DETERMINING THE LOCATION OF GENES IN DROSOPHILA MEANOGASTER BY OBSERVING PHENOTYPIC RATIOS OF OFFSPRING July 7, 2014

ABSTRACT The purpose of this study was to find where genes for specific traits are located, either on the autosomes or sex chromosomes. We determined this by examining two different D. melanogaster populations for differences in the phenotypic ratios of offspring. D. melanogaster were chosen for our study because they are easy to care for, and they have a short reproduction cycle. D. melanogaster have only eight chromosomes making them less complex organisms for study. Based on our results, we accepted our autosomal inheritance hypothesis because the ratio observed in the F2 generation was 3.23:1, which shows no statistical difference to our expected ratio of 3:1, indicating that the gene for wing presence is on an autosome. We also accepted the X-linked inheritance hypothesis because the ratio observed in the F2 generation was1.01:1, which showed no statistical difference to our expected ratio of 1:1, indicating that the gene for eye color is on the X chromosome.

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INTRODUCTION Genetics is the branch of biology concerned with heredity and variation (Cumming and Klug, 2000, p.5). Heredity is the passing of traits from a parent to an offspring. For example, handedness, the preference of using one hand over the other, is a trait that can be passed down from parents to their offspring in humans. Variation is the genetic differences among individuals of the same species. For example, offspring could either be left or right handed. These traits are passed through chromosomes. Chromosomes come in homologous pairs called autosomes, and one non-homologous pair called sex chromosomes. The number of chromosome pairs depends on the species. For example, humans have 23 pairs of chromosomes, 22 pairs of autosomes and one pair of sex chromosomes. Fruit flies have four pairs of chromosomes, three pairs of autosomes and one pair of sex chromosomes (Fig. 1). Chromosomes are coiled strands of DNA molecules found in every cell of the body of an organism. DNA molecules are the genetic codes that contain all the information needed to build and maintain a species (Cumming and Klug, 2000, p.6). DNA has segments that represent genes that code for specific traits in individuals (Fig. 2). These genes can either be on autosomes or sex chromosomes. In homologous chromosomes, autosomes, each pair is identical in length and in the location of the centromere, as well as the size and location of genes (Cumming and Klug, 2000, p.6). In non- homologous pairs, sex chromosomes, the chromosomes are not identical because in the pair, one chromosome is larger than the other, and they do not have the same genes. Each type of chromosome contains specific genes that determine the traits of an offspring.

1A 1B

Fig. 1. – This figure shows entire sets of chromosomes for humans and fruit flies. Figure 1A shows the 23 pairs of human chromosomes, 22 pairs of autosomes and one pair of sex chromosomes. Figure 1B shows the four pairs of fruit fly chromosomes, three pairs of autosomes and one pair of sex chromosomes.

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Chromosome Gene 1

Gene 2

DNA Fig. 2. – This figure shows a chromosome, the DNA that makes up the chromosome, and the gene segments of the DNA strand that code for specific traits.

Genes are segments of DNA molecules that code for specific traits (Cumming and Klug, 2000, p.7). Genes store information about traits and give variation among individuals. For example, humans have a gene location that determines handedness. An allele is an alternative form of a gene. The gene location in a chromosome pair will be the same, but the alleles can be different. The combination of alleles determines the expression of traits. For example, alleles will determine if an offspring is left handed or right handed. In Figure 3, the genes are for the same trait of handedness, but the chromosomes have different alleles for either left handedness or right handedness. The traits that are expressed depend on if the alleles are dominant or recessive. Dominant alleles, in this case right handedness, are those that are always expressed (Fig. 3). Dominant alleles also have the ability to mask other alleles. The alleles that are masked in the presence of a dominant allele are known as recessive. Recessive alleles, in this case left handedness, will only be displayed if there are two recessive alleles present and no dominant alleles present for the trait (Fig. 3). Since the offspring in Figure 3 has one dominant and one recessive allele, they will display the dominant trait of being right handed, with the recessive trait of left handedness being masked. The appearance of an offspring is determined by different combinations of the alleles present.

