Application of the Three-Point Cross in Drosophila melanogaster in Constructing Genetic Maps of the X Chromosome.

Application of the Three-Point Cross in Drosophila melanogaster in Constructing Genetic Maps of the X Chromosome.

Ingrid Schoonover
February 28, 2020
Genetics with Laboratory

Abstract

Drosophila melanogaster has long proved to be a useful model organism in furthering the understanding of genetic inheritance. This experiment tracks three traits (eye color morphology, wing morphology, and bristle morphology) in Drosophila melanogaster over three generations and constructs a three-point cross to determine the gene order and genetic distance of these three traits. These three traits were determined to be X-linked after analysis of the phenotypic outcomes of the parental and F1 generation crosses. The genetic map that constructed for the X-chromosome placed the order of the genes as white eye (w) > miniature wings (m) > forked bristles (f). The gene distance between white eye (w) and miniature wings (m) was calculated to be 30.9 map units with -10.7% error in respect to the established genetic map. The gene distance between miniature wings (m) and forked bristles (f) was calculated to be 30.9 map units with a +50% error with respect to the established genetic map. The overall gene distance between white eye (w) and forked bristles (f) being equal to 61.9 map units, with +12.1% in respect to the established genetic map.

Introduction

The fruit fly (Drosophila melanogaster) has been an excellent model organism for understanding genetic mechanisms due to the ease of culturing colonies, a rapid life cycle, and their genetic similarities to humans. The life cycle of Drosophila begins like other sexually reproducing species with the fusion of parental gametes following meiosis. Embryogenesis occurs after this process of fertilization, and Drosophila larvae emerge from their eggs within 24 hours and spend the next five days going through three larval stages (Hales et al., 2015). At the end of the third instar, the larvae form a pupal case and emerge as adult flies in an additional four to five days (Hales et al., 2015). The adult flies reach sexual maturity 12 hours after emerging from the pupae, which means that the entire life cycle from egg to adult only takes about 10 days to complete (Hales et al., 2015). This short life cycle makes it easy to study phenotypic and genotypic changes over generations, and their ability to produce a large number of offspring allows researchers to observe and quantify uncommon genetic events such as recombination.

They are also an ideal model organism for genetic studies because they are diploid organisms with two copies of each of their four chromosomes, and their sex chromosomes are heterogametic like most mammals (Cook et al., 2010) meaning that they are different for males and females, which offers an opportunity to understand sex-linked inheritance. Sexual determination in flies is determined by X dosage, flies with two X chromosomes will develop as phenotypical females, and flies with one X chromosome (usually XY, but sometimes XO) will develop as males (Hales et al., 2015). The large number of offspring produced in fruit fly crossings is also useful because over generations of thousands of flies some mutant alleles have been spontaneously produced. For example, the miniature locus (m) was discovered in 1910, this mutation produces flies with a small wing phenotype (Slatis and Willermet, 1953). Similarly, the white eye locus (w) and forked bristle locus (f) have been produced, these three loci and the ways in which they phenotypically present themselves in Drosophila melanogaster are the interest of this study. Specifically, this experiment will be attempting to produce a genetic map for the white eye locus, the miniature locus, and the forked bristle locus (f). This will be achieved by creating a three-point cross and then using the recombination frequency of the resulting offspring to calculate gene order, gene distances, and interference.

Some background on these three genes is crucial before explaining how this three-point cross will be achieved. In Drosophila the X and Y sex chromosomes are heterogametic, meaning they differ in appearance: the Y chromosome is shorter than the X chromosome, and most importantly they have nonhomologous regions (Griffiths et al., 2000). The genes that control eye color morphology, wing length morphology, and bristle morphology are all located on nonhomologous or differential regions of the X sex chromosome (Griffiths et al., 2000). The significance of this is that males only have one X chromosome so they will only have one copy of these genes, whereas females will have two copies because they have two X chromosomes (Cook et al., 2010). This structural and genetic difference between the sex chromosomes gives rise to the phenomenon of X-linked inheritance, which is when genes located close together are sorted together during meiosis rather than independently. This inheritance differs from the independent assortment of unlinked genes on autosomes where both the male and female offspring would have the same phenotypic frequencies, because with X-linked inheritance phenotypic frequencies will differ in male and female offspring (Griffiths et al., 2000).
The nature of X-linked inheritance is better understood after the dominance relationship of these three traits and their phenotypes is explained. The phenotypes of interest depend on whether Drosophila has the mutant or wildtype allele at the loci discussed earlier. The genes at the w locus control the expression of eye color, the dominant wildtype allele (w+) results in flies with red eyes, whereas flies homozygous with respect to the recessive mutant allele (w) have white eyes (Griffiths et al., 2000). The genes at the m locus control wing size, the dominant wildtype allele (m+) result in flies with normal length wings, and in contrast, flies homozygous with respect to the recessive mutant allele (m) have abnormally small a.k.a. miniature wings (Slatis and Willermet, 1953). The genes at the f locus control the appearance of bristles, the dominant wildtype allele (f+) results in flies with normal straight bristles, and the recessive mutant allele (f) produces forked bristles in homozygous flies (Carlini, 2020). The relationship of dominance between these alleles only matters for female flies with two X chromosomes because they will have two alleles for each trait. However, since males only have one X chromosome, they will express whatever allelic copy they inherited from their mothers. This also means that X-linked mutations cannot be passed from father to son. Similarly, in a crossing of triple mutant females to normal males, one would expect all of the male progeny to express the same mutant phenotype, but all of the female progeny would express the wildtype phenotype.

