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Kurt Worthmann April 21st, 2010 Biology 2: Concepts in Genetics Professor: Dr. Tacka Drosophila Lab Report

The Drosophila Project

Abstract Drosophila melanogaster is a small fruit fly that feeds on and lives around spoiled fruit. It is one of the most valuable organisms in genetics research and developmental biology. Drosophilas are popularly used in studying traits because they are practical, small and have a short life cycle of about two weeks (Manning et al, 2006). We are demonstrating exactly how Drosophila melanogaster are used to identify mechanisms of transmission genetics in eukaryotes. F1 and F2 generations were obtained by performing simple parental crosses. This was done so we could determine the mode of inheritance of the genetic trait of eye color. The mode of inheritance was different for all of the crosses but all deal with Mendels principles. Our results from the chi square analysis data all showed signs of our observed dating matching almost exactly to our expected data. We were able to accept our null hypothesis for all crosses.

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Intro Drosophila melanogaster are very useful tools in the study of genetics. In this experiment we are hoping to determine phenotypic ratios and dominant vs. recessive traits by cross breeding two different types of Drosophila; such as wild type mutant (red eye) with white eye drosophila and wild type mutant (red eye) with sepia eye drosophila. Drosophilas are most commonly used organism in genetic labs because they have a short life span and genetically speaking they are a very simple organism. The mode of inheritance among traits is easy to figure out as well by applying simple Mendelian tools in this organism. Nothing about Drosophila is complicating, they have very definable features and telling females apart from males is simple. Drosophilas are popular because they can live in many different climates, they can be found on every single continent except for Antarctica. These reasons are why many scientists choose to work with them. A similar study on drosophila was performed by a man named Bentley Glass . Mr. Glass was focused on the effects of genes to eye color. Of course his was more in depth than ours, focusing on pigmentation and proteins rather than just figuring out the mode of inheritance of a specific trait. But nonetheless his experiment relates to our because of the information he uses. Simple Mendelian aspects are generated through his experiment as well as ours. It is easy for us to look over his experiment and understand better how to apply all of Mendels rules to our experiment. This scientist discovered what pigment colors were caused by which gene and that the gene was located at the far right on chromosome II at locus 104.3. When crossing the wild-type red eye fly with the mutant sepia eye we will expect to see a 3:1 ratio in the F1 Generation. For every three wild type red eye we will expect to see one sepia eye. We would expect to see the same outcome for the F2 Generation. The trait for eye color is autosomal recessive in the mutant sepia eyed drosophila. Since the trait here is not sex linked we do not care about the amount of genders produced, only the eye color trait. The allele for red eye is dominant over white and a sepia eye because red eye is standard in flies where as sepia eye color is a recessive mutant allele. After collecting data from the entire class and performing a chi square analysis we were able to determine that our predictions are correct. When crossing the wild type red eye with the mutant white eyed drosophila we expect to see different ratios as compared to the wild type, sepia cross. This difference is because the trait for white type is sex linked recessive inherited. The cross of red males to white females would produce a ratio of 1:1, 1 red eye: 1 white eye in the F1 generation. In the F2 we would than see a ratio of 1:1:1:1 because the recessive white eye appears once on the female chromosome and once on the male chromosome causing two different white eyed genders. The same thing happens for the wild type, this is why we get the 1:1:1:1 ratio. You would think that the reciprocal cross would be the same but if you perform the punnett square you see that it doesnt. The reciprocal cross of white eyed males to wild type females in the F1 generation will have a different ratio. You will see all red eye type but equal amounts in the genders i.e. 2 females and 2 males. The F2 generation will produce a different ratio though of a 1:2:1. 1 red eye male: 2 red eye females: 1 white eye male. All of these predictions above will be determined with a chi square analysis test and from here we will be able to accept our hypotheses or reject them.

