Sordaria Lab
Sordaria Lab
Sordaria Lab
Owen Daugherty
Ms. Williams
Honors Biology
1 May 2017
Daugherty 2
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
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
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.
Daugherty 3
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
Daugherty 4
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
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
Daugherty 6
of the dish just enough to pour in the molten agar. Replace the lid immediately to prevent
contamination.
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
2. When ready for use, label two of the cornmeal-glucose-yeast agar dishes wild, two gray,
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
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
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
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.
1. Disinfect all work surfaces. Have the students wash their hands. Point out the location of the
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.
Daugherty 9
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 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%
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
Daugherty 10
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
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
Daugherty 11
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
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.
Daugherty 12
Works Cited
Bailey, Regina. "The 4 Concepts Related to Mendel's Law of Segregation." ThoughtCo. N.p.,
"The Functional Significance of Gene Location: Countering the Case for Biological Evolution."
"Fungi II - Phyla Ascomycota and Basidiomycota." Biology: Basic Concepts and Biodiversity.
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.