In the early 1900s, Thomas Hunt Morgan discovered while working with Drosophila models that crossing over can occur between homologous chromosomes during prophase I of meiosis, which is when regions of DNA exchange between the two chromosomes, producing recombinant combinations in the gametes (Lobo and Shaw, 2008). This revealed that the frequency at which recombination occurred between two genes was a result of the distance between those two genes on the chromosome (Lobo and Shaw, 2008). Recombination has the potential to disrupt the inheritance of linked genes when crossover occurs between two loci. Genes farther apart on a chromosome are more likely to experience a recombination event between them than genes closer together due to the statistical chance that a crossover event can occur at any particular region along the chromosome. (Lobo and Shaw, 2008). One of Morgan’s students Alfred Henry Sturtevant used this information to create the first genetic maps, which revealed the distance between genes in map units or Centimorgans (cM) which are equal to the recombination frequency (Lobo and Shaw, 2008). Genes are considered linked if they are less than 50 map units (or cM) apart with a recombination frequency of 0% to 50% and unlinked/independently assorting if they are greater than 50 map units apart with a recombination frequency of greater than 50% (Lobo and Shaw, 2008). Sturtevant discovered that double-crossover events between linked genes are also possible. Furthermore, through his research, he also showed that one crossover event can prevent a second crossover event from occurring in a process called interference (Lobo and Shaw, 2008).

The method for calculating gene order and genetic distance for three linked genes is with a three-point cross; the test cross of a triple heterozygous wildtype female (X^{w^+m^+f^+}/X^{wmf}) to a triple recessive mutant male (X^{wmf}/Y) (Griffiths et al., 2000). In order to produce flies with these genotypes, this experiment will first perform a parental cross by mating triple homozygous mutant female (X^{wmf}/X^{wmf}) to a triple homozygous wildtype male (X^{w^+m^+f^+}/Y). This parental cross produces the F1 generation of triple heterozygous wildtype females and triple recessive mutant males, the F1 generation is not the subject of interest because the phenotype will not be changed even if recombinant occurred between the mothers homozygous X chromosomes during meiosis. Next, the three-point cross will be performed by mating the F1 generation flies to produce the F2 generation. Since the female F1 flies are fully heterozygous, any recombination events between the three loci will produce new reciprocal phenotypes in the F2 generation (Griffiths et al., 2000). Recombination will not occur in the region with the three loci between the male’s sex chromosomes since this region on the X chromosome is non-homologous to the Y chromosome. There are eight possible phenotypes in the F2 generation since the male is either providing his Y chromosome with none of these traits or his X chromosome carrying the recessive alleles (Griffiths et al., 2000). If the genes were unlinked and sorted independently, then each of the eight phenotypes has an equal 1/8 or 12.5% chance of appearing in the offspring (Griffiths et al., 2000). On the other hand, if the genes are linked then the reciprocal parental phenotypes will appear most commonly among the offspring. The frequency at which the other three reciprocal phenotypes appear will reveal the gene order of the w, m, and f loci on the X chromosome, as well as the distance between them (Griffiths et al., 2000).

