Sordaria Genetics Lab

Anthony Rivetti

Miss Williams

Honors Biology

1 May 2017
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Introduction

The fungi species Sordaria fimicola was used in this lab, and two of its genes in

particular were observed. This species of fungus is part of the ascomycete phylum, characterized

by the carrying of spores in a sac, called an ascus (Ascomycete). The Sordaria genus falls

within this phylum and contains species that live in animal feces and produce fruiting bodies

called perithecia (Sordaria). Sordaria fimicola is one of the most common species of this genus

and produces eight ascospores within an ascus when it reproduces. These ascospores are

arranged in a specific pattern within the asci, reflecting the events that took place in meiotic cell

division (Meiosis). Therefore, it can be determined whether or not crossing over has occurred

by observing the patterns within each asci.

Sordaria fimicola cells are haploid for the majority of their lives, and they reproduce

sexually with each other. The fungus is found mostly in animal waste, particularly the feces of

herbivores (Sordaria Genetics). Sordaria benefits the ecosystem by breaking down this waste,

which allows nutrients to enter the soil and improve plant growth. This species begins its life

cycle as a haploid ascospore. Once an ascospore lands, it begins to germinate and branch off,

forming mycelia. For each mating type, a sac will form on the mycelium. The positive mating

type forms an ascogonium and the sac from the negative mating type is called an antheridium.

These sacs are filled with haploid nuclei, and during plasmogamy, the nuclei of both mating

types move into one sac. Then, a fruiting body called a perithecium forms, at the top of which are

multiple asci. Each asci is dikaryotic, containing two haploid nuclei, until karyogamy takes

place. Here, the nuclei fuse together, forming one diploid zygote. This zygote then undergoes

meiotic cell division, producing four different nuclei, and this is followed by mitotic cell
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division. The result is an ascus filled with eight haploid nuclei, each of which forms a cell

membrane and becomes an ascospore.

The end result of the life cycle of Sordaria allows for the locating of genes that code for

particular traits. This is because ascospores align in a linear way in an order that reflects the

events that occurred during meiosis (Meiosis). One trait for which the genes can be located

rather easily is color, as this is a simple characteristic to observe. In Sordaria fimicola, two genes

produce the color of ascospores, and each of these contains two alleles. These are the t and g

genes, and each contains a wild type allele (t+/g+) and a mutant type (t/g). The combination of

these two genes will determine the color of the ascospores. The g+ and t+ alleles combine to

produce black fungus, called the wild type. The mutant types form when t and g+ cross, which

forms tan, or t+ and g combine, producing gray ascospores. When both mutant alleles are

present, the ascospore appears clear.

In this lab, the tan fungus, consisting of the t and g+ alleles, and gray fungus, containing

the t+ and g alleles, were each crossed with black, or wild type, fungus (t+ and g+). These

respective tests enabled the location of the t gene and g gene to be found. Crossing tan with black

helps to find the location of the t gene because the g+ allele is constant in both fungi. Therefore,

crossing over only impacts the t gene. The same goes for the gray fungus test and the g gene,

with t+ remaining constant here. Crossing over can be seen when observing the ascospores

following meiosis and mitosis. By looking at the spore patterns, a process called tetrad analysis,

it can be determined whether or not crossing over has occurred (Thompson). If it has not, the

spores will be in a 4:4 ratio. This is because the alleles never switched places, thus they were

never separated from those of the same kind. If crossing over has occurred, the ascospores will

be in a 2:4:2 or 2:2:2:2 ratio. This is because the alleles on one chromosome switched places
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with the alleles on the homologous pair. This separated them in prophase I of meiosis, therefore

when the chromosomes and chromatids were separated in anaphase I and II, the alleles remained

separate. This analysis of chromosomes helps because it can be determined which chromatids

saw crossing over, and because the rate of crossing over can be found, which allows the distance

from the centromere to be calculated (Thompson).

The independent variable in this lab is the color of fungus, either tan or gray, that was

crossed with the black fungus. The dependent variable was the rate of crossing over for each

gene. The tan fungus test enables the rate of crossing over for the t gene to be found, and the test

with gray fungus allows the rate for the g gene to be found. These rates of crossing over can be

used to determine the distance from the center of the chromosome. A greater rate of crossing

over indicates a greater distance from the centromere. The purpose of this lab is observe asci to

determine if crossing over has occurred and the rate of crossing over for each gene. This

information can be used to find the distance of each gene from the center of its chromosome.

