Emily Pia Bio 110H Nate Jacobs October 29, 2012 Meiosis and Genetic Diversity in the Model

Organism, Sordaria fimicola Introduction Sordaria fimicola is a fungus belonging to the phylum Ascomycota, which indicates that the species follows the ascomycotes life cycle. The purpose of our lab was to examine the Sordaria life cycle, the way that Sordaria reproduce, and how their unique reproduction contributes to genetic diversity in the species. This required an understanding of the life cycle of Sordaria and the various types of recombination that could result from mating two different strains of the fungus. Sordaria can either reproduce asexually or sexually, and in this lab, we focused on at the sexual reproduction of Sordaria and the process of meiosis. Meiosis occurs when a gamete from each parent combines to form a zygote. During meiosis, nonsister chromatids can exchange genetic material through a process called crossing over. If the genotypes of the offspring resemble the genotypes of the parents, crossing over has not occurred. In contrast, if the genotypes of the offspring do not resemble the genotypes of the parents, crossing over has occurred. When looking specifically at Sordaria, mycelia of the two parents fuse, in a process termed plasmogamy, and eventually the two haploid nuclei fuse, in a process termed karyogamy. Then, the diploid cell can undergo mitosis, resulting in eight haploid ascospores lined up in each ascus which are held in perithecia. In this experiment, we used black wild type Sordaria and tan or gray mutant type Sordaria to show this process. When the two strains were combined in mating plates, sexual reproduction occurred and resulted in three different recombination patterns. If the ascospores followed a 2:4:2 or a 2:2:2:2 pattern, then we concluded that crossing over had taken place. If the ascospores followed a 4:4 patter, then we concluded that no crossing over had taken place. A 2:2:2:2 recombination pattern has four sets of alternating black and tan pairs. This pattern occurs when the homologous chromosomes line up next to each other in the nucleus and exchange material at the chiasmata. This occurs during metaphase one of meiosis. A 2:4:2 recombination pattern has either two black spores followed by four tan spores followed by two more black spores, or the reverse. This pattern occurs when the homologous chromosomes lay on top of each other during metaphase one of

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meiosis and exchange genetic material. A 4:4 no recombination pattern occurs when there is no exchange of genetic material between the homologous chromosomes. The Sordaria life cycle is shown below.

(Higher Fungi)

In a preliminary study to our experiment, researchers from the Imperial College of Science, Technology, and Medicine and the Institute of Evolution at the University of Haifa investigated the effects of climate on recombination frequencies in Sordaria fimicola strains. The research group took wild type Sordaria from two sides of an Evolution Canyon in Israel. This canyon was given its name because of the drastic differences in the opposing slopes that drive microevolution amongst the species that inhabit it. The climate of the north facing slope of the canyon is temperate, shady, and humid, while the climate of the south-facing slope of the canyon is harsh, sunny, and dry. When the researchers took Sordaria from both sides of the canyon, they found that rates of recombination were

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much higher on the harsh south facing slope than on the north facing slope. This led researchers to conclude that harsh climates serve as an environmental pressure that leads to more rapid recombination and microevolution.

In our lab, we looked at the recombination frequencies in Sordaria fimicola where all of the samples were kept at the room temperature. In this way, we could look at the recombination frequencies exhibited with no environmental pressures present, and use the data we collected as a baseline of the study aforementioned. The hypothesis that we developed is that there would be more Sordaria fimicola asci that exhibit recombinance than no recombinance. Therefore, we expected that greater than 50% of Sordaria asci would show either of the two recombination types. We developed this hypothesis based on the knowledge that the fitness of a species is largely dependent on the amount of genetic diversity in the population. It was possible, however, that our data would show that the majority of the Sordaria fimicola asci to exhibited no recombinance. To test our hypothesis, we made petri dishes containing samples of both the wild type black Sordaria with the mutant type tan Sordaria. We allowed the Sordaria to reproduce by incubating the plates for two weeks. After two weeks had passed, we took samples of areas that were most likely to exhibit recombinance and looked at the asci under a microscope and recorded the results. Materials and Methods Week One We obtained two Petri dishes and made four quadrants by placing four squares of agar containing fungal hyphae on the mating plates. Two of the agar squares were of the mutant type (we used the tan strain) and two agar squares were of the black wild type. The agar squares were placed hyphae side down, and the plate appeared as follows. We then incubated the plates for two weeks at room temperature to allow for growth, reproduction, and recombination.