Materials & Methods

Manipulation of Flies

Two methods of anesthesia were used on the flies: carbon dioxide gas and ether. Carbon dioxide gas was the preferred method of anesthesia while manipulating flies for a very short duration of time, such as while sorting groups by sex or while transferring them to new containers. This anesthetic does not cause any long-term harm to the flies which is ideal for breeding purposes. Carbon dioxide gas was administered to the flies by inserting a gas-emitting needle into the fly vials, sometimes the flies were placed on gas-emitting pads after this step to keep them “under” for longer periods of time (Carlini, 2020). This experiment also required the extensive viewing of individual flies under dissection microscopes for the purpose of determining their phenotypes (Carlini, 2020). For this purpose, the flies would be anesthetized with ether in an etherizer-chamber, which would put the flies into a very deep unconsciousness even after they removed from the chamber (Carlini, 2020). However, ether has the potential to harm the reproductive system of Drosophila, so this method was only administered to senior flies. After anesthetizing flies were manipulated with a small camel’s hair brush to prevent unnecessary harm or pain (Carlini, 2020). When flies were no longer required for the experiments, they were euthanized in a solution of 95% ethanol alcohol.

Mating Procedures, Phenotypic Descriptions, and Collection of Data

To start off the experiment the parental cross was performed by placing five wildtype (w+m+f+/red eyes, normal wings, normal bristles) males were placed into a small vial with four mutant (wmf/ white eyes, miniature wings, forked bristles) females during the beginning of January. The adult flies, having already mated were anesthetized and cleared from the vial on January 17, their phenotypes were recorded before they were euthanized.

The resulting F1 generation progeny from the parental cross consisting of 18 wildtype (w+m+f+/red eyes, normal wings, normal bristles) females and 6 mutant (wmf/ white eyes, miniature wings, forked bristles) males were anesthetized with carbon dioxide gas on January 24, and then separated into four groups. Each group was placed into its own small vial containing 10 mL instant Drosophila medium and 10 mL water (Carlini, 2020), groups #1 and #2 each consisted of four wildtype (w+m+f+) female flies and one (wmf) mutant male, while groups #3 and #4 each consisted of five wildtype (w+m+f+) females and two mutant (wmf) males. The purpose of separating the F1 generation flies into smaller groups was to facilitate the F1 cross (wildtype/w+m+f+ females x mutant/wmf males). Two weeks later on February 7, having given the flies a sufficient period to breed, the F1 adults were anesthetized with ether, cleared from their vials, and placed under a dissection microscope so that their phenotype and sex could be recorded.

The F2 generation progeny were given two weeks to mature before they too were cleared from the vials. On February 21 the adult F2 progeny that resulted from the F1 cross were anesthetized with ether, and then their phenotypes were recorded by observing them individually with the dissection microscope. There were eight possible phenotypes in the F2 generation, so each time a specific phenotypic combination was observed it was recorded in a notebook, the sex of each fly was also recorded. These eight phenotypes include: 1) (w+m+f+) wildtype eyes, wildtype wings, wildtype bristles; 2) (wmf) white eyes, miniature wings, forked bristles; 3) (w+mf) wildtype eyes, miniature wings, forked bristles; 4) (wm+f+) white eyes, wildtype wings, wildtype bristles; 5) (wmf+) white eyes, miniature wings, wildtype bristles; 6) (w+m+f) wildtype eyes, wildtype wings, forked bristles; 7) (w+mf+) wildtype eyes, miniature wings, wildtype bristles; and 8) (wm+f) white eyes, wildtype wings, forked bristles. All of the flies were disposed of in the 95% ethanol alcohol solution after the phenotypes were recorded.

Phenotypic and Sexual Descriptions

Female flies were distinguished from the males by their elongated and striped seven-segmented abdomen, males on the other hand had a black-tipped shorter five-segmented abdomen (Carlini, 2020). Additionally, sex was confirmed by checking for the presence of sex combs (short bristles) on the foreleg of the fly, males have these structures, but females do not (Carlini, 2020).

Furthermore, flies were also characterized by their phenotype in regard to eye color, wing length, and bristle type. The eye color phenotype was the easiest to identify; flies with red wildtype eyes were classified as (w+) and flies with white mutant eyes were classified as (w). The wing length phenotype was also fairly easy to determine; flies were classified with the wildtype wing phenotype (m+) if they possessed fairly long wings that extended past their abdomen, if they had short wings that did not extend past their abdomen then they were classified as the mutant miniature wing (m). Determining the bristle morphology was the most difficult phenotype to determine because both the mutants and wildtype bristles looked quite similar. However, there are some small differences that could be observed at low magnification: flies were classified as wildtype normal bristles (f+) if they had long bristles on their thorax that smoothly-curved towards the abdomen, they were classified as mutant forked bristles (f) if they had shorter forked and oddly-curved bristles on their thorax.

