Guiding Inquiries

1. What do you hypothesize is the inheritance pattern for each gene? (complete dom., incomplete dom., etc.). A hypothesis of complete dominance would lead to the expectation that ALL offspring of the parental cross (=F1 gen.) will be WT/WT, that is, show the dominant trait for both characters.
   1. The
2. Do you hypothesize your 2 genes are linked (on the same chromosome) or unlinked?
   1. A. A hypothesis of unlinked genes would lead to the expectation of independent assortment in the offspring of the dihybrid cross (=F2 gen.). This would lead to the expectation of a 9:3:3:1 ratio in the F2 generation.
   2. B. A hypothesis of unlinked genes/ independent assortment would also lead to the expectation of a map distance =~ 50%.
3. Do you hypothesize either or both of your genes are sex linked?

Longform

This study was an investigation in the patterns of inheritance of body and eye color genes in the fruit fly species Drosophila melanogaster. Over the course of five weeks, a parental cross between true-breeding wild type and double mutant fruit flies was prepared, then used to create two dihybrid and two test crosses between its F1 generation and true-bred mutants. These values were used to perform Chi^2 tests for significance and to compare the inheritance of each trait between sexes. The results from the study were supportive of hypotheses that the genes followed an inheritance pattern of complete dominance and were on autosomal chromosomes. However, the results were unsupportive of the hypothesis of the body and eye color genes being on different chromosomes and supported the possibility they showed linkage on the same chromosome.

Genetics is the study of heredity and of the variation in genes that are inherited through sexual reproduction (Naganuma & Roffey, 2018). Organisms in a species sexually reproduce through meiosis, which produces haploid eggs, or gametes. These eggs consist of one set of chromosomes whose number depends on the species. The gametes from one male and one female parent can merge into one diploid cell with two sets of chromosomes through fertilization.

Sexual reproduction has a high potential to create genetic diversity and variation. For example, only one set of chromosomes is inherited from each parent, and each chromosome can have variations of the genetic information they carry, known as alleles. These alleles contain different nucleotide sequences that may lead to the presence or absence of a trait in the phenotype, where variations of traits may be observed in a species. However, if the alleles have different degrees of dominance, the presence of one allele and the sequence it codes for may cause only that allele to present in the phenotype (Naganuma & Roffey, 2018). This pattern of variation, in which one inherited allele is “dominant” and appears in the phenotype whenever present, while the other is “recessive” and only appears when it is the only allele present in both chromosomes, is known as complete dominance.

However, complete dominance is not the only pattern of inheritance, nor is it its only determining factor. During meiosis, when chromosomes align on the metaphase plate at the center of the cell, they undergo the process of independent assortment: they align on the plate independently of the alignment of other chromosomes during metaphase, and they separate into two daughter cells independently at the end of it. This occurs in both metaphase I and II in meiosis, which can lead to increasing numbers of variation for each chromosome present in the organism.

The last variation of note is present after fertilization, once the sex of the offspring is determined. Sexual reproduction includes variation in sex, which is determined in different ways depending on the organism’s class. If the organism’s sex is determined through factors such as different sex chromosomes, as is seen in mammals, the inherited chromosomes may have different genes on each chromosome, resulting in the dependence or absence of dependence on multiple chromosomes to determine the phenotype of an individual. This may cause certain genes to be sex-linked: they are genes that are only present on the sex chromosomes and are not autosomal, or present on a chromosome multiple sexes have.

Genetics are studied in many ways via the controlled crossing of cultures and their genes. Cultures of plants and animals are studied via pairs such as parental crosses, where true-breeding organisms mate, dihybrid crosses, where two genetic characters and their mutations are studied across generations, and test crosses, where parental cross offspring and true double mutant organisms are crossed and studied. These studies are used to explain and support patterns of inheritance and variations in genetics for both one species and in general patterns across species. D. melanogaster, a popular species for the study of genetics, as well as other species within the Drosophila genus, show global differences in various forms of chromosomal polymorphism, where variation in phenotypes is prevalent and caused by different forms of chromosomal and DNA alteration (Singh, 2013). The genetic characters of Drosophila can also observed when affected by various epigenetic factors caused by their environment, ranging from natural exposure to temperature to artificial exposure to chemicals, organic methylation, and genetic assimilation (Schaefer & Nadeau, 2015).

In this genetics study, Drosophila melanogaster, a species of fruit flies, were studied based on specific mutations in their body and eye colors. The inheritance of these mutations can depend on many factors, such as patterns of inheritance, independent assortment, and autosomal inheritance. This study was performed based on hypotheses for each mentioned factor. It was hypothesized that the ebony body and sepia eye alleles were recessive genes, so offspring of the parental generations of Drosophila melanogaster would show the respective wild type phenotypes if the wild type alleles were present; both the ebony body and sepia eye alleles were on different chromosomes and would assort independently during meiosis without affecting the assortment of the other; and lastly, both the ebony body and sepia eye genes were autosomal and would have an equal chance of presenting in male and female fruit flies.

