Almarzooqi 1
Samar Almarzooqi
Biology 110 Section 907
10/29/2012
Lab Report
Differing Recombination Frequencies of Sordaria fimicola
Introduction:
As part of the research conducted by scientists about the evolution of sexual
reproductions, S. fimicola were observed from two differing slopes of the “Evolution
Canyons” in order to better understand the microevolution of these species. The two
slopes, the North Facing Slope (NFS) and South Facing Slope (SFS), experience differing
climate conditions. The NFS has a temperate climate with shade, cool and humid air
while the SFS experiences more extreme conditions that include solar radiations, high
temperature, and drought (Hass and Ward, 4). In order to better understand the
phenomena of increasing crossover frequency due to environmental pressures and the
mechanisms that control these crossover frequencies. S. fimicola were used in the
experiments because they have a short life cycle, produce asci with eight spores easy to
observe under a microscope, and have strains easy to isolate and grow in the lab (Hass
and Ward, 4). Strains from each slope were collected grown in a lab and then the wild
type spores were crossed with each other. Researchers found that the strains taken from
the SFS “showed higher mutation rates than did those isolated from the NFS” (Hass and
Ward, 5). From the results, it was concluded that genetic diversity occurs as a result of
the mutations and recombination crossover frequencies due to environmental pressures.
These observations led researchers to conduct more research focusing on “how
does the exposure to environmental stresses applied under controlled conditions change
the crossover frequency in populations of S. fimicola?” (Hass and Ward, 5) To answer the
overarching research question, further research in the laboratory needed to be conducted.
The frequency of each of the two types of crossover classes, 2:2:2:2 and 2:4:2, and also
the total overall combined frequency of crossover were further areas of evolution
researched. The experiment included exposing strains to both harsh conditions and also
controlled conditions. A control group is necessary because in order to determine direct
cause and effect, all other variables that could impact crossover frequency need to be
eliminated so that the only reason crossover frequency will change is due to the differing
environmental conditions.
The differing recombinations of asci that occur as part of the reproduction of S.
fimicola are 4:4, 2:2:2:2, or 2:4:2. 4:4 is a result of no recombination between the two
mating types. Which means that in Prophase I of Meiosis I, no crossover between the
non-sister chromatids on the homologous pairs occurs. In both 2:2:2:2 and 2:4:2,
recombination occurs. Therefore, crossing over between non-sister chromatids of
homologous pairs occurs in Prophase I of Meiosis I. Crossing over is when non-sister
chromatids of homologous pairs exchange segments of DNA which results in new
Almarzooqi 2
combinations of genes, which leads to more genetic diversity. A figure of each asci
possible due to reproduction is shown in the Results section.
In order to determine how differing pressures impact crossover frequencies and
overall genetic diversity for S. fimicola, an experiment similar to that the researchers
from “Evolution Canyons” was conducted. Each group was given a strain type, Grey or
Tan, to cross with the Wild Type strain. The asci produced from the crossovers were
observed under a microscope, and the crossover frequencies were tabulated by counting
20 asci and categorizing each asci into one of the three types, 4:4, 2:2:2:2, or 2:4:2. Class
data was combined with other sections to form complete course data that was used to
analyze the results. Each lab group focused on four research questions in order to answer
the overarching research question mentioned before.
Research Questions:
1. Does crossover occur during the color mutant x wild type crosses?
2. What is the overall crossover frequency of each of the color genes with the
centromere in organisms grown under standard laboratory growth conditions?
3. What are the frequencies of the two crossover types for each color strain?
4. Is there any variation in the ratio of the two crossover types between the two color
strains? (Hass and Ward 5)
Our group hypothesized that if each strain of S. fimicola were grown in the laboratory
under room temperatures, then the strain from an area with more harsh environmental
pressures would experience a high total crossover frequency compared to the strain
grown from an area with optimal condition. It was also hypothesized that the crossover
frequencies of each strain, 2:2:2:2 and 2:4:2, would occur in equal proportions. To
examine if our hypothesis was true, we conducted a crossover between Grey vs. Wild
Type strains and allowed them to incubate and grow. Under the microscope, the number
of 4:4, 2:2:2:2, and 2:4:2 asci were counted and tallied up. The total overall crossover
frequency (2:4:2 + 2:2:2:2) and each individual crossover frequency type were examined.
From previous understandings and research on genetic diversity, it was expected that the
spore color gene taken from an area that usually experiences extreme environmental
pressures would have a higher crossover frequency than those of the spore color gene
taken from a habitat with a more temperate environment. It was also expected that the
ratio between the two types of crossover frequencies (2:2:2:2 and 2:4:2) would be the
same for the two spore color genes. To analyze the data and determine if our hypothesis
is correct, our lab group calculated the frequencies of each crossover type and looking for
trends.
Materials and Methods:
In order to perform the lab, we needed two plates with different strains of
Sordaria, Tan and Wild Type, to use. We used a scalpel to scoop some of each strain and
place on a plate with mating agar. The mating agar was divided into four quadrants so
that the same spore strain were across from the other, not beside. The agar plates were
incubated for two weeks at room temperature and afterwards, some perithecia obtained
from the crosses between the two spore colors were observed under a compound light
Almarzooqi 3
microscope. The independent variable for the experiment was the spore color type (Grey
or Tan) and the dependent variable was the crossover frequency of each spore color.
