SSo
Fall
Investigation of the Crossing Frequencies Between Brown
and Black Spore Sordaria Fimicola
Dominic A. Galanti
Partners: Anurag Garikipati, Kayla Conway, and Troy MeriglianoPennsylvania State
UniversityBIOL 110HTA: Mark Goldy-BrownDue: 11/4/13
Sordaria Lab Report
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Introduction
Through research in evolution canyon it was learned through
experimentation that environmental factors can have and effect on the amount of
crossing over found in the inhabiting species. In order to learn more about the
process of crossing over in genetics more research was done and an experiment was
developed. For an efficient, economical experiment it was critical to find a species
which exhibits prime qualities for experimentation in the lab setting. These ideal
qualities of rapid generation time, small containment costs, ease of isolation, and
obvious trait exhibition were the deciding factors for the species to be chosen in the
experiment. The species which was analyzed through experimentation was Sordaria
fimicola, a fungi species which sporulated to produce different colored spores
depending on the strain. This species had been researched as well in the evolution
canyon, making it an ideal candidate for comparison. With an additional experiment
in the field the results from this experiment would help to decipher the relations of
crossing over and environmental factors, since this experiment is in a lab setting.
The importance of performing this experiment in a lab setting is that the variables
become limited whereas in an external environment factors such as temperature
and precipitation could influence the results of the experiment. By controlling the
variables to only the crossing over, this experiment will provide the genetic
mapping information for the spore color gene. Furthermore a laboratory trial
provides a baseline for examination and comparison to future or well documented
experiments. Through research in the lab procedure and Evolution Canyon it is
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known that certain spore combinations can be found in the asci of Sordaria. These
spore combinations are the result of different strains reproducing and crossing over
occurring in the meiosis phase.
In developing the experiment the lab procedure provided leading questions
to help formulate a proper trial. “What challenges arise in using the provided
procedure for mating different strains of Sordaria? What challenges arise in
preparing squashes of perithecia for scoring asci using the provided procedure? And
what evidence demonstrates that crossing‐over occurred between the spore color
gene and the centromere” were among the questions asked. The experiment
involved culturing two strains of sordaria fimicola and analyzing the spores
produced where they mate for crossing over. In doing this it was discovered that the
frequency of the two crossing over patterns, type B (2:4:2) and type C (2:2:2:2), was
31.2% and 30.0% respectively. Thus, this resulted in a total recombination of 61.2%
between the two sordaria fimicola strains.
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Methods
After the research aspect of this experiment a trial was setup. Petri dishes were
constructed from a nutrient agar agar solution and sterilized. These dishes were
then divided into four quadrants by a permanent marker on the underside of the
dish. This would help to identify the growth and crossing over of the two strains.
Once the dishes were divided into sectors, four, one centimeter squares of sordaria,
media were extracted from two master petri dishes, two from each dish, with a
sterile scalpel. One dish held a tan spore sordaria strain and the other a black/dark
spore sordaria strain. The extracted media was placed into the quadrants with each
square to it’s own section. Once sealed to prevent contamination the petri dishes
were incubated for two weeks to allow for growth and crossing over to occur.
Once incubated the dishes were examined for contaminants and those
without contaminants were analyzed. To analyze each dish a sterile inoculating loop
was used to transfer spores to glass slides in order to make squashes. With a light
pressure to the cover slip the perithecia burst allowing for observation of the asci
under a microscope. Through microscope observations the frequencies of each
pattern type were counted and recorded. By using this data the map distance and
number of recombinants could be determined. To determine these factors the
number of type B (2:4:2) and type C (2:2:2:2) were recorded as recombinants and
then divided by the total number of asci observed. This gave recombinant
frequency, which was divided by two and multiplied by one hundred to determine
map distance.
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Results
Figure 1: BIOL 110H Class Observations of Asci
Type A (4:4)
Type B (2:4:2) Type C (2:2:2:2)
6,358
5,105
4,908
Total
Observed
16,371
Total
Recombinants
B+C
10,013
Figure 2: Recombinant Frequency
Type B (2:4:2)
Type C (2:2:2:2)
Total Recombinants
5,105/16,371= 31.2%
4,908/16,371= 30.0%
10,013/16,371= 61.2%
As shown in figure 1 there were three patterns observed in this experiment,
types A, B, and C. Types B and C were the recombinants and led to the conclusions of
crossover frequency and map distance. The total crossing over was 61.2% leading to
the map distance answer of 30.5 cM or map units. This distance is determined by
multiplying the crossover frequency by one hundred and dividing by two. Given this
particular map distance it can be concluded that the gene for spore color is midway
between the centromere and the end of the chromosome. Given this data one can
conclude that crossing over does occur but not with the centromere because the
gene is not relatively close to the centromere. In addition through observations and
research it can be concluded that trends such as the patterns of types A and B asci
are the result of two parent sordaria strains crossing over, whereas other unusual
patterns not in accordance with these patterns are the result of other factors.
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Discussion
It was learned that the Sordaria fimicola fungus strain exhibits crossing over
under laboratory conditions. Although the results were not the same as those found
in the research, that was expected as the research was of Evolution Canyon where
more variables would have an effect on the fungi. In a lab the variables were limited
to what was being experimented thus, there was room for comparison. Although all
lab procedures were followed accordingly, human error was unavoidable. A few of
the petri dishes had grown mold and were not viable to examine, for this reason it is
important to make multiple cultures for an experiment in order to have at least one
reliable source. Under more rigorous sanitation practices and culturing methods the
procedure could have resulted in fewer dishes with mold. The use of a flow hood,
gloves, and a precisely sanitary environment would help to prevent mold grown in
the petri dishes(Stamets). Furthermore, the squashing of the perithecia posed a
difficulty. With too much pressure the asci would burst and one would have
difficulty examining spore patterns, with too little pressure the perithecia would
stay whole and the asci would not be visible. In addition to the difficulties there
were unexpected findings in some of the spore patterns including blue and green
spores. Was this the result of mold or a mutant gene in the sordaria strain? Further
experimentation with these spore colors would explain the purpose of those
additional colors and how they are produced. In conclusion of the evidence shown
in figures 1 and 2, by analyzing this data it is apparent that Sordaria fimicola
exhibits crossing over in a lab setting at different frequencies than that of Evolution
Canyon.
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Bibliography
Stamets, P.(2000) Growing Gournet and Medicinal Mushrooms. New York.
Ten Speed Press
Davidson, M. (n.d.). Fungus (Sordaria fimicola) Fruiting Bodies. Retrieved
from
http://micro.magnet.fsu.edu/primer/techniques/dic/dicgallery/sordariaperithecia
small.html
Nevo, E. (2009) Evolution in action across life in "Evolution Canyon" Israel. Trends
in Evolutionary Biology.
BIOL110H (n.d.). Bio 110 Laboratory Activity: Meiosis and Genetic Diversity in the
Model Organism, Sordaria fimicola.
Galanti, D,(2013) BIOL 110H Laboratory Notebook pg. 5-10. (11/4/13)
Goldy-Brown, M(2013) BIOL 110H Class Data. (11/4/13)
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