A Survey of the Mendelian Modes of Inheritance on Sepia-eyed and Apterous Fruit Flies
Student Name: Trissha Bautista
Group Members: Robert Nance, Ashley Pham, Alexa Figuerdo
Course: Biol 3452.505
Teaching Assistant: Jose Robledo
Date of Submission: March 21, 2018
Table of Contents
Abstract 2
Introduction 3
Research Objective and Aims 6
Research Designs and Methods 6
Materials and Methods 6
Data Collection and Results 7
Analysis of Results 8
Discussion and Conclusion 10
References 11
Abstract
Drosophila melanogaster is a good genetic model system used in various experiments due to its numerous advantages. To better understand modes of inheritance, D. melanogaster was used to predict the phenotypic ratio of offsprings produced. The hypothesis is that the offspring flies will follow known and already established phenotypic ratio while the null hypothesis states the opposite. Sixteen F1 generation of fruit flies exhibiting the sepia eyes and the apterous traits were crossed and observed in 2 vials. Through a dihybrid cross of the fly traits being examined, the typical Mendelian 9:3:3:1 ratio resulted. The chi-squared test was completed to establish the validity of the results. The statistical results failed to reject the null hypothesis because of a number of errors in the methods. Further studies on the sepia eyes and the apterous traits need to be completed to establish a good baseline for future experiments.
Introduction
Since the dawn of genetics studies on Mendel’s peas, genetic model organisms have been used to study the mechanisms by which inheritance operates. The study of these modes of inheritance is pertinent to understanding how biological processes work and can be related to evolutionary processes (Padilla 2018). Genetic model organisms have been used in research to create detailed genetic maps, so patterns of heredity can be understood better (“What are model organisms?” 2018). Examples of genetic model organisms used in research laboratories today include Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus. These genetic model organisms are used because of their rapid development, small size, and availability (Padilla 2018).
In this experiment, the genetic model organism used was D. melanogaster, more commonly known as the fruit fly. At only 3 mm in length, the fruit fly is small; however, the fly is large enough for the naked eye to examine (Padilla 2018). The sex of the adults is fairly easy to determine. Female flies bear a number of black bands on the tail end while male flies appear to have a solid black tail shown in Figure 1 below (Padilla 2018). Fruit flies are easy to care for as their diet is simple and have a short life cycle show in Figure 2.
Figure 1. Differences between the male and the female fruit flies (“The Biology Place” 2018).
Figure 2. Life cycle of the fruit fly (“Lecture 16” 2018).
There are two Mendelian laws of genetics that are of importance in this experiment. The first one is the Law of Random Segregation which states that in meiosis genes are distributed randomly to the gametes (Padilla 2018). This means that a heterozygous organism with Bb genotype, in which B represents the dominant allele and b represents the recessive allele, will have gametes with 50% of the dominant B allele or 50% of the recessive b allele. If one completes a monohybrid cross from two heterozygous organisms, then the genotypic ratio of homozygous dominant offsprings to heterozygous offsprings and to homozygous recessive offsprings will be 1:2:1.
The second law is the Law of Independent Assortment. This law states that unlinked gene pairs will be distributed independently of each other (Padilla 2018). If a dihybrid cross is completed on two heterozygous traits, then the phenotypic ratio will be 9:3:3:1. Table 1 shows the dihybrid cross completed for this experiment with R representing red eyes found in wild type fruit flies, r representing recessive sepia (brown) eyes, W representing winged flies, and w representing apterous or wingless flies. Like phenotypes are highlighted in the same color as other identical phenotypes to show the 9:3:3:1 ratio predicted by the Law of Independent Assortment. The sepia eye color gene and the apterous gene are not linked together genetically, making it appropriate to complete the dihybrid cross of the traits. As seen in Figure 3 below, the sepia eyes trait is located on chromosome 3 around marker 26.0 while the apterous trait is located on chromosome 2 around marker 55.2.
Table 1. Dihybrid Cross of D. melanogaster Sepia Eye Color and Apterous Alleles.
Alleles
RW
Rw
rW
rw
RW
RRWW
RRWw
RrWW
RrWw
Rw
RrWw
RRww
RrWw
Rrww
rW
RrWW
RrWw
rrWW
rrWw
rw
RrWw
Rrww
rrWw
rrww
Figure 3. A genetic linkage map of the chromosomes of D. melanogaster showing the location of the sepia eyes and the apterous traits (“Patterns of heredity” 2018).
