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Introduction:

Sordaria Fimicola is an ascomycete fungus, which is the largest phylum of fungus. The

genus is Sordaria, and it belongs to the Sordariaceae family. Sordaria is a haploid organism and

is in a vegetative state for most of its life cycle. Sordaria Fimicola starts as a single cell

organism, and within a day enters mitosis. Mitosis is a type of cell division in which the end

result is two identical cells. After mitosis, there are two identical haploid cells. As cells duplicate,

the non-sexual parts of the fungus, or the mycelium, start to form. The purpose of this mycelium

is to support the growth of the sexual portions of the fungus. The mycelium of two individuals

then meet, forming a diploid zygote. This diploid zygote goes through meiosis. Meiosis is a type

of cell division which results in the creation of four daughter sex cells, or spores. The diploid

zygote undergoes meiosis two times, producing eight ascospores, in linear arrangement within

an ascus, (Carolina Biological Supply Company). Ascospores are the product of the meiosis,

and asci are bag shaped structures that contain these ascospores. Ascospores are also the original

unicellular form of Sordaria Fimicola. Once this fungus is fully developed, it releases its

ascospores and dies shortly after. Sordaria Fimicola produces sexually. (Sciencing) There are two

different alleles that determine ascospore color, the common wild-type has black ascospores,

but strains with red, pink, grey, and tan ascospores also exist, (Meiosis and Recombination in

Sordaria Fimicola). For this lab, we used the wild-type allele, which has black ascospores, and

the grey and tan strands, which come from the mutant allele. Color ratios in the asci are what

determine rates of crossing over. Crossing over is the exchange of genes in homologous

chromosomes. A 4:4 ratio means that there are 4 black spores in a row,
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then 4 tan or grey spores in a row. If ascospores are found in this arrangement that means that

crossing over did not occur. If ascospores are found in the 2:2:2:2 arrangement or the 2:4:2

arrangement, then crossing over did occur. The 2:2:2:2 arrangement means that there are 2 black

spores, then 2 grey or black spores, then 2 black spores, then two grey or black spores in a row.

The 2:4:2 arrangement means that spores are found with 2 black spores, 4 tan or grey spores,

then 2 more black spores. (Class Power Point) The dependent variable of this lab is the crossed

over or non-crossed over Sordaria. The independent variable is the Sordaria that we started off

with. The purpose of this lab is to, determine if crossing over has occurred and to calculate the

gene to centromere distance, (Carolina Biological Supply Company).

Materials (Carolina Biological Supply Company).

Sordaria Fimicola, wild type

Sordaria Fimicola, mutant grey

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 burners

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Boiling water baths

Scalpel or spearpoint needle

Disinfectant such as phenol or 70% ethanol

Procedures (Carolina Biological Supply Company)

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 45 degrees C (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.
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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 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 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 grey and two tan stock culture dishes.

5 Incubate the dishes for 5 to 7 days out of direct sunlight at room temperature (22-25

degrees C) 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 the Sordaria crossing agar dishes "+/g" and the other half "+/tn" to indicate

crosses between the wild-type and mutant-grey (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 grey (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 with contain two blocks of the wild-type culture and two

blocks of either tan or grey 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 grey 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 students wash their hands. Point out the location of the

autoclavable disposal bag.

loops, and microscopes. 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 grey and tan

hybrid asci. Instruct the students to mentally note the position on the dish from which they
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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.

3 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.

4 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 grey

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.

5 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. 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.

6 Each group should count 100 to 200 asci. Collate class data in Table 1.

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

Results

Table #1: This table shows rates of crossing over between the tan and grey strains with the wild-

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

Asci (4:4) (2:4:2 or 2:2:2:2)

The two different kinds of strains crossed with the wild-type were grey and tan. For the

grey crossed by wild-type, there were 82 asci found that had no crossing over. There were 141

asci that did have crossing over and were found in either the 2:2:2:2 or 2:4:2 arrangements. Total,

there were 141 asci counted for the grey crossed with wild-type, and 63% of the asci found had

crossed over. The asci found were 31.5% away from the center of the chromosome. For the tan

crossed by wild-type, 91 asci were found that had no crossing over. 147 asci were found in the

2:2:2:2 or 2:4:2 arrangements, or with crossing over. We counted 238 total asci, and 62% of the

asci crossed over. The asci were 31% away from the centromere.

Discussion

Rates of crossing over help to determine the distance of the genes from the center of the

chromosomes. This is determined by loci, or chromosome positions. If loci are found linked

together, that means that they are on the same chromosome. The closer the loci are to each other,

the more likely they are to stay together during crossing over. The genes that we tested are

slightly more likely to be found crossing over than not crossing over because of the location of

the loci. The information that we gathered is useful because it helped us to understand the

location of these genes in comparison to the chromosomes. Knowing the location of genes is

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important because it plays a large role in how organisms traits evolve. It also gives scientists and

doctors a better understanding of chromosomal mutations and genetics. Our main source of error

in this lab was an inconsistency with finding rates of crossing over in the petri dishes. There were

a lot of petri dishes containing ascospores that showed no signs of crossing over. This made

microscopic examination frustrating and more difficult.

Thompson, Laura. "Sordaria." 30sorda. N.p., n.d. Web. 30 Apr. 2017.

Writer, Leaf Group. "Life Cycle of Sordaria Fimicola." Sciencing. Leaf Group, 24 Apr. 2017.

Web. 30 Apr. 2017.

"Welcome to Carolina Biological Supply." Carolina Biological Supply: World-Class Support for

Science & Math. N.p., n.d. Web. 30 Apr. 2017.

Katie Santamaria

Miss Williams

Honors Biology

29 April 2017