Fruit Fly: A Genetic Analysis

Experiments performed by Gregor Mendel on garden plants and honeybees let to great success in studying theories of inheritance. Mendel crossed true-breeding tall and dwarf pea plants and obtained all tall hybrid plants, called the F1 generation. After crossing the hybrid plants he obtained tall and dwarf plants in the ratio 3:1, called the F2 generation. He then concluded that the factor for tallness, a dominant factor, masked the expression of the factor for dwarfism called the recessive factor. He also called this cross a monohybrid cross. Mendel called these factors genes and the dominant and recessive forms, alleles. Those that inherited two identical copies of the genes were called homozygous and those that inherited two different alleles

were called heterozygous. He also concluded after seeing a mixture of dwarf and tall plants that the F1 produced two gametes during meiosis, one carrying the gene for tall and the other the gene for dwarf and that they segregate during the gamete formation. Mendel also carried out crosses of pea plants with two different traits called the dihybrid cross. He found out that the F2 generation had progeny with a mixture of traits in the ratio 9:3:3:1. He then concluded that the alleles of different genes assort independently of each other. (Snudstat and Simmons, 2011)
Other scientists later discovered that genes were found on chromosomes called the chromosomal theory of inheritance. To proof that genes are located on chromosomes, scientists had to locate a gene that is linked to a chromosome. Thomas Morgan discovered a particular mutant gene that is linked to eye color in his study of the fruit fly, Drosophila Melanogaster. After observing a mutation causing white eyes instead of normal red eyes, Morgan concluded that eye color mutation was inherited as a sexual recessive trait. Two spontaneous mutations causing rudimentary wings and yellow body color, which were inherited the same way, also appeared in the progeny. He found that the cell nuclei of female fruit flies contain two identical X-chromosomes while that of males had only a single X-chromosome together with another chromosome. He called the other chromosome Y-chromosome. He then concluded that males inherit the X-chromosomes from their mothers and the Y-chromosome from their fathers. Due to the association between the sex chromosome and the segregation of the allele for eye color, Morgan concluded that the genes are located on the chromosome and that each gene can be found on a particular chromosome. (Zetterstrom, 2008).
Some mutations are dominant while others are recessive.

In dominant mutations, the phenotypic effect is expressed in both heterozygotes and homozygotes while in recessive mutations the effect is only in homozygotes. Though most mutations are sex linked, many others are autosomal. The wingless condition is a mutation caused by a gene called apterous located on chromosome 2, which is a recessive gene and is only expressed in the homozygote fly. (Carolina Drosophila manual).

The fruit fly is a model system that is used to study genetics due its short life cycle, large number of offspring, easy culturing conditions and low cost. It produces a large sample size from which predictions can be determined about inheritance patterns. It has a simple chromosome organization, 3 pair of autosomal chromosomes and 1 pair of sex chromosome. The four stages of the fruit fly life cycle are egg, larva, pupa and adult. The generation time of Drosophila melanogaster is 2 weeks: 8 days in the egg and larval stages, and 6 days in the pupal stage. The adult may live up to 8 weeks under optimal

conditions.
In this experiment, F1 progeny from a cross between flies with two true breeding traits, each with one trait were provided by the instructor in a culture medium. The experiment was performed in three weeks and the F2 phenotypes were examined to predict and hypothesize the traits of the parental cross and the expected ratios of the F1 and F2 generations. The F1 cross was set up and progeny counted after 10 days and twice after every 4 days. F2 generations were examined and the inheritance pattern determined using crossing schemes and chi square analysis. If the inheritance pattern follows Mendel’s laws based on the chi square values, the hypothesis is accepted. Otherwise it is rejected.
Purpose
The purpose of this experiment is to learn the lifecycle of the fruit fly and identify the mutants produced in the offspring in order to understand the inheritance pattern of the fruit fly. Differences between monohybrid and dihybrid crosses and their ratios are also studied to explain the Mendel’s laws of dominance, segregation and independent assortment. Sex-linked and autosomal traits are identified and chi square analysis performed to test hypothesis.
Hypothesis/prediction.
Since the F1 generation showed no mutations other than the wild-type, the F2 generation was observed to have two mutations yellow body (a sex-linked trait) and apterous (an autosomal trait). Based on this, the parental cross was between a yellow body male and an apterous female. Below are the notations that will be used in performing the crosses:
X y/Y – mutant male fly – yellow body male
X y+/Y – wild-type male for body color
X y+/X y+ or X y+/X y – wild-type female for body color
+/+ - homozygous wild-type alleles for wings
+/ap – heterozygous wild-type alleles for wings
ap/ap – homozygous apterous
P1 Yellow body male x Apterous female
X y/Y;+/+ Xy+/ Xy+;ap/ap

Table 1.0 Punnet Square showing the genotypes of P1 gametes including the
expected genotypes of the F1 generation for wings

