Lab I: Three-Point Mapping in Drosophila melanogaster

Lab I: Three-Point Mapping in Drosophila melanogaster Makuo Aneke Partner: Christina Hwang BIO 365-004: Genetics with Laboratory TA: Dr. Hongmei Ma February 18, 2016

Abstract The purpose of this experiment was to determine the relationship between three traits found in Drosophila melanogaster, also known as fruit flies. The hypothesis for the experiment was that the traits studied (wing size ( m), eye color ( w), and bristle type ( f)) were X-linked and not independently assorted. This was done by breeding the fruit flies to obtain three generations (P 1, F 1, and F 2 ) that will aid in determining the mode of inheritance. Results from the experiment led to the conclusion that the three genes were X-linked. The gene order determined was wmf and the map distances were 44.6 m.u. (between w and m), 37.8 m.u. (between m and f), and ~70.4 m.u. (between w and f). The linkage map constructed correlated fairly with the recombination frequencies, however there were discrepancies involving the frequency of single crossovers.

Introduction One of the most important organisms that have been used to study genetics for several decades is Drosophila melanogaster, also called the fruit fly, known to spontaneously arise in the presence of ripened fruit. They are very useful for genetic analysis because they breed easily, they have short lifespans and reproduction periods, and they re easy to maintain and control in regards to food and temperature. Also, fruit flies are small enough to collect large populations yet large enough to distinguish wild type traits from mutant traits (Klug et. al. 2012). The specific traits studied in this experiment are wing size ( m), eye color ( w), and types of bristles ( f). Wild type fruit flies have a longer wing size (extending past the abdomen), red eye color, and long, sleek bristles seen on the posterior abdomen. Mutant fruit flies have short wings (not extending past the abdomen), white eye color, and forked/bent bristles (often the hardest to distinguish). These traits are known to be X-linked in Drosophila melanogaster rather than independently assorted. Unlike independently assorted genes, linked genes are located on homologous chromosomes and their locations on the chromosome influence heredity (Klug et. al. 2012). X-linked genes are traits that are exclusive to the X sex chromosome and often creates sex bias in offspring (Klug et. al. 2012). Other characteristics that are associated with linked genes are three-point crosses, map distances, and gene order. When three loci are involved, three-point crosses are used to diagram the number of offspring for each phenotype and the frequency of recombination between the two homologous chromosomes (Aggarwal et. al. 2015). The number of offspring are used to determine the order of the genes by calculating the frequency of parental genotypes (without any crossing over) single crossovers (between any two neighboring traits) and double crossovers (between two neighboring traits simultaneously).

Normally with linked traits, parental genotypes are most frequent while double crossovers are least frequent (Aggarwal et. al. 2015). The chance of recombination directly correlates with map distance, meaning that traits that are farther apart are more likely to participate in crossing over (Aggarwal et. al. 2015). It is calculated by dividing the recombination events (between two loci) by the total number of events. Once the chance of recombination is determined, it can automatically be used to determine distances between the genes (chance of recombination, % = map distance, m.u.). These values can then be used to construct a gene map to visualize where the genes are located on the chromosome (Klug et. al. 2012). The hypothesis for this experiment was that the genes studied in this experiment ( wfm) are X-linked and not independently assorted. The strategy was to successfully mate these fruit flies in order to determine linkage of mutant and wild type traits. The goal was to produce enough offspring in each generation to breed a large enough sample size in order to obtain more accurate results.

Methods and Materials The experiment began with the use of two fly stocks: wild type males (+++) and fully mutant females ( wfm). The white ( w) locus was identified by red (+) or white ( w) eye color, the forked ( f) locus was identified by straight (+) or the presence of forked/bent ( f) bristles along the posterior abdomen, and the miniature ( m) locus was identified by long wings past the abdomen (+) or miniature wings that do not pass the abdomen ( m). In the first week, the adults from the parental cross (+++ x wfm) were removed from their tubes. They were anesthetized using carbon dioxide, categorized by sex and genotype with the use of a dissecting microscope, then placed in a vial (containing 4 males and one female) to mate. Since the P 1 generation was already in the adult phase, they were given one week to breed and produce offspring for the F 1 generation. In the second week the F 1 generation, consisting of mutant males (X wfm ;Y) and heterozygous wild type females (X +++ ;X wfm ), was collected and transferred into new vials with fresh Drosophila medium. They were anesthetized using ether, categorized by sex and placed into four new vials, each containing five females and 3-5 males in order to produce a large F 2 population. These flies were given another week to mate. In the third week, the F 1 flies were cleared and the vial was checked for larvae. The larvae were then given two weeks to develop into adults. In the fifth week when the F 2 flies were all adults, they were cleared from their vials, anesthetized with ether, then phenotyped using a dissecting microscope. The F 1 cross was to produce offspring with 8 different phenotypes. Gene order and map distances were determined using the X 2 test, % of recombination, coefficient of coincidence, and interference.