Fig. 3. – This figure shows a homologous pair of chromosomes with the gene location for handedness. One chromosome has the dominant allele for right handedness, and the other chromosome has the recessive allele for left handedness. 3 P:\RMSC\RMS\Group Papers\FruitFly-2014.docx Saved: 10/2/2015 10:14 AM Printed: 10/2/2015 10:14 AM

The phenotype is the physical appearance of a trait that is determined by the alleles present in an offspring. For example, the phenotype would be if an offspring writes with his or her left hand or right hand. Hand preference is the expression of the alleles in the individual. During reproduction, each parent gives the offspring half of a set of chromosomes. When chromosomes are passed, the genes from each parent are passed as well, contributing a different set of alleles that determine the traits expressed in the offspring. Offspring can either be homozygous or heterozygous for traits. A homozygous dominant offspring (RR) contains two dominate alleles and will express the dominant trait of right handedness (Fig. 4A). A homozygous recessive offspring (rr) contains two recessive alleles. Since there are no dominant alleles to mask the recessive alleles, the individual will express the recessive trait of left handedness (Fig. 4B). A heterozygous offspring (Rr) contains both a dominate and recessive allele. Since a dominant allele is present, the recessive allele will be masked, making the individual right handed (Fig. 4C) .

4A 4B

R r

Homozygous dominant Homozygous recessive (RR) (rr)

R r

4C

R

Heterozygous (Rr)

r

Fig. 4. – This figure shows the different allele combinations for handedness on pairs of homologous chromosomes. Figure 4A shows a homozygous dominant (RR) pair. Figure 4B shows a homozygous recessive (rr) pair. Figure 4C shows a heterozygous (Rr) pair.

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Most genes are passed on the autosomes of an organism. Autosomes come in homologous pairs with the same size and location of genes. Because of this, offspring receive two alleles for each trait, one from each parent. Alleles are also passed on the sex chromosomes of an organism. There is only one pair of sex chromosomes in each set. Sex chromosomes determine the gender of the offspring. Theses chromosomes will be either be X or Y. Females will receive an X chromosome from both parents, while males will receive an X from the mother and Y from the father as shown in Figure 5. The X chromosome is much larger and has more genes than the Y chromosome. There is only a small homologous pairing region where the genes are the same on both chromosomes. For some traits, the X chromosome carries an allele that the Y chromosome does not (Fig.6). Females will receive two total alleles, one on each X chromosome. However, males will only receive one allele, which is on the X chromosome, because the Y chromosome is not large enough to hold a matching allele (Cain at el, 2008, Pg.289) (Fig. 6). The allele that is present on the male X chromosome must always be expressed since there is no matching allele on the Y chromosome.

Female sex chromosomes Male sex chromosomes

Fig. 5. – This figure shows the sex chromosomes for male and female offspring.

Fig. 6. – This figure shows the differences in the X and Y chromosome including size and number and location of genes present on each. The green segment on the X chromosome represents a gene that is present on the X chromosome, but not on the Y chromosome.

The purpose of this study is to find where genes for specific traits are located, either on the autosomes or sex chromosomes, by examining the phenotypic ratios of the offspring. We will accomplish this by comparing our observed ratios to the ratios determined by previous studies on autosomal and sex-linked inheritance. In previous studies it has been shown that if the gene for a trait is located on an autosome, the phenotypic outcome in the F2 generation will show a three dominant: one recessive ratio when the P1 (parental) generation has one homozygous

5 P:\RMSC\RMS\Group Papers\FruitFly-2014.docx Saved: 10/2/2015 10:14 AM Printed: 10/2/2015 10:14 AM dominant (RR) parent and one homozygous recessive (rr) parent (Fig. 7). In studies on X-linked inheritance, it has been shown that if the gene is located on the X chromosome, the phenotypic ratio in the F2 generation will show r r one dominant: one recessive ratio when the female in the P1 generation is homozygous recessive (X X ) and the male is dominant (XRY) (Cumming and Klug, 2000, p.93-94) (Fig. 8).

