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Introduction: (Carolina.com) The species of Sordoria is focused on in this lab report. The

species of Sordarias habitat of the three species of Sordaria that have been the principal

subjects in genetic studies is dung of herbivorous animals (Volk). The family that Sordaria is

located in is Sordariaceae. Also, sordaria is a genus of microscopic fungi, which is a wide

ranged group of species. Some general information about Sordaria is most of its lifecycle is

haploid, it reproduces sexually, and it helps the ecosystem to demonstrate the results of the

location of the chromosome, to the centromere. The life cycle of the Sordaria begins with the

four to eight ascopores that are located within the asci of the fungus. Ascomycetes are known as

sac fungi because of the characteristic shape of their asci (Davidson). These ascopores are

haploid cells. The ascopores then fall to the ground where they begin to grow. As these fungi the

ascopores grow, they start forming branches that make a multicellular organism made up of

haploid cells, which serves as a type of mating type. There is the positive mating type and the

negative mating type. Both mating types form sacs separate from each other that contain large

amounts of haploid nuclei with the correct mating type. These haploid nuclei came from Mitosis.

The sacs that are formed of the positive mating type are called Ascogonium and the sacs formed

of the negative mating type are called Antheridium. After these sacs are formed, a process occurs

which is called Plasmogamy. In Plasmogamy, the Ascogonium and Antheridium connect and

form a bridge where the haploid nuclei are able to combine together to form one sac. When two

haploid nuclei, one of each mating type, go into an individual cell, these then begin to branch off

creating the Perithecium which is a fruiting body. The branching that makes up the Perithecium

is called Mycelia. The Mycelia, which is the non-sexual part of the fungus. The mycelium will

eventually support the growth of the sexual aspects of the Sordaria fimicola. (Writer) The

branches are made up of dikaryotic hyphae which include two haploid nuclei. At the ends of
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these dikaryotic hyphae, an ascus forms which is another sac. Once the asci form, a process

called Karyogamy occurs in which the two haploid nuclei fuse together and form a diploid

nucleus. The cell then identifies as a diploid cell. The cells then undergo Meiosis, and then

Mitosis after that and by then there are eight haploid nuclei in one ascus.. The two genes that

will determine the ascopores color are the g and the t gene. Their other alleles are g+ and t+.

Certain combinations of these alleles will determine what color the ascopores are. The g and t

alleles combined creates a clear color; the g+ and t+ alleles create black; the g+ and t allele

create tan; the g and t+ allele create gray. To determine if the genes crossed over, the ratio of

colors would have to be 2:2:2:2. or 2:4:2. To determine if the genes didnt cross over, the ratio

would then have to be 4:4. The dependent variable within this experiment is the rates of crossing.

The independent variables are the color and order of the ascopores. A Sordaria fimicola dies

shortly after all of its ascopores have been released (Writer).

Materials:

* Sordaria fimicola, wild type

* Sordaria fimicola, mutant gray

* Sordaria fimicola, mutant tan

* Bottle cornmeal-glucose-yeast agar

* Autoclavable disposal bag

* 3 bottles Sordaria crossing gear

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* 20 sterile petri dishes

* Microscope

* Glass slides and cover slips

* Water dropping bottles

* Inoculating loops

* Bunsen burner

* Boiling water bath

* Scalpel or spearpoint needle

* Disenfectant

Procedures:

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 is melted.
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2. Cool the agar to 45 Degrees Celsius (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 disposable 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.

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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 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 (22-25 degrees

Celsius) 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 date, 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 date.

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 disposal bag.

2. Provide the students with water dropping bottles, glass slides, cover slips, inoculating oops,

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 perethecia and release the

rosettes of asci (Fig. 2). If too much pressure is applied, the

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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.

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 data in Table 1.

8. Chromosome maps for the two mutant genes are constructed by dividing the %MII by 2.
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Table1: This table is describing the number of how far away the genes are from the centromere

of the chromosome. Map Units finds this out, which is calculated by %MII/2. First, you need to

know which strains crossed over and which did not to calculate this.

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

Asci (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

Discussion: The rates of crossing over helped me determine the distance of the genes from the

center of the chromosome by dividing the MMII percentage by 2, to get the map units. These

genes are most likely to be crossed over because they are extremely close to the centromere.

Remember, the closer the genes are to the centromere, the more likely they will cross over. This

relates to the law of segregation by having a copy from both mom and dad with being close to

the centromere. This information is useful because we now know how we get a gene from each

mom and dad. It is important to know the location because you can then determine the certain

type of gene. These results are pretty accurate, but some possible errors are looking at a strain

multiple times, and missing strains under the microscope

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Works Cited:

Davidson, Michael W. Introduction to Optical Microscopy, Digital Imaging, and

Photomicrography. Molecular Expressions Microscopy Primer: Introduction to

Microscopy. N.p., 19 June 1998. Web. 01 May 2017.

"Sordaria Demonstration Cross Plate, Living." Carolina Biological Supply: World-Class

Support for Science & Math. N.p., n.d. Web. 01 May 2017.

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

Genetics-- Tom Volk's Fungus of the Month for March 2007. Thomas J. Volk, University

of Wisconsin-La Crosse., Mar. 2007. Web. 01 May 2017.

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

Web. 01 May 2017.