Data Analysis

In determining the gene order the non-crossover parental phenotypes were the first categories to be identified because these are the two most common reciprocal phenotypes among the offspring in the F2 generation: which were w^+m^+f^+ and wmf. The next step in determining the gene order was to identify the double-crossover (DCO) phenotypes, which were also easily identified because these are the two least common reciprocal phenotypes: which were w^+mf^+and wm^+f. Finally, the two reciprocal single-crossover phenotypes were identified as: w^+mf and wm^+f^+ for single crossover #1, as well as w^+m^+f and wmf^+ for single crossover #2. After identifying these four categories the next step was to draw out the parental chromosomes and simulate the mating outcomes with and without recombination events for each possible gene order. This trial and error process revealed that wmf was the only possible gene order that could produce the observed phenotypes.

Gene distance was calculated between adjacent genes (w and m, and then between m and f) using the phenotypic data collected from the F2 generation. These calculations, which are summarized by Equation 1 below, were computed by adding the number of offspring resulting from the single-crossover that produced the recombinant phenotype in question plus the offspring from the double-crossover events, and then dividing this number by the total number of offspring in the F2 generation and finally multiplying that number by 100 to get the distance in map units. Gene distance was also calculated between the genes on each end of the chromosome (w and f), these calculations are summarized in Equation 2 below. Essentially this distance was determined by adding up the sum of all the single crossover events, plus twice the sum of the double crossover, and then dividing this number by the total number of offspring in the F2 generation and finally multiplying that number by 100 to get the distance in map units.

Equation 1:

Equation 2:
To determine and quantify the degree of interference between wm crossovers and mf crossovers the coefficient of interference (C) was first calculated by divided the observed number of double-crossovers among the F2 generation by the expected number of double-crossovers. From this value, the coefficient of interference and interference were calculated (see Equations 3 and 4 below).

Equation 3:

Equation 4:

Results

The parental (P1) cross consisted of 4 triple homozygous mutant females (X^{wmf}/X^{wmf}) with 5 triple homozygous wildtype males (X^{w^+m^+f^+}/Y). The triple homozygous mutant females phenotypically appear as triple mutants, meaning that they had white eyes, miniature wings, and forked bristles. The triple homozygous wildtype males phenotypically appeared as completely normal with red eyes, long normal wings that extended past their abdomen, and normal bristles. This information is shown below in Table 1.

The purpose of the P1 cross was to produce a F1 generation with the genotypes required to perform a three-point cross. During prophase of meiosis I, the Drosophila’s four homologous chromosomes pair up to form tetrads. If crossing over occurs between the mutant female’s X chromosome there would no alterations to the genotype nor phenotype of the offspring because the two homologous X chromosomes carry the same genes. At the end of meiosis, the mutant mother flies can provide only gametes with a mutant X chromosome, and the father provides 50% gametes with a wildtype X chromosome and 50% gametes with a Y chromosome, which does not carry any of the traits. During fertilization, a paternal gamete fuses with a maternal gamete to form a zygote. Therefore, it was expected that 50% of the resulting F1 offspring would receive a mutant X chromosome from the mother and a Y chromosome from the father. These F1 offspring would have a triple homozygous mutant genotype (X^{wmf}/Y) and phenotypically appear as males with white eyes, miniature wings, and forked bristles. It follows then that the other 50% of the F1 offspring would receive a mutant X chromosome from the mother and a wildtype X chromosome from the father. These F1 offspring would have a triple heterozygous wildtype genotype (X^{w^+m^+f^+}/X^{wmf}) and phenotypically appear as females with red eyes, normal wings, and normal bristles. The expected phenotypes and genotypes of the F1 progeny from the parental cross are shown below in Figure 1.

The observed phenotypes in the F1 generation that resulted from the parental-cross matched what the expected phenotypes, meaning that there were only two phenotypes: females with red wildtype eyes, normal wildtype wings, and normal wildtype bristles (X^{w^+m^+f^+}/X^{wmf}); and males with white mutant eyes, miniature mutant wings, and forked mutant bristles (X^{wmf}/Y). However, the expected and observed numbers of each phenotype did not match. There were 24 total offspring in the F1 generation, 12 of them or 50% were expected to be w^+m^+f^+ females and 12 of them or 50% were expected to be wmf males. However, as shown below in Table 2, 18 of the 24 offspring or 75% of the F1 generation were phenotypical w^+m^+f^+ females, and the other 6 or 25% of the F1 generation were phenotypical wmf males.

