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 andplasmid purification, the tufA gene from an unknown green algae underwent geneticbarcoding which allows for environmental biologists to assess the biodiversity of organismsin a given ecosystem. The specimen was ultimately narrowed down to being a part of theUlva compressa clade, unable to determine the species of the specimen.

Introduction:

Genetic barcoding is one of the main ways used to assess biodiversity amongorganisms, especially if those organisms are similar in appearance. Algae is a typical type oforganism that genetic barcoding is used for due to their characteristics being somewhatsimilar to each other as the taxonomy specificity increases (Saunders G.W. 2005). In thisresearch project, the genetic composition of sea lettuce (Ulva spp.) is investigated todetermine the organism’s species. Ulva spp. have very similar visual characteristics; such astheir coloration, or their delicate blades. However their blades are broken down into twosubcategories: distromatic blades and monostromatic tubular structures. The identification ofthe 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 thebiodiversity of seaweed in certain environments. The most important reason why there is anincreased interest in the biodiversity of seaweed is to determine whether or not the speciespresent are invasive or endemic to that specific area. This is helpful in determining if theecosystem as a whole is doing well, as seaweed can be the determining factor on ecosystemhealth. Specifically, Ulva spp. is a competitively dominant algae in their ecosystem, therocky intertidal zone, due to their fast growth and high reproductive capability (SkipPomeroy, personal communication, October 2014). They are also the main prey item forspecies such as Littorina littorea, or the common periwinkle, who are predated on by largerorganisms; it can be extrapolated then, that the biodiversity of seaweed is an important aspectof 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 barcodingproject was Ulva spp. which was found in the lower rocky intertidal zone at low tide. Thistook place on September 8th, 2014 at approximately 12:10pm. Using a technique calledspecimen vouchering, the algae is preserved using various methods. In this case, the methodwas through photography; specifically while it was on herbarium paper prior to the dryingprocess. The unique specimen ID number for the vouchering process isBIO200.52.FA14.BJG33.

DNA isolation: A very small tissue sample (approximately the size of a dime) wascrushed up by adding liquid nitrogen to the sample in a mortar. After the mortar and pestlewas used to crush the sample, it had a paste-like texture to it. The DNA isolation techniquethat was used utilized the Qiagen DNeasy Plant mini Kit.

After the DNA was isolated, the quality and quantity was observed using Agarose GelElectrophoresis which separates DNA fragments by size. The DNA was stained with ethidiumbromide which made the DNA fluoresce under UV light. Using the Agarose Gel Electrophoresis labhandout 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 amplifiedexponentially 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 tufAforward primer #1 is GGNGCNGCNCAAATGGAYGG and the sequence of tufA reverse primer #2is CCTTCNCGAATMGCRAAWCGC. The amount of each primer added to the master mix was 0.25ul. Other materials added to the master mix included the Buffer, MgCl2, dNTPs, Taq polymerase, andwater, which brings the volume per reaction up to 20 ul. The amount of DNA added to the volume ofmaster mix per reaction was 5 ul which brought the final volume up to 25 ul. The PCR amplificationwas then put through agarose gel electrophoresis and it was determined whether or not the PCRamplification worked (Warren and Hagedorn. 2014).

Gene cloning: The PCR product is then cloned into an E. coli vector using an InvitrogenTOPO TA Cloning Kit, the PCR product was inserted into the plasmid successfully. After the cellswere incubated at 37°C for one hour, 100ul of the cells were placed on a plate that containedampicillin and X-gal which will help identify which colonies contain the plasmid that holds the PCRinsert. (Warren and Hagedorn. 2014).

Plasmid purification: After the cells were placed on a plate that contained ampicillin andX-gal, colonies were grown in overnight cultures. Once there were enough colonies, one colony thatcontained the plasmid was isolated using the Qiagen QIAprep Spin Miniprep kit. The entireprocedure can be found in the plasmid isolation and analysis lab handout and the QIAprep Miniprephandbook published in 2003. Once the plasmid DNA was purified, it was analyzed by cutting theinsert out of the plasmid vector through the use of EcoRI restriction enzyme. The presence of thetufA gene was determined using agarose gel electrophoresis (Warren and Hagedorn. 2014).

Sequence analysis: The plasmid DNA with the PCR insert was sent to University ofRhode Island’s sequencing facility for automated sequencing. The returned sequence was then editedby removing all N’s and all of the nucleotide sequence before the tufA forward primer #1. This newlyedited sequence was then entered into a number of databases, including a website that searchesGenBank for similar sequences, and a website that translates the nucleotide sequence into an aminoacid sequence. The nucleotide sequence was compared to two additional species sequences, both ofwhich 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.

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.

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.

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.

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 09November 2014.

2. Hofmann, L. C., Nettleton, J. C., Neefus, C. D. & Mathieson, C. Arthur. 2010. Crypticdiversity of Ulva (Ulvales, Chlorophyta) in the Great Bay Estuarine System (AtlanticUSA): 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 ofacrochaete 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 preliminaryappraisal holds promise for future applications Phil. Trans. R. Soc. B vol. 360 no. 14621879-1888

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 thebioinformatics lab handouts.

6. Watson, D. & Norton, T. 1985. Dietary preferences of the common periwinkle,Littorina littorea (L.). Journal of Experimental Marine Biology and Ecology.