Allele Shuffling of Single Gene Traits in Drosophila
Lacey Meek
Introduction: the purpose of this lab was to help students understand and predict the passage of traits from one generation to the next through mapping the passage of single gene traits indrosophila. The use of drosophila was determined by their small size, easy maintenance, quick reproduction and maturation period, and the large number of offspring they produce.
Null Hypothesis: If the p-value is greater than 5%, then allele shuffling can be said to be random.
This experiment began by scoring parent generation flies after breeding them and placing the larvae in separate tubes. Flies were anesthetized using FlyNap© with a small application brush that was placed between the foam top and the tube until flies collapsed. After scoring, with use of a dissection microscope, flies were euthanized by placing them in alcohol. After a period of two weeks, matured larvae were anesthetized with FlyNap©, and with the use of a dissection microscope, five males and five females were separated and put into a separate tube for re-breeding of specific traits. These traits were: a sepia and wild eyed cross, a vestigial/wild and sepia/wild cross, and a white-eyed and wild-eyed cross. The rest of the flies were euthanized by placing them in alcohol. The flies separated were placed into new tubes with mashed potatoes, paired by cross and left to breed. After breeding and while the young were still larvae, parental F2 flies were removed from the tubes, leaving their offspring to mature. When matured, FlyNap© was again used to anesthetized the flies. They were then scored, and euthanized in alcohol. The data from the scorings of each group was pooled and written as a table. Students took computers and created tables with chi-square values in order to understand the relationship between expected data and observed data, to prove whether or not the null hypothesis was correct.
The results were as follows:
| Cross 1: Sepia and Wild Eyed | |||||
| Sese X Sese | |||||
| Phenotype | Observed | Expected | Chi-square Value | p-Value | Significant |
| Red-eyed | 871 | 905.25 | 1.29584 | > 5% | No |
| Sepia-eyes | 336 | 301.75 | 3.881753 | > 5% | No |
| Total | 1207 | 1207 | 5.177593 | > 5% | No |
| Cross 2: Vestigial/Wild and Sepia/ Wild | |||||
| Vgvg/Sese | |||||
| Observed | Expected | Chi-square Value | p-Value | Significant | |
| Vestigial Red | 197 | 258 | 14.4225 | < 5% | Yes |
| Vestigial Sepia | 231 | 86 | 244.477 | < 5% | Yes |
| Normal Red | 708 | 774 | 5.62791 | > 5% | No |
| Normal Sepia | 240 | 258 | 1.25581 | > 5% | No |
| Total | 1376 | 1376 | 265.78322 | < 5% | Yes |
| Cross 3: White-eyed and Wild-eyed | |||||
| Ww X Ww | |||||
| Phenotype | Observed | Expected | Chi-square Value | p-Value | Significant |
| White-Eyed Males | 228 | 216.5 | 0.610855 | > 5% | No |
| White-Eyed Females | 184 | 216.5 | 4.87875 | > 5% | No |
| Wild-Eyed Males | 240 | 216.5 | 2.55081 | > 5% | No |
| Wild-Eyed Females | 214 | 216.5 | 0.028868 | > 5% | No |
| Total | 866 | 866 | 8.069283 | < 5% | Yes |
Conclusion: The purpose of this lab was to help students understand and predict the passage of traits from one generation to the next through mapping the passage of single gene traits indrosophila. The use of drosophila was determined by their small size, easy maintenance, quick reproduction and maturation period, and the large number of offspring they produce. The students worked from a null hypothesis: if the p-value is greater than 5%, then allele shuffling can be said to be random. Students bred and scored flies in a parent generation. They then bred the F2 offspring and scored them again, pooling data in order to track the progression of genetic traits.
The data collected rarely varied widely from the expected numbers, with the exception of the second cross, where numbers of vestigial winged flies both with red and sepia eyes varied quite a bit from the expected equation. In the first cross, 871 red eyed flies were counted and 336 sepia eyed flies were counted. The expected numbers for red eyed flies was 905.25, and the expected number for sepia eyes was 301.75. This made the chi-square values approximately 1.3 and 3.9, both making the p-value greater than 5%, and therefore insignificant. In the second cross, vestigial red eyed flies were counted to be 197 when the expected was 258 and the chi-square value was 14.42. Vestigial sepia eyed flies were counted to be 231 when 86 were expected, and the chi-square value was 244.48. Normal red eyed flies were counted to be 708 when the expected was 774, and sepia red eyes eyes were counted at 240 when the expected was 258. The chi-square values for these counts in order were 5.63 and 1.26. In both vestigial results, the p-values were less than 5%, making them significant and threatened to disprove the hypothesis. In the third and final cross, there were 228 white eyed males counted when the expected was 216.5 making the chi-square value 0.61. 184 white eyed females were counted when the expected number was 216.5, making the chi-square value 4.88. Red eyed males were counted at 240 when the expected was 216.5. The chi-square value was 0.03. 214 Red eyed females were observed as well when 216.5 were expected. The individual p-values were greater than 5% but the overall was less than 5%, which is also significant.
The hypothesis stated that if the p-value was greater than 5%, then allele shuffling could be said to be random. In cross one, all p-values are greater than five, which supported the hypothesis. In cross-two, two of the results were less than 5%, as well as the overall result. These results were significant because they contrast the hypothesis. They could have been the result of a mistake made during the breeding, or because of the habitat they were in. To thoroughly prove or disprove the hypothesis, it would be best to recreate the test and add new data to what we'd already found. In the third cross, all of the values were greater than 5% except for the overall value, which was less than 5%. This is significant as well, especially when the fact that this was an overall value is considered, and should be looked into more thoroughly because of it.
The results of this experiment vary drastically, though they do overall support the hypothesis stated. The importance of this contrast is great, because it states that an outside force effected the results somehow. According to the Punnett squares that were calculated before breeding, there should have been far more flies with normal wings than vestigial, suggesting that the vestigial flies had mutated in some way beyond their wings, or that an error was made in the experiment. However, overall, the hypothesis is supported.
The results are in support of the hypothesis. However, the contrasts leave quite a bit of room for doubt. The numbers of observed vestigial flies were far too much higher than their expected numbers to leave room for confidence. According to the expected numbers, this was not a random outburst of vestigial winged flies but something that was influenced, whether preventable or no. Most preventable errors include the collection of too many vestigial flies for breeding, the loss of winged flies as they were transferred from one tube to the next, habitat quality, and loss of data from one of the experimenting groups. In the first and second cases, a second experiment should be conducted. In the third case, more research should be conducted on the habitats of these flies, and a controllable environment should be created where light may be blocked out or flies may be exposed to different temperatures. Vestigial flies could also have another mutated trait that makes them hardier than their winged counterparts. To have more conclusive results, it would be recommended that the experiment was repeated and that data used to compare against the data of this experiment.