The study of the modes of inheritance in fruit flies is crucial as 75% of the disease-causing gene in humans (Reiter et al. 2001). Due to that degree of homology, further studies of the fruit fly is called for to further understand how modes of inheritance may work in the expression of disease-causing genes in humans or other animals. The problem that the team is trying to solve is finding out whether or not the data will follow the ratio that is expected per the table provided above. The hypothesis is that the statistical results of the experiment will follow the characteristic phenotypic ratio of 9:3:3:1 from the dihybrid cross. The null hypothesis is that the statistical results of the experiment will not follow the expected ratio of 9:3:3:1 from the dihybrid cross shown above.
Research Objective and Aims
The objectives of the experiment are to understand phenotypic analysis and to understand the mode of inheritance from the data of the cross completed. To determine whether or not the results follow the 9:3:3:1 trend and to reject or accept the null hypothesis, fruit flies were grown, mated, and observed and a chi-squared test of the resulting data was completed towards the end of the experiment. The results were compared to similar, already completed research projects to see the validity of the experiment.
Research Designs and Methods
Materials and Methods
To complete this experiment, the materials that were used include four vials and vial labels, foam vial plugs, flynap anesthetic agent, plastic mesh, one anesthetizing vial and wand, one paintbrush, yeast pellets, fruit fly food flakes, and 1% propionic acid. The vials, vial labels, vial plugs, and plastic mesh were used to create the home of the flies. Yeast pellets, food flakes, and 1% propionic acid were used to provide food for the flies. Finally, the anesthetizing vial, anesthetizing wand, and paintbrush were used to anesthetize the flies for sexing and observation purposes.
First, two fly homes containing food had to be prepared. Yeast pellets were mixed with the food flakes and a few drops of 1% propionic acid in each of the two vials until the color of the mixture turned blue and the consistency remained clay-like. While waiting for the mixture become firm, the plastic mesh was cut and then was placed in the vial. The mixture had to be firm enough and not watery and be neatly placed at the bottom of each of the vials to ensure that the flies do not get stuck in it and die. The vials were labeled A and B.
The F1 generation vial was given to the team and then placed in the freezer for 1 to 2 minutes to immobilize the flies. Two drops of flynap were dabbed on to the foam plug of the anesthetizing vial using the anesthetizing wand. Only two drops were used as flynap is extremely potent and could kill the flies instead of just anesthetizing them. Once the F1 flies were immobilized, they were transferred into the anesthetizing chamber of the anesthetizing vial and then the stopper was placed to prevent the flies from escaping. After waiting for approximately 10 minutes, the flies were asleep. The flies were then sexed and four male flies and four female flies were placed in each fly home prepared. The fly homes were placed in the lockers horizontally to ensure that the flies do not get stuck on the food mixture at the bottom.
Data Collection and Results
Both vials had four female flies and four male flies each. The phenotypes of the flies were not noted, so a chi-squared test based on the sexes of the flies cannot be completed. On the first day, one fly was found to be stuck on the food mixture in Vial B. On the second day, all of the flies in Vial A were alive, but only three flies were alive in Vial B. We continued the observing both vials and only one fly remained in Vial B and five flies in Vial A after two weeks. The failure of the other flies to thrive in Vial B led us to think that too much acid was used in the food mixture, killing the flies. Luckily, the remaining fly in Vial B was a female and was able to lay eggs. Larvae began growing in both vials after two weeks instead of just a few days after placing the flies in the new homes, leading the team to believe that the flies were still young when they were sexed. Because we had trouble with mating and maintaining the flies, we let the F1 flies stay in the fly homes instead of removing them. By the end of the fifth week of observation of the flies, the flies were anesthetized in order to identify the phenotypes that the F2 generation exhibited. The recessive flies (sepia-eyed and apterous) were given to the teaching assistant. The results are tallied in Table 2 below and the timeline of the experiment is shown in Figure 4 below.
Table 2. Results of the Experiment Grouped by Phenotypes Observed.
Phenotype
Number of Flies
Red eyes with wings
71
Red eyes and apterous
19
Sepia eyes with wings
20
Sepia eyes and apterous
9
Total number of flies
119
Figure 4. Timeline of events of the experimental methods.
