Sordaria Genetics

Molly Thomas

Miss Williams

Honors Biology

1 May 2017
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Introduction

The fungus that was studied in this lab was Sordaria fimicola. It is a part of the

Sordariomycetes family. Commonly it is used for genetic observations. It can be found in the

dung of herbivores and rotting vegetation most places in the world. Most of its life is spent as a

haploid organism and reproduces sexually. Its life cycle begins with an ascospore, which is a

Sordaria haploid cell. It gets blown by the wind and falls to the ground. There it germinates and

undergoes mitosis which creates identical haploid cells, as it grows into multicellular haploid

organism it forms branches. It has 2 mating types which are positive and negative. Each mating

type forms sack full of haploid nuclei that got there through mitosis. Sacks from the positive type

are called Ascogoniu. Sacks from the negative type are called Antheridium. Plasmogamy occurs,

which is when the two sacks connect forming a bridge allowing haploid nuclei from the two

mating types to mix all together in one sack. Then two nuclei from different mating types come

together in one cell, begin to branch off cell by cell. Cytokinesis occurs forming branches, these

cells are called dikaryote cells which contain to haploid nuclei that are not fused together.

Ascocarp is a fruiting body of a fungus and for this species of fungus it is called the Perithecium.

It is made up of all the branches, hyphae, put together. At the end of the branches is another sac

that forms called an ascus which is still dykatyotic. Karyogamy is when the two haploid nuclei

come together to form a diploid nucleus making the cell diploid. Meiosis occurs in the diploid

cell and by the end of Meiosis, there are four haploid nuclei. Mitosis occurs for each of the four

haploid nuclei and end up with eight haploid nuceli in one ascus. Each becomes its own haploid

cell inside of accus which are called ascospores, they break out and the cycle repeats.

There are two genes that determine the ascospore color. The natural spore color is black.

They are the t and g gene. There are four different color possibilities which are tan, grey, clear,
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and black, which is the wild type. Since the fungus is normally in the haploid for there is no

recessive or dominance. So, g+t+ produces black spores, gt+ produces gray spores, g+t produces

tan spores, and gt produces clear spores. After crossing over if the ratio is 4:4 then no crossing

over occurred. But if the ration is 2:4:2 or 2:2:2:2 then crossing over did occur. If the ratio of

crossing over to not crossing will show the rate of crossing over. The independent variable was

the color of the spores and the dependent variable was the number of ascospores that did or did

not cross over. The purpose of the lab is to use the ratio of asci that crossed over to determine

how far away the genes are from the center of the chromosome. Genes that are close together

often cross over together and by finding how far away the genes are we can see if they might

cross over together, by mapping out the location of the two genes.

Materials (Sordaria Lab Packet.)

The materials in this kit are sufficient for 14 groups of students. The materials are supplied for

use with the exercise in this kit only. Carolina Biological Supply Company disclaims all

responsibility for any other uses of the materials.

Included in the kit

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

Needed but not supplied

microscopes

glass slides and cover slips

water dropping bottles

inoculating loops

Bunsen burner

*boiling water bath

scalpel or spearpoint needle

disinfectant such as phenol or 70% ethanol

*If a water bath is not available, a container of boiling water may be substituted.

Procedures (Sordaria Lab Packet.)

Laboratory Preparation

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
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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 disposal bag.

Preparation of Stock Cultures

1. Disinfect the work surface and wash your hands.

2. When ready for use, label two of the conrmeal-glucose-yeast agar dishes wild, two

gray and two tan.

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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 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 +/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.

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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

1. Disinfect all work surfaces. Have the students wash their hands. Point out the location of

the autoclavable disposable 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 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 (Fig. 2). If too much pressure is applied, the ascospores will be forced
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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 date in Table 1.

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

Table 1: Shows the No. MII Asci that did not cross over, the No. MII Asci that did cross over,

the total Asci, the percent that crossed over, and the map units away from the center of the

chromosome for grey and tan.

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

Table 1 represents the data gathered from the lab. In the lab 82 grey asci did not cross

over and 141 grey asci did cross over. The total number of grey asci found was 223. To find the

percent that crossed over the number of asci that crossed over was divided by the total number of

asci. By doing this the percent of grey asci that crossed over was 63%. That perecent was used to

find the distance from the center of the chromosome in map units. It was 31.5 map units away.

The same thing was done with the tan asci and 91 asci did not cross over while 147 did cross

over. Out of the total number of 238, 62% did cross over. Meaning that it was 31 map units from

the center of the chromosome.

Discussion

The results from the lab showed the rate of crossing over. A gene is more likely to cross

over the farther it is away from the center of the chromosome. Since we found out how likely a

the genes were to cross over it can be used to found how far away in map units the gene is from

the center of the chromosome. The grey were 31.5 map units away and the tan were 31 map units

away. Genes that are close together often cross over together. So it would be very likely for them
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to both cross over together if they are on the same ends of the chromosome. If grey is 31.5 map

units up and tan as 31 map units down they would be very far away from each other and would

not be likely to cross over. If they are on the same side then yes it could be likely that they would

cross over together. Finding out where genes are and how they cross over can be helpful to

know. It could be helpful in identifying how mutations occur and how genes are passed down. If

you know how the genes are crossed you can use that information to control it to get the more

desirable traits. The results from the lab were not necessarily the most accurate. There was not

enough data for it to be reliable. More data would be required to get more accurate results about

500 per category.

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Works Cited

Revolvy, LLC. ""Sordaria+fimicola" on Revolvy.com." All Revolvy Quizzes. N.p., n.d. Web. 30

Apr. 2017.

Sordaria Lab Packet. 1999. Carolina Biological Supply Company: USA. The Law of

Segregation (article). Khan Academy. N.p., n.d. Web. 30 Apr. 2017.

Volk, Tom. Sordaria Fimicola, a Fungus Used in Genetics Tome Volks Fungus of the Month

For March 2007. N.p., n.d. Web. 30 Apr. 2017.