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Introduction

There are many different species of fungi. The specific type that will be talked about in

this lab is called sordaria fimicola. Sordaria is in the fungi genus and the sordariaceae family.

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

may also be found in introductory laboratory settings where it is manipulated and examined for

educational purposes (Davidson). The majority of its life, a the sordaria is found in the haploid

form. Sordaria, in genetics, undergoes a strict method of sexual reproduction. This specific type

of fungi is known as sac fungi because of the shape of their asci. According to dictionary.com,

ascus, or asci, is the sac in ascomycetes in which sexual spores are formed. These asci each

contain four to eight ascospores in the sexual stage. This helps the ecosystem because it classifies

the certain type of fungi and where it belongs in its ecosystem. To map out two separate genes to

figure out where they are located on the chromosome, the sordaria fimicola goes through a life

cycle.

The life cycle sordaria goes through has many parts to it. The beginning of the life cycle

starts with an ascogonium and antheridium which are both sacs containing haploid nuclei

because of mitosis. The ascogonium sac is the positive mating type and the antheridium is the

negative mating type. From stage one, the two sacs go through a process called plasmogamy.

Plasmogamy is when the two sacs connect, or attach, forming a bridge allowing for the different

haploid nuclei to mix together, which is how the sacs are found in stage two. After plasmogamy

occurs, two haploid nuclei go into an individual cell and begin to branch off forming more cells.

In stage three, the group of new cells being created through plasmogamy makes a fruiting body

called an asocarp. This asocarp contains branching that is made up of dikaryotic cells, called

dikaryotic hyphae. A dikaryotic cell contains two haploid nuclei that have not been fused
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together. From this stage, the new sacs that were formed, attached to the dikaryotic hyphae,

begin to go through their own process. These sacs are called asci and start as dikaryotic. The asci

then go through a process called karyogomy, during stage four. Karyogomy is the process in

which two haploid nuclei fuse together to form a diploid nucleus. After the asci have a diploid

nucleus, they go through meiosis which is the sixth stage of the life cycle. After meiosis, mitosis

occurs in each of the four, new haploid nuclei during stage seven. This creates eight ascospores

in each ascus. From there, in stage eight, each ascospore becomes its own haploid cell and

eventually breaks out of the asci. In stage nine, the final stage, germination occurs in the mycelia

(Sordaria Genetics). Mycelia is the mass of branched, tubular filaments (hyphae) of fungi

(Encyclopdia Britannica). During this stage, it produces spores, directly or through special

fruiting bodies (Encyclopdia Britannica).

The sordaria has two genes that determine the ascospore color and their alleles which are

the t and g genes. These genes can be combined to make four different colors. The g+t+ genes

collaborate to produce black spores. The gt+ genes collaborate to produce gray spores. The g+t

genes collaborate to produce tan spores. Lastly, the gt genes collaborate to produce clear spores.

Each ascus has a series of eight ascospores within itself. By observing the color and order of the

colors of all eight of these ascospores, you will be shown if the asci went through the process of

crossing over or not. If an asci did undergo the crossing over process, the eight ascospores will

be found in a 2:2:2:2 ratio or a 2:4:2 ratio. If you have found the ascospores in a 2:2:2:2 ratio,

you could see two black, followed by two gray or tan, then two black, and two gray or tan. But,

these ratios can be found starting with the gray or tan color as well. The same thing occurs when

there is a 2:4:2 ratio of colors. If the ascus did not undergo the crossing over process, its
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ascospores will be found in a 4:4 color ratio, which can start with black and followed by gray or

tan, or can start with gray or tan and be followed by black.

Within every scientific experiment there are independent and dependent variables. In this

experiment, the independent variable is the different combinations of the g and t genes, whether

was gray and black or tan and black. The dependent variable is the ratio of the colors of the

ascospores. The purpose of this experiment is to find out where two genes are located on a fungi

chromosome.

Materials ("Sordaria Genetics)

Sordaria fimicola, wild type

Sordaria fimicola, mutant grey
Sordaria fimicola, mutant tan
Bottle cornmeal-glucose-yeast agar
Autoclavable disposable bag
3 bottles Sordaria crossing agar
20 sterile petri dishes
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

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.

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 45C (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 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

grey, 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 (22-25C)

until perithecia have formed at the periphery if 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 +/tn 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 positioned

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.

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 ot +/g) they are

removing perithecia. Refer to Figure 1 for the most probable location of hybrid asci on
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the dishes. Notice the locations are different for gray and tan 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.

