Kudlac 1 Kaitie Kudlac March 24, 2015 Professor Ma Genetics 356 The Determination of the Genetic Order and Genetic Map for the Eye Color, Wing Size, and Bristle Morphology in Drosophila melanogaster Abstract: In this experiment, Drosophila melanogaster was used as a model organism to look at the concept of linkage and X linked inheritance through the traits of eye color, wing size, and bristle morphology. Through the production of an F1 and F2 generation starting with a P generation of a recessive mutant female and a wild type dominant male, eight various phenotypes were yielded in order to determine recombination frequencies and map distances. Ultimately, the gene order was determined, as well as the map distances, and though the established distances did not exactly correspond to those determined in the lab, the correct gene order was determined and the genes or traits were established as linked or located on the X chromosome through a chi square analysis. We determined the map distances as eye color to bristle morphology being 88.89 map units, eye color to wing size as 45.6 map units, and wing size to bristle morphology as 43.3 map units. Introduction: The main focus and purpose of this lab is to understand the concept of linkage and gene mapping in Drosophila or fruit flies. More specifically, to determine the location of the wing size, eye color, and bristle morphology through the frequency of recombinants
Kudlac 2 on a genetic map of a fruit fly. Through these maps, large numbers of genes are related through the basis of the frequency of crossing over between the various genes. These goals will be met through working with two fly stocks, wild type and mutant. The first cross that is made is between a recessive mutant female crossed with a wild type normal male. This generation is also known as the parent generation or P generation, making the parents a female fly with all recessive mutant genes and a male fly with all wild type dominant genes. This then yielded a F1 generation in which the males were all recessive mutants and the females were wild type. An F2 generation was then bred and created by crossing a wild type female and recessive male. This cross then yielded eight different phenotypes whose recombination frequencies were then used to determine the location of the genes in a genetic map. In order to create a genetic map and attempt to understand the linkage concept in a short time, an organism, known, as a model organism is required. A model organism is an experimental organism helpful to research and whose genetics is studied on the idea that the findings can then be applied to other organisms such as chimpanzees or humans. In this lab we use Drosophila melanogaster to experiment with X linked chromosomes, following Thomas Morgan s groundbreaking studies on fruit flies. Drosophila melanogaster is an excellent model organism as it exhibits a variety of traits and features that make it easy to study. These features include being relatively easy to breed, comprising a relatively short life cycle, reproducing with large numbers of offspring, having a fairly straightforward genetic analysis, traits, such as the ones that are studied in this lab are also easily recognizable, and finally the differences between the sexes of the flies are easy to distinguish. In this lab there are three different genes or traits being
Kudlac 3 studied. The first is the white locus (w), which affects eye color. The dominant or wild type for this locus is the red eye color while the recessive trait for this locus is white-eye color. The second gene is the forked locus (f), which affects bristle morphology. Flies with the recessive forked mutation will have short bristles that will have forked or split ends while the dominant or wild type flies will have smooth curved bristles. The third and final gene that this lab is examining is wing size (m). Mutant flies or flies that express the recessive allele will have miniature wings that do not extend past the tip of the abdomen while wild type flies or flies that express the dominant trait will have normal size wings. One of the most important concepts that are being examined in this lab is the idea of X- linked traits. Not only do the X and Y sex chromosomes carry the genes that determine sex they also carry other genes for other characteristics, as is seen in Drosophila melanogaster. Because males have an X and Y chromosome and females have two X chromosomes, both males and females can inherit X- linked traits since they each have an X chromosome. Because men only have one X chromosome, if the mother has the disease or trait, the male has a 100% chance of also getting the trait since they have to inherit their mother s X chromosome since they inherit the Y chromosome from the father. Females are a little bit different. Since they inherit one X chromosome from their mother and one X chromosome from the father, if the mother is affected they will also get the disease since both X chromosomes contain the disease but additionally if the father has the disease they will also get the disease since they get the father s only X chromosome (O Neil, 2012). Another case is if the mother is a carrier for the trait. If the mother is a carrier than each child has a 50% chance of getting the disease because only one of the X chromosome s is affected. However if the mother has the disease and the
Kudlac 4 father does not, a female child will not get the disease necessarily but instead will become a carrier since they will get one X chromosome that contains the mutated gene from the mother and one normal X chromosome from the father (NIH, 2015). X linked traits differ from autosomal in that autosomal dominant tend to occur in every generation of an affected family and occurs when one mutated copy of the gene is enough to be affected with the disorder (NIH, 2015). This normally occurs when the affected person has one affected parent. X linked dominant on the other hand is caused by mutations with genes on the X chromosome (O Neil, 2012). Females are often more frequently affected than males. Families that have an X- linked dominant disorder often have affected males and females in each generation. One of the major characteristics of this X linked inheritance pattern is that fathers cannot pass these traits to their sons. Autosomal recessive is different from autosomal dominant in that two mutated copies of the gene are present and the parents each carry a single copy of the mutated gene or are known as carriers. These disorders are not seen in every generation (O Neil, 2012). X- linked recessive as opposed to autosomal recessive is different in that males are more frequently affected than females and the chances of passing the disorder on differ from male to female, similar to an X- linked dominant trait. Families with an X- linked recessive disorder often have affected males as opposed to affected females in each generation (NIH, 2015). As earlier described, three different genes and traits are examined in this lab, leading to a three point genetic map or a three point cross. The three point cross is executed by crossing a wild type male, fruit fly in this case, with a recessive female fruit fly. This will produce an F1 generation with recessive males and wild type females. The F1 generation flies are then crossed eventually yielding eight different phenotypes in the