Analysis of Results
To see if the experimental results follow the hypothesis, a chi-squared test was completed using Equation 1 below with the observed results denoted by O and the expected results denoted by E. The results are tallied in Table 3 below.
χ2 = ∑(O−E)2/E Equation 1
Table 3. Chi-Squared Test Results.
Ratio
Phenotype Observed
O
E
O-E
(O-E)2
(O-E)2/E
9/16
Red eyes with wings
71
66.9375
4.063
16.50
0.247
3/16
Red eyes and apterous
19
22.31
-3.312
10.97
0.492
3/16
Sepia eyes with wings
20
22.3125
-2.310
5.336
0.239
1/16
Sepia eyes and apterous
9
7.4375
1.563
2.441
0.328
Total
119
119
1.306
The degrees of freedom was calculated by subtracting the number of categories (n) for the results by 1 (Equation 2). There are four categories, so degrees of freedom equals three.
Degrees of freedom = n – 1 Equation 2
Finally, a table of χ2 values was used and the result from the chi-squared test was located in the Table 4 based on the degrees of freedom of the resulting data. We found that our chi-squared value lies between the p-values of 0.5 and 0.7.
Table 4. Probability Value Versus Degrees of Freedom Table Used to Locate Chi-Squared Test Result (“Chi-Square Test” 2018).
Discussion and Conclusion
The hypothesis states that the results will resemble the predicted 9:3:3:1 ratio for the dihybrid cross while the null hypothesis states otherwise. The range of the p-value obtained from the chi-squared test, 0.5 to 0.7, lies towards the left of the table and shows that the null hypothesis is to be accepted. This means that the hypothesis that the phenotypic ratios will follow the typical Mendelian 9:3:3:1 ratio is not to be accepted. Because the p-value was between 0.5 and 0.7, the chi-squared test proved that at least a 50% deviation exists with the results.
The 50% deviation means that the errors that were encountered in the results cannot be attributed just to random chance. From the beginning, errors can already be pinpointed in the research methods. First, the phenotypes of the F1 generation were not noted. This means that the phenotypes of the next generation cannot be predicted more accurately. Next, multiple fly deaths were attributed to an uneven mixture of propionic acid, the food flakes and the yeast. Lastly, the F1 generation was kept in the same tube as the next generation which definitely skews the resulting number of flies and does not ensure pure F2 generation as the F1 and the F2 flies could have mated with each other. To begin with, the team was told that the originating vial contained purely F1 flies, but that vial could have had larvae growing already or the P0 generation living in it. Another possible error stems from the fact that the flies were incubated in an environment that is slightly cooler than room temperature. This might have caused the slow development of the flies, producing the small sample size of 119 flies in the end. A similar experiment on sepia-eyed and apterous flies failed to reject the null hypothesis (Wiles and Hargadon 2013). The authors attributed to the errors to small sample size. To correct these errors, one must be cognizant of the phenotypes, breeding or generations, and sexes of the flies and ensure warm and proper environments for fly development.
Future experiments may explore if the traits examined are sex-linked or not. One could also attempt to figure out at what temperatures the flies develop optimally as well as the genotypic ratios associated with the F1 generation flies based on the phenotypes of the offsprings. As species evolve, so does genetics. When genetics changes, then diseases change along with it. The study of the genetics of D. melanogaster paved way to an in depth understanding of evolutionary relationships between humans and other animals and is continuing to do so.
References
Chi-square test, 2018 Biocyclopedia.com.
Drosophila development, 2018 Zoology.ubc.ca.
Padilla P., 2018 Genetics Laboratory Manual Spring 2018. Denton, Texas.
Patterns of heredity, 2018 Biologycorner.com.
Reiter L., Potocki L., Chien S., Gribskov M., Bier E., 2001 A Systematic Analysis of Human Disease-Associated Gene Sequences In Drosophila melanogaster. Genome Research 11: 1114-1125.
The biology place, 2018 Phschool.com.
What are model organisms?, 2018 Yourgenome.org.
Wiles S., Hargadon K., 2013 Inheritance patterns in monohybrid and dihybrid crosses for sepia eye color and apterous (wingless) mutations in Drosophila melanogaster. H-SC Journal of the Sciences II.