Nathan Merritt March 19, 2006

Mendelian Inheritance Patterns in Drosophila Melanogaster

Abstract:
To determine the genotypes of an initial (parental) generation of Drosophila Melanogaster by observing both the F1 and F2 generations and applying Chai Square analysis to the F2 offspring. Knowledge of Mendelian inheritance patterns will be used to determine, to a 95% certainty, the genotypes and phenotypes of both the parental and F1 generations. Three Drosophila Melanogaster crosses will be observed separately as they interbreed to create two generations of offspring. The F2 generation of each cross will be closely examined and quantitative pheneotypical data will be taken.

Introduction:
To the peoples of the late 1800's, the system that regulated inheritance and inherited characteristics was still a total mystery. The popular belief at the time was that the two parent's traits were "blended" together to produce a hybrid offspring with half the traits of each parents. This theory was popularized by what we now know as incomplete dominance of alleles, a prime example of which is the pea plant: where red and white "parent" flowers give rise to pink offspring. It appeared to people at that time that the traits of the parents were blended together much like two glasses of juice. Scientists and breeders however, often observed data that directly contradicted this theory of blending. The same two pea plants, when allowed to breed for another generation, produced red and white colored flowers as well as the pink. Had the blending theory been correct, one could expect only pink pea plants from the union of two pink plants. Similar data were observed by breeders of dogs and horses, who often followed blood lines across generations. Gregor Mendel, a monk and mathematician in the mid 1800's, was the first to apply quantitative methods to the problem of inheritance. He choose specific observable traits in pea plants, and then inbred the plants until he had created a pure line of each trait. By using true-breeding traits, that is traits that get passed on and expressed in offspring, Mendel was able to trace the motion of genetic information among his pea plants. Mendel developed techniques to prevent the peas from self-pollinating, enabling him to control both parents in a cross with absolute certainty. Mendel's experiments combined plants in which a single characteristic was expressed in one parent and not observed in the other. He noted that every single offspring of the first cross, the F1 generation in his language, expressed only one of the two traits. For example, when the parental, or P, generation was a white flower and a purple flower, every single offspring was purple. It appeared to Mendel, and other scientists of the time, that the "whiteness" was swallowed up and disappeared entirely after the first cross.
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Mendel's genius was in his thorough scientific method. Instead of discarding the entirely purple F1 generation, Mendel inbred them a second time to create a second generation of offspring, commonly referred to as the F2 generation. Interestingly, there were white flowers in the F2 generation, something clearly not possible had the model of blending been correct. Mendel recorded immense amounts of data from these crosses and noticed that mathematically the ratio of white plants to purple plants approached 1 : 3 the larger the data sets became. Repetition with other traits revealed that all the other chosen characteristics followed the same pattern, in which one trait re-emerged in the F2 at a ratio of 1 to 3. The consistent appearance of the data led Mendel to dub the trait expressed in the F1 generation by all the plants as the "dominant" trait, and the trait that re-appeared the "recessive." Although he had no knowledge of genetics, indeed the field would not even be invented for many, many years, Mendel's hypothesis was that the more commonly expressed trait dominated the recessive trait, and would be expressed whenever it was present. Conversely, the recessive trait could only be expressed when two "recessives" (as Mendel had no knowledge of alleles) were present and there was no dominant to override them. This conclusion explained what we now call complete dominance, but left Mendel struggling to model the aforementioned red, white and pink pea plants. He eventually proposed that sometimes a trait could be controlled by both alleles, allowing for the observed "blending" of half white pigment and half red. Drosophila Melanogaster have a germination time of 10-14 days, and are completely sexually mature less than 24 hours after hatching. This short maturation period, as well as the large number of eggs each female can lay, make the Drosophila Melanogaster, or fruit fly, an extremely useful tool for genetic study. Immense sample populations can be bred in relatively short periods of time, allowing for both statistical research and frequent genetic mutation. Males can be differentiated from females by a set of sex combs on their front legs. Also, females generally have a lighter and larger abdomen.

