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Sordaria Genetics

Trevor Mendola

Ms. Williams

Honors Biology

30 April 2017
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Introduction:

Sordaria fimicola is a species of fungi that is a member of the phylum Ascomycota and

the family Sordariaceae (Volk). It spends most of its life cycle as a haploid, but does spend small

portions of its life span as a diploid and a dikaryote (MEIOSIS AND RECOMBINATION IN

SORDARIA FIMICOLA). Sordaria fimicola is most often found growing in animal feces or

rotting vegetation (MEIOSIS AND RECOMBINATION IN SORDARIA FIMICOLA). The

life cycle of this fungus begins as a haploid cell, called an ascospore. These ascospores germinate

and form long branch-like structures called hyphae. Sordaria fimicola have mating types,

positive and negative. Hyphae with positive mating types form sacs called ascogonia, which

have many nuclei. Hyphae with negative mating types form sacs called antheridia, which also

have many nuclei. Through a process called plasmogamy, these sacs form a branch, known as a

trichogyne, connecting them together and creating a dikaryotic cell. These hyphae all together

form an ascocarp. At the tips of each hyphae are sacs dikaryotic sacs called asci. Through a

process called karyogamy, these nuclei combine, forming a diploid zygote cell. Meiosis then

occurs, creating four individual haploid nuclei. Those nuclei then undergo mitosis resulting in a

total of eight nuclei, also known as ascospores. The ascocarps release these ascospores and the

life cycle starts over again (MEIOSIS AND RECOMBINATION IN SORDARIA

FIMICOLA). These ascospores can have different colors based on which forms of genes are

present in the DNA of the cell. These genes are the tn gene, which has the forms tn and tn+, and

the g gene, which has the forms g and g+. An ascospore with a tn allele and a g allele will be

clear. An ascospore with a tn+ allele and a g allele will be gray. An ascospore with a tn allele and

a g+ allele will be tan. An ascospore with a tn+ allele and a g+ allele will be black. When a

wild-type (black) and mutant-tan (or mutant-gray) strain mate, one gene (either tn or g) will be
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constant, allowing you to see if the other gene crossed over. Because of this, you can calculate

approximately how far away a certain gene is from the certain of the chromosome (See Figure 3

and 4 in the Procedures section). The independent variable for the experiment was the ascospore color

type (mutant-tan or mutant-gray) and the dependent variable was the rate of crossing over for each

ascospore color type. The purpose of this lab is to learn about genetics and observe rates of

crossing over. Sordaria fimicola is a good test subject because of how it reproduces and how

observing ascospore color makes it very easy to tell if crossing over occurred (Volk).

Materials (Carolina Biological Supply Company):

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
boiling water bath (if a water bath is not available, a container of boiling water may be

substituted)
scalpel or spearpoint needle
disinfectant such as phenol or 70% ethanol

Procedures (Carolina Biological Supply Company):

Laboratory Preparation

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 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 bottles 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 a refrigerator.
8. Dispose of the bottles in the autoclavable disposal bag.

Preparation of the 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

cornmea-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.
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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 surface. 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 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 incubation until the 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.

Figure 1 (Carolina Biological Supply Company):

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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

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 perithecia 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 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 of with hybrid asci containing spores of

two different colors.

Figure 2 (Carolina Biological Supply Company):
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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 the 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.

Figure 3 (Carolina Biological Supply Company):

Figure 4 (Carolina Biological Supply Company):

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Results:
Table 1:
Strains No. of MI No. of MII Total %MII (No. Map Units
Crossed Asci (4:4) Asci (2:4:2 or Asci MII/Total) (%MII/2)
2:2:2:2)
(g) x (+) 82 141 223 63% 31.5
(tn) x (+) 91 147 238 62% 31

The results show how often the tn gene or the g gene crossed over in the hybrid asci.

For the hybrids of the mutant-gray and wild-type, 223 asci were observed. No crossing over was

found in 82 asci, while crossing over was found in 141 asci, meaning 63% of the asci showed the

g gene crossing over. With this information, you can also estimate about how far the g gene is

from the center of the chromosome. It was determined that the g gene was about 31.5 map units

away from the center of the chromosomes. In the case of mutant-tan and wild-type hybrids, 238

asci were observed. A 4:4 ratio of asci was found in 91 asci, while a 2:4:2 or 2:2:2:2 ratio was

found in 147 asci, meaning 62% of the asci showed the tn gene crossing over. It was calculated

that the tn gene was about 31 map units away from the center of the chromosomes.

Discussion:

These results can be interpreted to take an educated guess as to how far away the genes

are from the chromosome. Higher rates of crossing over likely point to the gene being further
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from the center of the chromosome and lower rates point to the gene being closer to the center of

the chromosome. With data from our experiments, we can assume the gene was further away

from the center of the chromosome. This data is not completely accurate however, many

participants used a bit too much force trying to break the fruiting bodies, resulting in the asci

bursting and releasing ascospores. Also, not enough data was collected, which of course means

that the data that was collected is not perfect. In further trials of this lab, more data should be

collected to have true results.

Works Cited

Sordaria Genetics. 1999. Carolina Biological Supply Company: USA.

MEIOSIS AND RECOMBINATION IN SORDARIA FIMICOLA. Bethlehem, Pennsylvania:

Lehigh University, n.d. PDF.

Volk, Tom. "Sordaria Fimicola, a Fungus Used in Genetics." University of Wisconsin - Botany.

N.p., Mar. 2007. Web. <http://botit.botany.wisc.edu/toms_fungi/mar2007.html>.