Through genetic barcoding of the tufA gene, an unknown green algae specimen was identified as belonging to the Ulva compressa clade. DNA was isolated from the specimen and the tufA gene was amplified via PCR. The gene was cloned and the nucleotide sequence analyzed and compared to sequences in GenBank. Phylogenetic analysis indicated the specimen was most similar to Ulva sp. BER-2007, but could not be identified to the species level. While in the U. compressa clade, further testing is needed to confirm its identity as a potential new species of Ulva in Narragansett Bay.

1. Genetic barcoding of Green Algae Ulva sp. for algal
inventory of Narragansett Bay
Benjamin Gibson
Abstract:
Through the use of DNA isolation, PCR amplification, plasmid cloning and
plasmid purification, the tufA gene from an unknown green algae underwent genetic
barcoding which allows for environmental biologists to assess the biodiversity of organisms
in a given ecosystem. The specimen was ultimately narrowed down to being a part of the
Ulva compressa clade, unable to determine the species of the specimen.
Introduction:
Genetic barcoding is one of the main ways used to assess biodiversity among
organisms, especially if those organisms are similar in appearance. Algae is a typical type of
organism that genetic barcoding is used for due to their characteristics being somewhat
similar to each other as the taxonomy specificity increases (Saunders G.W. 2005). In this
research project, the genetic composition of sea lettuce (Ulva spp.) is investigated to
determine the organism’s species. Ulva spp. have very similar visual characteristics; such as
their coloration, or their delicate blades. However their blades are broken down into two
subcategories: distromatic blades and monostromatic tubular structures. The identification of
the algal species Ulva is extremely difficult due to shared characteristics between species
(Hofmann et al. 2010).
The importance of this research is because of the recent interest in assessing the
biodiversity of seaweed in certain environments. The most important reason why there is an
increased interest in the biodiversity of seaweed is to determine whether or not the species
present are invasive or endemic to that specific area. This is helpful in determining if the
ecosystem as a whole is doing well, as seaweed can be the determining factor on ecosystem
health. Specifically, Ulva spp. is a competitively dominant algae in their ecosystem, the
rocky intertidal zone, due to their fast growth and high reproductive capability (Skip
Pomeroy, personal communication, October 2014). They are also the main prey item for
species such as Littorina littorea, or the common periwinkle, who are predated on by larger
organisms; it can be extrapolated then, that the biodiversity of seaweed is an important aspect
of the environment as they impact the majority of the rocky intertidal zone (Watson &
Norton. 1985).
Materials and Methods:
Collection of specimens: The specimen collected for this genetic barcoding
project was Ulva spp. which was found in the lower rocky intertidal zone at low tide. This
took place on September 8th, 2014 at approximately 12:10pm. Using a technique called
specimen vouchering, the algae is preserved using various methods. In this case, the method
was through photography; specifically while it was on herbarium paper prior to the drying
process. The unique specimen ID number for the vouchering process is
BIO200.52.FA14.BJG33.
DNA isolation: A very small tissue sample (approximately the size of a dime) was
crushed up by adding liquid nitrogen to the sample in a mortar. After the mortar and pestle
was used to crush the sample, it had a paste-like texture to it. The DNA isolation technique
that was used utilized the Qiagen DNeasy Plant mini Kit.

