11th ICRS Abstract book - Nova Southeastern University
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11th ICRS Abstract book - Nova Southeastern University

11th ICRS Abstract book - Nova Southeastern University 11th ICRS Abstract book - Nova Southeastern University

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Poster Mini-Symposium 6: Ecological and Evolutionary Genomics of Coral Reef Organisms 6.152 Validation of Reference Genes for Quantitative Real-time RT-PCR Normalization In Stress-Induced Pocillopora meandrina. Lauren PAGARIGAN* 1 , Misaki TAKABAYASHI 1 1 University of Hawaii Hilo, Hilo, HI Normalization of quantitative real time RT-PCR (qRT-PCR) is needed to obtain accurate estimates of gene expression. Today, the most commonly used method to normalize target genes is through the use of reference or housekeeping genes. Reference genes however, need extensive validation to show constitutive expression under different conditions and between tissue types. qRT-PCR has only been used for a handful of anthozoa gene expression studies and even smaller amount of gene expression studies evaluating natural and anthropogenic stressors. In these studies, often only one reference gene was used. Commonly used reference genes for anthozoa include beta-actin, 18S, and EF1 alpha. In this study we used common reference genes found in past studies (18S, B-actin, EF1 alpha) in addition to POU homeodomain protein brn1 and putative senescence associated protein for the evaluation of quality reference genes for anthozoa exposed to stress. This study characterized the expression of these five candidate reference genes for the scleractinian coral, P.meandrina, after a chronic exposure to ammonium and heat shock treatments. All five reference genes exhibited variable expression. GeNorm was used to choose three housekeeping genes with the most stable expression ratio in our samples for the calculation of an RT-PCR normalization factor that is more stable than using one reference gene alone. 6.153 Field Characterization Of Stress Gene Expression Patterns in Hermatypic Corals Exposed To Effluent From The Delray Beach Outfall Tonya SHEARER* 1 , Sara EDGE 2 1 School of Biology, Georgia Institute of Technology, Atlanta, GA, 2 Robertson Coral Reef Program, Harbor Branch Oceanographic Institute, Ft. Pierce, FL Degradation of coral reef systems is an increasing concern throughout the world with multiple synergistic factors contributing to the decline of reef habitats. Nutrient enrichment is one of many stressors that has been demonstrated to directly and indirectly negatively impact coral reef organisms in controlled laboratory and in situ experiments. Recently, a local environmental group has correlated nutrient-rich effluent from Delray Beach (South Florida) ocean outfall with persistent blooms of cyanobacteria at nearby, Gulf Stream Reef (GSR). In order to determine whether nutrient enrichment directly (by suppression/enhancement of physiological functions) and/or indirectly (by physical contact with blooming cyanobacteria) influences expression of stress genes, Montastraea cavernosa colonies have been sampled from GSR and reef habitats upstream and downstream from this outfall. Using microarray technology, differential expression of stress genes in corals from potentially impacted sites relative to unaffected sites was measured to determine if different populations are reacting to various levels of stress and whether a nearby wastewater treatment outfall may be contributing to the deterioration of certain reef communities. 