Molecular Sequencing and Morphological Analysis for Determination of Pilobolus
Species
Bailey Graebner
Shane Crean
Dr. Dale Beach
Department of Biological and Environmental Sciences, Longwood University, Farmville, Virginia
23909
December 5, 2014
Abstract: With the ambition of confirming the collected isolate as genus Pilobolus and
furthermore identifying species, morphological characteristics were analyzed.
Morphological observations were obtained in shape, size, and measuring the lengths and
widths of multiple spores for two sporangium, which ultimately classified the isolate as
Pilobolus crystallinus, according to Zycha. In order to confirm the theory that the isolate
is of family Pilobolaceae, genus Pilobolus, and species crystallinus, molecular methods
were conducted to obtain DNA sequences of the isolate. The extraction and preparation
of genomic DNA for the rRNA and Beta-Tubulin genes began the molecular
methodology. PCR amplifications for the ITS rRNA and Beta-Tubulin genes were
assembled and tested for quality using an agarose gel to determine the amount of base
pairs for the genomic DNA. The rRNA gene was directly sequenced and showed the
isolate as either P. crystallinus or P. roridus. The Beta-Tubulin gene sustained a ligation
reaction to further the process and was cloned for DNA in preparation for a colony PCR
to collect the DNA coding sequence. The sequences from the rRNA and Beta-Tubulin
genes allowed for a multiple sequencing analysis and for the further examination of the
phylogenetic relationships among the isolates. The multiple sequencing analysis showed
a closer relationship for the isolate with a known P. crystallinus strand, supporting the
morphological sporangium measurements and leading to the conclusion of the isolate as
Pilobolus crystallinus.
Introduction
The genus Pilobolus classifies under the family Pilobolaceae, along with the
genera Pilaria and Utharomyces. The three genera are similar in having phototrophic
sporangiophores, reproducing asexually, growing within feces, and containing a vast
number of spores in each sporangium. Similar to that of Utharomyces, Pilobolus acquires
the trait of subsporangial swelling, which is vital to the life cycle of Pilobolus.
Figure 1: Structure of Pilobolus
Figure 2: Classification of Pilobolus
The lifecycle of Pilobolus starts within the feces of cows and horses, where hyphae begin
to develop throughout the dung. After a period of time, trophocysts start to develop along
the hyphae, producing colors of orange and yellow, and grow directly from the hyphae in
a tube-like structure. Mature trophocysts act as bases for the developing sporangiophores
and sporangium: the sporangiohore grows in a tube-like structure, and the sporangium is
located on the surface of the produced sporangiophore. Each sporangium, similar to that
of Pilaria and Utharomyces, contains a vast number of spores, approximately twenty
thousand, which are formed by a coding technique allowing spore walls to surround each
nuclei within the sporangium. Meanwhile, the black sporangium is supported by the
sporangiophore and subsporangial swelling starts to occur. The swelling creates a
pressure that acts against the sporangium in a manner so that the sporangium is
discharged from the structure. The sporangial discharge is aimed among a sporangium
trajectory that is influenced by the organism’s attraction to the sunlight. The sporangium
trajectory normally occurs on a nearby plant, where grazing cows or horses then
consume, digest, and excrete the new spores within the produced waste. This ultimately
will then lead to the development of hyphae within the newly produced feces, allowing
for the repetition for the lifecycle of Pilobolus.
Figure 3: Pilobolus Life Cycle
The life cycle is critical to confirming the isolate as Pilobolus, for if subsporangial
swelling occurs, the isolate is that of Pilobolus. The objective of discovering the species
required a morphological analysis of the spores and obtaining molecular sequences,
which were also utilized to confirm the isolate as genus Pilobolus.
Materials and Methods
Isolates thought to be of Pilobolus species were collected from a farm located in
Cumberland, Virginia. Three cultures were grown individually in a sealed petri dish and
were incubated for one week in a 12-hour light and 12-hour dark cycle at a temperature
of approximately 20-22°C to allow time for the isolates to grow. Sporangia had become
attached to the lid of the petri dish during the growth period, which were evidence that
subsporangial swelling had occurred. The sporangia were obtained utilizing a needle and
were then transferred onto slides to use the Ocular micrometer to measure the sporangial
sizes. The two sporangia that were transferred from the culture were measured;
furthermore, 10 spores from the first sporangium and 14 spores from the second
sporangium were measured individually in length, width, and the ratio of length to width.
Averages were then calculated for each measurement to provide morphological evidence
to compare against the measurements of Zycha and determine the species of the isolate.
