genus is Sordaria. Sordaria can be found worldwide in the feces of herbivores (Maryland

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1 Solverson!1 Introduction The specific species of Sordaria being worked with in this lab is Sordaria fimicola. Sordaria fimicola is a species of microscopic fungus. It is from the family Sordariaceae, and its genus is Sordaria. Sordaria can be found worldwide in the feces of herbivores (Maryland Biodiversity Project). It is commonly used in experimentation and studied in school, partially because it can be used to verify Mendel's laws of segregation directly, not just statistically (Volk, Sordaria fimicola). Sordaria is a type of fungus, and Fungi play an important role in energy cycling within, and between, ecosystems (Leaf Group Writer). This species of Sordaria is haploid for most of its life cycle. An ascospore is a single haploid cell. It begins to germinate and starts to form branches called Mycelia. There are two different mating types of Sordaria: the positive mating type and the negative mating type. Both of these are multicellular and haploid. In sexual reproduction, both mating types begin to form sacks that are full of haploid cells of that same mating type. The sack from the positive mating type is called Ascogonium, and the sack from the negative mating type is called Antheridium. Then, a process called Plasmogamy occurs, which is when the sacks from both mating types connect and the haploids from both types move into one sack. The Sordaria continues to branch off and as this occurs cytokinesis also occurs. As a result of this, one haploid nuclei from each mating type is in each new cell. As the Sordaria continues to branch off and cytokinesis continues to occur, the branches mix together and form a fruiting body. The name of this fruiting body for the specific type of Sordaria being used in this experiment is Perithecium. This Perithecium is Dikaryotic, which means that the cells nuclei have not yet merged to form a single nucleus. The Perithecium is filled with Asci, which are little sacks that contain one haploid

2 Solverson!2 nuclei from each mating type. These nuclei then undergo Karyogamy, which is when the two haploid nuclei combine to form a single diploid nucleus. This diploid nucleus then undergoes meiosis, and it forms four different haploid nuclei. Then, each of these four haploid nuclei undergo mitosis. There are now eight haploid nuclei all in the same Ascus. These nuclei form cell membranes and become their own haploid cells. These cells are called ascospores. The Perithecium breaks open, and the Asci are released. Then the Asci break open and the ascospores are released. The process then repeats (Sordaria Genetics). This experiment works with two genes: the t gene and the g gene. Each of these genes have two alleles. These two genes work together to determine the color of the fungus/ascospores. There are four possible colors: Tan, Gray, Black (wild type), and Clear. The two alleles for the t gene are t+ and t. The two alleles for the g gene are g+ and g. The t+ and g+ are for the wild type (black), and the regular t and g are for the mutant colors, which is simply any color other than black. The combination of these genes decides the color. The combination of g+ and t+ produce black spores. The combination of g and t+ produce gray. The combination of g+ and t produce tan. Lastly, the combination of g and t produce clear spores. In this experiment, the colors being used and mated together are gray and black, and tan and black. The gray and black mixing determines the rate of crossing over of the g gene and the tan and black mixing determines the rate of crossing over of the t gene (Sordaria Genetics). The independent variable in this lab is the color of fungus being mated with black: tan or gray. The dependent variable is the rate of crossing over, which is determined by the color being used. From the rate of crossing over the distance of the genes t and g from the centromere, or

3 Solverson!3 center of the chromosome, can be determined. Thus, the distance from map units from the centromere is also a dependent variable. The purpose of this lab is to be able to tell if crossing over occurred for each gene. This is determined by observing the Asci of Sordaria. If the ratio of colors is 4:4 no crossing over occurred. If the ratio is either 2:4:2 or 2:2:2:2 crossing over did occur. If all ascospores are the same color the Sordaria mated with the same color, so it can not be determined whether crossing over did or did not occur. By looking at the rate of crossing over, we can determine the exact location of these genes on the chromosomes and their distance in map units from the centromere of the chromosome. (Sordaria Genetics)

4 Solverson!4 Materials (Sordaria Genetics) Included in the kit Sordaria fimicola, wild type Sordaria fimicola, mutant gray Sordaria fimicola, mutant tan bottle cornmeal-glucose-yeast agar autoclavable disposal bag 3 bottles Sordaria crossing agar 20 sterile petri dishes Needed but not supplied microscopes glass slides and cover slips water dropping bottles inoculating loops Bunsen burner *boiling water bath scalpel or spearpoint needle disinfectant such as phenol or 70% ethanol *If a water bath is not available, a container of boiling water may be substituted.