The F1 generation flies were mated to one another to produce the F2 generation. A total of 181 offspring were recorded for the F2 generation, with eight unique phenotypes. The phenotypes and numbers of all the F2 offspring from the F1 wildtype female x F1 mutant male cross are shown below in Table 3. A total of 71 flies (36 females and 35 males) displayed the w^+m^+f^+ phenotype, which was characterized by red wildtype eyes, normal wildtype wings, and normal wildtype bristles. A total of 17 flies (7 females and 10 males) displayed the wmf phenotype, which was characterized by white mutant eyes, miniature mutant wings, and forked mutant bristles. A total of 7 flies (3 females and 4 males) displayed the w^+mf phenotype, which was characterized by red wildtype eyes, miniature mutant wings, and forked mutant bristles. A total of 30 flies (15 females and 15 males) displayed the wm^+f^+ phenotype, which was characterized by white mutant eyes, normal wildtype wings, and normal wildtype bristles. A total of 10 flies (5 females and 5 males) displayed the w^+m^+f phenotype, which was characterized by red wildtype eyes, normal wildtype wings, and forked mutant bristles. A total of 27 flies (10 females and 17 males) displayed the wmf^+ phenotype, which was characterized by white mutant eyes, miniature mutant wings, and normal wildtype bristles. A total of 12 flies (7 females and 5 males) displayed the w^+mf^+ phenotype, which was characterized by red wildtype eyes, miniature mutant wings, and normal wildtype bristles. A total of 7 flies (5 females and 2 males) displayed the wm^+f phenotype, which was characterized by white mutant eyes, normal wildtype wings, and forked mutant bristles.

The eight possible phenotypes did not occur in equal proportion, instead, one reciprocal phenotype dominated among the F2 generation, and three other reciprocal phenotypes appeared in a lower frequency, which is consistent with X-linked inheritance. The most common reciprocal phenotype was w^+m^+f^+/\ wmf, a total of 48.6% of the offspring displayed one of these phenotypes. This is called the parental phenotype, and this is what occurs when there is no recombination (see Figure 2a below). Two other reciprocal phenotypes occurred in equal frequency (20.4% of each reciprocal phenotype). One of these reciprocal phenotypes was w^+mf/wm^+f^+, which occurred when a single crossover recombination event occurred between the w and m loci(shown in Figure 2b below). The other one of these reciprocal phenotypes was wmf^+/w^+m^+f, which occurred when a single crossover recombination event occurred between the m and f loci (shown below in Figure 2c). The least common reciprocal phenotype was w^+mf^+/wm^+f, which was observed in only 10.5% of the F2 offspring. This uncommon phenotype occurred when there was a double crossover recombination event, meaning that there was a crossover between w and m, and also between m and f (shown below in Figure 2d).

To determine if these three traits were linked or unlinked and independently assorting two chi-square tests were performed. The first chi-square test looked at each of the three traits separately, each of these three chi-square tests would have one degree of freedom since there are two phenotypic classes. This means that the chi-square critical value at a 5% significance threshold (P=0.05) is 3.841. The null hypothesis for these three chi-square tests is that these genes are independently assorting. The null hypothesis can be rejected if the chi^2 value is greater than 3.841.

If only looking at the eye color morphology for the F1 heterozygous wildtype red-eyed females (X^{w^+}/X^w) x F1 homozygous mutant white-eyed males (X^w/Y) cross, then half of the 181 offspring in the F2 generation or 90.5 flies would be expected to display the wildtype red-eye phenotype (w+) and the other half would be expected to display the mutant white-eye phenotype (w). However, the actual phenotypes varied: 100 flies had wild-type red eyes and 81 had mutant white eyes. This calculated out to a chi-square value equal to 1.99. Since the calculated chi^2 value equal to 1.99 for wild type versus white eyes falls below the critical chi^2 value of 3.841, critical chi^2 value of 3.841 for one degree of freedom and P=0.05, meaning that the null hypothesis cannot be rejected. This means that the flies observed eye color phenotypes could match the 1:1 ratio expected by independent assortment and that the observed variation from the expected values could be due to chance alone (see Table 4, Trait 1 below).

If only looking at the bristle morphology for the F1 heterozygous wildtype bristled females (X^{f^+}/X^f) x F1 homozygous mutant forked bristled males (X^f/Y) cross, then half of the 181 offspring in the F2 generation or 90.5 flies would be expected to display the wildtype bristle phenotype (f+) and the other half would be expected to display the mutant forked bristle phenotype (f). However, the actual phenotypes varied from what was expected: 140 flies had wild-type bristles and 41 had mutant forked bristles. This calculated out to a chi-square value equal to 54.15. This calculated chi^2 value equal to 54.15 for wild type versus forked bristles is greater than the critical chi^2 value of 3.841 for one degree of freedom and P=0.05, meaning that the null hypothesis can be rejected. This means that the variation observed in the flies' bristle phenotypes does not match the 1:1 ratio that would be expected if these genes were independently assorting, so it can be concluded that these genes are linked (see Table 4, Trait 2 below).