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Methods and Materials

Preparing Vials of Media We obtained a sterile transparent glass vial from the side bench. Placed one cup of Carolina Instant Drosophila Medium Formula 4-24 into the glass vial. Added one cup of distilled water to the vial of medium. Placed a cotton plug in the opening of the vial. Then allowed the media to absorb for about 5 minutes before adding flies. Anesthetizing the Fruit Flies We obtained a sterile transparent glass vial from the side bench. From a different bench we grabbed a container of miniature Drosophila melanogaster provided by Carolina Supplies Company. Then we transported flies from the container of Drosophila melanogaster to the empty glass vial. The flies came out of the container by performing a slight tap with my index finger. The vial was then covered with a cotton plug. We collected a vial of anesthetizing solution (FlyNap from Carolina Supplies Co.). An anesthetic wand was dipped into the FlyNap solution than inserted into the glass vial which obtained the flies. The anesthetic wand was placed in the vial for only 15 seconds. We than waited one minute for the flies to stop moving/flying. Making Subcultures The unconscious flies were then placed onto a plain white index card. A dissecting microscope was taken from the shelf. The index card with the flies was placed under the microscope. We used a magnification of 100um. We differentiated between females and males. We did this by knowing that males have a solid black abdomen and that females have a black striped abdomen. 9 females and 7 males were collected and placed into the glass vial which contained the media gently with a paint brush. We left the vial lying horizontally so the flies didnt get absorbed by the media while they were still knocked out. Once the flies woke up they were left standing vertically under a light to make sure that they stayed at room temperature. We labeled the vial with a piece of scotch tape. On the tape we wrote our names, the type of flies, and the date. Sexing Fruit Flies in the Pupa Case We first removed all adult wild type red eye flies from the glass vial and terminated them. Now that we were only left with Pupa in the vial so we were ready to sex them. We were able to determine the sex of the mature pupa without looking at them under the microscope. The male pupa have sex combs where as the female pupa have none. This is how we differentiated between the two sexes. We selected 5 virgin females from the vial. We did this by marking them with a glass marker on the side of the vial.

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Making Parental Crosses to Obtain F1 Offspring We prepared a new culture vial with media by repeating the above steps. We than selected 5 male sepia eye flies from the container of miniature Drosophila melanogaster provided by Carolina Supplies Company. We did this by anesthetizing them and following the steps previously done. 6 female virgin pupas from my wild type red eye subculture were added into the vial of media. We added the 5 sepia eye males as well. We laid the vial horizontally until the 5 males woke up and became active again. The vile was then placed under a lamp and the vial was placed upright. We kept our eyes on the flies and waited for them to mate. Making F1 x F1 crosses to Obtain F2 Offspring We prepared a new culture vial with media by repeating the above steps. At this time my female red eye flies and male sepia flies have mated. Pupa had collected on the sides of the glass vial. It was time to sex the female and male adult flies from the F1 culture. We lightly anesthetized the flies so that we could view them under a dissecting scope. Once we sexed them we added 5 virgin females and 5 males from the F1 generation into the vial that was prepared with media. After the flies had woken up we placed the vial vertical under the lamp. We waited for the flies to mate and we recorded our observations in out notebook. We than did a chi square analysis to see if we could accept our null hypothesis. We also did punnet squares to figure out the phenotypic ratios of the F1 and F2 generations.

Results Table 1.1 Wild Type Male x White Eyed Virgin Female F1 Generation XW Xr XW Xr XW Y XW XW Xr XW Y

XW Xr = 2 Red Eye Females XW Y = 2 White Eye Males

Table 1.1 demonstrates the true-breeding parental cross between wild type (red eye) males with white eyed virgin females. In this punnett square we see a ratio of 1:1. 2 red females and 2 red males were the results. We dont see any white eyed drosophila because the wild type red eye is dominant over the white eye. We expected to see a 1:1 and that is what we obtained from the parental cross.

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Table 1.2 Wild Type Male x White Eyed Virgin Female F2 Generation XW XW XW XW XW Y Xr Xr XW Xr Y

XW XW = 1 White Eye Female Xr XW = 1 Red Eye Female XW Y = 1 White Eye Male Xr Y = 1 Red Eye Male Table 1.2 demonstrates the cross between the F1 generation (wild type male x white eyed virgin female). From crossing the F1 generation we were able to obtain the F2 generation. For this outcome we expected to see a ratio of 1:1:1:1, White eye female: Red eye female: White eye male: Red eye Male. This ratio occurred because the white eye trait is sex-linked inherited. So white eye will occur when it is homozygous recessive or paired with a male because eye color is only expressed on the X chromosome. Table 1.3 Wild Type Virgin Female x White Eyed Male F1 Generation XW Xr Xr XW Xr XW Xr Y Xr Y Xr Y

XW Xr = 2 Red Eye Females Xr Y = 2 Red Eye Males Table 1.3 demonstrates the true-breeding parental cross between wild type (red eye) virgin females with white eyed males. This ratio of a 1:1 was expected in the F1 generation because wild type is dominant over white eye. Also the ratio of male to female is 1:1 because this trait is sex linked and it is not autosomal recessive which has no effect on variation in the number of genders observed.