2. After the DNA was isolated, the quality and quantity was observed using Agarose Gel
Electrophoresis which separates DNA fragments by size. The DNA was stained with ethidium
bromide which made the DNA fluoresce under UV light. Using the Agarose Gel Electrophoresis lab
handout the quality and quantity of the DNA was determined (Warren & Hagedorn. 2014).
PCR amplification: Using PCR amplification, the isolated DNA is able to be cloned and amplified
exponentially to ensure there is enough of the target segment. Due to the sample being a green algae,
this was done using the tufA forward primer #1 and the tufA reverse primer #2. The sequence of tufA
forward primer #1 is GGNGCNGCNCAAATGGAYGG and the sequence of tufA reverse primer #2
is CCTTCNCGAATMGCRAAWCGC. The amount of each primer added to the master mix was 0.25
ul. Other materials added to the master mix included the Buffer, MgCl2, dNTPs, Taq polymerase, and
water, which brings the volume per reaction up to 20 ul. The amount of DNA added to the volume of
master mix per reaction was 5 ul which brought the final volume up to 25 ul. The PCR amplification
was then put through agarose gel electrophoresis and it was determined whether or not the PCR
amplification worked (Warren and Hagedorn. 2014).
Gene cloning: The PCR product is then cloned into an E. coli vector using an Invitrogen
TOPO TA Cloning Kit, the PCR product was inserted into the plasmid successfully. After the cells
were incubated at 37°C for one hour, 100ul of the cells were placed on a plate that contained
ampicillin and X-gal which will help identify which colonies contain the plasmid that holds the PCR
insert. (Warren and Hagedorn. 2014).
Plasmid purification: After the cells were placed on a plate that contained ampicillin and
X-gal, colonies were grown in overnight cultures. Once there were enough colonies, one colony that
contained the plasmid was isolated using the Qiagen QIAprep Spin Miniprep kit. The entire
procedure can be found in the plasmid isolation and analysis lab handout and the QIAprep Miniprep
handbook published in 2003. Once the plasmid DNA was purified, it was analyzed by cutting the
insert out of the plasmid vector through the use of EcoRI restriction enzyme. The presence of the
tufA gene was determined using agarose gel electrophoresis (Warren and Hagedorn. 2014).
Sequence analysis: The plasmid DNA with the PCR insert was sent to University of
Rhode Island’s sequencing facility for automated sequencing. The returned sequence was then edited
by removing all N’s and all of the nucleotide sequence before the tufA forward primer #1. This newly
edited sequence was then entered into a number of databases, including a website that searches
GenBank for similar sequences, and a website that translates the nucleotide sequence into an amino
acid sequence. The nucleotide sequence was compared to two additional species sequences, both of
which were found in the list of sequences provided by Professors Kerri Warren and Tara Hagedorn.
Results:
Specimen collection: The specimen
was collected underneath the Roger
Williams University learning
platform in the lower intertidal zone
on September 8th, 2014 at
approximately 12:10pm which was
during low tide (fig. 1). At first
observation, the specimen appears to
be characteristically similar to Ulva
lactuca.
Figure 1. Preserved
green algae specimen.

3. DNA isolation: The isolated DNA was
intact and fairly abundant (fig. 2). There
was no need to dilute the DNA and there
were no additional steps to take to
increase DNA abundance. After isolating
the DNA, the target was amplified using
PCR amplification.
Figure 2. Agarose gel electrophoresis of total
genomic algal DNA. Gel 3, lane 7. Lane 2
contains the DNA ladder
PCR amplification: The PCR
amplification was a success, using
agarose gel electrophoresis. In the gel,
there was a single band that was slender
(fig. 3). The segment of DNA that was
amplified was the tufA gene which is a
common gene in all green algae.
Figure 3. (below) Agarose gel
electrophoresis of amplified PCR
product which was the tufA gene.
Gel 1, in the lane marked with an
arrow.
Gene cloning: Once the PCR product
was placed into the plasmid of E. coli,
100 ul of the cells were placed on a plate
containing X-gal and ampicillin. The
blue colonies are blue because they make
a B-galactosidase enzyme that helps the
cells metabolize the X-gal. The only way
to acquire the enzyme is if they have the
Bgalactosidase gene. If the vector insert
is present, it will intercept the
Bgalactosidase gene which stops the
Bgalactosidase enzyme from being made
resulting in the white colored colonies.
The colonies that are blue do not contain
the vector insert, whereas the white
colonies contain the plasmid with the
inserted PCR product. There were a total
of 26 blue colonies and 74 white colonies
(fig. 4).
Figure 4. E. coli colonies. The white
colonies indicate that the PCR insert was
incorporated into the plasmid, and
transformed into the bacteria. The blue
colonies indicate that the PCR insert was not
incorporated into the plasmid.