Liquid cultures of the Pilobolus isolates were then prepared and utilized to
prepare genomic DNA from “grinding up” the hyphae. DNA extraction was conducted
by following the procedure of the MoBio UltraClean Soil Kit. This procedure was
followed directly with the exception that the tubes were not placed in a heating block.
After the process, the DNA suspension was stored at -20°C.
The “cleaned,” genomic DNA prepared was then used to assemble a 50 microliter
PCR in order to obtain the rRNA and the Beta-Tubulin gene from the isolates. Two tubes
were obtained to place 21 microliters of water and 25 microliters of master mix (Tris-
HCl, MgCl, KCl, dNTP, and TAQ) into each tube. The assembled PCR reactions were
then placed in a thermal profile, a series of temperatures and times (altered slightly from
standardized protocol): 4 minutes at 94°C, followed by a cycle of 30 seconds at 94°C, 1
minute at 50°C, and one minute and 30 seconds at 72°C, which was repeated 30 times
consecutively. Then the PCR reactions underwent 7 minutes under the conditions of 72°C
and continued to stay in the thermal profile at 12°C over night. Ten microliters of each
reaction were loaded onto an agarose gel to verify the product size. A PCR clean-up
protocol from the manufacturer was then followed for the rRNA reaction to ensure that
the “clean” DNA was sufficient for sequencing.
A ligation reaction (directed by Invitrogen K2020 Kit) was then prepared for the
Beta-Tubulin reaction. 2 microliters of the Beta-Tubulin was added to a tube along with 1
microliter of 10X Ligation Buffer, 2 microliters of vector (PCR 2.1), and 2 microliters of
water for a 10-microliter reaction. The ligation was then incubated in a thermal cycler
block for approximately 16 hours (overnight) then frozen until transfer. The transfer
portion of the protocol followed that of the manufacturer’s. The transformation of the
ligation was to complete the cloning of Beta Tubulin fragment. The PCR vector 2.1 was
utilized for cloning. Aseptic techniques were used for the cloned Beta-Tubulin to spread
onto LB-Amp-XGAL (40%) plates. Colonies of white color were selected to perform a
PCR. The PCR consisted of 12.5μl of 2X master mix, 1μl of primer mix, 11.5 μl of water,
and the cells. The colony PCR then allowed for direct sequencing results. A multiple
sequencing analysis was then conducted, using the sequencing results, for the rRNA
genes and the Beta-Tubulin genes once the sequencing results had been delivered.
Conclusions
The measurements of the isolate’s morphology classify the isolate as Pilobolus
crystallinus, according to Zycha’s measurements: the length measures as 5μm-12μm and
the width as 3μm-7μm. As represented in Figure 4, 10 spores were measured for the first
selected sporangium and 14 spores for the second sporangium. The averages for the
spores of the first sporangium concluded as 5.0 μm for length, 5.20 μm, and 1.06μm for
the width to length ratio. The averages for the spores of the second sporangium
concluded as 5.07 μm for the length, 4.43 μm for the width, and 0.84 μm for the width to
length ratio. The intermediate and elliptical shape of the spores that were observed also
classifies the isolate as Pilobolus crystallinus on a morphological level.
Figure 4: Morphological data concluded from spore
measurements. Values in averaging the results of the two
sporangium:
Length: 5.035 μm
Width: 4.815 μm
Width/Length: 0.95 μm
Figure 5: Agaraose Gel Results of
rRNA and Beta-Tubulin: rRNA and
Beta-Tubulin showed correct
amount of base pairs (700/700 and
900/900 respectively).
The morphological data of the spores supports the results of the ribosomal RNA
sequencing: The rRNA and Beta- Tubulin genomic DNA was amplified through an
agarose gel to ensure the correct number of base pairs was obtained (see figure 5). The
results from the amplification of the rRNA and Beta-Tubulin PCR gel demonstrated that
the sizes of the bands for rRNA and Beta-Tubulin were 700 and 900, respectively. The
BLAST1 data results provided numerical information that explains the sequence as 85%
match of identities to P. crystallinus and 86% match of identities to P. roridus. The
multiple sequencing analysis of the rRNA (see figure 7) isolates showed that the isolate
(M7_Culture_rRNA) was more-closely related to the strain of Pilobolus crystallinus.
Figure 6: The BLAST data results show the amount of identities in which the sequencing of the isolate
matched to known strains of P. crystallinus and P. roridus.
