Sordaria Recombination in Response to X-Ray Radiation

Rea Mittal, Coreena Chan

Bio 110H Section 001
Priyanka Solanki and Haley Randolph
October 26, 2015

Mittal 2
Introduction
Spiny mouse were observed in Evolution Canyon, a natural laboratory with two slopes with
distinctively different environments. One, dubbed the African slope or south facing slope (SFS), is dry
and grassy with an overall yellow-brown appearance. The European slope, or the north facing slope
(NFS), is temperate with dark gray soil and dense foliage, giving it an overall gray appearance. The spiny
mouse exist on both slopes, facing different environmentally selective pressures, which affected their hair
melanin contents. Crosses between the SFS and NFS slopes had a higher cross-over frequency than the
cross over frequencies of mice on the same slopes, meaning that harsh environmental pressures can lead
to higher recombination rates (Singaravelan et al., 2010). Spiny mice on the SFS also had an observed
higher cross over frequency than the spiny mouse on the NFS. This is most likely due to the increased UV
radiation of the SFS. This side of the canyon physically has more exposure to the sun, therefore the
increased sunlight has sub sequential environmental effects.
Recombination, or crossing over, is the exchange of DNA sequences during meiosis that can
generate genetic diversity (Burpee et al., 2015). Recombination in response to different environmentally
selective pressures can allow for the survival of recombinants if the altered function of the protein is
beneficial. Mutations, caused by external pressures such as radiation, can also similarly increase genetic
diversity (Sudpraset et al., 2006). Both processes are displayed in this experiment. The Sordaria spores
grown in the lab are specifically exposed to x-ray radiation. By applying this selective pressure ourselves,
the experiment is assured to have resultant recombinant Sordaria. Sordaria spores grown in nature may
also be exposed to similar selective pressures, but the pressures most likely wont be as focused as the
pressures in this experiement. Also, experimenters wouldnt be able to control all the environmentally
selective pressures if the Sordaria was grown in nature. In the lab, the experimenters can control for all
other types of selective pressures, therefore more accurate associations can be interpreted from the results
since there wont be confounding variables.
Three types of asci can arise in Sordaria. The wild type black and tan ascospores can arrange
themselves in variations of Type A (4:4), Type B (2:4:2), and Type C (2:2:2:2). All three asci types go

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through a similar initial process. The haploid parent spores go through plasmogamy, where the cytoplasm
combines to form one cell, to create a dikaryon cell, where there are two nuclei in one cell. The dikaryon
cell goes through karyogamy which creates one diploid nucleus from the two haploid nuclei. The cell then
goes through meiosis, creating four haploid cells. Then the cell goes through mitosis, creating eight
haploid cells. The different asci types created have variations in crossing over in the meiosis stage of this
life cycle. Type A, the non-recombinant asci, have chromosomes that dont cross over in meiosis I. After
the chromosomes replicate, and a tetrad has formed and lined up on the metaphase plate, the
chromosomes dont cross over. Therefore, the four offspring made in meiosis II are in a 2:2 ratio, with the
similar genotypes next to each other. Then, in mitosis, eight asci are formed and they are in a 4:4 ratio
because the four cells created earlier were directly replicated to make these daughter cells. Type B asci are
recombinant, so they cross over in meiosis I. When the tetrads are on the metaphase plate and ready to
separate, the chromosomes have crossed over so that when the four daughter cells are made, they are
arranged with the same spore type on the ends and the other spore type in the middle in a 1:2:1 formation.
Therefore, the eight identical daughter cells after mitosis are in the 2:4:2 formation. In type C, the
chromosomes cross over in a different arrangement, where the father chromatid and mother chromatid
closest to the metaphase plate exchange DNA, so that the spores are in alternative alignment (1:1:1:1)
before they split in anaphase I and create four daughter cells. After mitosis, eight daughter cells are
created where two identical cells are created from one of the four parent cells after Meiosis 1. Therefore,
Type C recombinant asci are in a 2:2:2:2 ratio (Burpee et al., 2015)
Four basic research questions exist. Firstly, what challenges arise in using the standard procedure
for mating different strains of Sordaria and how might they be addressed? What challenged arise in
preparing squashed of perithecia for scoring asci and how might they be addressed? Does cross-over
occur between the spore color strain and wild type? What is the cross over frequency of the spore color
gene with the centromere in organisms grown under standard laboratory growth conditions. These
questions will be examined throughout the experiment.