If only looking at the wing morphology for the F1 heterozygous wildtype normal winged females (X^{m^+}/X^m) x F1 homozygous mutant miniature winged males (X^m/Y) cross, then half of the 181 offspring in the F2 generation or 90.5 flies would be expected to display the wildtype wing phenotype (m+) and the other half would be expected to display the mutant miniature wing phenotype (m). However, the actual phenotypes varied from the expected phenotypes: 118 flies had wild-type wings and 63 had mutant miniature wings. This calculated out to a chi-square value equal to 16.71. This calculated chi^2 value equal to 16.71 for wild type versus miniature wings is greater than the critical chi^2 value of 3.841 for one degree of freedom and P=0.05, meaning that the null hypothesis can be rejected. This means that the variation observed in the flies wing phenotypes does not match the 1:1 ratio that would be expected if these genes were independently assorting, additionally, it can be concluded that these genes are linked (see Table 4, Trait 3 below).

Overall, the observed bristle morphology in the F2 generation deviated the greatest from what was expected, with a chi^2 value of 54.15. The eye color morphology had the least deviation from the expected in the F2 generation, with a chi^2 value of 1.99. All of this information is shown below in Table 4.

The second chi-square test looked at each of the three traits together, a trihybrid test cross of the F1 parents (fully heterozygous wildtype females (X^{w^+m^+f^+}/X^{wmf}) x fully homozygous mutant males (X^{wmf}/Y)) was performed to calculate the expected frequencies of each phenotype. The F1 parental gametes can combine in eight possible ways to create eight unique phenotypes in the F2 generation. If these traits are not linked and segregate via independent assortment during meiosis, then each of the eight phenotypes should be expected in equal frequency (12.5% or 1/8 chance), meaning that 22.625 offspring should be expected in each phenotypic category. With eight phenotypic categories, there are seven degrees of freedom. This means that the chi-square critical value at a 5% significance threshold (P=0.05) is 14.07. The null hypothesis for this chi-square test is that these three genes are independently assorting. Thus, the null hypothesis can be rejected if the chi^2 value is greater than 14.07. As was reported in Table 3, the eight phenotypic ratios did not match this 1:1:1:1:1:1:1:1 ratio with 22.626 flies in each category. Instead, the expected versus observed phenotypes varied dramatically, each phenotypic category had as little as 7 observations or as many as 71 (see Tables 3 and 5). With this data the chi^2 value was determined to be equal to 141.80, these calculations are shown below in Table 5. This calculated chi^2 value equal to 141.80 for all three traits (eye, wing, and bristle morphology) is greater than the critical chi^2 value of 14.07 for seven degrees of freedom and P=0.05, meaning that the null hypothesis can be rejected. This means that the variation observed eight phenotypic categories for eye, wing, and bristle morphology does not match the 1:1:1:1:1:1:1:1 ratio that would be expected if these three genes were independently assorting, additionally it can be concluded that these genes are linked (see Table 5 below).

The genetic map shown below in Figure 3 was generated by calculating the gene order and gene distance of the three loci. The gene order for the w, m, and f loci were determined by identifying w+m+f+ and wmf as the reciprocal parental phenotypes, w+mf+ and wm+f as the reciprocal double crossover phenotypes, and w+mf and wm+f+ as the single-crossover reciprocal phenotypes for recombination between w and m, and finally w+m+f and wmf+ as single-crossover reciprocal phenotypes for recombination between m and f. After identifying these phenotypic categories, a trial-and-error stimulated mating of the F1 parents revealed that the m locus was between the w and f locus. This gene order: w, m, and then f was the only gene order on the X chromosome that could have produced the phenotypes observed in the F2 generation of Drosophila.
The gene distance between w and m was determined to be 30.9 map units since there were 19 double crossovers and 37 single crossovers between w and m. These calculations are shown below in Equation 5.

Equation 5:

The gene distance between m and f was also determined to be 30.9 map units since there were 19 double crossovers and were 37 single crossovers between m and f. These calculations are shown below in Equation 6.

Equation 6:

Finally, the gene distance between w and f was determined by adding up the sum of all the single crossover events, plus twice the sum of the double crossover, and then dividing this sum by the total number of individuals in the F2 generation. These calculations are shown below in Equation 7.

Equation 7:

Interference occurs when a recombination event in a particular region of the chromosome prevents another recombination event from occurring on another region of the chromosome, the net result of interference is that the expected and observed frequencies of double-crossover events are not equal.

