Sordaria Genetics

Dominique Smaldino

Miss Williams

Honors Biology 10

1 May 2017
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Introduction:

Sordaria is a genus of chiefly dung-inhabiting ascomycetous fungi (order Sphaeriales)

having scattered hairy-necked perithecia with dark continuous ascospores (Merriam-Webster,

Sordaria). The type of sordaria that will be used to complete this lab is called sordaria fimicola.

It is a species of dung ascomycete that is very common. It produces eight ascospores within one

ascus. The ascospores are created by a process called meiosis. This particular species of sordaria

produces sexually all of the time and it is haploid for most of its life cycle. You can usually find

this type of sordaria [growing] on decaying organic material (Fungus Fruiting Bodies).

Now, the life cycle of sordaria starts with an ascospore that goes through a process called

germination, which is just a process of something coming to life. After this, there are two

branches that have been formed that are called the mycelia, which are haploid. There are two

different mycelia, both of which are multicellular mating types. One is filled with positive nuclei,

and the other with negative nuclei. If the sordaria wants to sexually reproduce, then each mating

type will form a sack, one is positive and one is negative. The positive one creates what is called

Ascogonium and the negative one creates Antheridium. From there, plasmogamy, which is the

fusion of the protoplasts of cells (dictionary.com), takes place and the two sacks that were

created bond together, and all of the nuclei moves to one sack. The ascocarp then starts to form.

An ascocarp is a fungal fruiting body. The ascocarp is made up of a bunch of branches and the

branches are made up of cells which contain two haploid nuclei, which means it is dikaryotic.

The specific type of ascocarp that is formed here is called perithecium. The ends of the branches

will create asci, which are dikaryotic sacks. In each of these asci, there are two haploid nuclei,

one from each mating type. Karyogamy then occurs, which fuses the two haploid nuclei together

to create a diploid nucleus. The diploid nucleus then undergoes meiosis to form four haploid
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nuclei just in one ascus. Then, the four new haploid nuclei undergo mitosis to create eight

haploid nuclei in one ascus. The nuclei start to form membranes and become individual, haploid

ascospores and the entire process starts again.

For this lab, we are looking at two genes of the sordaria. The two genes are the t gene and

the g gene, both of which have two alleles. The two alleles for the first gene are t + (wild type)

and t (mutant). The two alleles for the second gene are g + (wild type) and g (mutant). When

crossing over occurs, you can create different colors. G + and t + produces black, g and t plus

produces gray, g + and t produces tan, and g and t produces clear. The purpose of this lab is to

look at two genes, determine their color and their ratio, and then because of their arrangement in

the ascus, we will be able to calculate the rate of crossing over. From there, we will be able to

find what the distance from the gene to the centromere is. When crossing over occurs, we should

be able to see it under the microscope. There should be a ratio of colors in the asci. If there is

either a 2:2:2:2 or a 2:4:2 ratio that is seen under the microscope, then we know that crossing

over has occurred, because there are more than just one type of gene in one ascus. Lastly, the

dependent variable in this lab would be the ratio of the ascospore colors. The independent

variable would be the mating crosses. The ratio of the ascospore colors depends on the mating

crosses because the crosses were manipulated to show us the ratio of the colors in the ascospore.

Materials (Sordaria Genetics Lab):

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

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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

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 Genetics Lab):

Laboratory Preparation
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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 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

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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 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.
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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 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 2: (Sordaria Genetics Lab)

Cross plate of Sordaria indicating locations of inoculation blocks and the most probable
locations (arrows) of hybrid asci. Note that the locations are different for the different
ascospore mutants (modified from Koch, 1973).
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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 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

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.

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Figure 2: (Sordaria Genetics Lab)

Squashed perithecium of Sordaria exposing rosette of hybrid asci.

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.

Figure 3: (Sordaria Genetics Lab)

Production of MI (Meiosis I) asci where no crossing over has occurred.
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Figure 4: (Sordaria Genetics Lab)

Production of MII (Meiosis II) asci where crossing over has occurred.

Results:

Table 1: (This shows the type of strains crossed, the number of asci that went through Meiosis I
and Meiosis II, the total asci, the % of asci that went through Meiosis II, and the map units).
No. of MII
Strains No. of MI Asci %MII (No. Map Units
Asci (2:4:2 or Total Asci
Crossed (4:4) MII/Total) (%MII/2)
2:2:2:2)

(g)
82 141 223 63% 31.5
(+)

(tn)
91 147 238 62% 31
(+)

The table above is showing the results that we got from this lab. There were 82 asci that

were not crossed over in the grey strain. There were 141 asci that did not go through crossing

over in the grey strain. Also in the grey strain, there were 223 total asci, 63% of which were

crossed over, and the map unit was 31.5. For the tan strain, there were 91 asci that did not cross
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over, 147 asci that did cross over, 238 total asci, 62% of which were crossed over, and there were

31 map units.

Discussion:

The results we found were able to help us determine the distance of the genes from the

center of the chromosome. We had many more asci that were crossed over. Our results showed

that for the grey strain, 63% of the asci were crossed over, and for the tan strain, 62% of them

were crossed over. We know that when a gene is further from the center of the chromosome, it is

more likely to undergo crossing over. And since we had more that crossed over, we can infer that

they were most likely far from the center. Next, if the genes are closer together, than they are

more likely to be crossed over. Since both of the genes map units, or distance from the center, are

extremely close (31 and 31.5), then we can conclude that these genes are most likely to be

crossed over. Although, they could be on completely different chromosomes, there is no way for

us to surely know that though. There are actually many reasons behind why knowing this

information could be useful. In many well-studied genetic systems, including humans, an

accurate genetic location makes it feasible to proceed with a molecular biological analysis of the

gene and to eventually learn what protein the gene encodes. Also, knowledge of gene location is

useful in constructing special strains for specific experimental purposes (Linkage Maps). So,

knowing the location of the genes can help scientists in their studies with genes, along with many

other things. Our results were not very accurate. The gene for the tan spore is about 26 map

units from the centromere, while the gene for grey spores is about 60 map units (Sordaria Lab

Packet). So, although our results for the tan spore were a bit better than the grey spore, they still

were not very accurate. There could have been many sources of error. They may not have been
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cultured for long enough. Also, when we had to break the asci apart to see it under the

microscope, we could have broken it too much so we could not see the genes clearly, or we could

have not broken it enough.

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Works Cited:

Griffiths, Anthony JF. "Linkage Maps." Modern Genetic Analysis. U.S. National Library of

Medicine, 01 Jan. 1999. Web. 30 Apr. 2017.

"Plasmogamy." Dictionary.com. Dictionary.com, n.d. Web. 24 Apr. 2017.

"Sordaria Fimicola." Molecular Expressions Microscopy Primer: Specialized Microscopy

Techniques - Differential Interference Contrast Image Gallery - Fungus (Sordaria

Fimicola) Fruiting Bodies. N.p., 22 Apr. 2003. Web. 24 Apr. 2017.

Sordaria Lab Packet. 1999. Carolina Biological Supply Company: USA.

"Sordaria." Merriam-Webster. Merriam-Webster, n.d. Web. 24 Apr. 2017.