Greenspan 1

The Effects of X-ray Radiation on the Recombination Frequency in Sordaria fimicola

Blake Greenspan
Penn State University
Biology 110H Section 45L
Introduction

The study of genotypic and phenotypic responses to the environment is ubiquitous

throughout biology. It is known that new genotypes and phenotypes rise from genetic diversity.

Genetic diversity is largely a byproduct of crossing over during meiosis which contributes to new

recombinant genotypes. Crossing-over during prophase I allows for genetic material to be

exchanged between homologous chromosomes. Cross-over occurs randomly along the length of

the chromosomes which results in further genetic diversity as genetic material is exchanged. As a

result, some phenotypic differences can be subtle, but genotypic differences can be present

which can be observed to distinguish crossing-over events.

S. fimicola, an ascomycete fungus, is an excellent organism to use to test the factors

controlling cross-over frequency because it is known as a model organism. S. fimicola have been

the subject of a great amount of research from the aspects of its life cycle to its genetics (Burpee

et al., 2018). Furthermore, its rapid life cycle, ease to maintain and breed in a lab, and its

production of perithecia containing asci with eight spores contribute to its use as a model

organism (Burpee et al., 2018). Perithecia are sacks containing 8 asci that have color. There are

two colors of spores: black (wild-type) and tan (mutant). The tan color rises only from mutation,

and it has no better fitness than wild-type (Lamb et al., 1998). The spores can help researchers

understand the events of meiosis. This is because the eight spores allow for researchers to study

the frequency of crossover in these organisms due to environmental pressure. The colors of the

asci are easy to distinguish under a microscope as either black or tan which helps to study

crossing-over events. During reproduction, three types of asci can arise. When crossing-over
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does not occur, a 4:4 ratio will be the result known as type A. However, when crossing-over

occurs, a 2:4:2 ratio (type B) or a 2:2:2:2 ratio (type C) occurs. Physically, the type of asci

created depends on which chromatids cross-over. When the homologous chromosomes interact

in a 3D plane, the inner chromatids cross-over and produce type B or type C depending on the

chromatids that exchange genetic information. Thus, “Lamb et al. (1998)” studied S. fimicola

and the responses to environmental pressures such as UV radiation to better understand the

factors controlling cross-over frequency.

A special evolutionary cannon offers an insight into the different environmental pressures

exerted on species. In Israel, contrasting environments in the same location allow for two

different environmental pressures to be exerted on the same species (Nevo, 2001). There are two

slopes: the north facing slope (NFS) and the south facing slope (SFS). The south facing slope

receives greater UV radiation and higher temperatures compared to the north facing slopes.

Considering the environmental differences in the two slopes in Evolution Cannon, researchers

were able to cross samples from each slope using samples from 3 different locations on each

slope (Saleem et al., 2001). The researchers hoped to gain insight into the frequency of cross-

over between the crosses due to ultraviolet rays. Thus, wild-type (WT) and tan spore color

strains were collected from each side and crossed. They crossed WT and tan spore type among

the same slope (NFS v. NFS and SFS v. SFS) and across slopes (NFS v. SFS). They found that

there were differences in cross-over frequency between slopes. The difference in cross-over

frequency among slopes occurred but not at a significant frequency. Thus, they concluded that

environmental pressures potentially lead to differences in the frequency of cross-over and an

increase genetic diversity (Lamb et al., 1998).

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Many questions about the factors controlling cross-over events were not answered in the

experiment, and no studies specifically looked at the effects of other environmental pressures on

cross-over. Therefore, in order to test the effects of x-ray radiation, we performed a cross

between S. fimicola that have been exposed to radiation and S. fimicola that have not been

exposed to radiation. Specifically, a cross between high radiated wild-type and low radiated tan

type. In addition, an untreated control was used to establish reference point. We hypothesized

that exposure to x-ray radiation would increase the frequency of cross-over in S. fimicola. And,

the results showed that there was an increase in the frequency of cross-over among the

treatments compared to the control.