Eggs Sperm
+ +
ap +/ap +/ap
ap +/ap +/ap

Table 2.0 Punnet Square showing the genotypes of P1 gametes including the
expected genotypes of the F1 generation for body color

Eggs Sperm
X y Y
Xy+ Xy+/ X y Xy+/Y
Xy+ Xy+/ X y Xy+/ Y

As shown on the two tables above the expected F1 ratio is 1:1:1:1 all heterozygous wild-type. A reciprocal cross of both the body type and wings will show yellow males with wings in the F1 generation and wild-type females but yellow males were not observed in the F1 flies provided.
F1 Wild-type male x Wild-type female
X y+/Y;+/ap Xy+/ Xy;+/ap
Table 3.0 Punnet Square showing the genotypes of F1 gametes including the
expected genotypes of the F2 generation for wings

Eggs Sperm
+ ap
+ +/+ +/ap
ap +/ap ap/ap

Table 4.0 Punnet Square showing the genotypes of F1 gametes including the
expected genotypes of the F2 generation for body color

Eggs Sperm
X y+ Y
Xy+ Xy+/ X y+ Xy+/Y
Xy Xy+/ X y Xy/ Y
Fig 1.0 Branch diagram showing the genotypes of the F1 gametes and the expected genotypes of the F2 generation and their corresponding ratios.
Sex Segregation of Ratios Genotypes Phenotypes
Chromosomes genes for wings
¼ +/+ = 1/16 Xy+/ X y+;+/+ wild-type
¼ Xy+/ X y+ 2/4 +/ap = 2/16 Xy+/ X y+; +/ap wild-type
¼ ap/ap = 1/16 Xy+/ X y+; ap/ap apterous
¼ +/+ = 1/16 Xy+/ X y;+/+ wild-type
¼ Xy+/ X y 2/4 +/ap = 2/16 Xy+/ X y;+/ap wild-type
¼ ap/ap = 1/16 Xy+/ X y;ap/ap apterous
¼ +/+ = 1/16 Xy+/Y;+/+ wild-type
¼ Xy+/Y 2/4 +/ap = 2/16 Xy+/Y;+/ap wild-type
¼ ap/ap = 1/16 Xy+/Y;ap/ap apterous
¼ +/+ = 1/16 Xy/ Y;+/+ yellow body
¼ Xy/ Y 2/4 +/ap = 2/16 Xy/ Y;+/ap yellow body
¼ ap/ap = 1/16 Xy/ Y;ap/ap yellow body apterous

Table 5.0 shows the expected ratios obtained from the branch diagram cross of figure 1.0. Indicates the computed ratios by adding the similar phenotypes from the different branches obtained
Phenotypes Females Males Overall
Wild-Type 6/16 3/16 9/16
Wild-Type apterous 2/16 1/16 3/16
Yellow body 0 3/16 3/16
Yellow body apterous 0 1/16 1/16
The F2 generation is expected to have 9 wild-type, 3 wild-type apterous, 3 yellow body and 1 yellow body apterous, a phenotypic ratio of 9:3:3:1. The phenotypic ratio of the females is expected to be 6:2 and that of the males is expected to be 3:1:3:1.
Methods
Materials
Caroline drosophila manual fine sorting brushes
transparent vials markers
plastic foams CO2 gas
microscope fly food
Flynap white sheet of paper

Observing parents and setting up F1 Cross
3 vials of F1 flies that are progenies of unknown P cross were obtained from instructor. The name of the stock including the date and the person who set up the cross were recorded. 3 empty vials and foam stoppers were obtained. Two drops of flynap were added to the inside of each of the foam stoppers and placed in the 3 vials; these are called napping chambers. The F1 adult flies from each of the 3 vials were transferred into the napping chambers by tapping the bottom of the culture against the palm of the hand and quickly inverting the vial to the napping chamber. The foam stopper with the flynap was placed immediately over the napping chamber to avoid flies flying off. When the flies were completely put to sleep a stereomicroscope was set up and the flies were placed on a white sheet of paper. The F1 flies were identified using sorting brushes. The phenotypes of the males and females were recorded. To set up an F1 cross, 3 new empty vials were obtained, equal amounts of instant fly food and distilled water mixed gently and allowed to solidify. The F1 flies were transferred into the 3 new culture media, the date and name of each student recorded. The flies were later placed in an incubator at 25°C.
Fly breeding and data collection
After 7 days, three more vials were set up for back-up and the original F1 flies provided by instructor were morgue and put away in a fly trash container. 14 days after, the flies were anaesthetized, counted and phenotypes including the mutants were noted. Another count was done 21 days later and the phenotypes including the mutants were also noted. A chi square table was set up for males, females and overall to test the hypothesis.
Results
35 females and 30 males were counted in the F1 flies provided and their phenotypes were all wild-type. A total of 425 flies were counted for the sample and the counts were done on three separate days.
Table 6.0 F2 Generation showing number of flies counted and the different phenotypes
Date Counted Wild-type Wild-type apterous Yellow body Yellow body apterous
Male Female Male Female Male Female Male Female Overall
11/11/14 50 98 5 9 21 4 187
11/18/14 34 97 21 25 39 216
11/20/14 5 1 2 8 6 22
Total 89 196 26 36 68 10 425

Table shows the different counts performed. Columns show the sexes and the phenotypes and the rows show the dates. The overall column shows the total for each day the count was done.