Materials and Methods:

Quantities of the following were used throughout the duration of the experiment for each of the three crosses. Sterile plastic vials with foam covers Fruit fly nutrition compound (with preservatives) Glass microscope plates Ice packs Light microscopes Fine-bristle paintbrushes The following were received prior to the beginning of the lab. Three Drosophila Melanogaster crosses, labeled A, B and C - transported in separate sealed plastic vials Recording materials Drosophila Melanogaster anaesthetic
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Electric refrigeration unit and styrofoam insulating boxes Three work groups were created and each assigned to a Drosophila Melanogaster cross vial. Upon receiving the vials, each work group allowed the parental (initial) generation to mate and lay eggs in their vials. After five days, the parental flies where moved to new vials, each containing five males and five females. This was done in an effort to expand the total population, allowing for statistically significant conclusions to be drawn. The parental generation was observed under light microscope for basic physical characteristics before being moved to fresh vials. After the second round of F1 eggs had been lain the parental generation was removed from the vials and disposed of, in an effort to protect the genetic pool. Had the parental generation been allowed to breed with the newly hatched F1 generation no conclusions could have been drawn from the final F2 data. The F1 generation of eggs was set aside and allowed to hatch. Again, all live flies were removed from the vials to avoid unwanted cross breeding. Flies were moved using a paintbrush to chilled glass microscope plates, where the F1 generation was generally observed under light microscope, however no quantitative data were taken. The F1 flies were relocated to vials containing five males and five females, these vials were again set aside to allow the F1 flies to mate and lay F2 generation eggs. After the F2 eggs had been laid, all F1 flies were removed from their vials and disposed of. After 11 days, the F2 generation of eggs began hatching. The newly born flies were moved out of their birth vials and frozen, allowing researchers to extensively catalog their expressed physical features. Data were taken on the sex of the flies, as well as any other varying physical characteristics in each cross. After a complete census the F2 generation was discarded. Data were posted, and each work group analyzed their own data as well as the data for the other crosses. After all data were collected, Chai Square analysis was applied to determine whether or not the Drosophila Melanogaster crosses had followed the predicted Mendelian inheritance patterns.

Results and data:

Cross A data: Generation Parental (P): First Offspring (F1): Second Offspring (F2): Male Red Eye Normal Wing Red Eye Red Eye: 88 Sepia Eye: 43 Female Sepia Eye Normal Wing Red Eye Red Eye: 82 Sepia Eye: 32

Cross B data: Generation Parental (P): Male Sepia Eye Normal Wing Female Red Eye Normal Wing
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First Offspring (F1):

Red Eye Normal Wing Sepia Eye & Normal Wing: 27 Sepia Eye & Vestigial Wing: 3 Red Eye & Normal Wing: 67 Red Eye & Vestigial Wing: 28 Cross C data:

Red Eye Normal Wing Sepia Eye & Normal Wing: 27 Sepia Eye & Vestigial Wing: 1 Red Eye & Normal Wing: 50 Red Eye & Vestigial Wing: 6

Second Offspring (F2):

Generation Parental (P): First Offspring (F1): Second Offspring (F2):

Male White Eye Normal Wing White and Red Eye Red Eye: 66 White Eye: 119

Female White Eye Normal Wing White and Red Eye Red Eye: 50 White Eye: 67

Chai Square analysis for Cross A: Hypothesis: In Cross A, the Sepia Eye allele is recessive to the Red Eye allele Total number of flies counted in Cross A is 244. According to Mendelian Genetics, the recessive allele should account for one quarter of the total F2 progeny, while the dominant is three quarters. Therefore, we expect 61 sepia eyed flies, and 183 red eyed flies. Because an eye color can only be either red or sepia (black) in this cross, there is only one degree of freedom. Operating at a 95% of certainty with one degree of freedom, our Chai Square number is 3.84. For the hypothesis to be true at this probability, our calculated value must be lower than this number, where a calculated value of 0 indicates a perfect statistical match. Observed Sepia eye, Male plus Female = 43 + 31 = 74. Observed Red eye, Male plus Female = 88 + 82 = 170. Using the Chai Square formula, the sum of all: (Expected - Observed)^2 / Expected ((61-74)^2 / 61) + ((183-170)^2 / 183) = 1.984 + 0.923 = 2.907. Therefore, our calculated chai square value is 2.907 to three significant figures. This is less than the official chai square value for 95% certainty and one degree of freedom, therefore we can accept the hypothesis. The allele for Sepia Eye is recessive to the allele for Red Eye in the Drosophila Melanogaster Cross A.