Methods

To begin the experiment, a cross between normal wild-type spores and normal tan type

spores was created as the control. This was to establish a reference point when comparing the

frequency of cross-over between the samples. Spores were treated with no radiation (none), 100

grays of x-ray radiation (low), or 500 grays of x-ray radiation (high). Treatment crosses included

high wild-type and low tan, high wild-type and high tan, high wild-type and none tan, low wild-

type and high tan, low wild-type and low-tan, low wild-type and none tan, none wild-type and

high tan, and none wild-type and low tan. We performed a treatment cross between high radiated

wild-type and low radiated tan type.

To obtain the results of the cross, an agar plate was set up with the two varying fungal

samples. The differing samples were placed adjacent to one another (the same fungal samples

were diagonal from one another). The plate was set up this way to maximize crossing-over

events and to ensure a sufficient hybrid zone. After 14 days of growth, a squash was prepared. A

sample was taken from the hybrid zone and placed on a viewing plate. A sample was taken from
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the hybrid zone to increase the chance of observing crossing-over events. Next, the sample was

burst to ensure that the asci could be seen under the microscope. Then, the number of non-

recombinant types and recombinant types (type B and type C) were counted. The asci were

scored meaning we defined the asci as recombinant or not. After, the frequency of cross-over

was determined (Burpee et al., 2018).

Results

Asci Counted in Experiment (Table 1)

Type A Asci Type B Asci Type C Asci Total Asci Recombinant

(4:4) (2:4:2) (2:2:2:2) Frequency
Control 266 165 135 566 .530

WT High v 35 22 27 84 .583
Tan High
WT High v 34 31 35 100 .660
Tan Low
WT Low v 15 16 19 50 .700
Tan Low
WT High v 13 15 22 50 .740
Tan None
WT Low v 49 24 27 100 .510
Tan None
WT None v 13 21 16 50 .740
Tan High
WT None v 14 11 19 44 .682
Tan Low
WT Low v 42 33 25 100 .580
Tan High
Table 1: The table presents all the quantitative data obtained from the experiment. The number of each
type of asci for each treatment is displayed. In addition, the total asci counted in each treatment was
calculated. The equation, (# of Type B asci + # of type C asci) / total number of asci, was used to
determine the recombinant frequency. Finally, the term “high” indicates a culture exposed to high
amounts of x-ray radiation (500 grays), the term “low” indicates a culture exposed to low amounts of x-
ray radiation (100 grays), and the term “none” indicates a culture exposed to no exposure of x-ray
radiation.
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Frequency of Asci Types in Experiment (Table 2)

Treatment Frequency of Frequency of Frequency of Ratio Type

Recombinant Type B Asci Type C Asci B/TypeC
Asci
Control .530 .292 .239 1.221:1

WT High v Tan .583 .262 .321 .816:1

High
WT High v Tan .660 .310 .350 .886:1
Low
WT Low v Tan .700 .320 .380 .842:1
Low
WT High v Tan .740 .300 .440 .682:1
None
WT Low v Tan .510 .240 .270 .889:1
None
WT None v Tan .740 .420 .320 1.313:1
High
WT None v Tan .682 .250 .432 .579:1
Low
WT Low v Tan .580 .330 .250 1.320:1
High
Table 2: The table displays the frequency of Type B asci and Type C asci found in the various treatments.
The equations, # of type B asci / total number of asci and # of type C asci / total number of asci, were
used to determine the frequency of the type of asci. Also, the ratio of the B and C was calculated.
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Bar Graph of Recombinant Frequency and Treatment Cross (Figure 1)

Recombinant Frequency and Treatment Cross

0.8 0.74 0.74
0.7 0.682
0.66
Recombiant Frequency

0.7 0.583 0.58

0.6 0.53 0.51
0.5
0.4
0.3
0.2
0.1
0
Control WT High WT High WT Low WT High WT Low WT WT WT Low
x Tan x Tan x Tan x Tan x Tan None x None x x Tan
High Low Low None None Tan High Tan Low High
Treatment Cross

Figure 1: The graph shows the different recombinant frequencies of the nine treatment groups.

The graph is intended to help visually differentiate the frequencies; however, the exact amount is

at the top of the bar for precise data interpretation.