6.154 Quantification Of Algal Endosymbionts (Symbiodinium) in Coral Using Real-Time Pcr Jos MIEOG* 1 , Madeleine VAN OPPEN 2 , Ray BERKELMANS 2 , Wytze STAM 1 , Jeanine OLSEN 1 1 University of Groningen, Haren, Netherlands, 2 Australian Institute of Marine Science, Townsville, Australia Understanding the flexibility of the endosymbioses between scleractinian corals and single-cell algae of the genus Symbiodinium will provide valuable insights into the future of coral reefs. Here, a real-time PCR assay is presented to accurately determine the cell densities of Symbiodinium clades C and D in the hard coral Acropora millepora, which can be extended to other coral-symbiont associations in future. The assay targets single- to low-copy genes of the actin family of both the coral and algal symbiont. Symbiont densities are expressed as the ratio of Symbiodinium cells to each host cell (S/H ratio, error within 30%), but can also be normalized to coral surface area. Greater accuracy in estimating ratios of associations involving multiple clades (error within 60%) is achieved compared with similar assays based on highcopy ribosomal DNA loci (error within an order of magnitude). Healthy adult A. millepora containing ~1.4 x 10 6 zooxanthellae per cm 2 (as determined by haemocytometer counts) had S/H ratios of ca. 0.1, i.e., ~1 symbiont cell per 10 host cells. In severely bleached colonies this ratio decreased to less then 0.005. Because of its capacity to accurately determine both densities and ratios of multiple symbionts within one sample, the assay will open the door for novel research into the mechanisms of symbiont shuffling and switching. 6.155 Functional Potential Of Microbial Communities Associated With pseudopterogorgia Americana From The Southern Coast Of Puerto Rico Nikole E. KIMES* 1,2 , Joy D. VAN NOSTRAND 3 , Jizhong Z. ZHOU 3 , Ernesto WEIL 4 , Pamela J. MORRIS 1,2 1 Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC, 2 Hollings Marine Laboratory, Charleston, SC, 3 Department of Botany and Microbiology, University of Oklahoma, Norman, OK, 4 Department of Marine Sciences, University of Puerto Rico, Mayaquez, Mayaquez, Puerto Rico Coral-associated microbial communities are increasingly recognized as important components of the coral holobiont that influence coral health and disease. It has been suggested that these communities contribute to the health of the coral ecosystem through energy and nutrient cycling, as well as provide corals with protection against pathogens. There are few studies, however, that directly address the functional role of these communities and their ecological potential. In the present study, we examined the functional gene potential of the coralassociated microbial community found in the surface mucopolysaccharide layer (SML) of a common Caribbean coral, Pseudopterogorgia americana. SML samples were collected in March of 2006 off the southern coast of Puerto Rico at La Paguera. DNA was extracted and amplified for use in a functional gene array, the GeoChip, which contains 24,000 probes targeting 10,000 functional genes associated with key biogeochemical processes. Our preliminary data indicate that this microbial community possesses 1189 genes known to play a role in numerous ecological processes. These processes include carbon degradation/fixation (159 genes), dissimilatory sulfite reduction (94 genes), metal homeostasis (203 genes), methane generation and oxidation (50 genes), nitrogen fixation/reduction (145 genes), ammonification (94 genes), and organic chemical degradation (444 genes). This data, strongly suggests that the coral-associated microbial community found in the SML of P. americana may play an active role in maintaining the coral holobiont through cycling of key nutrients, metals and organic contaminants. In on-going studies, we are evaluating the functional potential of healthy as well as diseased Montastrea faveolata SML samples from the same region. 301