Conclusion:
Cross A nicely exemplifies a Mendelian mono-hybrid cross. In a mono-hybrid cross, the final result is a one to three ratio of the expressed recessive trait to the expressed dominant trait. This fits with the observed Cross A F2 data, and allows us to conjecture that the F1 and P generation must also follow the mono-hybrid cross pattern. By this
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logic, the F2 generation must be composed exclusively of organisms with the b+b genome, that is they must all be heterozygous dominant for the desired trait. This notation indicates one copy of the wild-type gene for eye color, b+ (expressed as red eyes) and one copy of the mutant allele, notated as b and expressed as black eyes in the flies. Further study shows that all observed members of the F1 generation had red eyes, and since we know their genome is b+b, we can conclude that b+ (red eyes) is dominant over the sepia eye allele. Carrying this logic up to the parental generation, the only way for an even distribution of b+ and b is if both parents are homozygous, one recessive and one dominant. In meiosis, when both parents are homozygotes for a given trait there is no chance involved in how the offspring will receive alleles. Each parent will give one copy of the gene, so a F1 genotype of b+b clearly indicates that the parents are homozygous. In this case, we know also know from earlier observation that only male flies of the F1 generation have red eyes, therefore we can finally conclude the genotype of both members of Cross A: (Male) b+b+ XX (Female) bb Cross B is interesting because it can not be completely classified as a Mendelian dihybrid cross. The Sepia/Red gene is expressed as the recessive Sepia eye in 28% of the F2 generation, strongly suggesting that the F1 is a monohybrid cross. It follows then that the parental generation in Cross B contains male homozygous recessive and female homozygous dominant organisms for the Sepia/Red eye color allele. Due to the law of independent assortment, these data do not allow any conclusions to be drawn about the gene controlling wing size. Cross B is unusual in that the second set of genes do not exactly fit into a textbook Mendelian model. The allele assortment still follows Mendel's laws of inheritance, but it begins differently. As there is clearly a mutated allele present in the F2, somewhere in both the F1 and parental generations the same allele for vestigial wings must exist. Since the vestigial wings were not observed in either generations, the allele for vestigial wing must be recessive to the wild-type allele for straight wings. Furthermore, the only way for there to be no homozygous recessive flies in the F1 generation is if only one of the parents in any cross carried the recessive allele. Otherwise there is a chance that two recessives would be passed on, resulting in a recessive F1 fly. Assuming that the observations were taken correctly, the data strongly indicate that the parental genotype is as follows, noting that it is not possible to determine the sex of the parent that carried the recessive allele: (Male) bbv+v XX (Female) b+b+v+v+ or alternatively: (Male) bbv+v+ XX (Female) b+b+v+v Cross C is a Mendelian monohybrid cross of a sex-linked characteristic. The mutated allele causing white eyes was shown by Morgan to be located on the X chromosome of the Drosophila Melanogaster. With this in mind, we know that the only way to have both red

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and white eyed males in the F1 generation is to have red eyed heterozygous females in the parental. As males receive only one X chromosome, and it always comes from their mother, the mother must be heterozygous in order for there to be a chance that she gives out either a w (white eyes, mutant) or w+ (red eyes, wild type). The data indicate that there were only white eyed females in the parental, but this does not hold up against the assertion that there were both red and white eyed males in the F1. Upon examining the F2, we notice that there are white eyed females. Morgan's research demonstrated the white eyed allele to be recessive to the wild type, so the only way a female could be white eyed was if she was homozygous recessive for the white eyed allele. This could only happen if both parents had a recessive allele in their genotypes, proving that both male and female white eyed flies must have existed in the F1. By examining all three generations together we can conclude that the genotype of the parental generation is as follows: (Male) w- XX (Female) ww and w+w

Bibliography:
Ward's AP Biology Lab 7, Ward's Natural Science Establishment, Inc. 2002.

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