The lab that follows was designed to show the transfer and expression of genes. The experiments were performed using Drosophila Melanogaster. The Drosophila were crossed through the F2 generation to show the path of the genes. The initial cross was between two stocks of flies. The first stock was homozygous for the wild traits (red eyes and rounded wings. This stock was designated with a + symbol. The second stock was homozygous for two mutations (brown eyes and a double rounded wing type). This stock was designated ds. These two stocks made up the parent generation. The parent cross was designated ds x +.
The hypothesis was that eye color and wing shape were under genetic control of two separate genes that expressed complete dominance with brick red eyes and single rounded wings being dominate over the mutant types. The traits were believed to be autosomal and not linked.
The following materials were used during the course of this experiment. Parent stocks of Drosophila Melanogaster, food media (dry mix, and water), yeast, binocular microscopes, fly-nap (anesthetizing solution), cold chambers (for anesthetizing), incubator, brushes, white index cards, two sets of vials (one for anesthetizing and one for holding the flies) and foam plugs. The first step in setting up this lab was to get the holding vials ready. Equal portions of dry media and water were added to the clean vials to form the media. Then a few flakes (5-10) of yeast were added to provide food for the newly hatched larva.
Next the parent stocks were sexed and scored. The flies were anesthetized by using cold chambers to prevent them from flying until they could be scored. Sex was determined by placing the Drosophila on a white index card and viewing them under binocular microscopes. A small paint brush was used to move the flies without harming them while checking for the distinctive characteristics (i.e. sex combs) to determine the gender of the flies. Phenotypes were also determined by using the binocular microscopes. The + females were then placed in the same vial with the ds males. The reciprocal cross was also performed. Each vial was then sealed with a foam plug. The vials were then placed in an incubator.
Approximately five days after set up the F1 generation larvae appeared. Then eight days after set up the F1 larvae began to pupate. At this point the parent generation adults were released to prevent any mating between generations that would affect the outcome. When the F1 generation emerged as adults they were anesthetized using the chemical fly-nap. They were then sexed and scored in the same manner as the parents stocks. Seventy females and fifty males were counted. All adults displayed the wild phenotype. The F1 generation flies were then placed in a new vial.
The same process was followed for the F1 generation until the F2 larvae began to pupate. At this point the F1 adults were released. When the F2 generation emerged as adults they were anesthetized using fly-nap and were then sexed and scored. After the F2 generations were scored they were then eliminated to prevent recounts or cross breeding.
When the P2 (ds) x P1 (+) test cross is performed to produce the F1 generation there is an expected ratio of 1:1 for the heterozygous genotype. After performing this initial cross 120 offspring were collected, sexed and scored. Of the seventy females and fifty males, all 120 were heterozygous dominate as shown in the figures below (fig. 1)
The F1 generation was then selfed to produce the F2 generation. The expected ratio for the F2 generation was a 9:3:3:1 with the Wild (+), Double-Rounded wings, Brown eyes, and Double Mutant being the respective phenotypes. The F1 cross yielded 293 offspring. Using the 9:3:3:1 ration the expected numbers were 164.8125: 54.9375: 54.9375: 18.3125. The actual results obtained were 163: 57: 51: 22 as shown below (fig. 2.)
Fig 1: F1 Results
|Phenotype||Red Eyes, Rounded Wings||Red Eyes, Rounded Wings|
Fig. 2: F2 Results
The observed ratios did not fit the expected ratios exactly so a Chi-Square analysis was performed to ensure that the ratios observed still fit the expected. The Chi-Square test and results are shown below (fig. 3).
Fig. 3: Chi-Square Analysis
(163-164.8125)2 / 164.8125 = (-1.8125)2 / 164.8125 = 3.2851562 / 164.8125 = .0199326
(57-54.9375) 2 / 54.9375 = (2.0625) 2 / 54.9375 = 4.2539062 / 54.9375 = .0774317
(51-54.9375) 2 / 54.9375 = (-3.9375) 2 / 54.9375 = 15.503906 / 54.9375 = .2822098
(22-18.3125) 2 / 18.3125 = (3.6875) 2 / 18.3125 = 13.597656 / 18.3125 = .7425341
1.1221082 = x2
df = 4-1 = 3
.05 = 7.82
.01 = 11.35
The results of the Chi-Square showed that the outcome was non-significant and could then be accepted. Because the hypothesis was shown to be correct no further testing for linkage was necessary.
As mentioned above the hypothesis for this experiment was that eye color and wing shape are controlled by different alleles that show complete dominance with brick-red eyes being dominate over brown eyes and single rounded wings being dominate over double rounded wings. The results of the experiments showed this hypothesis to be correct. The initial test cross of the parent generation produced a F1 generation with an expected 100% heterozygous genotype. The results shown in Figure 1 above show this outcome to be true. Furthermore, the fact that all of the F1 generation displayed the wild phenotype shows that the red-eyes and rounded-wing genes were the dominant alleles.
The F2 generations also held true to the initial hypothesis by displaying the expected 9:3:3:1 ratio. However, it should also be noted that of the 293 flies reported in the findings there were also twelve known flies that were undeterminable due to unfolded wings or incomplete emergence from the pupa. Also to be noted is the fact that although 293 flies were produced the vials still contained unhatched pupa and larvae along with a few adults that had become logged in the media. The experiment was ended at 293 adults in the F2 generation due to time constraints and to prevent possible mating of the F2 generation that might skew the results of this test. It is believed, however, that despite the relatively few unknowns and the undeveloped adults, the outcome would still have yielded similar results had it been drawn out to include all of the F2 generation.
The data outlined above shows a pattern that fits the starting hypothesis and conventional outcomes for a dihybrid cross with two genes that each express complete dominance. One aspect that has not yet been mentioned is the placement of the genes. The hypothesis also stated that it was believed that the traits were not sex-linked. The proof of this lies in the outcome of the crosses. In this experiment the males of the parent generation were the carriers of the mutant gene. Had this been the case all of the F1 generation would appear as wild types (assuming the wild is dominate), which is what was expected (fig 4). However in the F2 only males would appear to have the mutation. And the ratio of the mutant phenotype would only be 1:3 (mutant:wild shown in fig. 5). It is important to remember that the reciprocal cross was also performed. Had the genes been sexed linked the reciprocal cross would have yielded different results. The F1 generation would have produced a ratio of 1:1 with all of the males bearing the mutant phenotype (fig 6). This was shown not to be the case.
Fig. 5: Expected Results of F1 Cross if Sex Linkage Occurred
Fig. 4: Expected Results of Parent Cross if Sex Linkage Occurred
|X ds||X+X ds||X+X ds|
Fig. 6: Expected Results of Reciprocal Parent Cross if Sex Linkage Occurred
The Hypothesis further stated that the genes were not linked. Because the Chi-Square analysis of the F2 results was non-significant this is shown to be true. Had the results of the Chi-Square been show to be highly significant further testing could have been performed to determine the linkage, however, this was not necessary.
In conclusion, the above data was presented to show the methods used and the results obtained from an experiment designed to test the hypothesis that the genes for eye color and wing shape in Drosophila Melanogaster were under the genetic control of two separate autosomal genes both bearing complete dominance with red eyes and single rounded wings being dominated over brown eyes and double rounded wings, also that these genes were not linked. It is believed that the data contained in this report shows this hypothesis to be true.