Both table 1 and table 2 display the data and calculations that were found during the

experiment. The results indicate that the average recombinant frequency of all treatment plates

(64.9%) were higher than the control plates (53.0%). The average of all recombinant frequencies

for the treatment groups was 13.9% higher than the control. An analysis of each treatment cross

shows that the cross of low radiated wild-type and non-radiated tan (51.0%) was the only

treatment where the recombination frequency was lower than the control (53.0%). However, all

other treatments’ recombinant frequencies were higher than the control’s recombinant frequency.

In addition, the ratios of type B and type C for each treatment were found to be close to 1:1 with

the average of the ratios being 9.16:1.

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Discussion

The purpose of this study was to determine if environmental pressures, specifically x-ray

radiation, increased the cross-over frequency in in S. fimicola. We found that there is evidence of

crossing-over between the gene for spore color and the centromere in S. fimicola because of the

increase in recombinants. Furthermore, we found that there are differences in the cross-over

frequency of the control and those treated with the environmental stressor of x-ray radiation. The

results indicate that the average recombinant frequency of all treatment crosses (64.9%) were

higher than the control cross (53.0%) by 13.9%. This evidence supports our hypothesis that x-ray

radiation can influence the frequency of cross-over therefore increasing cross-over events. In

addition, we found that the ratio between the frequency of recombinant asci B and recombinant

asci C were nearly 1:1. This means that both types of recombinants are equally as likely during

the events of crossing-over. The increase in recombinants indicates that new genotypes are being

created. Therefore, the new genotypes contribute to an increase in genetic diversity which

increases the chances of a greater fitness for S. fimicola to better survive against x-ray radiation.

In general, all treatments, except one, experience a higher cross-over frequency than the

control. This indicates that the environmental pressure influenced that rates of cross-over. Thus,

this evidence supports the hypothesis that environmental factors, specifically x-ray radiation,

influence crossing over in S. fimicola. However, it is surprising that not all environmental

pressures produce the same cross-over frequency. Unless it was due to error, some treatments

had a greater recombination frequency than other treatments. This signals that some pressures

might be greater than others, and different pressures can produce a greater recombination. On the

same note, it was unexpected that there was no relationship between the intensity of radiation

and cross-over frequency.

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It is possible that we experienced some error throughout the experiment. One possible error

was other unknown pressures influencing cross-over. The influences could affect the outcomes

of meiosis and other processes that we were monitoring which ultimately would skew the data.

Another source of error was the lack of growth from the samples. Upon observation, many of the

asci were underdeveloped. This means that we should have let our samples grow for a longer

time to develop. Undeveloped asci don’t show color, and it is hard to find/count the frequency of

recombinant; thus, our sample size was very low. A low sample size is another source of error in

the experiment. Since the asci counted were minimal, this increases the chance of error in the

experiment. Therefore, a larger sample size should have been used to increase the confidence of

the results.

Overall, we found that environmental pressures can influence crossing-over events during

meiosis in S. fimicola. Other researchers can use this data to compare different environmental

pressures and the frequency of recombination. They can find similarities or differences to further

test how the frequency of crossing-over is affected by environmental pressures. One future

experiment could test if humidity influences the frequency of recombination. The same process

could be used as this experiment except the pressure would be three varying moisture levels

(none, low moisture, and high moisture). Once again, this experiment could offer insight into the

frequency of cross-over with an environmental pressure present.

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References

Burpee, D., Cyr, R., Hass, C., Ward, A., and Woodward, D. 2018. A Laboratory Manual for

Biology 110 Biology: Basic Concepts and Biodiversity. Department of Biology, The

Pennsylvania University, University Park, PA.

Lamb, B. C., Saleem, M., Scott, W., Thapa, N., & Nevo, E. (1998). Inherited and

Environmentally Induced Differences in Mutation Frequencies Between Wild Strains of

Sordaria fimicola from “Evolution Canyon”. Genetics Society of America,149, 87-99.

Nevo, E. 2001. Evolution of genome-phenome diversity under environmental stress. Proceedings

of the National Academy of Sciences, 98(11): 6233-6240.

Saleem, M., Lamb, B.C., and Nevo, E. 2001. Inherited Differences in Crossing Over and Gene

Conversion Frequencies Between Wild Strains of Sordaria fimicola from “Evolution

Canyon”. Genetics 159: 1573-1593.