes are listed. They depend on the sex of the parents used in your initial crosses. Red-eyed males XY crossed with white-eyed females XX, should produce all XX red eyes females and all XY white-eyed males in the F1 generation. White-eyed males XY crossed with purebred red-eyed females XX will produce red-eyed females, XX and red-eyed males XY in the F1 generation.
Determine possibilities for the F2 generation. Record in your data and class data. Determine the X values.
Sex Linked Cross
The gene for eye color is carried on the X chromosome. As with humans, Drosophila males are XY, and females are XX. The possible F1 and F2 phenotypes are listed. They depend on the sex of the parents used in your initial crosses. Red-eyed males XY crossed with white-eyed females XX, should produce all XX red eyes females and all XY white-eyed males in the F1 generation. White-eyed males XY crossed with purebred red-eyed females XX will produce red-eyed females, XX and red-eyed males XY in the F1 generation.
Determine possibilities for the F2 generation. Record in your data and class data. Determine the X values.
The null hypothesis for the F2 generation would be 25% of males would have red eyes, 25% of males would have white, 50% of females would have red, and 0% of females would have white. The chi square value is 0.44 and the degrees of freedom is 1. The maximum p value for 0.05 at 1 degree of freedom is 3.84. 0.44 is less than 3.84 meaning we accept the null hypothesis.
Dihybrid Cross
The gene for normal wings is dominant (V) to vestigial wings (v). Mate a homozygous red-eyed, vestigial-winged fruit fly with a sepia-eyed, normal winged fruit fly. The expected F, phenotype would be all red-eyed, normal winged fruit flies. The expected F2 would be 9 red-normal : 3 red-vestigial : 3 sepia-normal : 1 sepia-vestigial. Record your data and class data. Determine the X values.
The null hypothesis would be a 9:3:3:1 ratio. The chi square value is 2.9706 and the degrees of freedom is 3. The maximum p value for 0.05 at 3 degrees of freedom is 7.81 which is more than 2.9706. This means that we accept the null hypothesis.
Study Questions
1. If the genes analyzed by the dihybrid cross were linked on the same chromosomes, how would that affect your results?
If the analyzed genes were on the same chromosome, there would be a larger number of traits being passed on together. For example, if red eyes and normal wings were a linked gene on the same chromosome, then there would be an increase in amount of flies with these genes.
2. Why is it important that the parental females be virgins?
The parental flies must be virgin because female fruit flies store sperm when they mate for future use. If the parental flies were not virgins, then the outcome would be compromised since there would be a mix of unwanted genes going around.
3. Why are the adult flies removed?
The adult flies are removed so that they do not mate with the next two (F1 and F2) generations of flies. This prevents any unwanted changes in teh gene pool and doesn't affect the probability of genotypes and phenotypes for the next generation.
4. Why are Drosophila favorite experimental models for genetics?
Drosophila are great for experiments based on genetics because it doesn't take a lot to take care of them (just leave them in a tube with media...), as well as their short lifespan and quick and plentiful (disgusting) breeding. This allows for generations to be born quickly and thus makes the experiment go faster.
5. Why is it necessary to analyze a large number of offspring before making conclusions about genotypic and phenotypic ratios?
It is necessary to analyze large populations before making conclusions in order to determine if the phenotype expressed in constant through out the population or if it is just a stroke of luck. This is also why chi square analysis is done, in order to check the probability of these things happening.
6. Why did you collect data for both males and females?
Data was collected for both males and females in order to determine if a phenotype is sex linked or not. If the gene was sex linked then the outcome of the data would be skewed according to the sex linked gene.
7. If you were a molecular biologist and had obtained two gene clones for eye color one isolated from pure bred wild type red eyed flies, the other from the mutant sepia eyed flies, how would you analyze these genes to identify the type of gene mutation?
In order to find the type of gene mutation, I would first find the order of nucleotides of both genes. This would allow me to compare the nucleotides and see what mutated and what is different. If there were extra nucleotides, then mutation was caused by insertion, while if there was nucleotides missing then deletion took place..
8. What possible genotypes and phenotypes would you observe if the F2 flies from the dihybrid cross were allowed to randomly mate?
If the F2 generation were allowed to mate randomly, then the phenotypes in the F3 would be similar to the phenotypes in the F2 generation with flies that have traits similar to the F2. There would be flies with red eyes and normal wings, red eyes/vestigial, sepia/normal, and sepia/vestigial.