Slight pressure needed to be applied to the perithecia so that they would burst and
the individual asci with eight spores could be seen. Each group member counted a total of
20 asci and categorized each one as 4:4, 2:2:2:2, or 2:4:2. Class data was tallied for both
the Grey and Tan spore colors and then the course data was tabulated and distributed to
students for their use. All of the data was analyzed by generating crossover frequencies
for 2:2:2:2 and 2:4:2 recombination by taking the number of each type of recombination
and dividing it by the total number of asci observed for that spore strain. The total
crossover frequencies were calculated by taking the combined total of recombinations,
2:2:2:2 and 2:4:2, and dividing it by the total number of asci observed for each spore
color strain. The map distance, which is the distance from the gene to the centromere on a
chromosome, was calculated for each spore color strain by taking the overall crossover
frequencies of 2:4:2 and 2:4:2 and dividing it by 2. It is understood that 1 map unit = 1%,
so the map distance for each spore color strain can be calculated (Graziano).
Theoretically, a gene with a larger map distance will be more likely to experience a
crossover in Prophase I (Hass and Ward, 7).
Procedure was taken from “Meiosis and Genetic Diversity in the Model
Organism, Sordaria” lab handout.
Results:
Table 1. Individual Data for Tan Spore Color
Nonrecombinant
# of Type A
Asci
(4:4)
10
Recombinant
# of Type B
Asci
(2:4:2)
4
# of Type C
Asci
(2:2:2:2)
9
Total # of
Asci
Total #
Recombinant
Asci
(B + C)
20
10
Individual data shows that the total recombinant asci are in proportion with the nonrecombinant asci, 10 to 10. Also, the number of 2:2:2:2 asci counted are more than the
number of 2:4:2 recombinant type asci counted.
Almarzooqi 4
Table 2. Combined Course Data Analysis:
Nonrecombinant
# of Type A
Asci
(4:4)
Recombinant
# of
Type B
Asci
(2:4:2)
Tan Spore Color
5669
4301
# of
Type C
Asci
(2:2:2:2)
3976
Total #
Recombinant
Asci
(B + C)
Total
# of
Asci
Frequency of
Recombinant
Asci
(B + C)/total
# asci)
Frequency
of Type B
Asci
(B/total #
asci)
Frequency
of Type C
Asci
(C/total #
asci)
Ratio
B/C
59.4%
30.8%
28.5%
1.08
57.4%
29.5%
27.9%
1.05
13,946
8,277
Gray Spore Color
3012
2081
1973
7,066
4,054
Course data shows that the frequency of recombinant 2:4:2 is greater than the frequency
of 2:2:2:2 asci for both the Grey and tan spore color strains. The overall frequency of
recombinant asci was greater for the tan spore color strains.
Combined Course Data was obtained through Angel on the Bio 110 Section 907 page.
Calculations:
Frequency of Recombinant Asci
Frequency of Type B
Frequency of Type C
Ratio
Map Distance=
(4301 + 3976)/ 13, 949 x 100 = 59.4%
(4301/13946) x 100= 30.8%
(3976/13,946) x 100= 28.5%
(4301/3976) = 1.08
%𝑐𝑟𝑜𝑠𝑠𝑜𝑣𝑒𝑟
2
Tan Spore Color = 59.4%/2 = 29.7% = 29.7 map units
Grey Spore Color = 57.4%/2 = 28.7%= 28.7 map units
The results from the cross between both wild type vs. tan spore color and grey spore
color for the course data shows that there is a difference between the frequencies of each
type of recombination type within each cross. For the tan spore color, the frequency of
2:2:2:2 was 28.5% and the frequency of 2:4:2 was 30.8%, and for grey spore color, the
frequency of 2:2:2:2 recombination was 27.9% and the frequency of 2:4:2 recombination
was 29.5%. The tan spore color cross shows that the ratio of 2:4:2 to 2:2:2:2 is 1.08 and
ratio of 2:4:2 to 2:2:2:2 for Grey Spore Color is 1.05 which means that overall, the
frequency of each type of recombination does not occur in equal proportions. The overall
frequency of recombination for each spore color, tan and grey, was 59.4% and 57.4%
respectively.
Almarzooqi 5
The types of recombination are pictured below with what was observed under the
compound light microscope.
Figure 1: 4:4 (No Recombination)
Figure 2: 2:2:2:2 Recombination
Occurs when crossing over occurs between non-sister chromatids. Independent
Assortment of the crossed over chromosome strand means that it goes to the other side,
leading to 8 spores in a 2:2:2:2 fashion.
Figure 3: 2:4:2 Recombination
Occurs when crossing over occurs and due to independent assortment, the spores are
arranged in a 2:4:2 fashion.
