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Sordaria Genetics

Jessie Potter

Ms. Williams

Honors Biology

1 May 2017
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Introduction

For this lab, the species of sordaria used was sordaria fimicola, a type of fungus frequently

found in fecal matter (Writer). Sordaria fimicola can be found in the Sordariaceae family and

the genus of the species is sordaria. The fungus spends the majority of its life cycle as haploid. It

can reproduce both sexually and asexually and is most commonly found in

the feces of herbivores (Sordaria Fimicola). As mentioned before, sordaria fimicola is a type

of fungi and all fungi play a vital role in the ecosystem. Fungi help to break down dead

organisms and biological waste, freeing nutrients for use by other organisms and clearing away

their remains. Fungi also act in partnership with some plants and algae, and are often vital to the

survival of these organisms ("What Is the Role of Fungi in the Ecosystem?").

The life cycle of sordaria fimicola starts out with an ascospore, or a single, haploid cell,

landing on the ground and beginning to germinate, which means to grow or develop, as

defined by Dictionary.com. When it germinates, it forms branches called mycelia. Sordaria

fimicola has two different mating types, the positive mating type and the negative mating type.

Both mating types are multicellular haploid organisms and want to sexually reproduce, so they

each form a sac full of haploid nuclei. The negative mating type sac is called an antheridium and

the positive mating type is called an ascogonium. Next, plasmogamy occurs, which is when the

antheridium and the ascogonium combine or connect and the haploid nuclei all go into the same

sac. The haploid nuclei then pair up, with one from each mating type, and form individual cells

called dikaryotic cells, which is a cell that has two haploid nuclei not yet fused together. These

cells then make up the branches of the fruiting body that is formed called an ascocarp. The name

for the ascocarp of our particular species of sordaria is called the perithecium. At the end of the

perithecium, asci are formed. Asci are sacs that contain two, unfused haploid nuclei and the
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singular form of asci is ascus. Next, karyogamy takes place, which is when the two haloid nuclei

inside an ascus fuse together and form a diploid nucleus. Meiosis then occurs and we are left

with four different haploid nuclei. These four haploid nuclei now undergo mitosis and form eight

haploid nuclei all in one ascus. The eight haploid nuclei start to form membranes and become

eight haploid cells. These eight haploid cells are called ascospores. The perithecium bursts open

and the asci it contained go flying. Each asci contains eight ascospores and when the asci break

apart, the release the eight ascospores, which then land on the ground and begin to germinate,

starting the entire process over again (Class Presentation).

There are two genes that determine spore color in sordaria fimicola. These genes are known as

the g-gene and the t-gene, and they each contain two alleles. The two alleles for the g-gene are g+

and g, while the two alleles for the t-gene are t+ and t. The combination of these alleles are

ultimately what decides the color, and there are four different possible combinations. The first

combination, which is g+t+, will produce black spores, which are the wild type. The next

combination is gt+ and this produces grey spores. The combination of g+t will produce tan spores

and finally, the combination of gt will produce clear spores (Class Presentation).

After the four haploid nuclei undergo mitosis and form the eight haploid nuclei inside an

ascus, we can look at the ascus under a microscope and determine the color ratio of the

ascospores. If the ascospores being worked with were tan and black and had a 2:2:2:2 ratio,

which means that the color of the ascospores went black, black, tan, tan, black, black, tan, tan,

then it can be concluded that crossing over occurred. The other ratio that indicates crossing over

has occurred is a 2:4:2 ratio, which means that the order of color would go black, black, tan, tan,

tan, tan, black, black. There is one other ratio possible, which is the 4:4 ratio. However, if this

ratio occurs, that means that crossing over did not take place. We can use these color ratios to
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determine the rates of crossing over as if at the end of the lab, more asci have either the 2:2:2:2

or the 2:4:2 ratio than have the 4:4 ratio, it can be concluded that we had higher rates of crossing

over (Class Presentation).

In this lab, there were both dependent and independent variables. The dependent variable is

the location of the gene on the chromosome, as this is what is being tested in the experiment. The

independent variable is whether gray or tan sordaria was used along with the black sordaria,

which was the constant. The overall purpose of this lab is to determine if crossing over took

place and to find out where the genes are located on the chromosome. We can determine this by

looking at the rates of crossing over.

