inventory of Narragansett Bay

Transcription

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 8 th, 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 BIO 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, MgCl 2, 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 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 8 th, 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 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 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 AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. searched on 09 November Hofmann, L. C., Nettleton, J. C., Neefus, C. D. & Mathieson, C. Arthur 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, Rinkel, B. E., Hayes, P., Gueidan, C. & Brodie, J a molecular phylogeny of acrochaete and other endophytic green algae (ulvales, chlorophyta)1. J. Phycol. 48: Saunders, G. W Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications Phil. Trans. R. Soc. B vol. 360 no Warren, K. & Hagedorn, T 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 Dietary preferences of the common periwinkle, Littorina littorea (L.). Journal of Experimental Marine Biology and Ecology.