Kudlac 5 F2 generation. Then with the F2 generation flies, the parental and recombinants must be identified. The parental phenotype is most often the phenotype with the largest number of flies. Conversely, the double crossover phenotype is the phenotype with the lowest number of flies, and the single crossover is somewhere in between. Once these phenotypes have been determined, the gene order then needs to be established. This is normally done through the double crossovers since these are the alleles in the middle that are flipped. After this the distance between the two genes is then determined through the recombination frequency, which is calculated by dividing the number of recombinant progeny by the total progeny. The double crossovers need to be counted twice since they are part of both sets of gene recombinants. Recombination frequencies need to be calculated for all three different gene combinations. The recombination frequency is directly linked to the distance of the gene loci in centimorgans or map units. Once the recombination frequencies are calculated a genetic map can be drawn, because the higher the recombination frequency the further apart the genes are or vice versa. In other words the greater the distance between the two genes the greater chance that the genes will cross over or have recombination. The official genetic map for Drosophila melanogaster indicates that correct order of the specific genes that are studied in this lab report are w (eye color), m (wing size), and f( bristle morphology). The distance between w and m is 34.6 map units, the distance between m and f is 20.6 map units, and the distance between w and f is 55.2 map units (Chromosome Mapping in Eukaryotes, 2015). Materials and Methods: Because the main portion of this procedure and the main work in this lab was counting and analyzing flies, one of the most important techniques with the manipulation
Kudlac 6 of the flies. One of the main skills in this lab is counting and characterizing the various generations of flies, however, to do this the flies must be anaesthetized. To do this, the flies are either placed on a fly bed with CO2 gas or they are anaesthetized through a small CO2 needle that is placed in the vial to put them to sleep before they are counted outside of the vial. Once the flies are anaesthetized, they are then placed under a dissecting scope where they are counted according to the different phenotypes that they present. In order to obtain these different generations, the flies were allowed to mate for up to two weeks. As the typical life cycle for Drosophila melanogaster is twelve days to reach maturity, this two-week incubation period allows for slower developing flies to reach maturity so they are sexually active and ready to produce the subsequent generation. It also allows for a more accurate fly count. While Dr. Walters completed the parental cross, we removed the parental flies from the vials so that we would only initially count the F1 generation flies. Once the F1 generation flies were counted, approximately 5 males and 5 females were placed into each tube in order to promote reproduction and sexual activity. Ultimately, only two different generations of flies will be counted and characterized, the F1 generation and the F2 generation. In the P generation there are only two phenotypes, females that are mutant recessive and males that are wild type dominant. In the F1 generation there are also two phenotypes to be both distinguished and counted; the flies are either males which are mutant recessive or females that are dominant wild type. The F2 cross is a little bit different. There are eight different phenotypes that are possible for the flies. These phenotypes include red eyes, normal wings, and curved bristles, red eyes, normal wings, and forked bristles, red eyes, miniature wings, and curved bristles, red eyes, miniature
Kudlac 7 wings, and forked bristles, white eyes, miniature wings, and forked bristles, white eyes, miniature wings, and curved bristles, white eyes, normal wings, and forked bristles, and finally white eyes, normal wings, and curved bristles. At this stage it is no longer important to determine the sex of the flies since this is the stopping point of the lab. However, these traits or groups of phenotypes can appear in either males or females. Collectively, these phenotypes will be determined using a dissecting scope. For the rest of the lab report, the eye color trait will be referred to as w, the forked bristle morphology trait will be referred to as f, and the wing size trait will be referred to as m. The letters will indicate the recessive trait while the + will indicate the wild type trait. Once the flies are counted and characterized, the final portion of the experiment is to determine the gene order and the map distance calculations using the number of flies from the F2 generation. The first step in determining the gene order is to determine which phenotypes are the parentals and which phenotypes are the double crossovers. Most often, the phenotype with the largest number of flies is the parental while the phenotype with the smallest number of flies is the double crossover; the single crossovers are in the in between zone. After these different groups are distinguished, recombination frequencies for each different gene combination are calculated to determine the percent recombination. The percent recombination is able to determine the gene order because the percentage is equal to the gene distance in map units or centimorgans. Then by using the fact that the double crossovers are the alleles in the middle that are flipped, the gene order can be determined based on the distances between the two genes and the double crossover gene being in the middle of the two alleles.
