Sordaria Genetics

Allison Keeling

Ms. Williams

Honors Biology

1 May 2017
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Introduction

In this lab, the fungus Sordaria was used to test the rate of crossing over in order to locate

the distance of two genes in relation to its centromere. Specifically, Sordaria fimicola was used.

Sordaria fimicola is a type of fungus that can be found growing in rotting vegetation and animal

dung (Meiosis and Recombination). The Sordaria fimicola can be found most of the time in its

haploid form because the only time it is in its diploid form is after two haploid nuclei fuse

together and right before it undergoes meiosis. The Sordaria fimicola can sexually reproduce

with itself, it is not able to reproduce asexually reproduce. Fungus is very important to the

ecosystem because, they are essential to the recycling of nutrients in all terrestrial habitats

because they are the dominant decomposers of the complex components of plant debris, such as

cellulose and lignin (Kendrick). Basically, fungus is used as a decomposers of living things like

plants and animals.

The life cycle of Sordaria fimicola starts with it in its ascospore form, or its haploid form.

It then lands on the ground and begins to germinate and form branches called mycelia. These

mycelia can either by one of the two mating types, which are positive and negative mating types.

Sordaria fimicola do not have normal mating types such as male and female as mammals do,

they have positive and negative. These mycelia can also be referred to as monokaryotic hypha.

These hypha form sacs where, the nucleus divides by mitosis and these nuclei distribute

themselves in the growing hypha (Sordaria Genetics). These sacs are called the Ascogonium,

which is the positive mating type, and the Antheridium, which is the negative mating type. These

sacs go through plasmogany, where the sacs are connected through a long, hairlike hypha, the

trichogyne (Sordaria Genetics). The trichogyne stems from the Ascogonium to Antheridium,

this allows for the positive and negative nuclei to become mixed together in the same sac. The
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haploid nuclei in the sac began to pair off and form dikaryotic cells, meaning the nuclei have not

fused together yet. These cells then make up the branches of the Ascocarp, which is the fungal

fruiting body. For Sordaria fimicola the fruiting body is more specifically called the perithecium

(Class Presentation). Asci them begin to form on the top the perithecium, and karyogamy begins

to take place. This is when the haploid nuclei fuse together to form a diploid nucleus. After the

diploid nuclei are formed they undergo meiotic cell division with the purpose of forming four

different haploid nuclei. The four nuclei will then undergo mitosis to form eight haploid cells,

which are called ascospores. The ascospores are contained inside the perithecium, so when it

bursts open the ascospores fly out. In a real-life situation, the ascospores would fly out and land

on the ground where they would begin to germinate and the whole process would begin again

(Class Presentation).

The two alleles that were being tested in this lab and that determine the color of the

Sordaria fimicola is the t-gene and g-gene. These genes each contain two alleles, which are the

t+ and t, and the g+ and g (Class Presentation). These alleles work in conjunction to determine

the color of the Sordaria fimicola. Black fungi are formed by the g+t+, the gray is formed by the

gt+, the tan is formed by the g+t, and the clear is formed by the gt (Class Presentation). In this

lab, the black or wild fungi was used as a constant, meaning it had mated with the gray and the

tan.

During the lab, the students would have to break the perithecium by smashing it between

the slide and the cover slip using their finger or pencil eraser. The purpose of the perithecium

bursting open was so the students could count which ascospores had participated in crossing over

and which ascospores had remained the same. They would do this by looking at the ascospores

under the microscope. The students could tell this by the way they looked. When the four nuclei
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underwent mitotic cell division and formed eight haploid nuclei inside of an ascus, the ratio of

these cells would determine the color of the fungi. The two combinations were tan and black, and

gray and black, and they could both have the same ratios. They could have the ratio of 2:2:2:2,

2:4:2, or 4:4 (Class Presentation), An example of 2:2:2:2 would be the order of tan, tan, black,

black, tan, tan, black, black, an example of 2:4:2 would be gray, gray, black, black, black, black,

gray, gray, and an example of 4:4 would be black, black, black, black, tan, tan, tan, tan. The

2:2:2:2 and the 2:4:2 ratios show that crossing over had occurred, while the 4:4 ratio shows that

crossing over had not occurred (Class Presentation). The students were told to count ascospores

with these ratios to determine the rate of crossing over. If more of the 2:2:2:2 and the 2:4:2 ratios

were recorded than the 4:4 ratio than it shows higher rates of crossing over.

There were independent and dependent variables in this lab. The dependent variable can

be identified as the location of the genes or the map units away from the centromere because the

purpose of the lab was to find the location of the genes. The independent variables were the gray

and tan because they were each used with the black. The different fungi colors were the

independent because they were manipulated to find the dependent variable. The purpose of the

lab was to determine where the g-gene and t-gene were located on the chromosome and where

their position was in relation to the centromere. This was determined through the rate of crossing

over (Class Presentation).