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Table 1.4 Wild Type Virgin Female x White Eyed Male F2 Generation Xr XW Xr XW Xr Xr Xr Y XW Y Xr Y

XW Xr and Xr Xr= 2 Red Eye Females XW Y = White Eye Male Xr Y Red Eye Male Table 1.4 depicts the F2 generation of the F1 cross between wild type virgin females with white eyed male drosophila. In this cross our F2 generation had a ratio of 1:2:1 which is explained in and confirmed by Mendels first law of segregation. We expected to see this after obtaining our F1 results. Since white eye didnt show up in the F1 we knew it had to show up once in the F2 because its recessive.

Table 1.5 Wild Type Male x Sepia Virgin Female F1 Generation R s Rs R Rs

Rs

Rs

Rs= Red Eye Drosophila Table 1.5 portrays the true-breeding parental cross between wild type males and sepia virgin females. Since wild type is dominant over the mutant allele we expected not to see this mutation of sepia eyes appear in the F1 generation.

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Table 1.6 Wild Type Male x Sepia Virgin Female F2 Generation R R RR s Rs

Rs

ss

RR and Rs = 3 Red Eye Drosophila ss = Sepia Eyed Drosophila Table 1.6 demonstrates the cross between the F1 generation. In this punnett square we see the classic F2 Mendelian ratio of 3:1. This is because this particular mutation (sepia) is autosomal recessive and not sex linked like the white eye mutation. Since it is recessive it will show up in later generations and never in the F1. It was easy to develop this correct ratio of 3:1 because of the small sample size that we were working with. If the sample size was to be bigger we would have had more room for error.

Table 1.7 Wild Type Virgin Female x Sepia Male F1 Generation s R Rs s Rs

Rs

Rs

Rs= Red Eye Drosophila Table 1.7 portrays the true-breeding parental cross between wild type virgin females and sepia eyed males. Since wild type is dominant over the mutant allele we expected not to see this mutation of sepia eyes appear in the F1 generation.

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Table 1.8 Wild Type Virgin Female x Sepia Male F2 Generation R R RR s Rs

Rs

ss RR and Rs = 3 Red Eye Drosophila ss = Sepia Eyed Drosophila

Table 1.8 demonstrates the cross between the F1 generation. In this punnett square we see the classic F2 Mendelian ratio of 3:1. This is because this particular mutation (sepia) is autosomal recessive and not sex linked like the white eye mutation. Since it is recessive it will show up in later generations and never in the F1. It was easy to develop this correct ratio of 3:1 because of the small sample size that we were working with. If the sample size was to be bigger we would have had more room for error.

Table 2.1 F1 Generations (Males + Females) Data Drosophila Crosses Virgin Wild x Sepia Male Virgin Sepia x Wild Male Virgin Wild x White Male Virgin White x Wild Male White Eyed 0 0 0 0 Sepia 0 0 0 0 Wild Type 41 53 49 67

Table 2.1 portrays the number of flies collected from the F1 generation. This is no reason for the difference in the numbers of Drosophila. No mutant type flies appeared because the wild type gene is dominant over both of the mutant genes. This is exactly what we expected to see in the F1 generation.

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Table 2.2 F2 Generations Data Original Cross Cross True Breeding F1 Virgin Wild x Sepia Male Virgin Sepia x Wild Male Virgin Wild x White Male Virgin White x Wild Male New Cross Cross of Two F1s Cross of Two F1s Cross of Two F1s Cross of Two F1s Cross of Two F1s White Eyed Females 0 0 0 13 Males 0 0 25 15 Sepia Females 9 7 0 0 Males 7 5 0 0 Wild Type Females 17 19 35 20 Males 21 17 21 19

Table 2.2 illustrates the data and number of flies collected from the F2 generation. We observe a larger increase of numbers in wild type as compared to any of the mutant eye colors because the wild type is dominant over the recessive mutant types. These numbers were obtained so that we could further evaluate them with chi square tests to see if we observed what we expected. This data is the most crucial information to the entire lab experiment. Table 3.1 Wild Type Virgin Females x White Eyed Males F2 Generation Chi Square Analysis D2 D2/e

Phenotypes Red Eye Males Red Eye Females White Eye Males White Eye Females Total

Observed 21 35 25 0 81

Expected 20.25 40.5 20.25 0 81

D 0.75 -5.5 4.75 0

.5625 30.25 22.56

0

.0278 .7469 1.114

undefined 1.88

2 = 1.88

degrees of freedom (n-1) = 3

0.70>P> 0.50

Table 3.1 depicts the chi square analysis of the F2 generation of the cross between wild type virgin females and white eyed males. In this analysis we see that the 4 categorical variables dont differ much from the expected. This shows that what we observed is almost accurate to that of what we should have received. Since the probability is right around the conventionally accepted significance level of .05 we are able to accept our results and accept our null hypothesis which states that we expect to see a genotypic ratio of 1:2:1. Our genotypic ratio of 1:2:1 also fits Mendels first law of segregation, giving us more proof that this part of the experiment was successful.