Table 7.0 Comparison of the expected phenotypes with the actual phenotypes of F2 generation
Date Expected Phenotypic Ratio Expected Number Observed Number
Phenotype Male Female Male Female Male Female
Wild-Type 3/16 6/16 72 174 89 196
Wild-Type apterous 1/16 2/16 24 58 26 36
Yellow body 3/16 72 0 68 0
Yellow body apterous 1/16 25 0 10 0
Total 193 232 193 232
Table shows the expected phenotypic ratio of both males and females including the expected number based on the total count of 425 flies.

Chi Square Analysis
Table 8.0 Chi Square analysis for F2 Male fruit flies
Phenotypic class Observed Expected O-E (O-E)2 (O-E)2/E
Wild-Type 89 72 17 289 4.01
Wild-Type apterous 26 24 2 4 0.17
Yellow body 68 72 -4 16 0.22
Yellow body apterous 10 25 -15 225 9.00
Chi Square = 13.40
Table shows the chi square conducted to verify the hypothesis.

Observed is the count that was done for the F2 male phenotypes and expected is the numbers based on the expected ratio 3:1:3:1.

The number of classes (n) = 4 so the degree of freedom n-1 = 4-1=3. Chi square value is 13.40. Using a significance level of 0.05, the p value is 0.003847. The result is significant at p ˂ 0.05

Table 9.0 Chi Square analysis for F2 female fruit flies

Phenotypic class Observed Expected O-E (O-E)2 (O-E)2/E

Wild-Type 196 174 22 484 2.78

Wild-Type apterous 36 58 -22 484 8.34

Chi Square = 11.13

Table shows the chi square conducted to verify the hypothesis. Observed is the count that was done for the F2 female phenotypes and expected is the numbers based on the expected ratio 6:2.

The number of classes (n) = 2 so the degree of freedom n-1 = 2-1=1. Chi square value is 11.13. Using a significance level of 0.05, the p value is 0.000849. The result is significant at p ˂ 0.05

Table 10.0 Chi Square analysis for overall F2

Phenotypic class Observed Expected O-E (O-E)2 (O-E)2/E

Wild-Type 285 246 39 1521 6.18

Wild-Type apterous 62 82 -20 400 4.88

Yellow body 68 72 -4 16 0.22

Yellow body apterous 10 25 -15 225 9.00

Chi Square =

20.28
Table shows the chi square conducted to verify the hypothesis. Observed is the count that was done for both F2 male and female phenotypes and expected is the numbers based on the expected ratio 9:3:3:1.

The number of classes (n) = 4 so the degree of freedom n-1 = 4-1=3. Chi square value is 20.28. Using a significance level of 0.05, the p value is less than 0.000149. The result is significant at p ˂ 0.05
All expected phenotypes were obtained: 285 wild-type, 62 wild-type apterous, 68 yellow body flies and 10 yellow body apterous. No yellow body and yellow body apterous females were observed.
Discussion
Explanation of results:
The results demonstrate that the wild-type allele (+/+) for wings is the dominant allele for wings since the F1 all showed wild-type (+/ap). Apterous is therefore a recessive mutation. Phenotypes observed for body color confirmed that the mutation is sex-linked and it is transmitted from mothers to sons. All expected phenotypic classes were observed in the males but only wild-type and apterous was observed in the females as expected. No yellow body females were observed. However, the p value for males is 0.003847, p value of females is 0.000849 and the overall p value is 0.000149. There were discrepancies between the expected numbers and the observed numbers and the results of the p values are significant at p ˂ 0.05. This shows that the results are not due to chance. The phenotypic ratio differed from the expected 9:3:3:1 and the does not support the hypothesis about the inheritance pattern. The hypothesis is therefore rejected.
Conclusions:
The fruit fly is a good model system to study inheritance pattern and observe Mendel’s laws. The experiment was performed successfully, the life cycle of fruit fly was successfully observed. Mendel’s laws of dominance, segregation and independent assortment were observed. Phenotypes were observed and counts successfully made from which analysis was done to draw conclusions. However, there are a couple of setbacks in the experiment that could have significantly affected the results. The sample size was not large enough because some of the flies got stuck in the medium while tapping for transfer. A better medium could be designed that can be easier to handle the flies since they are very small animals. It is also difficult to differentiate the phenotypes observed under the microscope because the flies are so young.