Poster Mini-Symposium 6: Ecological and Evolutionary Genomics of Coral Reef Organisms 6.156 Using BACs to compare the genomes of two Caribbean corals, Acropora palmata and Montastraea faveolata Collin CLOSEK* 1 , Christian VOOLSTRA 1 , Michael DESALVO 1 , Shinichi SUNAGAWA 1 , Alejandro REYES 1 , Monica MEDINA 1 1 Natural Sciences, University of California, Merced, Merced, CA We have constructed Bacterial Artificial Chromosome (BAC) libraries of two Caribbean coral species, Acropora palmata and Montastraea faveolata. These BAC clones allow the possibility of having stable large insert libraries for genomic studies. The constructed libraries are estimated to have a10X coverage, and an average insert size of 130kb. We are currently using 454 sequencing of several homologous BAC clones. Our target chromosomal regions were chosen because they contain genes that were differentially expressed in transcriptome experiments with multiple developmental stages and stress conditions (i.e. thermal bleaching, disease). We intend to use these data for cnidarian comparative genomics (i.e. identify potential regulatory regions, gene annotation, synteny analysis). To our knowledge, this is the first study in which large genomic regions of corals belonging to the complex and robust lineages will be sequenced. We can now take advantage of the availability of the complete genomes for the sea anemone Nematostella vectensis and the hydrozoan Hydra magnipapillata to study not only scleractinian, but also cnidrian genome evolution. This study is a building block towards the completion of these corals’ genomes. 6.157 The Symbiodinium Genome Sequence: A Progress Report Mary Alice COFFROTH* 1 , Thomas DOAK 2 , Jodi SCHWARZ 3 , Mónica MEDINA 4 1 Geology, University at Buffalo, Buffalo, NY, 2 Dept of Biology, Indiana University, Bloomington, IN, 3 Biology, Vassar College, Poughkeepsie, NY, 4 School of Natural Sciences, UC-Merced, Merced, CA We (TG Doak, RB Moore, C Delwiche, O Hoegh-Guldberg, & MA Coffroth) submitted two proposals to the National Health Genome Research Institute (NHGRI) in fall 2003, one for a BAC library for Symbiodinium, and one for complete genomic sequence of one or more isolates. As a result of the BAC white paper, NHGRI approved funding for BAC library construction. This project will result in an arrayed set of BAC clones, with matching blots that researchers can request for probing. In response to the sequencing white paper, NHGRI approved exploratory sequencing, to determine the feasibility of sequencing a complete genome. This is ongoing at the Washington University St. Louis Genome Center under the direction of Sandra Clifton. You can obtain the genome white paper here: http://www.auburn.edu/~santosr/pdf/Symbiodinum_WPl+Appendix_v2.pdf. For the initial efforts, we have focused on a Caribbean clade B strain isolated by Mary Alice Coffroth, which is also the subject of an EST project that is ongoing at the Joint Genome Institute, directed by Mόnica Medina. Modern annotation of genomes depends both on good EST sets for gene finding, and on the comparison of related genomes. We hope to include an additional clade isolate, and are looking for collaborators who can provide DNA for a strain that also has an ongoing EST project. We will report on the ongoing efforts to provide DNA in adequate quantity and quantity to keep these genome projects moving. This aspect of a genome project is not specifically funded, but rather depends on volunteer efforts by the interested community of researchers. Thus, we are very much interested in talking to researchers with expertise in these areas. We have hoped that one salient outcome of this project will be a focused meeting of people doing molecular biology in dinoflagellates, irrespective of what dinoflagellate. 6.158 Development Of Improved Diversity Arrays Technology (idart) For Whole-Genome Genotyping Of Corals Shi WANG* 1 , Naila ALIEVA 1 , Lingling ZHANG 1 , Elisha MEYER 1 , Galina AGLYAMOVA 1 , Tom JUENGER 1 , Mikhail MATZ 1 1 Section of Integrative Biology, School of Biological Sciences, University of Texas at Austin, Austin, TX Molecular marker technologies are useful tools for assessing important ecological and evolutionary issues such as genetic diversity, gene flow and speciation. We are currently developing a novel microarray-based whole-genome genotyping technology called iDART. iDART make it possible to simultaneously discover and genotype as many as 200-300 codominant genetic markers in a single hybridization experiment, and thus is easily applicable to non-model organisms such as corals. We have recently demonstrated the feasibility of this approach in the model study and currently developing the DNA chips for Acropora millepora and several other corals, along with the procedures for genotyping individual coral larvae. The first iDART application will be the genome-wide association study of the stress resilience and dispersal potential in the coral A. millepora. The spatial and population genetic structure of the identified genomic loci will be analyzed for the evidence of recent natural selection related to the ongoing climate change. 6.159 Diversity in Populations Of Free-Living symbiodinium From A Caribbean And Pacific Reef Mackenzie MANNING* 1 , Ruth GATES 1 1 Hawaii Institute of Marine Biology, Kaneohe, HI The presence and diversity of free-living dinoflagellates belonging to the endosymbiotic genus Symbiodinium was explored in seawater samples collected above coral reefs in Kāne‘ohe Bay O‘ahu, Hawai‘i, USA, and Puerto Morelos, Quintana Roo, Mexico. Two genetic markers were used to assess Symbiodinium diversity in the water column; the internal transcribed spacer 2 region of the nuclear ribosomal array (ITS2), and a hyper-variable region in Domain V of the large subunit (23S) of the chloroplast ribosomal array (cp23S-HVR). Sequencing of cloned gene fragments reveals that clade B, C, D and H Symbiodinium are detectable in the seawater samples. In addition to the previously described types B1, C3, C15, C21 and D1, novel Symbiodinium sequences belonging to clades B and C were also retrieved. The majority of Symbiodinium sequences recovered from Kāne‘ohe Bay belonged to clade C and those from Puerto Morelos to clade B, a pattern that reflects the dominant types of Symbiodinium found in endosymbiosis with scleractinian corals in these two areas. This study represents the first direct assessment of Symbiodinium diversity in waters adjacent to coral reefs located in the Caribbean and the Pacific and confirms the presence of Symbiodinium in this compartment of the ecosystem. These data provide context for future studies examining spatial and temporal patterns in the availability of Symbiodinium in the water column, work that will ultimately promote a greater understanding of the interactions between symbiotic dinoflagellates and their environmentally sensitive benthic hosts. 302

