Sordaria Genetics

Sam Mager

Ms. Williams

Honors Biology

1 May 2017
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Introduction

Sordaria Fimicola, also known as just Sordaria, is a common fungus species of Dung

Ascomycete (Carolina Biological Supply Company, 1999.) It is a part of the Sordariaceae family

(gbiforg.) Sordaria likes dung so it often sends its ascospores onto plants in hope of being eaten

by herbivores. It is often used in genetics due to its specific method of reproduction and because

it is very simple to work with.

The life cycle of Sordaria starts off with a single haploid ascospore. The ascospore then

germinates, creating a multicellular branch-like fungi made up of haploid cells. If the Sordaria

wants to sexually reproduce, it forms a sac- like structure with haploid nuclei that formed from

mitosis. The sac created by the positive mating type is known as the Ascogonium and the sac

created by the negative mating type is known as the Antheridium. The Sordaria undergoes

Plasmogamy, and the two sacs connect, with all of the nuclei moving into one of the two sacs.

Next, the cells of the Sordaria begin branching out. Within each cell, one positive and one

negative nuclei pair up. These cells are dikaryotic because they each contain two haploid nuclei.

The branching out cells make up the ascocarp, which is the fruiting body of the Sordaria, known

as the Parethicia (fsu.org.) At the tip of each branch is an ascus. Each ascus contains one nuclei

of each mating type. Next, the process of Karyogamy takes place. The two nuclei inside of the

ascus fuse together, forming a diploid nucleus. Although the Sordaria is haploid for the majority

of its life cycle, for this brief moment it becomes diploid. The diploid nucleus then undergoes

meiosis, creating four haploid nuclei. Those four haploid nuclei then undergo mitosis, resulting

in eight haploid nuclei. These eight haploid nuclei turn into the eight ascospores that are in each

asci. Inside of a Perithecia, there are many asci. When the Perithecia is mature, it releases all of

its asci, which in turn, release all of their ascospores. The asci tries to release the ascospores
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towards light. The ascospores want to land near plants so that they can be eaten by herbivores,

thus completing their life cycle.

By looking at the ratio of the colors of the ascospores, it can be determined if crossing

over occurred. There are two different genes that determine the color of the ascospores; the t

gene and the g gene. Since Sordaria is haploid, each chromosome only has one allele from each

gene. The different combinations that can possibly occur are g and t (clear ascospore), g+ and t

(tan ascospore), t+ and g (gray ascospore), and t+ and g+ (black ascospore.) In our experiment,

when black and gray were mated, it allowed for the g gene to be focused on because both black

and gray have t+ genes. When black and tan were mated, it allowed for the t gene to be focused

on because both black and tan contain g+ alleles. The purpose of the experiment is to determine

where each gene lies on the chromosome by looking at the ratio of ascospores where crossing

over occurred to ascospores where crossing over did not occur, which would be the dependent

variable. The independent variable was which colors are combined, which affects the dependent

variable. The asci were observed through a microscope. If they had a color ratio of 4:4, crossing

over did not occur. If there is either a 2:2:2:2 or a 2:4:2 color ratio, then crossing over did occur.

By looking at the amount of asci that crossed over to the asci that did not cross over, it can be

determined where the gene is located on the chromosome.

Materials (Carolina Biological Supply Company, 1999)

The materials in this kit are sufficient for 14 groups of students. The materials are

supplied for use with the exercise in this kit only. Carolina Biological Supply Company

disclaims all responsibility for any other uses of these materials.

Included in the kit:

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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

Also needed:

Microscopes

Glass Slides and cover slips

Water dropping bottles

Inoculating loops

Bunsen burner

Boiling water bath

Scalpel or spear point needle

Disinfectant such as phenol or 70% ethanol

Procedures (Carolina Biological Supply Company, 1999)

Preparation of Agar Dishes

1. Slightly loosen the bottle caps and set the bottles in a boiling water bath to melt the agar.

Mark the bottle caps with the type of agar within the bottles. 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.
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2. Cool the agar to 45 degrees 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 the 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 & 5 with the Sordaria crossing agar, distributing the remaining agar

among the four 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.

Preparation of Stock Cultures

1. Disinfect the work surface and wash your hands

2. When ready for use, label two of the cornmeal-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 spear point needle, remove a

portion of the culture containing perithecia (black pepper grain appearance) and transfer
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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 5-7 days out of direct sunlight at room temperature (22-25 degrees

C) until perithecia have formed at the periphery of the dishes.

During the 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 +/tn to

indicate the positions of the wild type (+) and gray (g) or tan (tn) cultures.

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 spear point 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 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.
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During Laboratory 2: Microscopic Examination

1. Disinfect all work surfaces. Have the students wash their hands. Point out the location of

the autoclavable disposal 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 perithencia. Refer to Figure 1 for the most probably 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 of the dish from which they prepared the slide.

When students locate an area on the dish where hybrid asci are found, they can share this

information with the other class members.

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
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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 1.

8. Chromosome maps for the two mutant genes are constructed by diving the %MII by 2.

Results

Table 1; Rates of crossing over for the t and g genes with calculated

map units
Strains # of MI Asci # of MII Asci (2:4:2 or Total % Map

Crossed (4:4) 2:2:2:2) Asci MII Units

(g) x (+) 82 141 223 63 31.5

%
(tn) x (+) 91 147 238 62 31

As stated in the table above, the two strains that were crossed were gray and black [(g) x

(+)] and tan and black [(tn) x (+)]. The number of Asci that did not undergo Crossing over was

82 for the gray and black strains and 91 for the tan and black strains. The number of Asci that did

undergo crossing over was 141 for the gray and black strains and 147 for the tan and black

strains. The total number of hybrid asci for the gray and black strains was 223 and 238 for the tan

and black strains. The percent of asci that underwent crossing over was 63% for the gray and

black strains and 62% for the tan and black strains. The map units from the center of the
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chromosome for the black and gray strains was 31.5 with the map units for the tan and black

strains being 31.

Discussion

Overall, the results for both strains were pretty similar. There were more hybrid asci

found for the tan and black strands than with the gray and black strands. However, the percent of

asci from both that underwent crossing over is nearly identical. The percent of asci that crossed

over for the gray and black strains was only one percent higher than the percent of asci that

underwent crossing over for the tan and black strains. The map units from the center of the

chromosome was also very similar for both strains, with the g gene being located 31.5 map units

from the center of the chromosome and the t gene being located 31 map units from the center of

the chromosome. This mean that the g gene is more likely to undergo crossing over than the t

gene, although is only by a very little amount. However, there is no guarantee that these genes

even reside on the same chromosome. There is also no way to tell in which direction from the

center of the chromosome each gene lies. Therefore, it is unknown whether or not these genes

cross over together. These results are fairly accurate but it is possible that there were ascospores

that were missed. If I were to do this experiment again, I would take more time to look in the

microscopes.
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Work Cited

Carolina Biological Supply Company, 1999

"Fungus (Sordaria FImicola) Fruiting Bodies." Molecular Expressions Microscopy Primer:

Specialized Microscopy Techniques - Differential Interference Contrast Image Gallery -

Fungus (Sordaria Fimicola) Fruiting Bodies. N.p., n.d. Web. 30 Apr. 2017.

Gbif. "Sordaria Fimicola (Roberge Ex Desm...." Sordaria Fimicola (Roberge Ex Desm.) Ces. &

De Not., 1863 - Checklist View. N.p., n.d. Web. 30 Apr. 2017.