r r R r

RR R Rr Rr R RR Rr

rr R Rr Rr r Rr rr

P1 Generation

Fig. 7. – This figure shows the outcomes of offspring for each generation when the P1 generation has one homozygous dominant (RR) parent and one homozygous recessive (rr) parent, and the gene is located on an autosome. The red circles show the three dominant offspring, and the blue circle shows the one recessive offspring, giving the phenotypic ratio of 3:1. R r X Y X Y

r R r r R R r R r r X X X X Y X X X Y X X X R

X Y r R r r r r r r X X X X Y X X X X Y

Fig. 8. – This figure shows the outcomes of offspring for each generation when the P1 generation has one homozygous recessive female (XrXr) parent and one dominant male (XRY) parent, and the gene is located on the X chromosome. The red circles show the two dominant offspring, and the blue circles show the two recessive offspring, giving the phenotypic ratio of 1:1.

For our study Drosophila Melanogaster were chosen because they are easy to care for, and they have a short reproductive cycle (Fig. 9). The short reproductive cycle allows for a large sample size to be gathered in a short amount of time, giving us more accurate data for our time frame. D. melanogaster have only eight chromosomes, four chromosomes from each parent. Due to only having eight chromosomes, they are less

6 P:\RMSC\RMS\Group Papers\FruitFly-2014.docx Saved: 10/2/2015 10:14 AM Printed: 10/2/2015 10:14 AM complex organisms, making them good study subjects for genetics. D. melanogaster were also chosen because of their clear and obvious differences in phenotype, which made data collection easier and more accurate. They were also chosen because we were able to purchase the parental generations we needed with the specifications necessary for obtaining our desired observed phenotypic ratios.

Fig. 9. – This figure shows an example of the D. melanogaster (fruit flies) that were used in our study.

The autosomal inheritance hypothesis is the phenotypic ratios in the D. melanogaster population in the F2 generation will be 3:1 when the P1 generation has one homozygous dominant (RR) parent and one homozygous recessive (rr) parent, indicating the gene is on an autosome. The X-linked inheritance hypothesis is the phenotypic ratios in the D. melanogaster population in the F2 generation will be 1:1 when the P1 generation has one homozygous recessive female (XrXr) parent and one dominant male (XRY) parent, indicating the gene is on the X chromosome.

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METHODS

To begin our study, we purchased two sets of F1 generation larvae. The P1 generation, the parental generation, were bred by Carolina® Biological Supply Company. One set had a homozygous dominant (RR) parent and a homozygous recessive (rr) parent. The other set had a homozygous recessive female (XrXr) parent R and a dominant male (X Y) parent. We received the F1 generation, first filial generation, which were the offspring of the P1 generation. The F1 generation flies were crossed by our instructors, allowing the D. melanogaster to produce the F2, second filial, generation. The F2 generation displayed varying phenotypic ratios (3:1 or 1:1), depending on the location of the specific gene. These flies were used for our data collection and analysis. During our experiment, The D. melanogaster were separated based on gender (Fig. 10). The male genitalia is surrounded by heavy, dark bristles which does not occur on females (Flagg, 2000, p.12). The abdomen is darker in males with one thick stripe, while females have a lighter abdomen with multiple evenly spaced stripes. The tip of the abdomen is more rounded in males than in females. In general, male D. melanogaster are smaller than females. We also looked at the P1 generation to see the wild-type characteristics for D. melanogaster. Wild-type is the normal traits found in the population. They had several common characteristics, such as red eyes, six legs, two wings, and leg bristles (Fig. 11).

Fig. 10. – This figure shows the differences in male and female D. melanogaster that were used to separate the flies by gender.

Fig. 11. – This figure shows the wild-type characteristics of the D. melanogaster population: red eyes, six legs, two wings, and leg bristles.