Results Table 1 shows the phenotype and number of flies in the F 1 generation. It consisted of 16 mutant males (X wfm ;Y) and 20 heterozygous wild type females (X +++ ;X wfm ). The P 1 generation had consisted of four wild type males (X +++ ;Y) and one fully mutant female (X wfm ;X wfm ) which was all that was needed to produce enough F 1 offspring. Table 1: Phenotypes and Number of Offspring in the F1 Generation Phenotype Number of flies Males X wfm ;Y 16 Females X +++ ; X wfm 20 The table shows the expected phenotypes of the P 1 cross. Females and males of the F 1 generation were able to be distinguished by eye color (red for females, white for males). Table 2 shows the phenotypes and number of flies of the F 2 generation for both males and females. Table 2: Numbers and Phenotypes of F 2 Offspring Resulting from the F 1 Cross Phenotype Number of males Number of females +++ 160 157 wfm 28 57 +fm 17 27 w++ 60 82 ++m 37 60 wf+ 10 15 +f+ 33 26 w+m 81 96 Total = 426 Total = 520 The greatest number of flies fell into the category of fully wild type for eye color, bristles, and wings while the least number of flies were mutant for eye color and bristles and wild type for wings. The sex of the flies is roughly a 4:5 ratio of males to females.

Table 3 shows the X 2 analysis for each of the traits in the F 2 population by themselves. The expected ratio for this analysis was that of a monohybrid testcross for each trait inherited independently; each trait having a 50% chance of being inherited. Table 3: Chi-Square Test of Independent Assortment for the Three Traits (wfm) Trait 1 (wild-type vs. white eyes) Phenotype Observed (O) Expected (E) (O-E) 2 O-E) 2 /E wild-type (red) 517 473 1936 4.09 mutant (white) 429 473 1936 4.09 Trait 2 (wild-type vs. forked bristles) Σ = X 2 = 8.18 Phenotype Observed (O) Expected (E) O-E) 2 O-E) 2 /E wild-type (straight) 733 473 67,600 142.9 mutant (forked) 213 473 67,600 142.9 Trait 3 (wild-type vs. miniature wings) Σ = X 2 = 284.2 Phenotype Observed (O) Expected (E) O-E) 2 O-E) 2 /E wild-type (long) 543 473 4900 10.36 mutant (short) 403 473 4900 10.36 Σ = X 2 = 20.72 The trait with the highest deviation from the expected value was the forked (f) bristle trait. The trait with the lowest deviation from expected value was the white (w) eye color trait. However, all X 2 values are very high. Table 4 similarly shows the X 2 analysis for each different phenotype. Tables 4: Chi-Square Test of Independent Assortment for each Phenotype Phenotype Observed (O) Expected (E) (O-E)^2 (O-E)^2/E +++ 317 118.25 39501.56 334.05 wfm 85 118.25 1105.56 9.35 +fm 44 118.25 5513.06 46.62 w++ 142 118.25 564.06 4.77 ++m 97 118.25 451.56 3.82 wf+ 25 118.25 8695.56 73.54 +f+ 59 118.25 3510.56 29.69 w+m 177 118.25 3451.56 29.19 X2 = 531.02

The expected values were derived from a trihybrid testcross of the three traits assorting independently; each having a 12.5% chance of being inherited. Some phenotypes are close to the expected value, but the overall X 2 value is extremely high. Figure 1 shows the map distances of the three traits derived by calculating the chance of recombination. Figure 1: Genetic Map of Traits w <--------------44.6 m.u.--------------> m <---------37.8 m.u.---------> f <----------------------------~70.4 m.u.-----------------------------------> These values were calculated based on the observed values for each of crossover. The map shows a greater chance of recombination between w and f than between f and m. The calculated coefficient of coincidence was calculated by dividing the observed double cross-over frequency (12.9%) by the expected double cross-over (16.9%). This gave a value of 0.763. The interference is then calculated by subtracting the coefficient of coincidence from 1, which yields a value of 0.237. The interference value is greater than zero. Therefore, positive interference occurred, meaning that fewer double crossover events were observed than expected.

