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Introduction:

Sordaria, more specifically known as Sordaria fimicola, is a common type of dung

ascomycete. This fungus usually grows on decaying organic material, but is also used in

laboratories for various educational purposes. Sordaria is classified in the class of Pyrenometes,

along with [other fungi like] Xylaria polymorpha, dead mans fingers, [and] Hypomyces

lactifluorum, the lobster mushroom (Sordaria fimicola, a fungus used in genetics). Sordaria

also can be four different colors, tan, gray, clear, and black which is known as the wild type. The

fungus has two different genes with two alleles per gene. These two genes are the t gene and

the g gene. The two alleles for each gene are g and g+ for the g gene and t and t+

for the t gene. The different combinations of these genes are what makes the Sordaria a

specific color. If the g+ and t+ alleles combine they produce black spores, if the g and t+

alleles combine they produce gray spores, if the g+ and t alleles combine they produce tan

spores, and if the g and t alleles combine they produce clear spores.

Sordaria does not asexually reproduce and starts, and remains for most of its life, in

haploid form. To start reproducing the haploid Sordaria cell undergoes Mitotic Cell Division to

create long strings/chains of cells. After the fungus is in the long strings/chains, they start to form

sacks with different nuclei in the sacs. Sordaria has two different mating types, positive and

negative, and both sacs have a different mating type of nuclei. These two sacs fuse together to

form one Dikaryotic cell. A Dikaryotic cell is one cell that has two haploid nuclei, but the nuclei

are not fused together. As the sacs fuse, the Dikaryotic cell begins to create long chains of eight

nuclei, known as ascospores. These ascospores then combine to develop into an ascocarp

(perithecium) which is the fruiting body for Sordaria. Once the Sordaria has fully matured in the

ascocarp, eight ascospores contained in a clear sac are released. The ascospores that are released
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are haploid cells like the one that began the Sordaria life cycle. These newly created ascospores,

are the original unicellular version of Sordaria fimicola (Life Cycle of Sordaria Fimicola).

After looking at our ascospores under a microscope we can determine whether or not

crossing over took place. If there is a 2:2:2:2 (ex. 2 black:2 tan:2 black:2 tan) or 2:4:2 (ex. 2:

black:tan:2black) color ratio of the eight ascospores then crossing over did take place. If there is

a 4:4 (ex. 4 black:4 tan) color ratio of the eight ascospores then crossing over did not take place.

Taking the number of crossed over ascospores and not crossed over ascospores we can determine

the rate of crossing over for the Sordaria.

The independent variables in this lab are the Sordaria asci that we started out with. The

dependent variables are the crossed over or not crossed over Sordaria asci after the fungus

sexually reproduced. The purpose for this lab is to look at different Asci and determine if

crossing over took place and to find the specific location for the g gene and the t gene on the

chromosome.

Materials (Sordaria Genetics)

1. Sordaria fimicola, wild type

2. Sordaria fimicola, mutant grey

3. Sordaria fimicola, mutant tan
4. Bottle cornmeal-glucose-yeast agar
5. Autoclavable disposable bag
6. 3 bottles Sordaria crossing agar
7. 20 sterile petri dishes
8. Microscopes
9. Glass slides and cover slips
10. Water dropping bottles
11. Inoculating loops
12. Bunsen burner
13. Boiling water bath
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14. Scalpel or spearpoint needle

15. 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.

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
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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.

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

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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

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.
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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 One: This table includes the total number of asci not crossed over, the total number of asci

crossed over, the total number of asci found, the percentage of asci crossed over, and the total

map units the genes are located from the center of the chromosome.

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

Crossed Asci (4:4) Asci (2:4:2 (No. (%MII/2)

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

This chart shows the different number of crossed over and not crossed over asci for each strain,

the total number of asci found for each strain, the percentage of asci crossed over, and the map units away

from the center of the chromosome for each stain. For the gray and black strains that reproduced 82 asci

did not cross over while 141 asci did cross over. That means that 63% of the asci crossed over. After some

calculations the g gene is 31.5 map units away from the center of the chromosome. For the tan and

black strains 91 asci did not cross over, while 147 asci did cross over. That means 62% of the asci crossed

over for this strain. After some more calculations, the t gene is located 31 map units away from the

center of the chromosome.

Discussion

Genes are more likely to cross over is they are farther away from the center of the

chromosome. Therefore, the higher the rate of crossing over means that the gene is farther away

from the center of the chromosome. For the g gene, the g allele and the g+ allele, 63% of
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the alleles crossed over. Using the equation %MII/2 we are able to get the number of map units

away from the center of the chromosome. We divide the number by two because the center of the

chromosome divides the chromosome into two equal parts. We do the exact same thing for the

t gene. For the t gene, the t allele and the t+ allele, 62% of the genes crossed over. After

using the equation, %MII/2, it is shown that the t gene is 31 map units away from the center of

the chromosome. One of the main concepts of the law of segregation is that organisms have two

different alleles per trait. This is law relates to this lab because we know that each asci has two

different alleles for its color. It just depends on the combination of these alleles to determine its

color. Knowing the location of a gene on a chromosome is important in general because it can

help doctors know more about chromosome mutations and diseases. If doctors notice that two

genes are flipped from their normal position, then they can conclude a chromosome mutation

like inversion occurred. The results for this lab are fairly accurate because they were done in a

controlled environment with an experienced teacher aiding the process. However, professional

scientists in a state-of-the-art lab would obviously get more accurate information than us in a

classroom. Our sources of error include things like maybe counting the same hybrid ascospore

twice unknowingly or taking samples for the microscope from the wrong part of the petri dish

and agar.
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Works Cited

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

www.thoughtco.com/mendels-law-of-segregation-373472. Accessed 30 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,

micro.magnet.fsu.edu/primer/techniques/dic/dicgallery/sordariaperitheciasmall.html.

Accessed 30 Apr. 2017.

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

sciencing.com/life-cycle-sordaria-fimicola-6909851.html. Accessed 30 Apr. 2017.

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

Volk, Tom. Sordaria fimicola, a fungus used in genetics. Sordaria fimicola, a fungus used in

genetics-- Tom Volk's Fungus of the Month for March 2007,

botit.botany.wisc.edu/toms_fungi/mar2007.html. Accessed 30 Apr. 2017.