Materials (Sordaria Genetics)

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
Microscopes
Glass slides and cover slips
Water dropping bottles
Inoculating loops
Bunsen burner
Boling water bath
Scalpel or spearpoint needle
Disinfectant such as phenol or 70% ethanol

Procedure (Sordaria Genetics)

Preparation of Agar Dishes


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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 has

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 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 of 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 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 the wild-type Sordaria fimicola, and flame the mouth over a Bunsen
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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

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 of the dishes.

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 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


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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 or +/g) they are

removing the 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 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 the 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.


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6 After locating a rosette of hybrid asci, use high power to observe the ascospores and

determine is 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. 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.

7 Each group should count 100 to 200 asci. Collate class data.

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

Results

Table #1- In this table, the rate of crossing over and the distance of the gene from the nucleus

are both displayed, along with total numbers of asci counted for both the tan and gray asci.

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


Crossed Asci (4:4) Asci (2:4:2 or (No. MII/total) %MII/2
2:2:2:2)
(g) x (+) 82 141 223 63% 31.5

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

Table 1 is showing results from the experiment. The first row shows the results of the gray and

black spores while the second row shows the tan and black spores. The first column, labeled No.
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of MI Asci (4:4), shows the number of asci that had a 4:4 ratio, which means they didnt undergo

crossing over. The second column, labeled No. of MII Asci (2:4:2 or 2:2:2:2), shows the number

of asci that had either a 2:4:2 or 2:2:2:2 ratio, meaning that they did undergo crossing over. The

third column shows the total asci counted during the experiment for each set of colors and the

fourth column displays the percent of asci that underwent crossing over. The percentage was

calculated by taking the number of asci that did undergo crossing over and dividing it by the total

number of asci. Finally, the fifth column shows the map units, or how far the gene is located

from the center of the chromosome. This was calculated by taking the percent and diving by two.

The results of the experiment show that more asci underwent crossing over than didnt undergo

it for both sets of colors. The tan and black spores had nine more asci that didnt undergo

crossing over and six more asci that did undergo crossing over, making their total number of asci

fifteen more than the gray and black spores. The tan and black spores also had a map unit of 31,

making the t-gene closer to the center of the chromosome than the g-gene, as the gray and black

spores had a map unit of 31.5.

Discussion

As mentioned earlier, the results show that the t-gene is located closer to the center of the

chromosome than the g-gene is. This was determined as the t-gene was located 31 map units

away from the center of the chromosome while the g-gene was located 31.5 map units away.

However, this contradicts prior information that the further a gene is away from the center of the

chromosome, the more likely it is to cross over as even though the t-gene is closer, the tan and

black asci crossed over more times than the gray and black did. The two genes though are only .5

map units away from each other, so they are most likely to be crossed over as the closer two

genes are, the more likely they are to be crossed over together. Knowing the location of genes on
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a chromosome is also extremely important as a gene's location on a chromosome plays a

significant role in shaping how an organism's traits vary and evolve (Genes Location).

The results of this experiment are not very accurate as demonstrated by the fact that even

though the t-gene is closer to the center of the chromosome, the tan and black asci crossed over

more times than the gray and black did, and the higher the rate of crossing over, the further away

the gene is from the centromere, or the center of the chromosome. The data collected was

produced by all of the honors biology classes and there could be many reasons why it was

skewed. One of them could be that the students mistakenly counted more or less asci in the

crossing over category than there really were or counted more or less asci in the non-crossing

over category than there really were. Another source of error could have been that one group

with tan and black asci worked hard all period to gather as much data as they could while another

group with gray and black asci could have slacked a little and not gathered as much data, making

the tan and black group have more asci crossed over even though it was closer to the center of

the chromosome.
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Works Cited

"Gene's Location on Chromosome Plays Big Role in Shaping How an Organism's Traits

Evolve." ScienceDaily. ScienceDaily, n.d. Web. 30 Apr. 2017.

"Germinate." Dictionary.com. Dictionary.com, n.d. Web. 30 Apr. 2017.

"Sordaria Fimicola - Details." Encyclopedia of Life. N.p., n.d. Web. 30 Apr. 2017.

Sordaria Genetics. Carolina Biological Supply Company, 1999. Print

"What Is the Role of Fungi in the Ecosystem?" Reference. IAC Publishing, n.d. Web. 30 Apr. 2017.

Writer, Leaf Group. "Life Cycle of Sordaria Fimicola." Sciencing. Leaf Group, 24 Apr. 2017. Web. 30

Apr. 2017.