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The F2 generation was used for our data collection and analysis. The F2 generation were the offspring of the F1 generation. To test our hypotheses, the D. melanogaster had to be examined and sorted based on phenotypic differences. First, we tapped the bottles on a hard surface so the D. melanogaster would move to the bottom of the vial. Then, we transferred them into an empty vial and closed it with a plug. We inserted a wand with Fly Nap® into the vials to anesthetize the D. melanogaster. We waited five minutes for them to fall asleep and put them onto a blank note card, making it easier to observe them under a microscope. The D. melanogaster were first sorted by gender then by visible phenotypic differences.

Each set of F2 generation flies displayed different phenotypes. Data was collected about the number of males and females who had wild-type characteristics or non-wild-type characteristics. The first set of flies showed visible differences in wing presence, expressing either wings or no wings (Fig. 12A). The second set showed visible difference in eye color, expressing either red eyes or white eyes (Fig. 12B). After determining which D. melanogaster had the wild-type and non-wild-type characteristics, we calculated the ratios of wild-type: non wild- type for each set of flies. Finally, we used Chi Square Analysis to determine if there were statistical differences between the expected ratios and our observed ratios.

4A 4B

Fig. 12. – This figure shows the phenotypic differences that we observed in our sets of flies. Figure 12A shows an example of flies with white eyes and red eyes. Figure 12B shows an example of a fly without wings.

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RESULTS The first set of flies we observed and recorded were flies with and without wings (Table 1). In our study there were 563 males with wings and 167 males without wings. There were 705 females with wings, and there were 225 females without wings. Out of a sample size of 1660 there were 1268 flies with wings and 392 without wings.

Table 1. – This table shows the scores of D. melanogaster with and without wings

Wings No Wings

Males 563 167

Females 705 225

Total 1268 392

Total flies 1660

Then we calculated the expected results and ratios for each type of inheritance so we could compare them to our observed results and ratios (Table 2). To calculate our 1:1 ratio expected results we had to take our total number of flies, which was 1660, and multiply it by .5. We multiplied the total number of flies by .5 because a 1:1 ratio is equal to 50% of the total flies observed. We got 830 for flies both with and without wings. Then we had to perform a Chi Square Analysis for our observed and expected ratios. For this 1:1 ratio our Chi Square value was 462.3. Since our Chi Square value for our 1:1 ratio was greater than our critical value of 3.84, there was a statistical difference between our observed and expected ratios. To calculate our 3:1 expected results we had to take our total number of flies, which was 1660, and multiply it first by .75 to get the expected number of flies with wings. We calculated 1245 flies with wings. Then we multiplied the total number of flies by .25 to get the total number of flies without wings. We calculated 415 flies without wings. We multiplied by .75 and .25 because the expected 3:1 ratio is equal to 75% and 25% of the total flies observed. Then we had to perform a Chi Square Analysis for our observed and expected ratios. For this 3:1 ratio our Chi Square value was 1.7. Since our Chi Square value for our 3:1 ratio was less than our critical value of 3.84, there was no statistical difference between our observed and expected ratios.

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Table 2. – This table shows the observed and expected results and ratios for D. melanogaster with and without wings

Observed Expected Expected

Wings 1268 830 1245

No Wings 392 830 415

Ratios 3.23:1 1:1 3:1

Second we observed and recorded the set of flies with red eyes and white eyes (Table 3). In our study there were 292 males with red eyes and 545 males with white eyes. There were 588 females with red eyes, and there were 329 females with white eyes. Out of a sample size of 1754 there were 880 total flies with red eyes and 874 total flies with white eyes.

