Evolution Canyons occur naturally as a valley between two mountains where one slope, the

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1 Meiosis and Genetic Diversity in Sordaria fimicola Haley DeMartin Lab Section 001 Due October 27, 2014 Introduction Several Evolution Canyons exist in Lower Nahal Oren, Mount Carmel, Israel. These Evolution Canyons occur naturally as a valley between two mountains where one slope, the South Facing Slope (SFS) experiences harsh conditions such as strong sunlight, very dry air, and drastic temperature changes. The other side, the North Facing Slope (NFS) experiences a much milder, temperate climate. Sordaria fimicola grow on both slopes, and researchers want to find out if the crossing over between the color genes on non-sister occurred more frequently on one slope versus the other due to differing climates. Researchers crossed tan mutant strains of Sordaria with black wild type strains from both slopes, because when the two strains reproduce sexually plasmogamy and karyogamy occur and then the mutant color gene on one chromatid can cross with the wild type color gene on a non-sister chromatid. The resulting crossover event can be seen when looking at the order of colored ascospores in each ascus under the microscope. Specifically, researchers crossed mutant strains with NFS-NFS and compared the cross-over events, then they crossed mutant strains with SFS-SFS and compared the cross-over events, and finally they crossed the mutant strains with NFS-SFS and compared the amount of cross-over events. They found some differences in cross-over frequencies between Sordaria on the same slope, however, they found much higher differences in cross-over frequencies between the NFS and SFS. This proves that harsh environmental conditions can lead to changes in the cellular mechanisms that control crossing over, promoting more genetic diversity in harsh climates 1.

2 DeMartin 2 The map distance, the distance from the centromere to the spore color gene, is able to be calculated after the cross-over frequencies are determined. To increase precision, the Sordaria fimicola were grown in a controlled laboratory environment that mimics the conditions of Evolution Canyon. The cross-over frequencies of the spore color gene from populations that have been grown under ideal lab conditions are necessary as a baseline comparison for the crossover frequencies that occur in the Sordaria that grow under harsh lab-recreated environmental conditions. The researchers are testing the hypothesis that harsh environmental pressures can increase the frequency of crossing over. Therefore, scientists cannot claim that there are more cross-over events in harsh environments if they do not have comparable data from Sordaria that was grown in ideal conditions 1. A perithecium contains many asci that can be seen under a microscope once the perithecium has been squashed open. The ascospores are in rows of eight and show where crossing over has taken place. First, two spores of different color mate; one black wild type and one tan mutant. Next they fuse their cytoplasm together and go through plasmogamy. They are haploid cells until karyogamy takes place, and their nuclei fuse. Next, each 2n ascus goes through meiosis and during meiosis I, crossing over of non-sister chromatids occurs, or not. Then, meiosis II occurs where the two homologous chromosomes eventually split apart into their four daughter asci (refer to Diagram 1). No genetic variation has occurred here (because there was no crossing over of non-sister chromatids between the centrosome and the color gene) except random assortment because the chromosomes with the wild type gene could be on the left or the right of the chromosomes with the mutant gene but it would not matter because the ratio would still be the same.

3 DeMartin 3 After meiosis, mitosis occurs and the four one n asci duplicate themselves to make eight asci. The first four will be one color and the second four will be the other color. This is what makes the 4:4 ratio. For every asci the same thing happens however often times crossing over occurs in meiosis I. If the two homologous chromosomes are oriented next to each other, like in Diagram 2, and crossing over occurs then one would observe a 2:2:2:2 ratio. This is because the right sister chromatid will cross with the left sister chromatid on the homologous chromosome that is to the right of the first chromosome. Then during meiosis when the sister chromatids break apart the one on the left will code for one color, the one on the right will code for the opposite color creating a 1:1:1:1 ratio until finally, mitosis occurs and it turns into a 2:2:2:2 ratio. However, if the homologous chromosomes are on top of each other the ratio will be 2:4:2 because during meiosis both left side non sister chromatids will have crossed over with each other. This results in after meiosis one asci with one color, then two asci with the other color, then one more asci with the same color as the first (1:2:1 ratio). Finally mitosis occurs and it creates a 2:4:2 ratio. Diagram 1: Meiosis I and Meiosis II 2

4 DeMartin 4 Diagram 2: Crossing Over During Meiosis 2 Crosses between the tan and wild type spores that are non-recombinant are a 4:4, and recombinant 2:4:2 and 2:2:2:2. To find cross-over frequencies examined, one could observe these patterns by looking under the microscope for the order of the asci. One would look under the microscope and count the number of asci of each combination. Then a researcher would use the cross-over frequency equation (crossover frequency equals recombinants divided by total) to find the cross-over frequency, and ultimately find the map distance, or the distance from the color gene to the centromere. From this data, researchers can find out if Sordaria grown in harsher conditions have a higher cross-over frequency than Sordaria grown in milder conditions. Materials and Methods Sordaria of two different color strains were grown in the laboratory and then were crossed in order to obtain baseline data for crossover frequency and map distance. First, the group set up an agar plate used for mating, and they drew lines on the bottom of the plate dividing it into four equal quadrants. In the first quadrant and the quadrant touching that one with only a corner, wild type was written. Then, the other two quadrants were labeled tan. Next, the work surface and the scalpel was disinfected. From the wild type plate, a 0.5 cm 2 square was cut out and placed it hyphae side down on the quadrant labeled wild type. This step got repeated

