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Sordaria Fimicola Genetics

Owen Daugherty

Ms. Williams

Honors Biology

1 May 2017
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Introduction:

In this experiment, the species of Sordaria being used is Sordaria fimicola, which is an

ascomycete fungus that normally grows on decaying organic material [and breaks this material

down, which is its main role in the ecosystem] (Davidson). In a natural environment, Sordaria

fimicola is very commonly found throughout the world in the feces of herbivores. (Sordaria

Fimicola Details.). Ascomycetes are known as sac fungi because of the characteristic shape

of their asci, which each contain four to eight ascospores in the sexual stage. The specific

attributes of the asci and the method of release of the ascospores is what primarily determines

which subgroup ascomycete species are placed in. (Davidson). These ascospores have two

mating types, + (positive) and (negative). Some Sordaria spcecies are able to asexually

reproduce, but Sordaria fimicola sexually reproduces. To sexually reproduce, the sacs that are

formed, called ascogonium and antheridium, go through a process called plasmogamy to

combine the + and ascospores into one sac. The majority of the short lifespan of a Sordaria

fimicola is spent living as a haploid cell in multicellular combinations of branches, but there are

times where it is also found as a dikaryot or diploid.

The lifecycle of Sordaria fimicola lasts a rather short time, usually a little bit longer than

one week. An image of the lifecycle can be seen in Figure 1 with a description beneath it. The

asexual reproduction that is referenced in this photo does not apply to Sordaria fimicola, so it can

be ignored. The sexual reproduction lifecycle is the lifecycle that Sordaria fimicola goes

through.
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Figure 1: Ascomyetes Life Cycle


Both + and haploid cells gather together to form multicellular mating branches. These mating

branches form sacs with haploid cells in them, with the + types sac being called an ascogonium

and the types sac being called an antheridium. The ascogonium and antheridium will

eventually interact with one another and combine to form one sac, creating a dikaryotic hyphae.

Multiple dikaryotic hyphae then combine together to form a perithecium, which is the fruiting

body of Sordaria fimicola. Asci containing dikaryotic nuclei are formed on this fruiting body,

and these dikaryotic nuclei eventually undergo karyogamy, formin together into one diploid

nucleus inside of an ascus. This single diploid nuclei then undergoes Meiosis inside of the ascus,

creating four haploid nuclei. Each haploid nucleus undergoes mitosis, causing a total of eight

haploid ascospores to be inside of an ascus. Parts of the perithecium, still containing the asci,

will burst open and release ascospores. These ascospores will germinate to form myceliea,

which grow into mating branches causing the process to restart itself (Newcombe).

In the species of Sordaria fimicola, two genes work together to determine one trait that is

being examined in this experiment: the color of the fungi. These to genes are the t gene and
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the g gene. Each gene has two different alleles. The t gene can either have a t or t+ allele,

and the g gene can either have a g or a g+ allele. In a haploid cell, these two genes will only

have one allele for each gene, which will determine the color of the fungi. There are four

different colors that a combination of these alleles can make. These colors are black, tan, gray,

and clear. These colors are determined by whatever mix of alleles the two genes provide. Here

are the four possible combinations of alleles for a haploid cell: black has t+ and g+, tan has t and

g+, gray has t+ and g, and clear has t and g. Using this trait, we can find out a general location of

how far away the genes are from the center of the chromosome of the Sordaria fimicola species,

which is the purpose of this experiment. This information can be found by looking at color ratios

inside of the asci. When either black and tan Sordaria fimicola or black and gray Sordaria

fimicola reproduce, their new cell created goes through crossing over during Meiosis. The color

black must be used because its alleles are g+ and t+, making it a constant when it crosses over

with the g and t+ gray or the g+ and t tan. This also makes the type of gene, either the g gene

or t gene, the independent variable. This single diploid cell eventually becomes eight haploid

cells because of the Meiosis and Mitosis that it goes through. If the ratio of these cells found in

an ascus are 2:4:2 or 2:2:2:2, that means crossing over occurred. If the ratio is 4:4, crossing over

did not occur. The ratio that is found is the dependent variable. These ratios are important

because it makes it possible to find the distance away from the center of the chromosome the

gene is. If there are more asci that show cells went through crossing over, the gene is located

farther from the center of the chromosome. If there is a small amount of asci that show cells

crossed over, the gene is closer to the center of the chromosome.

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
Microscopes
Glass slides and cover slips
Water dropping bottles
Inoculating loops
Bunsen burner
Boiling water bath (or container if boiling water)
Scalpel or spearpoint needle
Disinfectant

Procedure: (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 (22-25 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: This table shows results that were found either during the experiment or calculated after

the experiment using the found data.