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Researchers explored the various effects of low-level gamma radiation on DNA. They applied
different doses of gamma radiation on DNA, from 5 cGy to 500 cGy, and observed that there were
chromosomal aberrations and deletions in all of the different doses. They found that the dosage of
radiation was highly influential in how much damage the DNA took, where more radiation caused more
DNA strand breaks etc. The hOGG1 and XRCC1 genes (repair genes) were also expressed less because
the gamma radiation decreased the mRNA expression, determined experimentally by the reverse
transcriptase-polymerase chain reaction. This article concluded that high levels of radiation pose health
risks to organisms, particularly humans, because of the DNA damaging effects and reduced DNA
repairing capacity (Sudprasert et al., 2006). This study relates to this lab by noting how radiation can have
different effects on DNA structure and the subsequent organism. These effects can be positive, where
genetic diversity increases so some organisms can survive the radiation; negative, such as when lethal
mutations or breaks occur in the DNA so organisms can not function and die; or neutral, when mutations
occur where the introns are cut or when the mutation doesnt affect the amino acid/protein produced
(Burch et al., 2007).
Selective environmental pressures lead to more recombination in order to increase the likelihood
of survival in these new conditions through genetic diversity. Accordingly, X ray radiation causes more
recombination to occur (Burpee et al., 2015). X-ray radiation will be applied on the Sordaria and therefore
a higher cross-over frequency in the Sordaria exposed to the x-ray radiation (Type B and Type C asci),
rather than the controlled Sordaria, is expected.
Our specific experimental cross was wild type black x x-ray tan sordaria and the control cross,
which was wild type black x tan. To observe the cross over frequencies, the mated Sordaria will be
observed under a microscope. The Sordaria types will be observed and counted so that the recombinance
frequency can be calculated later. Type A nonrecombinant asci (4:4), Type B recombinant asci (2:4:2), and
Type C recombinant asci (2:2:2:2) will be seen. There will most likely be a higher ratio of recombinant
asci (Type B + C) compared to nonrecombinant asci (Type A) in the sordaria crosses exposed to radiation.
Due to the variable effects of the radiation, it will also be likely to see completely random mutations in

Mittal 5
the asci which can present as any of the types of asci (Type A, B, or C) or a random combination of them
(i.e. 1:2:3:2) that that cant be included in our recombinance results.

Methods
Following the information from the lab manual, the experimental treatment is an environmentally
selective pressure placed on the Sordaria, specifically X-ray radiation (Burpee et al., 2015). That
treatment allows comparison between the crossover frequency of Sordaria exposed to radiation to
Sordaria that were exposed to standard laboratory conditions. Wild type black Sordaria were crossed with
tan Sordaria to be the control plate. The experimental crosses included x-ray black Sordaria with tan
Sordaria, wild type black Sordaria with x-ray tan Sordaria, and x-ray black Sordaria with x-ray tan
Sordaria.
Each pair of students received two plates to set up. One plate was labeled control plate, and the
other was labeled experimental plate. The plate was split up into four equal sections, alternating the
strains so that for the control plate, the top left quadrant was wild type black, the top right quadrant was
tan type, the bottom left quadrant was tan type, and the bottom right quadrant was wild type black.
Different groups in Biology 110 Section 001H had different experimental crosses to monitor. The plates
already had agar in them, so the Sordaria were transferred from a sample plate of the type desired (ie wild
type black) to the quadrants specified for them on the plates. The x-ray black Sordaria had already been
exposed to radiation, so further radiation did not have to be applied. After the appropriate strains were
transferred to their designated locations, the lids were taped securely and then the plates sat for two weeks
so the Sordaria could mate. After two weeks, the Sordaria for observation were picked from the borders of
each type, essentially where mating was occurring between the different strains of Sordaria. The Sordaria
were then observed under a microscope with twenty asci scored per plate. The different types of asci
found were tallied so that the crossover frequencies could be calculated. Cross over frequencies were
calculated by counting the amounts of Type A, Type B, and Type C asci, and then dividing the total