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Table 3.2 Wild Type Males x White Eyed Virgin Females F2 Generation Chi Square Analysis D2 5.063 10.56 3.063 14.06 D2/e 0.3022 0.6306 0.1828 0.8396 1.953

Phenotypes Red Eye Males Red Eye Females White Eye Males White Eye Females Total

Observed 19 20 15 13 67

Expected 16.75 16.75 16.75 16.75 67

D 2.25 3.25 -1.75 -3.75

2 = 1.95

degrees of freedom (n-1) = 3

0.70>P> 0.50

Table 3.2 illustrates the chi square analysis of the F2 generation of the cross between wild type males and white eyed virgin females. In this data we expected to see a 1:1:1:1 ratio. Our observed numbers were slightly off of the expected but we still received a positive P value which was able to tell us that we were able to accept our null hypothesis. The testcrosses powerfully supported our hypothesis regarding genetic contributions to the dihybrid cross.

Table 3.3 Wild Type Males x Sepia Virgin Females F2 Generation Chi Square Analysis D2 0 0 D2/e 0 0 0

Phenotypes Wild Type Sepia Total

Observed 36 12 48

Expected 36 12 48

D 0 0

2 = 0

degrees of freedom (n-1) = 1

0.00>P> 0.00

Table 3.3 shows the chi square analysis of the F2 generation of the cross between wild type males and sepia eyed virgin females. This analysis turned out to be perfect. We observed what we expected which doesnt happen very often and shows that our experiment was successful. From this we can accept our null hypothesis saying that there will be a 3:1 ratio in the F2 generation of this particular cross because the mode of inheritance is autosomal recessive and not sex linked. Mendels first law of segregation proved to be accurate in this prediction.

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Table 3.4 Wild Type Virgin Females x Sepia Males F2 Generation Chi Square Analysis D2 6.250 6.250 D2/e 0.1543 0.4629 0.617

Phenotypes Wild Type Sepia Total

Observed 38 16 54

Expected 40.5 13.5 54

D -2.500 2.500

2 = 0.62

degrees of freedom (n-1) = 1

0.50>P> 0.30

Table 3.4 portrays the chi square analysis of the F2 generation of the cross between wild type virgin females and sepia eyed males. As you can see from this table our observed and expected arent too far off in numbers. In fact we were almost dead accurate. This cross was a success. We know this was a success because our P value tells us that we can accept our results. From this we can accept our null hypothesis saying that there will be a 3:1 ratio in the F2 generation of this particular cross because the mode of inheritance is autosomal recessive and not sex linked. Mendels first law of segregation proved to be accurate in this prediction.

Discussion The experiment went well and all of our data turned out to be correct. No error occurred and we received data that was testable and helped us determine our hypothesis. If anything we could have been done with this lab a few weeks in advance. A little slip up in the beginning made us have to start over. The parental wild type male flies never awoke from being anesthetized and died. We were thankful that this happened early on in the experiment and not later on or else our data would have been insufficient. I know that spring break really ruined peoples experiment because around that time is when we were crossing our F1s and some never took the parents out of the subcultures, causing them to have disturbed results. Luckily we were fortunate enough to stay ahead and perform that task at hand smoothly with only one minor bump in the road. Not only was our data sufficient but our hypotheses were what we predicted as well. Miraculously enough our one cross between wild type males and sepia females turned out to be 100% correct without errors. Our observed data exactly matched what we expected. Each hypothesis that we predicted is supported by our results. The P values from the chi square analysis charts shows you a percentage and we had nothing higher than a 7% rate of error. Ultimately our chi square analysis held the power to let us know whether or not our experiment was going to be a success or not. It turned out that it was. This lab was a learning experience for the whole class. It helped us understand how traits are transmitted through the use of a

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simple organism Drosophila melanogaster. It also taught us how to be organized and keep information in spatial order. Having an experiment like this one makes us all more comfortable with the much more difficult ones we will face in the future of our college careers.

Bibliographies Manning, Gerard. "Introduction to Drosophila." A quick and simple introduction to Drosophila melanogaster. 1 Oct. 2006. Web. 22 Feb. 2010. <http://www.ceolas.org/fly/intro.html>. "Sex-Linked Inheritance." Access Excellence @ the National Health Museum. The National Health Museum, 1999. Web. 24 Feb. 2010. <http://www.accessexcellence.org/RC/VL/GG/sex.php>. Glass, Bentley. The American Naturalist, Vol. 68, No. 715 (Mar. - Apr., 1934), pp. 107-114. Published by: The University of Chicago Press for The American Society of Naturalists.