4. Press the cover slip gently using the thumb or an eraser to crush the perithecia and release

the rosettes of asci. 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 and the ascospores will be

arranged in a 4:4 ration. If crossing over has occurred, segregation of the genes for spore

color do not segregate until Meiosis II and the arrangement of ascospores will be either

2:4:2 or 2:2:2:2.
7. Each group should count 100 to 200 asci. Collate class data in Table 1.
8. Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.

Results

Table 1: This table describes the number of asci that crossed over and the number of asci that did

not cross over. It also shows the rate of crossing over and the map units from the center of the

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

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

or 2:4:2)
(g) x (+) 82 141 223 63% 31.5
(tn) x (+) 91 147 238 62% 31

The two rows of this table are the two genes that are being classified with this lab, the t

gene and the g gene. The first column, pictured above, after the type of gene is the number of

asci that did not cross over. The second column is the number of asci that did undergo the

crossing over process. The number of g gene asci that did not cross over was 82 and the number

of g gene asci that crossed over was 141. The number of t gene asci that did not cross over is 91

and the number of t gene asci that crossed over was 147. As you can see, the total asci of the g

gene were 223 and the total asci of the t gene was 238. The percent of the g gene asci that

crossed over was 63%. The percent of t gene asci that crossed over was 62% which was very

close to the amount of g gene asci that went through the crossing over process. The final column

in the distance from the center of the chromosome, or the map units. Table 1 shows that the g

gene asci were 31.5 map units from the center of the chromosome. The t gene asci were only 31

map units from the center of the chromosome (See Table 1).

Discussion

The distance of the genes from the center of the chromosome will determine how likely

they are to crossover. Within the lab, the rates of crossing over played a large role in helping to

calculate how far each gene was from the center of the chromosome. It is important to know the

rates of crossing over to eventually figure out the map units of the genes. According to Philip

McClean, to determine the linkage distance simply divide the number of recombinant gametes
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into the total gametes analyzed. This quote is emphasizing the importance of knowing the

number of times the asci crossed over. This is because without this information, you will not be

able to calculate the map units of the genes from the center of the chromosome.

Knowing the map units of the genes from the center of the chromosome, you are able to

calculate if they are likely to cross over together or not. Considering that the genes map units

were so close, there is a possibility of them crossing over together. But, there is also a possibility

of them not crossing over together. This is because, although we found the map units from the

center, we are not sure which way the genes are from the center of the chromosome. For

example, if they were to cross over together, both genes could be found on the top half of the

chromosome. But, if they did not cross over together, one gene would be on the top half and the

other would be on the bottom half.

All of the information from this project is important because when scientists attempt to

find the placement of a gene on a chromosome, they will easily be able to. Knowing the location

of the genes on the chromosome is important because it plays a significant role in shaping how

an organism's traits vary and evolve, according to new findings by genome biologists (New

York University). These results are not one hundred percent accurate because we were only

searching for the rates of crossing over. They were also not completely accurate because of our

source of error, not collecting enough data. The rate of crossing over helps to calculate the

distance because the further the genes are from the center of the chromosome, the more likely

they are to cross over.

Looking more into this project, it is possible to relate it to the law of segregation.

According to khanacademy.org, the law of segregation is when an organism makes gametes,

each gamete receives just one gene copy, which is selected randomly. The law of segregation is
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shown throughout the project because with crossing over occurring, will affect the overall

results. As another example of the law of segregation, when two parents each give off different

traits, the result will depend on which trait each of them would have given to the child.

Works Cited

"Asci." Dictionary.com. Dictionary.com, n.d. Web. 22 Apr. 2017.

Davidson, Michael W. "Fungus (Sordaria Fimicola) Fruiting Bodies." Molecular Expressions

Microscopy Primer: Specialized Microscopy Techniques - Differential Interference

Contrast Image Gallery - Fungus (Sordaria Fimicola) Fruiting Bodies. N.p., 13 Nov.

2015. Web. 22 Apr. 2017.

McClean, Phillip. "Recombination and Estimating the Distance Between Genes." Genetic

Linkage. N.p., 1997. Web. 30 Apr. 2017.

New York University. "Gene's location on chromosome plays big role in shaping how an

organism's traits evolve." ScienceDaily. Web. 15 October 2010.

"Sordaria Genetics." Carolina Biological Supply Company, 1999. Print.

The Editors of Encyclopdia Britannica. "Mycelium." Encyclopdia Britannica. Encyclopdia

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Britannica, Inc., 20 July 1998. Web. 24 Apr. 2017.

"The Law of Segregation (article)." Khan Academy. N.p., n.d. Web. 30 Apr. 2017.

Volk, Thomas J. "Sordaria Fimicola, a Fungus Used in Genetics." Sordaria Fimicola, a Fungus

Used in Genetics-- Tom Volk's Fungus of the Month for March 2007. N.p., n.d. Web. 24

Apr. 2017.