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Introduction

Sordaria is an ascomycete fungus that normally grows on decaying organic material

(Davidson). Sordaria is a fungus because it is able to feed off of organic material which is

included in the basic definition of a fungus. The fungus has eight ascopores in each of its asci.

Sordaria is a fruit body and is often called S. fimicola (Davidson). The fungus is able to

produce these fruiting bodies for a limited time as the fungus has a short life cycle. The fungus is

a haploid organism for most of its life cycle. People are easily able to observe the cell division of

the fungus. It is most commonly found on dead organic matter and is often seen on the feces of

plants. The family of Sordaria is Sordariaceae and the genus is simply Sordaria. Sordaria is a

useful way to teach students how the life cycle of a fungus works. The fungus uses sexual

reproduction to reproduce. The purpose of this lab is to locate two genes in the fungi on a

chromosome. This will determine how far the genes are located from the centromere. Another

purpose of this lab is to determine the rate of crossing over. The rate of crossing over is

determined by how far away the gene is from the centromere of the chromosome.

The life cycle of the Sordaria begins with the eight ascopores that are located within the

asci of the fungus. These ascopores are single celled organisms and are haploid. These fall to the

ground where they begin to germinate or grow. As these fungi the ascopores germinate, they start

forming branches that make a multicellular organism made up of haploid cells. There are two

different mating types for these haploid cells. There is the positive mating type and the negative

mating type. Both mating types form sacs separate from each other that contain large quantities

of haploid nuclei with the correct mating type. These haploid nuclei came from Mitosis. The sacs

that are formed of the positive mating type are called Ascogonium and the sacs formed of the

negative mating type are called Antheridium. After these sacs are formed, a process called
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Plasmogamy takes place. In Plasmogamy, the Ascogonium and Antheridium connect and form a

bridge where the haploid nuclei are able to combine together to form one sac. When two haploid

nuclei, one of each mating type, go into an individual cell, these then begin to branch off creating

the Perithecium which is a fruiting body. The branching that makes up the Perithecium is called

Mycelia. The mycelias job is to extend the area in which a fungi can find nutrients (Munks).

The mycelium expands outward to look for water and nutrients which is transported to the

fruiting body (Munks). The branches are made up of dikaryotic hyphae which include two

haploid nuclei. At the ends of these dikaryotic hyphae, an ascus forms which is another sac. Once

the asci form, a process called Karyogamy occurs in which the two haploid nuclei fuse together

and form a diploid nucleus. The cell is now identified as a diploid cell. The cells then undergo

Meiosis and then Mitosis and by then there are eight haploid nuclei in one ascus. These haploid

nuclei are ascopores and these will eventually break out of the ascus and the process will be

repeated again.

The two genes that will determine the ascopores color are the g and the t gene. Their

other alleles are g+ and t+. Certain combinations of these alleles will determine what color the

ascopores are. The g and t alleles combined creates a clear color; the g+ and t+ alleles create

black; the g+ and t allele create tan; the g and t+ allele create gray. To determine if the genes

crossed over, the ratio of colors would have to be 2:4:2 or 2:2:2:2. To determine if the genes

didnt cross over, the ratio would then have to be 4:4. The dependent variable within this

experiment is the rates of crossing. The independent variables are the color and order of the

ascopores.

Materials (Sordaria Genetics)

Sordaria fimicola, wild type

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Sordaria fimicola, mutant gray

Sordaria fimicola, mutant tan
Bottle cornmeal-glucose-yeast agar
Autoclavable disposal bag
3 bottles Sordaria crossing gear
20 sterile petri dishes
Microscope
Glass slides and cover slips
Water dropping bottles
Inoculating loops
Bunsen burner
Boiling water bath
Scalpel or spearpoint needle
Disenfectant

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 is melted.

2. Cool the agar to 45 Degrees Celsius (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
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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 disposable 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 spearpoint needle, remove a portion of the culture

containing perithecia (black peppergrain 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 degrees-25

degrees Celsius) until perithecia have formed at the periphery of the dishes.
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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 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 date, 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 date.

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 disposal bag.

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2. Provide the students with water dropping bottles, glass slides, cover slips, inoculating oops,

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 other class members.

4. Press the cover slip gently using the thumb or an eraser to crush the perethecia 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 data in Table 1.
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8. Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.

Results

Table 1: Shows the number of asci that crossed and did not cross over in each test. Shows rate of

crossing over and how far away the g or t gene is from the centromere in map units.

Strains No. of MI No. of MII Total Asci %MII (No. Map Units

Crossed Asci (4:4) Asci (2:4:2 or MII/Total) %MII/2)

2:2:2:2)
Gray vs + 82 141 223 63% 31.5
Tan vs + 91 147 238 62% 31

Source: Sordaria Genetics. Carolina Biological Supply Company, 1999. Print

In the gray vs black results, there was a total of two hundred twenty-three asci. Eighty-

two of these asci did not cross over while the other one hundred forty-one did cross over. In the

tan vs black results, the total number of asci was two hundred and thirty-eight. Out of these asci,

ninety-one did not cross over, but one hundred forty-seven did. Sixty-three percent of the asci in

the gray vs black test crossed over and sixty-two percent in the tan vs black crossed over. The g

gene is thirty-one decimal five map units away from the centromere. The t gene is thirty-one map

units away.

Discussion

The further away a gene is from the centromere of a chromosome, the more likely it will

cross over. Therefore this information and the information collected in my results helped me to

determine approximately how far the gene is from the centromere of the chromosome. To find

the map units in both tests, the percentage of crossing over would be divided by two. Because
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there was a higher chance of crossing over in both tests, this meant that the genes would be

further away from the centromere. The genes have over a fifty percent chance to cross over

therefore there is a high possibility that the two genes will cross over. This information will help

us to determine the rate of crossing over during the life cycle of a Sordaria fungus. This will also

help us find the location of the genes on a chromosome. The location of genes on an organism

will determine that organisms traits. These results arent very adequate as to find a true

percentage of crossing over in a specific fungus, there would need to be a few hundred more asci

to make the results sufficient. It is also unknown if the gene is located up or down on the

chromosome as there is no way of telling that using this method. Therefore, the genes actual

location on the chromosome is unknown. There are a few sources of error that could have

occurred in this experiment. A student could have incorrectly counted up their asci or might have

not received a sufficient amount of information. The students could have also misidentified the

asci which would alter the resulting percentages.

Works Cited

Davidson, Michael W. Molecular Expressions Microscopy Primer: Specialized Microscopy

Techniques - Differential Interference Contrast Image Gallery - Fungus (Sordaria

Fimicola) Fruiting Bodies. N.p., n.d. Web. 23 Apr. 2017.

Munks, Brekke Peterson. "What Is Mycelium? - Definition & Function." Study.com. n.d. Web.

24 Apr. 2017.

"Genus Sordaria, Taxonomic Structure Skaphandrus. N.p., n.d. Web. 30 Apr. 2017.

Sordaria Genetics. Carolina Biological Supply Company, 1999. Print

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