Almarzooqi 6
Discussion:
The results from the experiment show that the frequency of each recombination
type, 2:2:2:2 and 2:4:2, do not occur in equal proportions as expected, and that the tan
spore color has an overall higher frequency of recombination compared to the grey spore
color, 59.4% compared to 57.4%. The difference between the two is 59.4%-57.4%=
2.0%, which is significant since the sample size of asci observed for the course is very
large; 21,012 total asci were counted by all students taking the course. From the lab
handout, we found out that due to the research conducted on the NFS and SFS of the
“Evolution Canyons”, it was deemed that spore strains from the SFS would experience
more recombination than the spore strains from the NFS since the SFS experienced more
extreme environmental pressures and the S. fimicola needed to adapt to the extreme
environment by having a higher genetic diversity (Hass and Ward, 4). From out results, it
can be concluded that the tan spore color S. fimicola was taken from an environment that
experiences more extreme environmental pressure like high temperatures and solar
radiation. The tan spore color strains have a total frequency of recombination of 59.4%
while the total frequency of recombination of the grey spore color strains calculated was
57.4%. Therefore, the tan spore color strain was obtained from an environment under
duress from extreme pressures inflicted on the organisms inhabiting it.
It can also be concluded that within each spore color strain cross, the
recombination type of 2:4:2 is more likely to occur than the 2:2:2:2 recombination. In
both the grey and tan spore color crosses, the total frequency of 2:2:2:2 was less than the
frequency of 2:4:2 recombination. For the tan spore color strain, the ratio of 2:4:2
recombination to 2:2:2:2 recombination was 1.08 and for the grey spore color strain, the
ratio of 2:4:2 recombination to 2:2:2:2 recombination was 1.05 (See Table 2 page 4). For
the individual data calculated, the number of 2:4:2 recombination was less than 2:2:2:2
recombination, 9 to 4 respectively. These results do not support the course data, whish
suggests that the frequency of 2:4:2 is greater than 2:2:2:2. This can be due to human
error since as an individual, I was not positive that I correctly counted the asci
recombination types.
The crossing over in Prophase 1 of Meiosis I is what contributed to the
recombination types seen in the asci. In the life cycle of Sordaria, the perithecium, which
are haploid, go through meiosis and then mitosis to produce the asci with 8 spores in
each. The 8 spores in the ascus reveal how the process of reproduction occurred since the
color of the spore can reveal whether or not crossing over occurred. The genetic diversity
of the Sordaria is easy to observe, which is why it is easy to see that the tan spore color
experienced a higher frequency of recombination, and therefore has a higher genetic
diversity.
The first part of our hypothesis stated that “if each strain of S. fimicola were
grown in the laboratory under room temperatures, then the strain from an area with more
harsh environmental pressures would experience a high total crossover frequency
compared to the strain grown from an area with optimal conditions,” was accepted and
validated from out results. Our experimental data shows that the total frequency of
Almarzooqi 7
recombination for the tan spore color is greater than the total frequency of recombination
for the grey spore color, 59.4% and 57.4% respectively. Since both strains were grown
under room temperatures with all other lurking variables that could impact the results
eliminated, the only source for the difference in frequency of recombination between the
two is their genetic evolution in the method of crossing over. The second part of our
hypothesis, which stated that the crossover frequencies of each strain, 2:2:2:2 and 2:4:2,
would occur in equal proportions, was not accepted from the results obtained. Overall,
the 2:4:2 recombination has a higher probability of occurring than the 2:2:2:2
recombination. From Table 2, it can be seen that for both the tan spore color strains and
grey spore color strains, the frequency of recombination for 2:4:2 to 2:2:2:2 was greater.
The ratio between the two for tan spore color is 1.08 and 1.05 for the grey spore color.
Since the ratios of 2:4:2 to 2:2:2:2 are very close between the two spore color strains, it
means that the frequency of recombination type is not different between the two and only
the total frequency of recombination changes when environmental pressures cause
genetic diversity through the increase in crossing over.
The difference between the two types of recombination is unexpected since it was
determined that the probability of each type occurring is expected in equal proportions.
Sources of error for the differences could be due to human error because as first year
Biology students, the recombination frequencies tallied could be wrong. Also, not all of
the asci on the slides were counted by each student, and it may be that each student
counted more of one type of recombination simply because they were easier to find on
the slide. Further research should be observed by conducting further experiments on the
sources and reasons behind the unequal ratios between 2:4:2 recombination and 2:2:2:2
recombination. Is one type more beneficial than the other, or are the results from our
course simply a source of human error, making the difference between the frequencies of
both recombinations calculated insufficient and unanalyzable?
Works Cited:
Meiosis and Genetic Diversity in the Model Organism, Sordaria. Written by Hass, C.
and Ward, A. 2010. Department of Biology, The Pennsylvania State University,
University Park, PA.
Cyr, R. 2002. Heredity and Life Cycles. In, Biology 110: Basic concepts and biodiversity
course website. Department of Biology, The Pennsylvania State University.
http://www.bio.psu.edu/
Graziano, Maria. “Sordaria Lab Lecture.” Class lecture, Pennsylvania State University,
State College, PA, October 15, 2012.