Mittal 6
amount of recombinant asci (Type B and Type C) by the total amount of asci scored (Type A, B, and C)
(Burpee et al., 2015).

Results
1a. Individual Data For Control (wild type black x tan)
Non- Recombinant: #

Recombinant: # of

Recombinant: # of

of Type A Asci (4:4)

Type B Asci (2:4:2)

Type C Asci (2:2:2:2)

Total # of Asci Scored

Total # of
Recombinant Asci
(B+C)

11

20

Total # of Asci Scored

Total # of

1b. Individual Data for Treatment (x-ray black x tan)

Non- Recombinant: #

Recombinant: # of

Recombinant: # of

of Type A Asci (4:4)

Type B Asci (2:4:2)

Type C Asci (2:2:2:2)

Recombinant Asci
(B+C)

20

14

Total # of Asci Scored

Total # of

2a. Small Group Data for Control (wild type black x tan)
Non- Recombinant: #

Recombinant: # of

Recombinant: # of

of Type A Asci (4:4)

Type B Asci (2:4:2)

Type C Asci (2:2:2:2)

Recombinant Asci
(B+C)

21

12

40

19

Total # of Asci Scored

Total # of

2c. Small Group Data for Treatment (xray type black x tan)
Non- Recombinant: #

Recombinant: # of

Recombinant: # of

of Type A Asci (4:4)

Type B Asci (2:4:2)

Type C Asci (2:2:2:2)

Recombinant Asci
(B+C)

15

10

3a/4a. Combined Section Data for Control

15

40

25

Mittal 7
Non-

Recombinant: #

Recombinant: #

Total #

Total # of

Frequency of

Frequency of

Frequency

Ratio

Recombinant: #

of Type B Asci

of Type C Asci

of Asci

Recombinant

Recombinant

Type B Asci

of Type C

B/C

of Type A Asci

(2:4:2)

(2:2:2:2)

Scored

Asci (B+C)

Asci

(B/total)

Asci

(4:4)

((B+C)/total)

108

70

64

242

178

.5537

(C/total)
.2893

.2645

1.0938

3b/4b Combined Section Data for Treatment (x-ray black x tan)

Non-

Recombinant: #

Recombinant: #

Total #

Total # of

Frequency of

Frequency

Frequency

Ratio

Recombinant: #

of Type B Asci

of Type C Asci

of Asci

Recombinant

Recombinant

of Type B

of Type C

B/C

of Type A Asci

(2:4:2)

(2:2:2:2)

Scored

Asci (B+C)

Asci

Asci

Asci

37

33

100

70

.7

.37

.33

(4:4)
30

1.1212

3b/4b Combined Section Data for Treatment (wild type black x x-ray tan)
Non-

Recombinant: #

Recombinant: #

Total #

Total # of

Frequency of

Frequency

Frequency

Ratio

Recombinant: #

of Type B Asci

of Type C Asci

of Asci

Recombinant

Recombinant

of Type B

of Type C

B/C

of Type A Asci

(2:4:2)

(2:2:2:2)

Scored

Asci (B+C)

Asci

Asci

Asci

17

23

60

40

.6667

.2833

.3833

.7391

(4:4)
20

3b/4b Combined Section Data for Treatment (x-ray black x x-ray tan)
Non-

Recombinant: #

Recombinant: #

Total #

Total # of

Frequency of

Frequency

Frequency

Ratio

Recombinant: #

of Type B Asci

of Type C Asci

of Asci

Recombinant

Recombinant

of Type B

of Type C

B/C

of Type A Asci

(2:4:2)

