Sordaria Lab Report. Sordaria Fimicola reproduce through the process of meiosis. This process entails

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1 Adil Sabir Bio 110H 18 October 2014 Sordaria Lab Report I. Introduction Sordaria Fimicola reproduce through the process of meiosis. This process entails prophase I, metaphase I, anaphase I, telophase I, prophase II, metaphase II, anaphase II, and telophase II. During the metaphase I stage, a crucial process called crossing over occurs. Crossing over is the process by which genes and genetic material are exchanged between homologous chromosomes and therefore create genetically variable offspring (Lab Manual Pg.33). Evolution Canyon is a natural microscale laboratory where the South Facing Slopes (SFS) receive more sunlight and the North Facing Slopes (NFS) are more temperate and have more shade. After testing and research, there was determined to be a higher crossover frequency in the SFS yet scientists were unsure whether this was because of their environmental conditions or another external factor (Lab Manual pg.36). According to Dr. Minou Nowrousian, Sordaria is a model organism to use because they reproduce quickly, have a short life cycle, have distinct parts that are easy to view under microscopes, and have multiple mutant strains that serve as markers for different meiotic events (Nowrousian, 2010). We performed this experiment and reproduced the Sordaria in identical conditions in order to determine if these results

2 deviated from those recorded in their natural habitat faced with different environmental factors. While our main research question was to see if there was a difference between the total recomb asci and crossover percentage between the Sordaria in Evolution Canyon and the Sordaria in our lab, our goal was to provide a baseline for future studies and experiments. While we cannot draw conclusions about crossover frequency in harsh conditions, we can, however, draw conclusions about our data as a baseline for experimental conditions. With our experimental data, we can determine if the difference in crossover frequencies occurred due to environmental factors and weather conditions or due to some other factor. We predict that the crossover frequencies will be identical under optimal, identical conditions. Of the Sordaria that were reproduced, there could have been three different types of asci created. The non recomb 4:4 (Type A) is when there are four tan spores next to four wild type spores. The recomb 2:4:2 (Type B) is when chromosomes two are stacked on top of each other and recombination creates two tan spores followed by four wild type spores and then two more tan spores or vice versa. The other recomb 2:2:2:2 (Type C) is when two chromosomes line up next to each other and recombination causes spore color alternates after every two spores. Some main research questions arise before conducting this experiment. What evidence demonstrates cross over between the spore color gene and centromere and what is the crossover frequen the spore color gene with the centromere in organisms grown under optimal conditions? How does environmental stress affect cross over frequency, structures associated with meiosis, and gene expression related to meiosis?

3 And finally, what is the map distance between the spore color gene and centromere? (Lab Manual Pg.37) To perform the experiment, we put four pieces of agar with fungal hyphae side downward into a petri dish. We had two strains of wild type and two strains of tan type and alternated strains around the dish. We then covered the dish and put surgical tape around the side to prevent materials entering and exiting the dish. We allowed the dishes to sit for a week so the fungi could reproduce and recombine (Lab Manual Pg.42). Then, we could take samples of the spores and squashed them, then view the asci under a microscope to record recombination ratios. We then used this data to determine crossover frequencies. II. Materials and Methods We began by taking a petri dish and dividing the agar gel within the dish into fourths. We then placed two wild type and two tan type Sordaria strains on alternating sections of the dish and sealed it off with surgical tape. After an incubation period of a week in optimal conditions (dark and moist at room temperature), the fungi were able to reproduce and recombine (Lab Manual Pg.42). After the incubation period, the fungi produced spores in the shape of small black balls. We then took spore samples using an inoculating loop and placed the spores on a glass slide with a drop of water. After, we took a plastic coverslip and pressed down on the slide in order to