5 DeMartin 5 for the other wild type quadrant. Then, a 0.5 cm 2 sample was taken from the tan plate as well, with a clean scalpel, and transferred hyphae side down onto the tan quadrant. This step was also repeated a second time. Finally, the plates were incubated for two weeks at room temperature and mating occured 1. First, the group obtained the mating plate, noticing the pattern of perithecia on the plate. With a disinfected inoculating loop, they scraped some perithecia from the center of one of the dividing lines and placed them in a drop of water on a microscope slide. Perithecia from the dividing lines were used because the perithecia there were most likely to go through sexual reproduction combining the wild type and tan spores 1. Next, the slide was covered with a coverslip. With a pencil eraser, the group pressed gently on the cover slip in an attempt to break open the perithecia without breaking apart the groups of asci. Then, the slide was put under the microscope and focused on a single asci group under 400X magnification. The group saw many asci sacs containing both black and tan spores. Each group member counted at least 20 asci groups and recorded the ratios they observed in the Individual Data Table. Next, the group combined their individual data and entered that data into the Small Group Data Table. Once all of the groups were finished, everyone compiled their data and put it in the Combined Section Data Table. Eventually each class merged their data and entered it into the Combined Course Data Table 1. To calculate cross-over frequencies the group used the equation: recombinant cross-over frequency equals the number of recombinant asci divided by the total number of asci observed. To calculate the map distance the equation: map distance equals the percent cross-over divided by two, was used because the map distance is the distance from the centromere to the spore color gene 1.

6 DeMartin 6 recombinant total = % of crossover 2 Results B+C A+B+C Table 1. Individual Data = crossover frequency = map distance This table provides the total number of each type of asci pattern each individual observed. # of Type B (2:4:2) # of Type C (2:2:2:2) Type B (B/total) Type C (C/total) (B+C/total) Table 2. Combined Section Data This table provides the total number of each type of asci pattern observed in the section. # of Type B (2:4:2) # of Type C (2:2:2:2) Type B (B/total) Type C (C/total) (B+C/total) Non- # of Type A (4:4) Non- # of Type A (4:4) Non- # of Type A (4:4) Table 3. Combined Course Data This table provides the total number of each type of asci pattern observed in the entire course. # of Type B (2:4:2) # of Type C (2:2:2:2) Type B (B/total) Type C (C/total) (B+C/total) 10,512 7,811 7,855 26,178 15, Due to the fact that recombinant asci are present in significant frequencies, this must mean that crossing over occurred between the spore color gene and the centromere. If crossing

7 DeMartin 7 over did not occur between the centromere and the spore color gene then there would be no visible crossing over (visible as recombinant types), only 4:4 patterns. Under standard laboratory growth conditions, the overall cross-over frequency between the spore color gene and the centromere is , or a 59.84% frequency of recombination. The calculated map distance between the spore color gene and the centromere is map units. This is because map distance equals the recombinants divided by two times the total all times one hundred = map units Discussion One unexpected finding was an infection growing on the plate. This is because it was difficult to keep everything sterile while making a mating environment for the Sordaria. Due to the fact that the equipment and lab work surface were not sterile, the Sordaria got an infection. The infection consisted of fluffy, spongy, white-looking material growing on top of the wild type strain of Sordaria. This was unfortunate because it limited the sections to take the perithecia from. Another challenge that arose was squashing the perithecia so that the asci could be viewed clearly under the microscope. The difficulty was determining how much pressure to use to squash the perithecia. If squashed too hard, the perithecia would break from their groups of eight and go flying out everywhere and be of no use. Or, if squashed too softly, the perithecia shell/sac would cover the asci and the asci could not be seen well enough to count their patterns. The fact that there were recombinant asci proves that crossover occurred between the spore color gene and the centromere. If crossing over did not occur then each offspring would be the wild type, parental phenotype. The cross-over frequency of the spore gene with the centromere in an organism grown under optimal growth conditions equals the number of

8 DeMartin 8 recombinant asci divided by the total number of asci. The map distance between the spore color gene and the centromere predicted by the crossover frequency under ideal growth conditions equals the number of recombinant asci divided by two times the total number of asci. The data observed was the frequency of recombination for Sordaria grown under ideal conditions. As far as answering the overall question of whether or not environmental stress affects cross-over frequency, structures associated with meiosis, and gene expression related to meiosis over time, it is still unknown from the data collected in this lab. All of the data in this lab only proves that recombination via meiosis occurs in an ideal laboratory situation. To answer the overall question, one would need to go through the exact same procedure and collect the same types of data but use Sordaria that is exposed to harsh environmental conditions. Then, one would compare the cross-over frequencies of the Sordaria grown under harsh conditions with the data obtained from the ideal-condition-grown Sordaria. Literature Cited 1. 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. 2. Schaeffer, Stephen W. "Cell Division 2: Life Cycles and Meiosis." Biology 110H Wiki. N.p., 1 Sept Web. 17 Nov Acknowledgments Hunter Mangel: Lab Partner Brandon Weinman-Follick: Teacher s Assistant