(2:2:2:2)

Scored

Asci (B+C)

Asci

Asci

Asci

29

25

80

54

.675

.3625

.3125

(4:4)
26

4b. Combined Section Data For All Treatments (x-ray black x tan, wild type black x x-ray tan, x -ay black x x-ray tan)

1.16

Mittal 8
Non-

Recombinant: #

Recombinant: #

Total #

Total # of

Frequency of

Frequency

Frequency

Ratio

Recombinant: #

of Type B Asci

of Type C Asci

of Asci

Recombinant

Recombinant

of Type B

of Type C

B/C

of Type A Asci

(2:4:2)

(2:2:2:2)

Scored

Asci (B+C)

Asci

Asci

Asci

83

81

240

164

.6833

.3458

.3375

(4:4)
76

Crossing over occurs between the spore color gene and the centromere. The DNA exchanged is
somewhere in that area, shown by the recombinance frequencies..
The overall crossover frequency between the spore color gene and the centromere in organisms
grown under standard laboratory growth conditions is 55.37%, found in the combined section data for the
control plate.
The overall crossover frequency between the spore color gene and the centromere in organisms
grown under experimental conditions is 68.33%, found by adding the total number of recombinant asci
from each treatment and dividing that by the total number of asci scored across all the treatments (Table
4b).

Discussion
The Sordaria presented an overall crossover frequency of 68.33% under x-ray radiation, and a
55.37% crossover frequency under standard laboratory conditions. The combined data frequency of
recombinant asci was roughly even across the three different treatment types, where the x-ray black
crossed with wild type tan had the highest crossover frequency rate (70%) by only a couple percentages.
Therefore, the radiation, a selective environmental pressure, applied on this specific cross resulted in a
higher recombinance frequency as hypothesized. Observing the data from the other treatments, this
conclusion from the x-ray black cross with tan Sordaria can be extrapolated to all the other treatment
types. External environmental pressures, whether with only one x-ray radiation strain or with two,
produced greater rates of recombination. Singh and her research group studied recombination frequency

1.0247

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in Drosophila Melanogaster in relation to an environmentally selective pressure, a parasitic infection.
While the dependent variable is the same, the crossover frequency due to a selective environmental
pressure, the environmental factor is different. Instead, the fruit flies were exposed to an infective parasite
(Serratia marcescens) as a selective factor. Four days after the parasitic treatment, and five days after
mating, they found an increased production of recombinant offspring with the fruit flies that were given
the infection treatment in comparison to the recombinance in their controls (Singh et al., 2015). This
study supports the hypothesis that recombination frequency increases due to selective environmental cues.
This study also helps extrapolate this information to different environmental pressures, including the
radiation tested as well as parasitic infections.
Looking at the different treatment types and their overarching recombination trends, the overall
crossover of Sordaria under any x-ray radiation was 68.33%, which was significantly higher than the
overall crossover frequency of 55.37% of Sordaria grown under normal conditions. This suggests that
applied radiation specifically increases recombination in organisms. A similar association was found in
Drosophila Melanogaster. Other researchers followed the rate of lethal mutations in the second
chromosome of Drosophila Melanogaster. The fruit flies were exposed to ultraviolet radiation (2537
Angstrom wavelength) when they were embryos and before they underwent meiosis. They found that the
frequency of lethal mutations increased when there was a rise in dosage of ultraviolet radiation (Muller et
al., 1954). The study was very precise in how efficiently and evenly the radiation was applied because
they were looking for the exact rate of mutations versus dosage of radiation. While this experiment only
had one dosage of x-ray radiation, Mullers study still supports the general claim that as radiation is
present, recombination is encouraged.
The combined data frequency of Type B recombination was slightly higher than the frequency of
Type C recombination in all of the crosses except for wild type black x x-ray tan. In the x-ray black x tan
cross, the ratio was Type B recombination to Type C recombination was subsequently 1.212, since Type B
recombination occurred at a higher frequency. However, the recombinance ratio of Type B to Type C in
our combined control data was 1.0938, roughly 1:1. Since recombination itself only had a frequency of