Discussion Normally, traits on nonhomologous chromosomes are inherited independently of each other s locations. However, traits that are linked on homologous chromosomes are often inherited together unless crossing over between the homologous chromosomes occur ((Klug et. al. 2012)). The hypothesis for this experiment was that the three traits would be X-linked and not inherited independently. Based on the results obtained from this experiment, this hypothesis can be supported. At the same time, Mendel s law of independent assortment would be rejected. Referring to table 1 and the phenotypes of the P 1 generation, the presence of fully wild type males in the P 1 generation disappeared in the F 1 generation. This occurred because their X chromosome was only inherited from the P 1 female (which was fully mutant) and their Y chromosome (which is without any of these traits and therefore uninfluential) was inherited from the male. Meanwhile, F 1 females inherited one fully wild type chromosome from the P 1 males and one fully mutant chromosome from the P 1 female. If these genes were autosomal, then the males of the F 1 generation would be heterozygous wild types along with the females because it would factor in an influential chromosome unlike the Y chromosome which contains none of the three traits (Rodell et. al. 2004). Table 2 shows the number and phenotypes of the F 2 males and females. There are significant differences between the numbers of offspring for each phenotype. If these traits had assorted independently, then each phenotype would have about the same number of offspring (⅛ of total) because the probability of inheriting each of the three traits would be ½, according to a trihybrid testcross (Rodell et. al. 2004). Table 4 validates this with a Chi-Square analysis for independent assortment for each phenotype. The calculation gives a X 2 that is well above the

accepted X 2 value of 14.067 (where df = 7 and p-value = 0.05) (Freund et. al. 2010). Table 3 shows a similar Chi-Square test, except it tests the independent assortment of each trait alone. Once again, the X 2 for each trait is above the accepted X 2 values (Freund et. al. 2010). High X 2 values such as the ones shown in tables 3 and 4 are enough to reject independent assortment and retain X-linkage. Figure 1 displays the established genetic map, map distances and gene order of the traits. The gene order, wmf, was determined by comparing the genotypes with the most offspring to the ones to the least offspring and making sure they matched (Klug et. al. 2012). The data generally correlates with the genetic map except it doesn t match the single crossovers. For example, since w_m is a larger distance than m_f, the observed recombination should be higher, however, it s lower. This could partially be due to mischaracterizing bristles on the flies because it is the trait that is hardest to determine and easily mistakable. Also, some of the vials with the F 1 cross did not produce enough offspring, so more had to be taken from a previously prepared stock. The gene order puts the gene for wing size ( m) in the middle of the other two traits. The map distance between w and m is 44.6 map units, the distance between m and f is 37.8 m.u. and the distance between w and f is 70.4 m.u. (which is slightly less than the addition of the first two distances because the double crossover is counted twice) (Klug et. al. 2012). Ultimately, the data obtained from this experiment generates the conclusion that the traits are X-linked and not independently assorted.

References Klug, W. S., Cummings, M. R., Spencer, C. A., and Palladino, M. A. (2012) Concepts of Genetics (10th edition). San Francisco, CA: Pearson Benjamin Cummings, Inc. Aggarwal, D. D., Rashkovetsky, E., Michalak, P., Cohen, I., Ronin, Y., Zhou, D., Korol, A. B. (2015). Experimental evolution of recombination and crossover interference in Drosophila caused by directional selection for stress-related traits. BMC Biology, 13, 101. doi: 10.1186/s12915-015-0206-5 Rodell, C. F., Schipper, M. R. and Keenan, D. K. (2004) Modes of Selection and Recombination Response in Drosophila melanogaster. J Hered 95 (1): 70-75. doi:10.1093/jhered/esh016 Freund, R. J., Wilson, W. J., and Mohr, D.L. (2010) Statistical Methods (3rd edition). Burlington, MA: Elsevier, Inc.