Materials (Sordaria Genetics)

- 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

- 20 sterile petri dishes

- Microscope
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- 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

Procedures (Sordaria Genetics)

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.

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.
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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.

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 (22-25 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.
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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.

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.

(tn) x (+)

(g) x (+)
g
or

or
g

Figure 1: Cross plate of Sordaria indicating locations of inoculation blocks and the most probable locations (arrows) of hybrid
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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 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 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 (Figure 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.

6. After locating a rosette of hybrid asci, use high

Figureto2:observe
power Squashed
the perithecium of Sordaria
ascospores and determineexposing
if
rosette of hybrid asci (Sordaria Genetics).
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crossing-over has occurred. If crossing-over has not occurred, segregation of the genes for spore

color has taken place during Meiosis I and the ascospores will be arranged in a 4:4 ratio (Figure

3). If crossing over has occurred, segregation of the genes for spore color do not segregate until

Meiosis II and the arrangement of ascospores will be either 2:4:2 or 2:2:2:2 (Figure 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.

Figure 3: Production of Meiosis I asci where no crossing over has occurred (Sordaria Genetics).

Results Figure 4: Production of Meiosis II asci where crossing over has occurred (Sordaria Genetics).
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Number of Number of Meiosis Percent of Distance

Stains Meiosis I II Asci/Crossing Total Meiosis II Asci from
Crossed Asci/No Crossing Over Occurred Asci (crossing over Centromere
Over (4:4) (2:4:2 or 2:2:2:2) occurred) in Map Units
(g) x (+) 82 141 223 63% 31.5
(tn) x (+) 91 147 238 62% 31
Table 1: Shows the number of asci observed and the rates of crossing over for the gray/black

cross and tan/black cross. The distance from the center of the chromosome is also shown.

The first row of data shows the results when the gray fungus was crossed with the wild,

or black, fungus. In this test, crossing over did not occur in 82 asci and did occur in 141. This

displays a crossing over rate of 63% for the g gene, which can be used to find the distance from

the center of the chromosome 31.5 map units. When the tan was crossed with the wild type, 91

asci did not undergo crossing over, while 147 asci did. This shows that the t gene has a 62%

crossing over rate, and is therefore 31 map units from the centromere.

Discussion

The results of this experiment show that the t and g gene sit at a similar distance from the

center of the chromosome. The rate of crossing over helped to determine these distances because

a greater rate indicates a greater distance from the center. The distance was found in map units by

dividing the rate of crossing over by two. Based on the results found, the g gene is slightly

farther from the center of the chromosome than the t gene. Because of this, they cross over at a

very similar rate, and therefore would likely cross over at the same time.

The purpose of this lab was to observe the rates of crossing over to find the locations of

two genes on their chromosomes. It can be important for scientists to know the locations of

genes for a number of reasons. Most importantly, this knowledge can enable doctors to locate the
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cause of many disabilities and mutations (Genetic Basics). This can be especially useful for

expecting parents, who are able to discover any genetic disorders their child may have so that

they can prepare to raise that child. Scientists can also use the knowledge of gene locations to

compare species and discover evolutionary relationships between them.

According to the teacher, these results were, for the most part, accurate. However, some

improvements could have been made. Firstly, more asci should have been counted, which would

have allowed for more accurate results. Also, a system that allowed the more precise locating of

the genes could have been helpful. This would enable the determining of whether or not the

genes were on the same chromosome or above/below the centromere. One possible mistake was

improper smashing of the perithecia. This could either not display some of the asci or break open

the asci, both leading to an incorrect number of asci counted. This would have changed the

recorded rate of crossing over and thus the distance found from the centromere.
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Works Cited

"Ascomycete." Dictionary.com. Dictionary.com, n.d. Web. 29 Apr. 2017.

"The Genetic Basics." National Institutes of Health. U.S. Department of Health and Human Services,

n.d. Web. 30 Apr. 2017.

"Meiosis and Recombination in Sordaria Fimicola." Lehigh University. N.d. Web. 29 Apr. 2017.

Sordaria Genetics. Burlington: Carolina Biological Supply, 1999. Print.

"Sordaria." Merriam-Webster. Merriam-Webster, n.d. Web. 29 Apr. 2017.

Thompson, Laura. "Mapping Genes in Sordaria." Furman University. N.p., n.d. Web. 29 Apr. 2017.