Table 3. – This table shows the scores of D. melanogaster with red eyes and with white eyes

Red Eyes White Eyes

Males 292 545

Females 588 329

Total 880 874

Total flies 1754

Then we calculated the expected results and ratios for each type of inheritance so we could compare them to our observed results and ratios (Table 4). Like our previous calculations, when calculating our 1:1 ratio results we had to take our total number of flies, which was 1754, and multiply it by .5. We got 877 for flies both with red eyes and white eyes. Then we had to perform a Chi Square Analysis for our observed and expected ratios. For this 1:1 ratio our Chi Square value was .02. Since our Chi Square value for our 1:1 ratio was less than our critical value of 3.84, there was no statistical difference between our observed and expected ratios. To calculate our 3:1 expected results we had to take our total number of flies, which was 1754, and multiply it first by .75 to get the expected number of flies with red eyes. We calculated1315.5 flies with red eyes. Then we multiplied the total number of flies by .25 to get the total number of flies with white eyes. We calculated 438.5 flies with white eyes. Then we had to perform a Chi Square Analysis for our observed and expected ratios.

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For this 3:1 ratio, our Chi Square value was 576.7. Since our Chi Square value for our 1:1 ratio is greater than our critical value of 3.84, there was a statistical difference between our observed and expected ratios.

Table 4. – This table shows the observed and expected results and ratios for D. melanogaster with red eyes and with white eyes.

Observed Expected Expected

Red Eyes 880 877 1315

White Eyes 874 877 439

Ratios 1.01:1 1:1 3:1

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CONCLUSIONS AND DISCUSSION The purpose of this study was to find where genes for specific traits are located, either on the autosomes or sex chromosomes, by examining the phenotypic results of the F2 generation. The autosomal inheritance hypothesis is the phenotypic ratio in the D. melanogaster population in the F2 generation will be 3:1 when the P1 generation has one homozygous dominant parent and one homozygous recessive parent, indicating the gene is on an autosome. The X-linked inheritance hypothesis is the phenotypic ratio in the D. melanogaster population in the

F2 generation will be 1:1 when the P1 generation has one homozygous recessive female parent and one dominant male parent, indicating the gene is on the X chromosome. We accepted both the X-linked inheritance hypothesis and the autosomal inheritance hypothesis. We accepted our autosomal inheritance hypothesis because the ratio observed in the F2 generation was 3.23:1, which shows no statistical difference to our expected ratio of 3:1 for autosomal inheritance, indicating that the gene is on an autosome. We also accepted the X-linked inheritance hypothesis because the ratio observed for the F2 generation was 1.01:1, which shows no statistical difference to our expected ratio 1:1 for X-linked inheritance, indicating that the gene is on the X chromosome. One limitation that could have affected our data was when transferring the D. melanogaster into another vial, a few escaped. Also, a few D. melanogaster were damaged when transferring them into the second vial. Others also got stuck in the medium. Another limitation was our inability to examine the unhatched larvae. Other limitations include incorrect identification of phenotypes when observing D. melanogaster. These all could have affected our total counts of flies for each phenotype, altering our phenotypic ratios. To improve this experiment, every D. melanogaster we examined for data should have been examined more closely to ensure correct identification. Another improvement could be if we had been able to count the unhatched larvae. This would have allowed for a larger sample size, which could give us more accurate data. Having more practice handling and sorting D. melanogaster before starting this experiment could have given us more experience and improved our accuracy and data collection. If we were to do this experiment again, we could also allow more self-more time to breed all the generations ourselves. Several questions for future studies have been considered by our group. For example, we wondered if the

F3 generation would have the same phenotypic ratios as the F1 or F2 generations. We also wanted to know if a different species of fly were used to conduct this experiment, would we have different ratios in our offspring? Also, looking at more than one trait per individual on the autosomes and sex chromosomes could be a future study to see if traits are able to affect one another. We would also like to find out if genes for eye color and wing presence are on the same chromosomes for all organisms.

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REFERENCES Cain, M., et al. (2008). Biology Eighth Edition. San Francisco, CA: Pearson Benjamin Cummings, Inc. Cummings, M. & Klug, W. (1997). Concepts of Genetics: Sixth Edition. Upper Saddle River, NJ: Prentice Hall, Inc. Flagg, R. (2005). Carolina® Drosophila Manual. Burlington, NC: Carolina® Biological Supply Company.

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