4 squash the spores which we then were able to view the recombination ratios of under a microscope (Lab Manual Pg.42). We added together the number of 2:2:2:2 and 2:4:2 alternating asci to get a total number of recomb asci and totaled the number on 4:4 non recomb asci and then were able to calculate the percent crossover and map distance by using the formulas: Percent Crossover = (# recomb asci/total # asci)x100 and Map Distance = % crossover/2. III. Results Table 1: Individual Data Non ina nt Total Total Type A (4:4) Type B (2:4:2) Type C (2:2:2:2) (Lab Manual pg.56) Table 2: Small Group Data Non ina nt Total Total Type A (4:4) Type B (2:4:2) Type C (2:2:2:2) (Lab Manual pg.56)

5 Table 3: Combined Section Data Non ina nt Total Total Type A (4:4) Type B (2:4:2) Type C (2:2:2:2) (Lab Manual pg.57) Table 4: Combined Course Data Non ina nt Total Total Type A (4:4) Type B (2:4:2) Type C (2:2:2:2) (Lab Manual pg.57) Table 5: Combined Section Data Analysis Non Re combin ant Total Total (B+C) (B +C/Tota Type B (B/Tota Type C (C/Tota Ratio B/C Type A (4:4) Type B (2:4:2) Type C (2:2:2:2 ) % 26.8% 30.4% (Lab Manual pg.58)

6 Table 6: Combined Course Data Analysis Non Re combin ant Total Total (B+C) (B +C/Tota Type B (B/Tota Type C (C/Tota Ratio B/C Type A (4:4) Type B (2:4:2) Type C (2:2:2:2 ) % 30% 29.9% (Lab Manual pg.58) % Crossover for Section = (# recomb asci/total # asci)x100 =57.2% Map Distance for Section= % crossover/2 =28.6 MU % Crossover for Course = (# recomb asci/total # asci)x100 =59.9% Map Distance for Course= % crossover/2 =29.95 MU IV. Discussion One challenge that occurred during the experiment was potential contamination if you did not tape the petri dish properly. Another was no source of spores if you did not put the hyphae side of the fungi facing downward, as well as improper growth if you do not keep the fungi in a

7 dark environment. Some problems we faced when preparing the squash slides were pushing down too hard on the slide so the perithecia scattered everywhere, or pushing down too lightly so the spores do not burst. We were able to determine that crossover did occur between the spore color gene and centromere since we counted a certain number of recomb asci and recombination occurs due to crossing over (Lab Manual pg.59). In addition, we found that there is a 57.2% crossover overall which is fairly close to 50% which is what we predicted. Although it is not 50%, we can't say this isn't typical for standard lab conditions. The deviation of this percentage could be due to random selection of asci. Since this percentage is so close to 50% and the actual percentages in Evolution Canyon have a mucher greater percentage in crossover in SFS than NFS, we can conclude that percent crossover, meiosis, and gene expression in Sordaria is variable. Our data that we collected can be useful in comparing against data collected from future experimental conditions. The map distance, which is half the percent crossover, is equal to The frequen crossover in Type B was 26.8% which is very close to the 30.4% in type C. The ratio of B to C was.88 which is very close to 1. The deviation of these percentages again could have been due to random selection of spores from the agar. Since these numbers are so close, we can determine that crossover frequency in Type B and Type C is essentially identical. We learned that gene expression and crossover frequency in Sordaria is variable. However, we only tested this hypothesis on just one type of Fungi. We can do further experiments to see if this was also true in more complex animals such as mammals, reptiles, or amphibians. If we could do further research and experiments on this topic, we could be able to

8 manipulate this field and learn more about gene expression and crossover frequency to have it benefit our society. References/Citations: Meiosis and Genetic Diversity in the Model Organism, Sordaria. Written by Hass, C., Richter, K., and Ward, A Department of Biology, The Pennsylvania State University, University Park, PA. Cyr, R Fungi I Evolution and Diversity, Phyla Chytridiomycota and Zygomycota. In, Biology 110: Basic concepts and biodiversity course website. Department of Biology, The Pennsylvania State University. Engh, Ines, Minou Nowrousian, and Ulrich Kück. N.p., 2 July Web. 22 Oct Biology 110 Laboratory Manual 2014