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
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20 sterile petri dishes

microscopes
glass slides and cover slips
water dropping bottles
inoculating loops
Bunsen burner
*boiling water bath
scalpel or spearpoint needle
disinfectant

Procedure (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 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 r 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 agar have solidified, the dishes may be stored for up to a week at room

temperature or in the refrigerator.

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8 Dispose of the bottles in the autoclavable disposal 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 (22-25C)

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 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

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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.

(Sordaria Genetics)

During Laboratory 2: Microscopic Examination

1. Disinfect all work surfaces. Have students was 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
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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 lower 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.

(Sordaria Genetics)
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

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the genes for spore color has taken place during Meiosis I and the ascospores will be

arranged in a 4:4ratio (Fig. 3). If crossing over has occurred, segregation of the genes for

spore color do not segregation until Meiosis II 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 Table1.
8. Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.

(Sordaria Genetics)

Results

Table 1: Shows the rate of crossing over through dividing the number of crossing over asci by

the total asci. Map units is calculated by dividing the percent by 2.

Strains No. of MI No. of MII Asci Total Asci %MII Map Units
Crossed Asci (4:4) (2:4:2 or 2:2:2:2) (No. MII/Total) %MII/2
(g) X (+) 82 141 223 63% 31.5 units
(tn) X (+) 91 147 238 62% 31 units
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The table displays the results from the lab. The number of the 4:4 and the 2:4:2 and

2:2:2:2 for both the gray and tan were combined results of the whole class. The first row showed

the results of the gray and black asci, and the second row showed the results of the tan and black

asci. The percent was determined by dividing the No. of MII Asci over the Total Asci. For gray

63% of the asci had crossed over and for tan 62% had crossed over. The gray had six less cross

over than the tan, but the tan had also had 15 more total asci than the gray. Then map units were

calculated by dividing the percent of crossing over by 2. For gray the number of map units was

31.5 and for tan the number of map units was 31. The map units of gray and tan were very close

to each other, but gray was .5 farther away from the centromere than the tan. This shows that

gray had experienced a higher rate of crossing over than the tan had.

Discussion

The results show that the gray and black had a higher rate of crossing over because it had

a 63% crossing over rate compared to the tan and black which had a 62% crossing over rate. The

rates of crossing over allowed the student to conclude that the t-gene is located closer to the

centromere because it is 31 map units away, compared to the g-gene which is 31.5 map units

away. The rates of crossing over and its distance from the centromere align with the fact that

genes located farther away from the centromere have a higher chance of crossing over than those

located closer (Ms. Williams). This is shown through the genes because the gray had a higher

percent of crossing over which caused it to be located farther away, while the tan is the same in

that fact it is just located closer. These genes are most likely to cross over together because genes

located close to each other are more likely to cross over together. In this lab, the t-gene and the g-
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gene are only .5 map units away from each other, which seems relatively close. They are also

located a far distance from the centromere, so they are already more likely to cross over.

The location of genes has become very important, maybe even more important than its

function. This is because knowing the relationship between gene location and trait variability

will help researchers understand the genetic basis of diseases and will also help breeders develop

economically useful plants and animals. Moreover, this new research makes it possible to

account for a genetic feature, long touted as evidence for biological evolution, from a creation

model perspective (Rana). The results of this experiment of the crossing over with Sordaria

fimicola, are somewhat accurate. This conclusion was made because the lab instructions state

that extensive research has shown that the gene for tan spore is about 26 map units from the

centromere, while the gene for gray spores is about 60 units (Sordaria Genetics). This shows

that the results were accurate in that the gray genes are located farther from the centromere than

the tan. The results could not be accurate in the amount of crossing over and non-crossing over

that had been recorded. Some students may have over counted or under counted the amount they

were seeing. The results were not consistent with lab instructions in that they were located .5

map units away from each other, while in the instructions they were 34 map units away. Another

source of error could have been that some groups could have collected more data than others

skewing the results.

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Works Cited

Kendrick, Bryce. "Fungi: Ecological Importance and Impact on Humans." Encyclopedia of Life

Sciences. Wiley, Oct. 2011. Web. 30 Apr. 2017.

Meiosis and Recombination in Sordaria Fimicola (n.d.): n. pag. Lehigh.edu. Lehigh.edu. Web.

Rana, Dr. Fazale. "The Functional Significance of Gene Location: Countering the Case for

Biological Evolution." Reasons to Believe. Reasons to Believe, 28 Oct. 2010. Web. 01

May 2017.

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

Volk, Tom. "Sordaria Fimicola, a Fungus Used in Genetics." Sordaria Fimicola, a Fungus Used

in Genetics. Wisconsin University, Mar. 2007. Web. 30 Apr. 2017.