Figure 7: Multiple sequencing analysis2 from rRNA of the
isolates with known P. crystallinus strain and P. roridus strain
The cloning of the Beta-Tubulin, necessary to sequence the gene, resulted in a
normal colony growth (of blue and white colonies). The Colony PCR reaction also
resulted in sequencing results, which were compared among the BLAST1 database. The
results showed that the Beta-Tubulin had 86% of its identities matching that of genus
Utharomyces (see figure 8). The sequence was then used to compare against the other
isolates and construct a phylogenetic tree2 (see figure 9). The Beta-Tubulin phylogenetic
tree showed that the relationship of the forward reaction of the PCR for the isolate
(Biol121-M7-5_M13F) and also for the reverse reaction (Biol121-M7-5_M13R).
Figure 8: Beta-tubulin BLAST1 results showing an 86% match to genus Utharomyces
Figure 9: Multiple sequencing analysis of Beta-Tubulin for the forward and reverse
reactions.
Discussion
The rRNA sequencing (from BLAST1) results concluded that the Pilobolus isolate
belonged to the species of Pilobolus crystallinus or Pilobolus roridus. It is evident that
Pilobolus crystallinus and Pilobolus roridus are similar on an evolutionary timeline, as a
result of a possible mutation or mechanism of natural selection. However, the multiple
sequencing analysis concluded that the isolate is more-closely related to P. crystallinus
rather than that of P. roridus. Furthermore, the morphological data of the isolate’s spores
distinctly identified the isolate as Pilobolus crystallinus, according to Zycha. In
comparison of the results, the evidence from the morphological data and the sequencing
results support the theory that the isolate is Pilobolus crystallinus.
Other experiments in developing this research would be to test other methods of
PCR to obtain more-defined results. For example, methods utilized in Wong’s
“Evaluation of Typing of Vibrioparahaemolyticus by Three PCR Methods Using Specific
Methods,” may serve beneficial to the research: Ribosomal gene spacer sequence (RS),
repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive
intergenic consensus sequence (ERIC) were used as PCR mechanisms to determine the
strand of the pathogen. This also involved the amplification of three sets of
oligonucleotide primers. For RS, a pair of 15-mer primers was designed as the root of the
spacer sequences of 16s and 23s ribosomal DNAs. For REP, the primers consisted of
multiple nucleotides with a pair of 18-mer primers. Finally for ERIC, a pair of 220-mer
primers was created based on the core repeated sequence. Utilizing different primers and
different mechanisms of PCR for the experiment may allow for improved results in that
these certain PCR mechanism may prove to be more effective.
Reflection
Some of the reoccurring problems that we encountered were that our numbers
were always below what the rest of the group had as their results. This was frustrating
because we thought we were completing the assignments correctly because we followed
the instructions. For example, when we made the DNA sample and then tested it to see
the quality of DNA, ours was always below the average. This caused some of our results
to be hindered, whether it seemed as if we were following the procedure or not.
However, we did believe our results were a legitimate product of a research
program because we were able to get sufficient sequence results (86%) and we followed
the correct procedure to get the results. The results could have been of better quality,
since there were occurrences in lab that, for time purposes, we shortened the
recommended time for certain steps in the procedures.
The project has helped us understand that a research program can be about
anything. Normally, the first time someone takes a science class you learn about the
scientific method, but this was the first time we were able to genuinely follow a model
similar to the scientific method from start to finish. The biggest part we learned and now
understand about a research program is how important the writing part of that is. It is one
thing to be able to do the experiment and understand it, but just as important is being able
to put all that information on paper for others to observe. The downside to the project is
that there was a limited range in which the each project could differentiate from each
other, although it was beneficial to compare results.
Sources/References
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Sheehan, Donald G. Ruch. “Phlyogeny of Pilobolaceae.” The Mycological
Society of America 10.3852 (2011): 36-44. Web.
O’Donnell, Kerry, and Francois M. Lutzoni, Todd J. Ward, Gerald L. Benny.
“Evolutionary Relationships among Mucoralean Fungi (Zygomycota): Evidence
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Phylogeny.fr: Robust Phylogenetic Analysis For The Non-Specialist. Réseau National de
Génopoles (RNG), 2003. Web. 11 Nov. 2014.
Wong, Hin-Chung, and Chih-Hsueh, Lin. “Evaluation of ‘Typing of
Vibrioparahaemolyticus by Three PCR Methods Using Specific Primers.” The
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1. Basic Local Alignment Search Tool (BLAST). National Library of Medicine, 2009.
Web. 4 Nov. 2014.
2. Phylogeny.fr: Robust Phylogenetic Analysis For The Non-Specialist. Réseau National
de Génopoles (RNG), 2003. Web. 11 Nov. 2014.