Poster Mini-Symposium 6: Ecological and Evolutionary Genomics of Coral Reef Organisms<br />

6.156<br />

Using BACs to compare the genomes of two Caribbean corals, Acropora palmata<br />

and Montastraea faveolata<br />

Collin CLOSEK* 1 , Christian VOOLSTRA 1 , Michael DESALVO 1 , Shinichi<br />

SUNAGAWA 1 , Alejandro REYES 1 , Monica MEDINA 1<br />

1 Natural Sciences, <strong>University</strong> of California, Merced, Merced, CA<br />

We have constructed Bacterial Artificial Chromosome (BAC) libraries of two Caribbean<br />

coral species, Acropora palmata and Montastraea faveolata. These BAC clones allow the<br />

possibility of having stable large insert libraries for genomic studies. The constructed<br />

libraries are estimated to have a10X coverage, and an average insert size of 130kb. We<br />

are currently using 454 sequencing of several homologous BAC clones. Our target<br />

chromosomal regions were chosen because they contain genes that were differentially<br />

expressed in transcriptome experiments with multiple developmental stages and stress<br />

conditions (i.e. thermal bleaching, disease). We intend to use these data for cnidarian<br />

comparative genomics (i.e. identify potential regulatory regions, gene annotation, synteny<br />

analysis). To our knowledge, this is the first study in which large genomic regions of<br />

corals belonging to the complex and robust lineages will be sequenced. We can now take<br />

advantage of the availability of the complete genomes for the sea anemone Nematostella<br />

vectensis and the hydrozoan Hydra magnipapillata to study not only scleractinian, but<br />

also cnidrian genome evolution. This study is a building block towards the completion of<br />

these corals’ genomes.<br />

6.157<br />

The Symbiodinium Genome Sequence: A Progress Report<br />

Mary Alice COFFROTH* 1 , Thomas DOAK 2 , Jodi SCHWARZ 3 , Mónica MEDINA 4<br />

1 Geology, <strong>University</strong> at Buffalo, Buffalo, NY, 2 Dept of Biology, Indiana <strong>University</strong>,<br />

Bloomington, IN, 3 Biology, Vassar College, Poughkeepsie, NY, 4 School of Natural<br />

Sciences, UC-Merced, Merced, CA<br />

We (TG Doak, RB Moore, C Delwiche, O Hoegh-Guldberg, & MA Coffroth) submitted<br />

two proposals to the National Health Genome Research Institute (NHGRI) in fall 2003,<br />

one for a BAC library for Symbiodinium, and one for complete genomic sequence of one<br />

or more isolates. As a result of the BAC white paper, NHGRI approved funding for BAC<br />

library construction. This project will result in an arrayed set of BAC clones, with<br />

matching blots that researchers can request for probing. In response to the sequencing<br />

white paper, NHGRI approved exploratory sequencing, to determine the feasibility of<br />

sequencing a complete genome. This is ongoing at the Washington <strong>University</strong> St. Louis<br />

Genome Center under the direction of Sandra Clifton. You can obtain the genome white<br />

paper here: http://www.auburn.edu/~santosr/pdf/Symbiodinum_WPl+Appendix_v2.pdf.<br />

For the initial efforts, we have focused on a Caribbean clade B strain isolated by Mary<br />

Alice Coffroth, which is also the subject of an EST project that is ongoing at the Joint<br />