5 Solverson!5 Procedures (Sordaria Genetics) Preparation of Agar Dishes 1. Slightly loosen the bottle caps and set the bottles in a boiling water bath to melt the agar. (Caution: Since the labels may come off the bottles during boiling, it is advisable to mark the bottle caps with the type of agar contained within.) Make sure the water level is even with the agar level. Swirl the bottles gently to be sure that all of the agar has melted. 2. Cool the agar to 45 C (the bottle should feel comfortably hot to the touch) by cooling the water bath to that temperature or by letting them sit for several minutes at room temperature. 3. Wipe down the work surface with a disinfectant such as phenol or 70% ethanol. Wash your hands. 4. Swirl the bottle of cornmeal-glucose-yeast agar, remove the cap, flame the mouth over a Bunsen burner for a few seconds and distribute the contents among six petri dishes. Lift the lid of the dish just enough to pour in the molten agar. Replace the lid immediately to prevent contamination. 5. Label each dish with the type of agar. 6. Repeat Steps 4 and 5 with the Sordaria crossing agar, distributing the remaining agar among the 14 dishes. 7. After all the agars have solidified, the dishes may be stored for up to a week at room temperature or in the refrigerator. 8. Dispose of the bottles in the autoclavable disposal bag.

6 Solverson!6 Preparation of Stock Cultures 1. Disinfect the work surface and wash your hands. 2. When ready for use, label two of the conrmeal-glucose-yeast agar dishes wild, two gray and two tan. 3. Using aseptic technique, inoculate the dishes with the appropriate culture. Remove the top from the tube of wild-type Sordaria fimicola, and flame the mouth over a Bunsen burner for a few seconds. With a flamed, cooled scalpel or spearpoint needle, remove a portion of the culture containing perithecia (black pepper grain appearance) and transfer to the middle of a cornmeal-glucose-yeast agar dish. Repeat this procedure to prepare another wild-type culture. 4. Using the other tubes, follow step 3 to prepare two gray and two tan stock culture dishes. 5. Incubate the dishes for 5 to 7 days out of direct sunlight at room temperature (22-25 C) until perithecia have formed at the periphery of the dishes. During Laboratory 1: Preparing the Crosses 1. Disinfect the work surfaces. Have the students wash their hands. 2. Label one half of the Sordaria crossing agar dishes +/g and the other half +/n to indicate crosses between the wild-type and mutant-gray (or wild-type and mutant-tan) strains. 3. Invert the dishes over Figure 1. Using a wax pencil or permanent marker, indicate the positions of wild type (+) and gray (g) or tan (tn) cultures. 4. Using a flamed, cooled, scalpel or spearpoint needle, cut the agar in the stock culture dishes into 0.5 cm cubes. Place the cubes upside down over the indicated positions on the surface

7 Solverson!7 of the crossing agar. Each plate will contain two blocks of the wild-type culture and two blocks of either tan or gray culture. 5. Incubate the dishes out of direct sunlight and at room temperature. 6. From 8 days after inoculation until forcible discharge of the spores, genetic data can be obtained. Usually, the cultures should be ready for microscopic examination in 8 to 10 days, but at cooler temperatures, 14 to 15 days may be required. In order to obtain accurate data, it is essential that mature ascospores be counted. If it is difficult to distinguish microscopically between the wild-type and gray or tan spores, the ascospores are too immature to collect data. Incubate the cross dishes for another day or two and observe again. During Laboratory 2: Microscopic Examination 1. Disinfect all work surfaces. Have the students wash their hands. Point out the location of the autoclavable disposable bag. 2. Provide the students with water dropping bottles, glass slides, cover slips, inoculating loops and microscopes. 3. Remove a few perithecia from the cross dishes with a flamed, cooled loop and prepare a wet mount. Have the students note from which cross plate ( +/tn or +/g ) they are removing perithecia. Refer to Figure 1 for the most probable location of hybrid asci on the dishes. Notice the locations are different for gray and tan hybrid asci. Instruct the students to mentally note the position on the dish from which they prepared their slide. When students locate an area on the dish where hybrid asci are found, they can share this information with the other class members.