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Week Three We retrieved the mating plates and recorded observations of our plates appearances in our lab notebooks. Then we used an inoculating loop to scrape perithecia from the center of the lines where recombination was likely to occur and placed it on a microscope slide with a drop of water to allow the perithecia to disperse instead of clumping. This allowed for us to see the individual asci better under the microscope. We put a coverslip on our slide and tapped the top to squash the perithecia and release the asci. Lastly, we looked at the Sordaria cultures under a compound microscope under 100x magnification. We looked for asci that contained more than one spore color and counted the asci that exhibited the various types of recombination. We recorded this information in a chart. After we had recorded our own data, our group data, our section data, and our course data, we calculated the total percent recombinant asci, the percent recombinant asci for each of the two recombinant types, and the mutant tan distance from the centromere. To calculate the total percent recombinant asci, we used the formula (B+C)/(A+B+C) x 100. For the two:four:two recombinant asci type, the formula we used was B/(A+B+C) x 100. For the two:two:two:two recombinant asci type, we used the formula C/(A+B+C). To calculate the map distance, we divided the percent recombination by two. Results Individual Data: Nonrecombinant Number of Type A Asci (4:4) 6

Recombinant Number of Type B Asci (2:4:2) 10 Number of Type C Asci (2:2:2:2) 6

Total Number of Asci

Total Number of Recombinant Asci (B+C) 16

22

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Combined Lab Group Data: Nonrecombinant Number of Type A Asci (4:4) 16

Recombinant Number of Type B Asci (2:4:2) 20 Number of Type C Asci (2:2:2:2) 11

Total Number of Asci

Total Number of Recombinant Asci (B+C) 31

47

Combined Section Data: Nonrecombinant Number of Type A Asci (4:4) Tan Spore Color 51 Gray Spore Color 35 18 26 79 44 47 35 133 82

Recombinant Number of Type B Asci (2:4:2) Number of Type C Asci (2:2:2:2)

Total Number of Asci

Total Recombinant Asci (B+C)

Combined Course Data: Nonrecombinant Number of Type A Asci (4:4) Tan Spore Color 5669 Gray Spore Color 3012 2081 1973 7066 4054 4301 3976 13946 8277

Recombinant Number of Type B Asci (2:4:2) Number of Type C Asci (2:2:2:2)

Total Number of Asci

Total Recombinant Asci (B+C)

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Combined Section Data Analysis:

Nonrecombinant

Recombinant # of Type C Asci (2:2:2:2) Total # of Asci Total # of Recombinant Asci (B+C)

Frequency of Recombinant Asci ((B+C)/total # of asci)

Frequency of Type B Asci (B/total # of asci)

Frequency of Type C Asci (C/total # of asci) Ratio B/C

# of Type A Asci (4:4)

# of Type B Asci (2:4:2)

Tan Spore Color 51 Gray Spore Color 47 35 133 82 61.7% 35.34% 26.32% 1.34:1

35

18

26

79

44

55.7%

22.78%

32.91%

.692:1

Combined Course Data Analysis:

Nonrecombinant

Recombinant # of Type C Asci (2:2:2:2) Total # of Asci Total # of Recombinant Asci (B+C)

Frequency of Recombinant Asci ((B+C)/total # of asci)

Frequency of Type B Asci (B/total # of asci)

Frequency of Type C Asci (C/total # of asci) Ratio B/C

# of Type A Asci (4:4)

# of Type B Asci (2:4:2)

Tan Spore Color

5669
Gray Spore Color

4301

3976

13946

8277

59.35%

30.84%

28.51%

1.08:1

3012

2081

1973

7066

4054

57.37%

29.45%

27.92%

1.05:1

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Tan Spore Color Map Units from Centromere Percent Crossing Over/2 = gene map units from centromere 59.35/ 2 = 29.68 map units from centromere

Gray Spore Color Map Units from Centromere Percent Crossing Over/2 = gene map units from centromere 57.37/2 = 28.69 map units from centromere