Genome Institute, directed by Mόnica Medina. Modern annotation of genomes depends<br />

both on good EST sets for gene finding, and on the comparison of related genomes. We<br />

hope to include an additional clade isolate, and are looking for collaborators who can<br />

provide DNA for a strain that also has an ongoing EST project. We will report on the<br />

ongoing efforts to provide DNA in adequate quantity and quantity to keep these genome<br />

projects moving. This aspect of a genome project is not specifically funded, but rather<br />

depends on volunteer efforts by the interested community of researchers. Thus, we are<br />

very much interested in talking to researchers with expertise in these areas. We have<br />

hoped that one salient outcome of this project will be a focused meeting of people doing<br />

molecular biology in dinoflagellates, irrespective of what dinoflagellate.<br />

6.158<br />

Development Of Improved Diversity Arrays Technology (idart) For Whole-Genome<br />

Genotyping Of Corals<br />

Shi WANG* 1 , Naila ALIEVA 1 , Lingling ZHANG 1 , Elisha MEYER 1 , Galina AGLYAMOVA 1 ,<br />

Tom JUENGER 1 , Mikhail MATZ 1<br />

1 Section of Integrative Biology, School of Biological Sciences, <strong>University</strong> of Texas at Austin,<br />

Austin, TX<br />

Molecular marker technologies are useful tools for assessing important ecological and<br />

evolutionary issues such as genetic diversity, gene flow and speciation. We are currently<br />

developing a novel microarray-based whole-genome genotyping technology called iDART.<br />

iDART make it possible to simultaneously discover and genotype as many as 200-300 codominant<br />

genetic markers in a single hybridization experiment, and thus is easily applicable to<br />

non-model organisms such as corals. We have recently demonstrated the feasibility of this<br />

approach in the model study and currently developing the DNA chips for Acropora millepora<br />

and several other corals, along with the procedures for genotyping individual coral larvae. The<br />

first iDART application will be the genome-wide association study of the stress resilience and<br />

dispersal potential in the coral A. millepora. The spatial and population genetic structure of the<br />

identified genomic loci will be analyzed for the evidence of recent natural selection related to<br />

the ongoing climate change.<br />

6.159<br />

Diversity in Populations Of Free-Living symbiodinium From A Caribbean And Pacific<br />

Reef<br />

Mackenzie MANNING* 1 , Ruth GATES 1<br />

1 Hawaii Institute of Marine Biology, Kaneohe, HI<br />

The presence and diversity of free-living dinoflagellates belonging to the endosymbiotic genus<br />

Symbiodinium was explored in seawater samples collected above coral reefs in Kāne‘ohe Bay<br />

O‘ahu, Hawai‘i, USA, and Puerto Morelos, Quintana Roo, Mexico. Two genetic markers were<br />

used to assess Symbiodinium diversity in the water column; the internal transcribed spacer 2<br />

region of the nuclear ribosomal array (ITS2), and a hyper-variable region in Domain V of the<br />

large subunit (23S) of the chloroplast ribosomal array (cp23S-HVR). Sequencing of cloned<br />

gene fragments reveals that clade B, C, D and H Symbiodinium are detectable in the seawater<br />

samples. In addition to the previously described types B1, C3, C15, C21 and D1, novel<br />

Symbiodinium sequences belonging to clades B and C were also retrieved. The majority of<br />

Symbiodinium sequences recovered from Kāne‘ohe Bay belonged to clade C and those from<br />

Puerto Morelos to clade B, a pattern that reflects the dominant types of Symbiodinium found in<br />

endosymbiosis with scleractinian corals in these two areas. This study represents the first direct<br />

assessment of Symbiodinium diversity in waters adjacent to coral reefs located in the Caribbean<br />

and the Pacific and confirms the presence of Symbiodinium in this compartment of the<br />

ecosystem. These data provide context for future studies examining spatial and temporal<br />

patterns in the availability of Symbiodinium in the water column, work that will ultimately<br />

promote a greater understanding of the interactions between symbiotic dinoflagellates and their<br />

environmentally sensitive benthic hosts.<br />

302

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