8 Solverson!8 4. Press the cover slip gently using the thumb or an eraser to crush the perithecia and release the rosettes of asci (Fig. 2). If too much pressure is applied, the ascospores will be forced out of the asci, making it impossible to collect data. A little practice will perfect the technique. 5. Using low power, examine the slide and locate rosettes of hybrid asci containing ascospores of two different colors. The wild-type ascospores appear black, while the gray and tan spores are a lighter color. Note: Many perithecia contain rosettes with ascospores of only one color. Persevere in searching until you locate perithecia with hybrid asci containing spores of two different colors. 6. After locating a rosette of hybrid asci, use high power to observe the ascospores and determine if crossing-over has occurred. If crossing-over has not occurred, segregation of the genes for spore color has taken place during Meiosis I (MI and the ascospores will be arranged in a 4:4 ratio (Fig. 3). If crossing over has occurred, segregation of the genes for spore color do not segregate until Meiosis II (MII) and the arrangement of ascospores will be either 2:4:2 or 2:2:2:2 (Fig. 4). 7. Each group should count 100 to 200 asci. Collate class date in Table Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.

9 Solverson!9 Results Strains Crossed Gray (g) and Black (+) Tan (tn) and Black (+) Table 1: Results of Tan and Black vs. Gray and Black Number of MI Asci (4:4) Number of MII Asci (2:4:2 or 2:2:2:2) Total Asci % MII (crossing over) Map Units % % 31 This table compares the results of the mating of gray and black Sordaria and tan and black Sordaria. It shows the number of Asci that did not cross over, the number of Asci that did cross over, the total number of Asci, the percent of the Asci that did cross over, and the map units of the specific genes from the centromere. The gray and black mixing determines the rate of crossing over of the g gene, and the tan and black mixing determines the rate of crossing over of the t gene. The g gene is 31.5 map units from the centromere, and the t gene is 31 map units from the centromere.

10 Solverson!10 Discussion The rate of crossing over helped determine the location of the genes on the chromosome because the distance, in map units, of the gene from the center of the chromosome is calculated by dividing the percent of ascospores that crossed over by two. Without knowing the number of ascospores that crossed over the percentage could not have been calculated and the location of the genes would not have been known. (Sordaria Genetics) These genes are likely to be crossed over as seen with the percentages in the table above % of the genes crossed over, which means that there is a good chance crossing over will occur. It will not always occur, but it will over half of the time. Many scientists believe that this is the reason why there is genetic variation and why brothers and sisters do not look identical. This is not what The Law of Segregation states, however. The Law of Segregation states that there is genetic variation because when an organism makes gametes, each gamete receives just one gene copy, which is selected randomly ("The Law of Segregation (article) ). Crossing over goes against this thought, saying that genetic variation happen because homologous pairs exchange alleles, not because traits are passed on randomly. The information about the locations of the g and t genes are useful to scientists who want to study these genes. Scientists can use the information about the location of the genes to find them and study them. This will help them better understand Sordaria and genes. The location of all genes on chromosomes is important for this same reason: so scientists can study them. From this studying, scientists can learn more about life itself, and the mysterious ways in which it works.

11 Solverson!11 The results of this experiment are not completely accurate. According to the Sordaria Genetics, extensive research has shown that the gene for tan spores is about 26 map units from the centromere, while the gene for gray spores is about 60 units distances greater than 33.3 map units cannot be directly calculated for the gene for gray spores. Thus, the experiment should have resulted in map units of 26 and 33.3, which are not the results recorded. They are close, but still different. The sources of error for this experiment may have been the fact that only 223 and 238 asci were observed for each color matching, while professional experiments would have observed much more. Also, some Asci may have been overlapped, and could have been counted twice.

12 Solverson!12 Works cited Leaf Group Writer. "What Do Fungi Contribute to the Ecosystem?" Sciencing. Leaf Group, 24 Apr Web. 30 Apr Volk, Tom. Sordaria Fimicola, a Fungus Used in Genetics-- Tom Volk's Fungus of the Month for March N.p., n.d. Web. 30 Apr Maryland Biodiversity Project - No Common Name (Sordaria Fimicola). N.p., n.d. Web. 30 Apr Sordaria Genetics Carolina Biological Supply Company: USA. "The Law of Segregation (article)." Khan Academy. N.p., n.d. Web. 30 Apr