4. Plasmid purification: Unfortunately
the plasmid from my specific sample
did not contain the expected gene,
however Nicholas Cmaylo and Cara
James both utilized the same
specimen and did have results. The
plasmid containing the gene is about
4,000 base pairs long (fig. 5).
Figure 5. (A) The DNA ladder used,
showing the amount of base pairs (bp) per
band level. (B) EcoRI gel plasmid
purification. Gel 2 Lane 3. (C) EcoRI gel
plasmid purification. Gel 1 Lane 7 labeled
with red arrow.
Sequence analysis of specimen (KW19BJG): The sequence of the original sample (KW19BJG)
confirmed that it was indeed a green algae. Using several sequences, the sequence of KW19BJG
was compared with the other tufA gene protein sequences provided by Professors Kerri Warren and
Tara Hagedorn (fig. 5).
Figure 5. Phylogenetic tree comparing the tufA gene protein sequences of several different species with the
original sample (KW19BJG).
The nucleotide BLAST results sent back a large quantity of sequences that the original specimen
was most closely related to. The number one sequence had an identical percentage of 99% and it
was Ulva sp. BER-2007 which doesn’t indicate the species name that the sequence belongs to.
However the most closely related sequence that had a species name associated with it was Ulva
compressa which had an identical percentage of 99% as well.

5. The nucleotide sequence of sample KW19BJG was compared to the sequences of two genetically
similar species of green algae deriving from nucleotide BLAST, two less similar species that are
common species found in this area, and two species of green algae that were found in the list of
sequences provided on bridges (fig. 6). The two species from the bridges list were chosen arbitrarily,
while the two less similar species were chosen based upon appearance. Ulva compressa is a tubular
type of sea lettuce whereas Ulva lactuca is a flat, more traditional type of sea lettuce.
Figure 6. Phylogenetic tree comparing the tufA gene nucleotide sequences of
several species of Ulva with the original sample (KW19BJG).
Discussion:
Using phylogenetic trees, KW19BJG was narrowed down to the Ulva compressa
clade. The clade contains two previously unknown species, Ulva sp. BER-2007 B125hi1 and
B25sm12, the former being the closest related species. The closest related species was found from a
sample that was isolated from the red alga Chondrus crispus collected from Sidmouth, South Devon,
England (Juliet Brodie, personal communication, November 19, 2014). Therefore, it is unlikely that
they are the exact same species. Using various reasons for process of elimination, it is determined
not to be any of the other species. Firstly, the location of the sampling site, being Narragansett Bay,
indicates that the specimen has to be native to the Atlantic Ocean. Secondly, through characterizing
the organism, dichotomous keys narrowed down the possibilities to Ulva fenestrata, Ulva fasciata
and Ulva lactuca. Using nucleotide sequence blasting, the most closely related species was one that
did not have a species name, as it was recently discovered: Ulva sp. BER-2007. It is most likely not
Ulva fenestrata because that is a pacific species of sea lettuce, which decreases the likelihood of it
being the mystery specimen; while Ulva fasciata and Ulva lactuca are not identical enough to the
sequence which makes either of them being the same species impossible. This leaves Ulva sp. BER-
2007 which is 99% identical to specimen KW19BJG. Therefore, this could represent an unidentified
species, but further testing with other molecular markers is required to confirm this. Without any
further testing using other genetic markers, such as the rbcL gene, the only conclusive evidence is
that the specimen is located within the Ulva compressa clade.

6. References
1. Guiry, M. D. & Guiry, G.M. 2014. AlgaeBase. World-wide electronic publication,
National University of Ireland, Galway. http://www.algaebase.org; searched on 09
November 2014.
2. Hofmann, L. C., Nettleton, J. C., Neefus, C. D. & Mathieson, C. Arthur. 2010. Cryptic
diversity of Ulva (Ulvales, Chlorophyta) in the Great Bay Estuarine System (Atlantic
USA): introduced and indigenous distromatic species, European Journal of Phycology,
45:3, 230-239
3. Rinkel, B. E., Hayes, P., Gueidan, C. & Brodie, J. 2012. a molecular phylogeny of
acrochaete and other endophytic green algae (ulvales, chlorophyta)1. J.
Phycol. 48:1020-7.
4. Saunders, G. W. 2005. Applying DNA barcoding to red macroalgae: a preliminary
appraisal holds promise for future applications Phil. Trans. R. Soc. B vol. 360 no. 1462
1879-1888
5. Warren, K. & Hagedorn, T. 2014. Lab handouts. This includes the: DNA Isolation,
Agarose Gel Electrophoresis, PCR, Bacteria plasmid cloning and transformation,
Bacterial colonies, plasmid DNA isolation, analyzing plasmid DNA and the
bioinformatics lab handouts.
6. Watson, D. & Norton, T. 1985. Dietary preferences of the common periwinkle,
Littorina littorea (L.). Journal of Experimental Marine Biology and Ecology.