This experiment was performed using cultures of Drosophila melanogaster, fruit flies, that were previously stocked by the school laboratory. Four glass vials, sterilized through autoclaving, were prepared to house the different Drosophila cultures. In each vial, one level cup of tap water was added to an equal amount of dry “Instant Drosophila Medium,” a commercial feeding medium made from plant materials. The new mixture was leveled out within the vial. Then, 3 to 4 grains of yeast were added on top of the mixture, and the vial was set aside.

At the same time, two stock vials of Drosophila melanogaster, which were both cleared during the previous morning, were immobilized under a fume hood using goggles and sterile gloves. One stock vial contained true-breeding wild type flies, while the other contained a culture of true-breeding double mutant flies with sepia eyes and ebony body colors. The stock vials were connected to empty glass vials that were designated as anesthesia vials. These vials were capped with foam plugs while an anesthetic wand was prepared and dipped into a container of Fly Nap®, a commercial anesthetic liquid. The wand was inserted between the foam plug and vial wall into the vial for approximately two to four minutes until all contained flies were immobilized. These flies were poured onto index cards and analyzed under dissecting microscopes to determine their sex and phenotype.

After all flies were analyzed, different amounts of fruit flies were placed into each prepared culture vial. In two of the culture vials, between six to eight virgin wild type females were placed with six double mutant males and were used to create a parental cross. These vials were then sealed and labeled as “wt x se,” denoting the phenotypes and crossover, the date and period that the culture was started, and with an identifying group name. In the other two vials, six double mutant females and six double mutant males were placed, and the vials were labeled as true-breeding, double mutant culture vials. All vials were sealed with sterile, porous foam plugs, grouped together with a rubber band, and placed in a fly incubator at 21 degrees Celsius for one week.

After one week, the vials were removed from the incubator and cleared outside of the laboratory to allow the adult flies to escape the vials. If the vial mediums appeared to be runny or moist, approximately half a teaspoon of dry medium was added into the vial. This procedure was repeated one week later before the next laboratory period. The vials were later anesthetized and the culture flies analyzed under dissecting microscopes for sex and phenotype as they were previously, while four new glass vials were prepared identically to the first vials. In two of these vials, six male and six female flies were collected from the culture that was created by the parental cross, the F1 generation, creating two dihybrid cross culture vials. Another six female flies were collected from a specific F1 culture where the vials had been cleared recently enough that the females were virgins and placed into each of the remaining two vials with six males from the true-breeding double mutant culture, creating two test cross culture vials. All four of the new vials were sealed with foam plugs, grouped together with a rubber band, and placed in the fly incubator at 21 degrees Celsius through the next week. After one week, the vials were cleared outside of the laboratory identical to the first four vials.

Manual data collection of the next generation, F2, was done two weeks after the vials were cleared. Across five different dates—November 6, November 8, November 13, November 15, and November 16—the same procedure was repeated to count the flies in the culture. Each vial was numbered and noted for the type of cross that was present in each vial. Then, the vials were opened and anesthetized under the fume hood identically to prior procedure. On each day, the flies were analyzed under a dissecting microscope to determine their sex and phenotype: male and female, then double wild type, double mutant, or showing either ebony body or sepia eye phenotype. These values were tabulated and were summed for the total values after November 16. This data was used to perform Chi^2 tests, determining the Chi^2 value and degrees of freedom of the dataset. The value was then compared to the Chi^2 probability table with the value under the probability column 0.05 and on the row of 3 degrees of freedom and was interpreted accordingly.

The results of this study support the hypothesis that the ebony body and sepia eye alleles in Drosophila melanogaster follow the pattern of complete dominance and are on autosomal chromosomes; however, they do not support the hypothesis that they are on separate chromosomes nor that they follow the pattern of independent assortment.

Following the parental cross performed at the beginning of the experiment, all offspring in the F1 generation had a wild type phenotype; none had sepia eyes or ebony bodies. However, in the following F2 generation, some offspring depicted one or both of the recessive mutations. While the F1 generation did not physically present with any of the mutations, due to the parental cross, their genotypes were heterozygous for both the wild type and mutant alleles in both genes. The wild type body and eye color alleles were dominant to the mutant alleles, but as the F1 offspring were carriers, their offspring were able to inherit and display the mutant alleles. This evidence supports the hypothesis of complete dominance. Figure 5 also shows that both the ebony body color and sepia eye color alleles presented in both males and females at similar percentages: of all organisms with the ebony body color phenotype, about 40% were males and 60% were females, while of all organisms with the sepia eye color phenotype, about 46% were male and 54% were female. As the difference in frequency is less than 20% in both comparisons, these ratios support the hypothesis that both body and eye color are autosomal traits; it is unlikely that they are on the sex chromosomes of Drosophila.

