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Victoria Wilson

Ms. Williams

1 May 2017
Introduction:

The fungus being studied in this lab is called Sordaria. Sordaria is a “genus of chiefly

dun-inhabiting ascomycetous fungi (order Sphaeriales) having scattered hairy-necked perithecia

with dark continuous ascospores” (“Merriam-Webster”). This genus is similar to Neurospora

and Podospora, and fungi belongs to the Sordariaceae family, and its type of fungus is

ascomycete fungus. It is normally found in the feces of herbivores and rotting vegetation. It

helps the ecosystems by helping the scientists studying the ecosystems. Sordaria has been used

as a model when studying the crossing over of chromosomes and their reproductive system. In

this species of fungi, most of its life cycle is haploid. To start off, ascospores are single haploid

cells. The life cycle begins when ascospores fly off the asci and eventually land. When they

land, they begin the process of germination. To germinate means that these ascospores will grow

branches because of mitosis. These branches are called mycelia. Within these branches are two

mating types. One type is called the positive type, and the other is called the negative type. Both

of these types are multicellular and haploid. This species of fungus does not asexually

reproduce. Therefore, if it wants to sexually reproduce, each mating type will form a sac of

haploid nuclei from that same type. The sack from the positive mating type is called the

ascogonium, and the sac from the positive mating type is called the antheridium. After this,

plasmogamy occurs. Plasogamy is when the nuclei from the ascogonium and antheridium

combine into the same sac. Inside this sac, two haploid nuclei from different mating types

combine to create cells. An ascocarp is a fungal fruiting body, and branches continuously form
off it from the two haploid nuclei combing to make cells. For this species, the ascocarp is called

Perithecium. The branches forming off of the Perithecium are diakaryotic. Diakaryotic means

that two haploid nuclei are not fused together. Also, asci will form at the ends of these branches.

The two haploid nuclei at the ends of the branches will be inside of the asci. Each asci contains

one haploid nuclei from each type of mating. The next process that occurs is karyogamy.

Karyogamy is when the two haploid nuclei fuse together to form a diploid nucleus. Now there is

a diploid nucleus in each ascus. This diploid nucleus undergoes Meiosis, which makes each asci

now contain four different haploid nuclei. Each of those four nuclei will then undergo Mitosis,

creating eight haploid nuclei in each ascus. These eight haploid nuclei form cell membranes and

become cells. These cells are the ascospores. With the type of fungus being used in this lab, this

process will take about 17 days. Another part of the fungi that will be observed in this lab are the

possible colors of the fungi. There are four different possible colors; gray, tan, clear, and black.

Black is considered the “wild type”. There are also two genes that the fungi work with, and each

gene has two alleles. The symbols t and t+ are alleles for the first gene, and g and g+ are alleles

for the second gene. Different combinations of the two genes create the four different colors.

The different combinations include: g+t+ create black, gt+ creates gray, g+t creates tan, and gt

creates clear. Only one form of the gene is seen because they are haploid. The purpose of this

lab is to look at the asci to see if crossing occurred for certain genes. By looking at these exact

rates of crossing over, the students can find the exact location and the map distance of the

chromosomes from the centromere. Knowing the combinations of alleles from each gene and

the colors that are expressed, the students will be able to find the distance of the chromosomes

from the centromere by using the color ratios in the asci to determine the rates of crossing over.
The different color ratios that showed that crossing over occurred are 2:2:2:2 and 2:4:2. The

ratio that shows that no crossing over occurred are 4:4. Any other ratios are neutral, or are not

counted. A student determines these ratios by looking at the colors of the eight haploids in each

ascus. For example, if the first two haploids are dark colored, the next four lightly colored, and

the final two dark colored, the ratio is 2:4:2. With this ratio, crossing over has occurred. The

independent variables are the different slides of Sordaria observed under the microscope. The

dependent variables, or what is being measured, are the number of asci counted as crossing over

or not crossing over (“In this Lab the Number of Asci is Being Counted”).

Materials:

Included in 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

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 spear point needle


Disinfectant such as phenol or 70% ethanol

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

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 has melted.

2.) Cool the agar to 45 degrees Celsius (the bottle should feel comfortably hot ot
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 the work of surface and wash your hands.

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
spear point 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 degrees-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
“+/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 spear point 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.

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 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 perothecia
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.

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 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.

7.) Each group should count 100 to 200 asci. Collate class data.

8.) Chromosomes maps for the two mutant genes are constructed by dividing the
% MII by 2.

Results:

Data

Strains No. of MI No. of MII Total Asci %MII (No. Map Units
Crossed Asci (4:4) Asci (2:4:2 or MII/Total) %MII/2
2:2:2:2)
(g) x (+) 82 141 223 63% 31.50%
(tn) x (+) 91 147 238 62% 31%
With the gray and black Sordaria, 223 asci total showed that crossing over did or did not
occur. A total of 82 asci showed that crossing over did not occur, and the other 141 asci showed
that crossing over did occur. The exact map distance of these genes from the centromere is
31.50%. With the tan and black Sordaria, 238 asci total showed that crossing over did or did not
occur. A total of 91 asci showed that crossing over did not occur, and the other 147 asci showed
that crossing over did occur. The exact map distance of these genes from the centromere is 31%.

Discussion:

The rates of crossing over helped determine the distance of the genes from the center of
the chromosome because in order to find the units in map distance of the genes from the
centromere, the number of asci that showed that crossing over did occur was needed. The
distance in map units was found by dividing the percent of MII by two. A map unit is “an
arbitrary unit of measure to express the distance between genes on a chromosome. It is
calculated from the percentage of recombinations that occur between specific genes so that 1%
of crossing over represents one unit on a genetic map, or approximately the number of new
combinations that can be detected. The measurement is accurate only for small distances
because double crossovers do not appear as new recombinations” (“The Free Dictionary”).
Based on the definition, these genes most likely will not be crossed over together because the
higher the percentage, the farther apart the genes are. The greater the distance in between the
genes, the greater the possibility of crossing over occurring. In this lab, the results were 31.50%
and 31%, which are low percentages and therefore will most likely not be crossed over together.
Generally, this information is useful in determining the color of the fungi. Knowing the location
of these genes are also important because even though crossing over could occur, it will most
likely not happen if the genes are close together, so this information also helps determine the
color of the fungi more accurately. These results are not very accurate. Sources of error could
have occurred while looking at the Sordaria underneath the microscopes. The group members
may have pressed on the cover slips too hard or too lightly, agar could have been in the fungi by
accident, or students could have miscounted when gathering their data. All of these errors
directly affect the results of the lab.
Works Cited:

Akshay24. "In This Lab the Number of Asci Is Being Counted the." In This Lab the Number of
Asci Is

Being Counted The Asci Have Spores That. N.p., n.d. Web. 28 Apr. 2017.

"Map Unit." The Free Dictionary. Farlex, n.d. Web. 28 Apr. 2017.

"Sordaria." Merriam-Webster. Merriam-Webster, n.d. Web. 28 Apr. 2017.

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