Kudlac 8 Results: The first cross that was conducted in this experiment was the P generation. This cross was between a recessive female who had white eyes, miniature wings, and forked bristles and she was crossed with a wildtpye male who had red eyes, normal wings, and curved bristles. These two flies are expected to produce a F1 generation of flies that will have males that are recessive mutants and females that are dominant wildtypes. Table 1: Phenotypes and Number of Offspring in the F1 Generation Phenotype Number of Offspring +++- red eyes, normal wings, curved 36 bristles Wmf- white eyes, miniature wings, and 26 forked bristles Though the sex of the flies is not shown in this table, all flies that have the recessive mutant alleles are males and all flies that have the wildtype dominant allele are female. This table indicates the phenotype of the F1 generation and the number of offspring that the P generation produced. These flies will then be crossed, in a test cross, to create the F2 generation of flies. Once the F1 flies are categorized and counted they are then used to mate the F2 generation. In order to mate the F2 generation a male recessive mutant with white eyes, miniature wings, and forked bristles is crossed with a dominant wildtype female with red eyes, normal wings, and curved bristles. This then produces the following fly counts and phenotypes.
Kudlac 9 Table 2: Numbers and Phenotypes of All F2 Offspring Resulting from the F1 Female +++ x wmf male Phenotype Number of Flies Red, normal wings, curvy bristles(+++) 23 Red, normal wings, forked bristles(++f) 12 Red, miniature wings, curvy bristles (+m+) 11 Red, miniature wings, forked bristles (+mf) 3 White, miniature wings, forked 7 bristles(wmf) White, miniature wings, curvy 2 bristles(wm+) White, normal wings, forked bristles(w+f) 10 White,normal wings, curvy bristles (w++) 13 This table indicates the eight different phenotypes that are produced as a result of the cross between a wildtype dominant female and a recessive mutant male. These numbers and these flies will be used to determine the genetic order and the genetic map for these three traits. The phenotype with the most flies is the parental and the phenotype with the smallest number of flies is the double crossover, the single crossovers are everything else that is not the parentals or double crossovers. However, instead of using the smallest number of flies for the double cross over, because we lost two vials of flies in this experiment, the double crossovers will be the phenotypes in which there are two traits that vary from the parentals. So in this case those will be +m+ and w+f. The parentals will be +++ and wmf. Normally the data would follow the number pattern as previously outlined, however, due to a loss of flies, the data does not adhere to the correct phenotypes. All recombinant calculations will be made based on this information. With these numbers, a chi square analysis will be used to determine if independent assortment exists within these genes or if these traits or genes are linked and inherited through sex chromosomes as opposed to autosomal chromosomes.
Kudlac 10 Table 3: Calculation of Chi Square using Expected Ratio of Dominant to Recessive for Each Trait in Monohybrid Test Cross Trait 1: Wild- Type (Red Eyes) vs. White Eyes Phenotype Observed Expected (O-E)^2 (O-E)^2/E Red Eyes 49 60.75 138.0625 2.273 White Eyes 32 20.25 138.0625 6.8179 This table shows the calculation of a chi-square that for the eye color gene. The chisquare that is calculated is equal to 9.091, which allows for the hypothesis to be rejected since the chi square value is more than 3.841 (when p is 0.05). Trait 2: Wild Type (Curved Bristles) vs. Forked Bristles Phenotype Observed Expected (O-E)^2 (O-E)^2/E Forked Bristles 32 20.25 138.0625 2.273 Curved Bristles 49 60.75 138.0625 6.8179 This table shows the chi-square calculation for the bristle morphology gene. The chi-square is equal to 9.091, which rejects the null hypothesis according to the degrees of freedom, which says the chi square value should be less than 3.841. Trait 3: Wild Type (Normal Size Wings) vs. Miniature Wings Phenotype Observed Expected (O-E)^2 (O-E)^2/E Miniature 23 20.25 7.5625 0.3734 Wings Normal Wings 58 60.75 7.5625 0.1244 This table shows the chi- square calculation for the wing size trait. The chi-square is equal to 0.4979, which is less than 3.841, so the hypothesis is accepted for this trait. While the charts above use the expected ratio of dominant to recessive individuals for each trait in a monohybrid cross, this lab looks more specifically at the trihybrid ratio and how the three genes are mapped in relation to each other. So rather than looking at the individual monohybrid cross, it would be more productive to look at the trihybrid cross.