However, the results of the Chi^2 tests performed on both the dihybrid crosses and the test cross reject the hypothesis of independent assortment for the body and eye color genes. In fourteen out of sixteen of the Chi^2 tests performed, the resulting values were higher than the critical value for the tests, 7.81. As the tests were compared to a probability value of 0.05, where there is a 5% chance at maximum where the results were produced by random chance, the differences in the Chi^2 test were deemed significant, and the hypothesis of independent assortment was rejected. This is supported by Figure 6, where a recombinant frequency of 31.36% was calculated from the test cross results. This frequency converts to a map distance of approximately 31.36 cM between the body and eye color genes. These results suggest the body and eye color genes are close enough together on the same chromosome that they are linked and are unlikely, though not impossible, to separate during crossing-over during meiosis.

One of the two Chi^2 test values that was lower than the critical value, the test value for double mutant organisms in vial 3’s dihybrid cross, was likely the result of human error. During the study, multiple organisms were counted that had white eyes instead of sepia eyes. This was likely caused by the sharing of anesthetic vials while setting up the individual crosses. The stock vials were true-breeding, so their offspring should not have been able to inherit white eyes from either the double mutant or wild type parents following the parental cross. However, vial 3 produced a total of 18 offspring with white eyes, while no other vials produced any offspring with white eyes during the ten-day study. Vial 3 was likely the only culture affected, but was still contaminated by another mutant. All of the flies with white eyes were not counted in the final results, so the presence of the white eye color allele may have lowered the Chi^2 test values for the vial.

Additionally, one of the test cross vials, vial 4, was unable to produce a fly culture for counting. No males were placed in the vial, so the females in the vial died out before counting was performed. This vial was unusable and was not included at all in the study results.

Aside from the aforementioned errors, the results of this study follow studies performed by Gregor Mendel and Thomas Hunt Morgan. The flies reproduced sexually through the creation of and fertilization of gametes. These gametes were created through meiosis and carried only one set of Drosophila chromosomes. As a result of this, all descendants of the true-breeding parental generation inherited one mutant allele and one wild type allele, which allowed subsequent generations to inherit one of these two sets and display different phenotypes in each cross. This observation represents the Mendelian law of segregation, which states that the two alleles of a gene in a parent’s chromosomes separate during meiosis and their gametes only carry one set of chromosomes, including one genetic allele (Naganuma & Roffey, 2018). The genes studied in this experiment also represent Morgan’s findings. Morgan determined that some genes do not follow Mendel’s law of independent assortment, which states that alleles for one gene segregate without reliance on other alleles. This law applies to alleles that are either on separate chromosomes or are far apart on the same chromosome. The closer two separate alleles are to one another, the less likely they are to separate during crossing-over, where a chiasma forms between homologs and causes genes to exchange between chromosomes, and the more likely they are to show linkage. In a pattern of linkage, a set of genes is more likely to be separated in daughter cells with the genotypes of the parents than it is to be separated during crossover and produce a different genotype, or recombination (Portin, 1993). In the case of D. melanogaster genes for body and eye color, their proximity, calculated in Figure 3 as approximately 31.36 cM, suggests they are unlikely to separate when a chiasma forms; however, the presence of recombinant flies in all crosses, including the test cross from which the map distance was derived, indicates this is not impossible by nature.

Genetic variation can cause differences in life and adaptation for many organisms in one species. Some characters produced by genetic variation and phenotypes are more useful than another; some can cause health or lifestyle issues that make one organism less likely to survive and reproduce in nature. In additional studies by Morgan which evaluated mutations in color and wing shape, many expressed differences in flight and in reproduction rates. Flies with rudimentary, balloon, albino, or no wings were suggested to be sterile or less viable for reproduction than those with normal wings or different mutations in color and length (Morgan, 1911). The patterns of inheritance and methods of genetic variation in D. melanogaster are also seen in other species and genes, including humans. These studies are fast, ethical methods of studying variation and are foundational for studying larger and more complex genes and evolution both in fruit flies and in other species, including the natural survival of a species and the traits its offspring inherit.

Future studies that could be done in this area of genetics include a comparison of dominant strengths between mutant alleles for body and eye color. The low presence of white eyes in vial 3 raises several questions. While its presence may have been attributed to a low number of contaminant flies in comparison to the double mutant flies being studied, it could also be hypothesized to be caused by different degrees of dominance. This extends to the other mutant character that may have been present in the contaminating fly, as all of the mutant stock vials contained double mutant flies that may have shown mutations in body color, eye color, and wing type. It would be interesting to compare the degrees of dominance between non-wild type alleles and to investigate why the other character, if different than the ebony body color, did not appear in any counts for vial 3.

This study was performed in order to observe the genetic inheritance of mutant and wild type alleles for body and eye color in Drosophila melanogaster. It was performed over five weeks and studied the degrees of dominance, map distance and recombinant frequency, and possibility of sex linkage in D. melanogaster body and eye color using ebony body and sepia eye color mutations. The results that were produced by this study support the hypotheses that the genes for body and eye color follow a pattern of complete dominance and are on autosomal chromosomes, but reject the hypothesis that they are on separate chromosomes and support a pattern of linkage between the two genes.