9. Assume that during a hypothetical monohybrid cross, the double recessive gene were lethal and expressed itself early in the fly development. Would that skew the results in a large population? Would the recessive eventually disappear from the population?
The results would be skewed in a large population because of the lethal nature of the double recessive, but it wouldn't completely disappear from the population. Instead it would have a low frequency and only survive through heterozygous individuals.
10. Based on the example in the background section, set up a Punnett Square to predict the genotypic and phenotypic ratios obtained from a Dihybrid cross in the F1 generation and F2 generation?
Study Questions
1. If the genes analyzed by the dihybrid cross were linked on the same chromosomes, how would that affect your results?
If the analyzed genes were on the same chromosome, there would be a larger number of traits being passed on together. For example, if red eyes and normal wings were a linked gene on the same chromosome, then there would be an increase in amount of flies with these genes.
2. Why is it important that the parental females be virgins?
The parental flies must be virgin because female fruit flies store sperm when they mate for future use. If the parental flies were not virgins, then the outcome would be compromised since there would be a mix of unwanted genes going around.
3. Why are the adult flies removed?
The adult flies are removed so that they do not mate with the next two (F1 and F2) generations of flies. This prevents any unwanted changes in teh gene pool and doesn't affect the probability of genotypes and phenotypes for the next generation.
4. Why are Drosophila favorite experimental models for genetics?
Drosophila are great for experiments based on genetics because it doesn't take a lot to take care of them (just leave them in a tube with media...), as well as their short lifespan and quick and plentiful (disgusting) breeding. This allows for generations to be born quickly and thus makes the experiment go faster.
5. Why is it necessary to analyze a large number of offspring before making conclusions about genotypic and phenotypic ratios?
It is necessary to analyze large populations before making conclusions in order to determine if the phenotype expressed in constant through out the population or if it is just a stroke of luck. This is also why chi square analysis is done, in order to check the probability of these things happening.
6. Why did you collect data for both males and females?
Data was collected for both males and females in order to determine if a phenotype is sex linked or not. If the gene was sex linked then the outcome of the data would be skewed according to the sex linked gene.
7. If you were a molecular biologist and had obtained two gene clones for eye color one isolated from pure bred wild type red eyed flies, the other from the mutant sepia eyed flies, how would you analyze these genes to identify the type of gene mutation?
In order to find the type of gene mutation, I would first find the order of nucleotides of both genes. This would allow me to compare the nucleotides and see what mutated and what is different. If there were extra nucleotides, then mutation was caused by insertion, while if there was nucleotides missing then deletion took place..
8. What possible genotypes and phenotypes would you observe if the F2 flies from the dihybrid cross were allowed to randomly mate?
If the F2 generation were allowed to mate randomly, then the phenotypes in the F3 would be similar to the phenotypes in the F2 generation with flies that have traits similar to the F2. There would be flies with red eyes and normal wings, red eyes/vestigial, sepia/normal, and sepia/vestigial.
9. Assume that during a hypothetical monohybrid cross, the double recessive gene were lethal and expressed itself early in the fly development. Would that skew the results in a large population? Would the recessive eventually disappear from the population?
The results would be skewed in a large population because of the lethal nature of the double recessive, but it wouldn't completely disappear from the population. Instead it would have a low frequency and only survive through heterozygous individuals.
10. Based on the example in the background section, set up a Punnett Square to predict the genotypic and phenotypic ratios obtained from a Dihybrid cross in the F1 generation and F2 generation?
11. Use the x2 statistic to determine the values for the monohybrid, dihybrid, and sex linked crosses for your individual data and or the class data. Are the hypotheses valid?
Yes the hypotheses are valid because the chi square x2 statistic was within the range of the p-value so we were able to accept the null hypotheses.
Conclusion:
In this experiment my hypothesis was proven correct since the null hypotheses were all accepted in each experiment and each different set of flies. This experiment showed that the genes that were said to be sex linked or autosomal dominant/recessive were being passed on according to how mendel's law states they work. Some ways this experiment could have gone wrong was if you checked that box, "ignore gender" during the data analysis for the sex-linked experiment. Next time this experiment could have been better done by taking/adding images from the online lab. The virtual lab was simple and mistakes could easily be corrected, as for the lab in real life... I KILLED All oUr females!! They wouldn't reproduce because I killed them or I accidentally let them go. I'm not going to lie to you, I really would kill all the flies in the world. My hatred for them burns with the force of a thOusand sUns.