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55.37%, it can be concluded that Type B crossovers are not strongly or inherently favored over Type C
crossovers. Of the approximately half of the Sordaria that recombined, about half of them crossed over in
Type Bs arrangement and the other half crossed over to produce a Type C asci, showing minimal
preference for either type. Researchers studied Sordaria Fimicola on Evolution Canyon in a similar way
to Singarevelens study, where Sordaria crossover frequencies were observed between and within
different segments of the north facing slope and the south facing slope. They showed that there was no
polarized segregation, or preferential segregation to one recombinant asci type over the other, by looking
at the sufficiently similar trends between the recombinant asci ratios, or octad trends. Further
supporting the earlier claims and background information, Saleem also found increased crossover
frequencies on the SFS (Saleem et al.,2001), the side exposed to greater radiation.
The results provide enough information to answer the four research questions introduced earlier.
Mating different strains of Sordaria posed a potential challenge because there were four different strains
of Sordaria to keep track of. However, through organizing the two plates into clearly labeled quadrants
where one strains borders were the plate and two quadrants of the the other strain type, this problem was
addressed. If some Sordaria on a plate didnt happen to mate, we simply neglected them from our
calculations. Preparing squashes of perithecia was a straightforward process however some of the
perithnecia did not break under the weight of the cover slip on the slide, so upon first observation, it was
difficult to find asci. This was addressed by applying slight pressure on the cover slip with a pen or pencil
to break the perithecia open so the asci could be observed. Crossing over didnt occur between spore color
strain and wild type. There were two different strains of spore color Sordaria: wild type black and wild
type tan Sordaria. Crossing over happened between either opposite spore color strains that were both wild
type, both exposed to x-ray radiation, or only one exposed to x-ray radiation and the other wild type.
Therefore, crossing over occurred between the spore color gene and the centromere instead. The
crossover frequency of the spore color gene with the centromere in Sordaria grown under standard
laboratory growth conditions was 55.37%, versus the 68.33% crossover frequency in Sordaria grown

Mittal 11
under experimental conditions. Subsequently, the hypothesis that there would be more recombinance in
the experimental Sordaria than in the controlled Sordaria was correct.
Other mutated spore patterns were found in the mated Sordaria, other than the expected Type A,
Type B, and Type C variations. For example, a Sordaria with a 2:2:1:1:1:1 arrangement was found. Even
though the radiation applied did create new asci types, it was due to spontaenous mutations and not
through encouraged recombination in meiosis. Therefore, these Sordaria were not included in the
recombinant results, even though the mutations did change the Sordaria from the nonrecombinant 4:4 asci
ratio. However, the error was that we dont know if those Sordaria would have been recombinant or non
recombinant and how that would have affected our results. Mold was also observed on some of the plates,
however mold was not the fungus of interest and could have had variable effects on the Sordaria growing
in the same environment. Another error in the experiment include the variability of our results,
considering we only gathered data for twenty asci per plate when there were many more available.
Therefore, gathering data for more asci would allow for more accurate calculations of recombinance
frequencies.
Future experiments furthering information from this experiment could incorporate the same
treatments applied here with different species. Sordaria is a fungus, therefore we can compare its rates of
crossover frequency under x-ray radiation to the crossover frequency rates in animals, plants, bacteria, etc
under the same treatments. The crossover frequency rates across different species of fungi that replicate in
similar patterns to Sordaria could be compared the the crossover frequencies in Sordaria. These
experiments would give more information about how x-ray radiation encourages recombination to occur,
and whether thats a phenomena unique only to fungi or can span different kingdoms.
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