Strains Number of Non- Number of Total Asci Percentage of Map Units


crossed crossed Asci (4:4 Crossed Asci crossed Asci From the
Ratio) (2:4:2 or Center of the
2:2:2:2 Chromosome
Ratio)
Gray and 82 141 223 63% 31.5 Map
Black Units
Tan and 91 147 238 62% 31 Map Units
Black

The results shown above indicate that there were a larger amount of asci with cells that

crossed-over compared to cells that did not cross over for both the experimented gray with black

and tan with black Sordaria fimicola. The gray and black mix had a crossing over rate of 63%

from a total of 223 counted asci, while the tan and black mix had very similar results with a 62%

crossing over rate from a total of 238 counted asci.

Discussion:

Using the results above, it is possible to find an accurate estimate the distance of how far

away the t gene and the g gene are from the center of the chromosome the gene is located

on. By taking the percentage of crossed asci (63% for the g gene and 62% for the t gene)

and using a formula developed by professionals, which is to simply divide the percentage by

two, a distance in map units can give an accurate measurement of how far away the genes are

from the center. The g gene was found to be around 31.5 map units away from the center of
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the chromosome and the t gene was found to be around 31 map units away from the center of

the chromosome.

These genes have a fairly good chance to cross over if they are on the same side of the

chromosome (either both genes are on the top or on the bottom) and would only be .5 map units

away from each other. If they were on opposite sides of the chromosome (one is on the top of

the chromosome and one is on the bottom), however, they would not cross over because they

would be over 60 map units apart.

This entire project relates to the law of segregation created by Gregor Mendel. This law

of segregation states that: 1. A gene can exist in more than one form or allele. 2. Organisms

inherit two alleles for each trait. 3. When sex cells are produced (by meiosis), allele pairs

separate leaving each cell with a single allele for each trait. 4. When the two alleles of a pair are

different, one is dominant and the other is recessive (Bailey). These genes do have more than

one allele and two alleles are inherited for this trait. The other two points of the law of

segregation are technically negated due to two genes making up one trait, but the general idea

still applies that each cell has one allele for each trait and there is a dominant color that stands

out.

Knowing the location of a gene can be very important due to recent discoveries in the

science world. It has been found in the relatively recent past that the location of a gene on a

chromosome can actually affect the variability of the trait that that gene determines. For

example, if a gene is found farther away from the center of the chromosome, there is more of a

chance that that trait has great variation in a certain species. One trait like this could be the

height of humans. If a trait is found closer to the center of a chromosome, that genes trait would

probably not have a lot a lot of variability or diversity. Knowing this information of how gene
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location affects diversity could help researchers begin to study the basis of diseases as well as

help breed plants and animals that would be more useful to the everyday lives of people

(Functional Significance of Gene Location). Knowing the location of genes can also help

determine whether or not certain genes cross over.

The accuracy of these results may not be completely reliable due to many different errors

that may have occurred. One source of error could have occurred when gathering the fungi to be

looked at. There were certain parts of the agar dish that the fungi should be taken from in order

to gather the best results. If fungi was taken from other of the dish, the results could have been

slightly skewed. Another error could have occurred while students counted asci. There is a

possibility that students may not have known what exactly to look for or counted colors equaling

a ratio that was not actually there. This could also affect the results.
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Works Cited

Bailey, Regina. "The 4 Concepts Related to Mendel's Law of Segregation." ThoughtCo. N.p.,

n.d. Web. 30 Apr. 2017.

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

N.p., 13 Nov. 2015. Web. 28 Apr. 2017.

"The Functional Significance of Gene Location: Countering the Case for Biological Evolution."

Reasons to Believe. N.p., n.d. Web. 30 Apr. 2017

"Fungi II - Phyla Ascomycota and Basidiomycota." Biology: Basic Concepts and Biodiversity.

N.p., n.d. Web. 30 Apr. 2017.

Newcombe, George, Jason Campbell, David Griffith, Melissa Baynes, Karen Launchbaugh, and

Rosemary Pendleton. "Revisiting the Life Cycle of Dung Fungi, Including Sordaria

Fimicola." PLOS ONE. Public Library of Science, n.d. Web. 29 Apr. 2017.

Sordaria Fimicola - Details." Encyclopedia of Life. N.p., n.d. Web. 28 Apr. 2017.

Sordaria Genetics Carolina Biological Supply Company. 1999.