Percent Error of Course Data (Actual Theoretical)/Theoretical x 100 = Percent Error Tan: (29.68-26.2)/26.2 x 100 = 13.28% Gray: (28.69-26.2)/26.2 x 100 = 9.50%

It is obvious from the data that crossing over occurred during the spore color strain crosses. The overall crossover frequency of the tan spore color gene was 59.35% and the distance of the tan spore color gene from the centromere was 29.68 map units. The overall crossover frequency of the gray spore color gene was 57.37% and the distance of the gray spore color gene from the centromere was 28.69 map units. Looking at the course data, there was little variation in the ratio of the two crossover types between the two spore color genes. The ratios were very close to one to one. Discussion This experiment showed how sexual reproduction and genetic recombination allows for genetic diversity. Our hypothesis was supported because in both mutant spore color crosses, recombination occurred greater than 50% of the time. From the data our course collected, 59.35% of Sordaria of the tan spore color gene exhibited recombinance and 57. 37% of Sordaria of the gray spore color gene exhibited recombinance. There seemed to be no variation in the ratio of the two crossover frequency types (2:4:2 vs. 2:2:2:2) as the ratios of the course data were very close to 1:1. Recombination is imperative to the success of a species because genetic variation in a population increases the overall

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fitness of a population. Because the Sordaria strains showed a majority recombination, we can conclude that the species is more fit than it would be had we found a majority of no recombination. The accepted crossover frequency between of both the tan and gray spore color strains with the wild type black strain was 52%. While our percentages were higher, both conclusions represent that the spore color genes are not linked. A percent recombination greater than 50% generally means that the genes are on separate chromosomes or very far apart on the same chromosome. (Cry) The accepted map distance from the centromere of the both the tan and gray spore color strains is 26.2. While our map distances are longer, again, they still represent the same idea that the genes are located pretty far away from the centromere. Our percent error for the crossover frequency of tan spore color strain with the black spore color strain was 13.28%. For the crossover frequency of the gray spore color strain with the black spore color strain, we found a percent error of 9.5%. These differences can most likely be contributed to our sources of error in the experiment. One source of error is that contamination was likely to occur. Any exposure to air or unsterile equipment handling the fungal cultures could have contaminated our samples and affected the growth and reproduction of the Sordaria. Another source of error is the difficulty both my group and class as a whole had when identifying the types of recombination. In some cases, squashes were not done properly limiting the amount of asci released. Other times there was too little water for the amount of Sordaria placed on the slide and the perithecia and asci were all overlapped. Sometimes the opposite occurred and too much water was on the slide for too little perithecia and the asci were too hard to find under the microscope. All three scenarios contributed to the trouble our class had identifying the type of recombinance the asci exhibited. Additionally, some people got so frustrated with the counting that they counted the number of recombinant asci in the pictures given to us instead of their own samples. This would contribute to the same data being recorded multiple times. Our experiment documented the natural rate of crossing over, as the fungi were allowed to grow and reproduce with limited environmental stresses. Because we have provided a baseline for the recombination in Sordaria fimicola, the information and data we collected can be used in further experiments. Other studies can use our information to compare how various environmental stresses alter the percent crossing over. For example, had this been a preliminary study for the Evolution

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Canyon experiment, the researchers could have compared the recombination frequencies of their Sordaria strains to the recombination frequencies of our Sordaria strains to see how climate affected their results. References Meiosis and Genetic Diversity in the Model Organism, Sordaria. Written by Hass, C. and Ward, A. 2010. Department of Biology, The Pennsylvania State University, University Park, PA. Cry, R. 2002. Chromosome Behavior and Gene Linkage. In, Biology 11: Basic concepts and biodiversity course website. Department of Biology, The Pennsylvania State University. http://www.bio.psu.edu/ Olive, Lindsay S. Genetics of Sordaria fimicola. I. Ascospore Color Mutants American Journal of Botany, Vol. 43, No. 2 (Feb., 1956), pp. 97-107 "Higher Fungi." Phungophobe's Phear -- "Higher Fungi" N.p., n.d. Web. 26 Oct. 2012. <http://www.biologie.uni-hamburg.de/b-online/library/plant_biology/Phungphear.html>.