Kudlac 11 Table 4: Calculation of Chi Square Assuming Three Traits are Assorting Independently (Ratios used from Trihybrid Test Cross) Phenotype Observed Expected (O-E)^2 (O-E)^2/E +++ 23 34.17 124.77 3.65 ++f 12 11.39 0.3721 0.0326 +m+ 11 11.39 0.1521 0.0133 +mf 3 3.79 0.6241 0.1647 Wmf 7 1.265 32.89 26 Wm+ 2 3.796 3.2256 0.8497 W+f 10 3.79 38.56 10.174 W++ 13 11.39 2.59 0.227 This table shows the chi square calculation based on the hypothesis that these three traits are assorting independently through the assumption of the expected rations used for the trihybrid test cross. The chi square value that was calculated based on this information was 41.111 leading to the rejection of the null hypothesis and indicating that these traits are linked and not independently assorted. Figure 1: Genetic Map: W 45.6 M 43.3 F Distance from W to F is 88.9 Legend Key: w- eye color, m- wing size, and f- bristle morphology
Kudlac 12 This genetic map displays the distance in map units between the three different traits or gene loci. Because the genes are generally far apart this allows for a greater chance of crossover and a greater propensity of recombination. The final piece of data that was collected for this experiment was the interference value. The interference value is calculated by subtracting 1 from the coefficient of coincidence. The coefficient of coincidence is calculated by dividing the observed double crossovers by the expected double crossovers. The coefficient of coincidence in this case is 1.383 so the interference value is negative.383. Discussion Generally the theoretical background of the predicted outcome of the experiment is that for any genetic cross, including this one, the further apart the genes are or the greater the distance between the two genes the larger chance of recombination and the higher recombination frequency between the two loci. In other words, because they genes or linked or rather if the genes are linked the greater the distance the more recombination is likely. The three traits were determined to be X-linked through the P generation and the F1 generation. Because the P generation started with a cross between a recessive mutant female and wildtype dominant male and yielded a F1 generation of all recessive mutant males and all dominant wildtype females, and because a male inherits his X chromosome from his mother, these traits are X linked because of the swap over the two generations. In other words because of the fact in the first generation that the males were dominant and recessive in the second generation, we can say that this trait is X linked or sex linked. The established genetic order for these three traits is w( eye color), m(wing size) and finally f( bristle morphology). The distance between w and m is 34.6 map units, the
Kudlac 13 distance between m and f is 20.6 map units and the distance between w and f is 55.2 map units. While these distances do not ultimately correspond to the distances that were determined in this lab they are relatively close. In the lab we determined the distance from w to f as 88.89 map units, the distance between w and m as 45.6 map units, and the distance between m and f as 43.3 map units. Though the distance were a little off, the genetic order that was determined was correct. Because our distances were not exactly the same as the established genetic map, there are a few reasons that might account for these discrepancies. One of the main reasons that our data was skewed was because between the F1 and the F2 generation two vials of our flies died. Because of this we did not have as many flies for our statistical analysis and these flies could have improved our numbers and increased or decreased the recombination values and thus changed the calculated distances for the genetic map. Another possible source of error was that ultimately we did not have large numbers of flies. In order for statistical analysis to be useful, a large total needs to be used so that small changes or errors are not detected in the overall result. A final possible source of error was that in some cases there were more single crossovers than double crossovers, attributing tot the difference in recombination frequency and genetic distances. In conclusion, the main findings of this experiment were that the traits of eye color, bristle morphology, and wing size are linked and are found on the X chromosome. They are in the order of wmf and are a total of 55.2 map units apart, though we found them to be 88.9 map units apart (Chromosome Mapping in Eukaryotes). References:
Kudlac 14 "Chapter 5 Chromosome Mapping in Eukaryotes." Chromosome Mapping. 1 Jan. 2015. Web. 26 Mar. 2015. NIH. "What Are the Different Ways in Which a Genetic Condition Can Be Inherited?" Genetics Home Reference. 1 Jan. 2015. Web. 26 Mar. 2015. O'Neil, Dennis. "Biological Basis of Heredity: Sex Linked Genes." Biological Basis of Heredity: Sex Linked Genes. 1 Jan. 2012. Web. 26 Mar. 2015.