Equation 8:

The expected number of double crossovers in the F2 generation is equal to the product of the observed single crossovers, multiplied by the total number of individuals in the F2 generation. The calculations, which determined that 17.236 double-crossovers should be expected for this sample are shown in equations 8 and 9.

Equation 9:

The coefficient of interference (C) is 1.09661 this was calculated in Equation 10 below by divided the observed number of double-crossovers (19) among the F2 generation by the expected number of double-crossovers (17.236).

Equation 10:

Interference was calculated in Equation 11 from the coefficient of interference.

Equation 11:

The interference was calculated to be equal to -0.0966, which means that there were 9.66% more double crossovers than expected in the F2 population. This negative interference is most likely explained by sampling error because the F2 population only consisted of 181 Drosophila individuals.

Discussion

In the duration of this experiment the three traits eye color morphology, wing morphology, and bristle morphology were determined to be X-linked through four methods. The first method was by comparing the phenotypes of males and female offspring in the F1 generation that resulted from the parental cross. If the traits were X-linked and since the parental cross consisted of a wildtype male mated to a mutant female, all of the female offspring were expected to have a wildtype phenotype since they would inherit one wildtype X chromosome and one mutant X chromosome, and all of the males were expected to have a mutant phenotype since they would only inherit one mutant X chromosome (refer to Figure 1). The observed phenotypes in the F1 generation matched the expected phenotypes, as shown in Table 2 all of the F1 females had wildtype phenotypes and all of the F1 males had mutant phenotypes. If these traits were located on one of the three pairs of autosomal chromosomes, then there would be no phenotypic differences between the male and female progeny of the F1 generation. The second method for determining if these traits are X-linked was with the chi-square test using data collected from the F2 generation that resulted from the F1 cross. The null hypothesis was that the three traits were unlinked and independently assorting. The chi-square test in Table 4 and Table 5 tested this hypothesis. Specifically, the chi-square test of independent assortment for all three traits considered together (Table 5) used the outcome of a trihybrid F1 test cross to predict that the eight phenotypes would appear in a ratio of 1:1:1:1:1:1:1:1, meaning that each phenotype had an equally likely 12.5% chance of occurring. However, the data collected from the F2 generation shown in Table 3 revealed that these phenotypes did not occur equally. Instead the phenotypic ratio was 71:17:7:30:10:27:12:7. The chi^2 value for this test equaled 141.80, and because there were 7 degrees of freedom the critical chi^2 value at P=0.05 was equal to 14.07. Since the calculated chi^2 value was greater than the critical chi^2 value (141.80 > 14.07) the null hypothesis was rejected, meaning that the variation between the expected and observed phenotypes could not be explained by independent assortment. This strengthened the conclusion that these traits were X-linked. The third method for determining the traits are X-linked was by identifying the trend in frequencies of reciprocal phenotypes, because in linked genes a reciprocal parent phenotype can occur in high frequency among the F2 generation, as well as an uncommon double-crossover reciprocal phenotype. Using the frequencies that each phenotype occurred, the parental reciprocal phenotypes were identified as: w+m+f+ and wmf, the double crossover reciprocal phenotypes were identified as: w+mf+ and wm+f, the reciprocal phenotypes for single-crossover between w and m were identified as: w+mf and wm+f+, and finally, the reciprocal phenotypes for single-crossover between m and f were identified as: w+m+f and wmf+ (see Figure 2). Finally, the fourth method for determining these traits are X-linked was by determining the recombination frequency. The recombination frequency is proportional to the gene distance in map units (m.u.), and genes are considered linked if they have a recombination frequency of less than 50% (distance between them is less than 50 map units). Equation 5 calculated the distance between the w locus and m locus to be equal to 30.9 map units, and equation 6 calculated the distance between the m locus and f locus to be equal to 30.9 map units. This means that the recombination frequency between the w and m and m and f loci is 30.9%, which is consistent with the rules of linked inheritance. So in summary, the genes that are responsible for the expression of white eye morphology, miniature wing morphology, and forked bristle morphology are X-linked because they are located on the portion of the X chromosome that is non-homologous to the Y chromosome, which means they can only be inherited through the X and not the Y chromosome.

