Sordaria Genetics

Braiden Wills

Ms. Williams

Honors Biology

1 May 2017
Introduction

Sordaria fimicola is an ascomycete fungus that is usually found growing in decaying

organic matter and animal feces. Sordaria belongs to the phylum Ascomycota, which is also

called sac fungi. The sac like structure after which this phylum of fungi is named is known as the

ascus, which contains the fungis four to eight ascospores (Lichtenstein). Asci are located at the

ends of branch like structures called mycelia, which are made up of haploid cells These branches

together make up the fruiting body of this fungus, which is known as the ascocarp (Sordaria

Fimicola Fruiting Bodies ). Sordaria fimicola is often examined in biology classes to teach

students about the life cycle of fungi and crossing over, because of its numerous similarities to

other types of fungi (Sordaria Fimicola, a Fungus Used in Genetics).

The life cycle of Sordaria fimicola begins with a single haploid cell known as an

ascospore that begins to germinate and self-replicate through the process of mitosis. As the

haploid cells divide, they begin to form branch like structures called mycelia. (Lichtenstein).

Mycelia can have two mating types, the + mating type and the mating type. Each mating type

forms sacs branching off of the mycelia which contain multiple haploid nuclei. The + mating

type sac is called the ascogonium and the mating type sac is called the antheridium. When both

mating type sacs are developed, a process called Plasmogamy occurs, and the two sacs combine.

Haploid nuclei from the different mating types mix into a single sac. Once this occurs, sets of

two haploid nuclei combine to form dikaryotic cells, which multiply and grow through mitosis to

make dikaryotic hyphae, which make up the ascocarp (Lichtenstein). At the end of each

dikaryotic hyphase, an ascus sac forms, which contains dikaryotic cells. Inside the ascus,

karyogamy occurs, and two haploid nuclei fuse together to form a single diploid nucleus. This

newly formed diploid cell undergoes meiosis and crossing over occurs. This process produces
four different haploid cells. These cells undergo mitosis, and because of this there are now eight

haploid nuclei in the ascus, which are each called ascospores. Ascospores are released from the

ascus and begin to germinate, starting the life cycle back over again (Lichtenstein).

There are two genes that determine the color of the ascospores in this species. These

genes are the t gene and the g gene. Each gene has two possible alleles, t and t+, and g and g+. A

combination of the g+ and t+ genes produces a black color. G and t+ will produce gray spores,

g+ and t produces tan and g and t produces clear (Sordaria Genetics). By looking at the color

ratio of the 8 ascospores in each ascus, you can tell if crossing over occurred durimg the making

of that ascus. If the color ratio is 2:2:2:2 or 2:4:2, crossing over has occurred. If the ratio is 4:4 or

all the ascospores are the same color, crossing over has not occurred (Sordaria Genetics).

For this study, the independent variable is the rate of crossing over, and the dependent

variable is the gene to centromere distance of the gene in map units. The purpose of this project

is to use the rate of crossing over to determine the location of the genes that determine spore

color on the chromosome.

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 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

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.

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.

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.

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

Results

Table 1: This table shows the number of spores for each cross that crossed over and did not cross

over, as well as the percent of crossing over and the map units of the gene to centrome distance

for each cross.

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

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

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

For the gray x wild type cross, there were 82 spores that did not cross over and 141 that

did. The percent of crossing over was 63%, meaning that the gene to centome distance in map

units is 31.5. For the tan x wild type cross, there were 91 spores that did not cross over and 147

that did. The percent of crossing over was 62% and the gene to centrome distance is 31 map

units.

Discussion

In this project, I was able to calculate the distance of the genes that affect spore color

from the centromere by first finding the rates of crossing over for each cross. Once I found this, I

could calculate the percent of ascospores that were crossed over put of the total number that was

found, and divide this percentage by two, which gave me the distance from the centromere in

map units. These genes are likely to be crossed over together because of the distance they are

away from the centromere. The farther away from the centromere, the higher the chance of

crossing over. In cells that crossing over occurs in, the genes segregate or separate during

meiosis II to form a 2:2:2:2 or a 2:4:2 color ratio. If crossing over did not occur, the genes

segregate during meiosis I to produce a 4:4 ratio.

Studying and researching to find the location of a gene on a chromosome on any

organism is important because the genes location helps determine how that organisms traits

vary and change over time. These results are probably not the most accurate because the sample
size was relatively small for the study. This is a possible source of error, if the sample size was

bigger, then the percent of crossing over would probably be more accurate. Also, it is possible

that the people swabbing the sordaria off of the agar took it from an area where hybrid asci is not

likely to be found, which could affect the results.

Works Cited

Fungus (Sordaria Fimicola) Fruiting Bodies." Molecular Expressions Microscopy Primer:

Specialized Microscopy Techniques - Differential Interference Contrast Image

Gallery - Fungus (Sordaria Fimicola) Fruiting Bodies. N.p., n.d. Web. 30 Apr.

2017.

Lichtenstein, Drew. "Life Cycle of Sordaria Fimicola." Sciencing. Leaf Group, 24 Apr. 2017.

Web. 30 Apr. 2017.

"Sordaria Fimicola, a Fungus Used in Genetics." Botany.wisc.edu. N.p., n.d. Web. 30 Apr. 2017.