The established gene map for these three traits confirms the gene order predicted in the experiments (refer to Figure 3), which is: white eyes (w), then miniature wings (m), and last forked bristles (f) This experiment predicted the gene distance between white eyes (w) and miniature wings (m) was 30.9 map units; while the established map showed that the true distance between these genes is 34.6 map units (Klug et al., 2014). This means that the gene distance calculated between the w and m loci was 10.7% less than the actual distance. This experiment also predicted the gene distance between miniature wings (m) and forked bristles (f) was 30.9 map units; while the established map showed that the true distance between these genes is actually 20.6 map units (Klug et al., 2014). This means that the gene distance between the m and f loci calculated with this data was 50% greater than the actual distance. Additionally, this experiment predicted that the distance between white eyes (w) and forked bristles (f) was 61.9 map units, but the establish map showed that the real distance was 55.2 map units (Klug et al., 2014). This means that the gene distance between w and f calculated with the F2 data was 12.1% greater than the actual distance.

However, it is important to identify the flaws within this experiment that might have impacted the findings. One significant flaw in the experiment was the small sample size, especially among the F2 generation. The established genetic map uses data collected from tens of thousands of F2 generations and thus the frequencies of recombination are stabilized amongst a very large sample size. This experiment on the other hand only used 181 flies to generate the F2 map, so abnormalities in recombination frequencies had a significant impact on all of the calculations. Furthermore, it is possible that the phenotypic categories of some of the F2 generation progeny were misidentified, especially for the bristle morphology which was difficult to characterize in some individuals. This possibility of human error in diagnosing bristle phenotypes was most likely the reason for the 50% error in gene distance between the m and f loci. Another problem with the experiment is that halfway through the viewing and cataloging of the F2 offspring some of the flies began to “wake up” from the deep ether-induced sleep and fly away, so these flies were not part of the dataset. Additionally, the difference in the number of individuals in the F2 generation with phenotypically normal characteristics and mutant characteristics might be due to an environmental characteristic such as media, the chemical composition of the water, temperature, light exposure, etc. that reduced the fitness of the mutant flies and prevented them from developing into adults.

So, in conclusion, the three traits, eye color morphology (red wildtype w+ versus white mutant w), wing morphology (normal wildtype m+ versus miniature mutant m), and bristle morphology (normal wildtype f+ versus forked mutant f) were determined to be X-linked. The gene order that was revealed by the phenotypic frequencies in the F2 generation of the F1 cross was: white eye locus (w) first, followed by the miniature wing locus (m), and last the forked bristle locus (f). The genetic map including the gene order and gene distances is shown in Figure 3. The calculations for the distances between each locus are shown in equations 5, 6, and 7. The distance between the white eye locus (w) and the miniature wing locus (m) was calculated from the recombination frequency which is equal to 30.9 map units. The distance between the miniature wing locus (m) and the forked bristle locus (f) was calculated from the recombination frequency which is equal to 30.9 map units. Finally, the distance between the locus (w) first, followed by the miniature wing locus (m), and last the forked bristle locus (f) was calculated and determined to be 61.9 map units.

References

Carlini, D. (2020) Lab #1: Three-Point Mapping in Drosophila. BIO-356 Genetics with Laboratory. American University Department of Biology: Washington, D.C.

Carlini, D. (2020) Lecture #9: Linkage & Mapping 1 BIO-356 Genetics with Laboratory. American University Department of Biology: Washington, D.C.

Carlini, D. (2020) Week 3 Lab Exercise: The Chi-Square Test. BIO-356 Genetics with Laboratory. American University Department of Biology: Washington, D.C.

Carlini, D. 2020. Working with Drosophila. BIO-356 Genetics with Laboratory. American University Department of Biology: Washington, D.C.

Cook, R. K., M.E. Deal, J.A. Deal, R.D. Garton, C.A. Brown, M.E. Ward, R.S. Andrade, E.P. Spana, T.C.

Kaufman, and K.R. Cook. (2010). A new resource for characterizing X-linked genes in Drosophila melanogaster: systematic coverage and subdivision of the X chromosome with nested, Y-linked duplications. Genetics, 186(4), 1095–1109.

Griffiths, A.J.F., J.H. Miller, D.T. Suzuki, R.C. Lewontin, and W.M. Gelbart. (2000) An Introduction to Genetic Analysis. (7th edition). New York: W.H. Freeman.

Hales, K.G., C.A. Korey, A.M. Larracuente, and D.M. Roberts. (2015). Genetics on the Fly: A Primer on the Drosophila Model System. Genetics, 201(3):815-842.

Klug, W.S., M.R. Cummings, C.A. Spencer, and M.A. Palladino. (2014). Concepts of Genetics. (11th edition). Pearson. (pp. 121).

Lobo, I. & Shaw, K. (2008) Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature Education 1(1):205.

Slatis, H.M., and D.A. Willermet. (1953). The Miniature Complex in Drosophila Melanogaster. Department of Genetics, McGill University, Montreal, Canada.

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