Summary of Professional Exchanges on Coral Genomics
The mountainous star coral, Montastraea faveolata, is a massive, mound-shaped coral colony found in the Gulf of Mexico Flower Gardens, the Florida Keys reef tract, throughout the Caribbean, and other areas of the tropical Atlantic. (Credit: Andrew Bruckner, NOAA Fisheries)
The discussion was initiated by a small group of research partners who asked the coral reef community (via the Coral-list) to provide letters of support for a proposed project to sequence the genome of the reef-building lobe coral, Porites lobata. The sequencing of a coral genome would provide a foundation for new avenues of coral scientific research, as well as provide a basis for technology development that could benefit coral reef resource management. Once the genome is sequenced, the work will be published and the entire genome would be made freely accessible to the public. Competition for funds and facilities for genome sequencing is keen and this might be the one chance to have a reef- building coral sequenced in the near future. Many other groups are preparing for large scale sequencing projects and there are only a finite number of laboratories and centers available that have the resources and methodologies to do this work. It is important for the community to come together and explain how and why the information gained from the sequencing would benefit the furtherance of coral research, and what the payoff would be for science and the public.
Responses to the initial posting to the Coral-List were mostly about which species or group of scleractinian corals would be the best candidate(s) for genome sequencing. The project had selected Porites lobata, in part, because of "its rising importance as a 'laboratory rat' in coral exotoxicology, coral cell biology, coral immunity and coral neurophysiology." P. lobata was also chosen because of its widespread distribution in the Indian and Pacific Oceans, the Red Sea, and Persian Gulf. Another important advantage of Porites over others, such as the acroporids (elkhorn, staghorn and table corals) and star corals, Montastraea spp, is that Porites lacks some of the various biochemical interfering substances that make it very difficult to apply molecular and biochemical techniques, without significant artifact which is present in many coral families. Finally, P. lobata and P. asteroides (mustard hill coral) show a high degree of similarity for many of their enzymes and genes, and it should be easy to adapt technologies that would utilize the gene sequence information of P. lobata (such as PCR, gene array, ELISA's, real-time PCR, and immuno-histology) to P. asteroides.
The elkhorn coral, Acropora palmata, is a tall, tree-like coral with flattened, fan-like branches. It is found in the tropical Atlantic. (Credit: Andrew Bruckner, NOAA Fisheries)
One participant in these discussions replied that the selection of Porites lobata as the candidate for sequencing came as a complete surprise. While having a coral genome sequenced would be of great benefit to science, it is critical to select a proper species. He would have preferred a species of Acropora or Montastraea, as they both seemed to be more advanced in molecular terms than Porites. If the species were to be a Caribbean species, another participant offered, one of the Montastraea annularis complex species (boulder star corals) would be his first choice, given that this is probably the most important coral today on the Caribbean reefs, and it is affected by multiple diseases. His second choice would be Acropora palmata for the same reasons.
Other participants quickly joined the discussion and suggested other candidate species for sequencing. One advocated Acropora arabia from Kuwait because it has adapted to a wide range of environmental stressors, such as wide temperature variations, high salinities, high turbidity, petroleum hydrocarbons on the surface, etc. Perhaps genetic insights could be gained to help other fast-growing acroporids to maintain their distribution better. Another participant added that there are several species of Porites and Acropora in the Persian Gulf area that have been subjected to, and withstood extreme ranges of temperature (17-35C) and high salinities (40-42+ ppt).
Another participant joined the discussion and questioned the long-term value of sequencing the genome of a threatened or endangered, or regionally local or endemic, species. The interpretation of the significance of the genomic results, in terms of vulnerability, or survival or distribution, would require a great deal more genetic information before one could begin to reap the benefits. A preferable strategy, he added, would be to select a widely distributed cosmopolitan species, and then look for significant differences in the more localized, specialized, or sensitive species. In that sense, P. lobata, or one of the widely distributed acroporids or pocilloporids (antler corals) would be as good a choice as any, although with a genus which contains many species, the question of species identification might be a problem. This participant viewed the project in a global, longer-term reef research and preservation framework. He has reservations about the short-term potential of genome research to come up with a 'silver bullet" that will fend off localized extinctions or reef collapses.
In response to these warnings, one participant summarized the two major benefits of sequencing a coral genome:
(1) a coral genome would be a major bonus for evolutionary genomics, since corals are representatives of the phylum Cnidaria , a sister group to all the currently sequenced metazoans, and (2) a basis will be created for molecular studies of how corals work. Of major interest for conservation biology would be the molecular mechanisms of stress and resistance, and also the molecular machinery of mutualism between host coral and zooxanthellae. Immediate profit would be availability of microarrays to monitor expressions of thousands of genes, which would be a great tool for the fine characterization of coral conditions and stresses.
Following this, he outlined the requirements and features he thought the "model" candidate species should possess. He compared the main candidates, Acropora sp, Montastraea sp, and Porites sp, to the model, which should have the following features:
A small genome. Most corals have genomes of similar or comparable sizes. The most common diploid number of chromosomes is 28. Therefore genome size, measured in numbers of chromosomes, not in nucleotide content, doesn't matter for most of the candidates. However, genome size is not a function of the number of chromosomes, but related to the size of the chromosomes which vary considerably between species, even within one genus.
- Should be easy to work with basic molecular techniques, such as DNA and RNA isolation. One participant with experience in molecular biology said that Acropora is difficult in this respect. Montastraea and Porites seem to be satifactory. Nucleic acid isolation and in situ hybridization and RNAi would work better the "meatier" the coral. In this respect, Montastraea is favored, especially M. cavernosa (great star coral), the fattest coral he ever worked with.
- Amenable to in situ hybridization techniques and RNAi techniques- to study gene expression patterns and knock the genes down, at least locally and temporarily. He did not know of any work with in situ hybridization and RNAi with corals. [In situ hybridization is a method of detecting the presence of specific nucleic acid sequences within a cytological preparation. RNAi (RNA interference) is a cellular mechanism that selectively negates the effect of any gene by destroying messenger RNA (mRNA). By destroying the targeted mRNA, improper protein synthesis, the cause of most disease, is interrupted, effectively "silencing" the target gene. The process is triggered by double-stranded RNA (dsRNA), where one strand is identical to the target mRNA sequence].
- Should be easily kept in the laboratory, preferably growing. All three candidates meet this criterion. Acropora grows fastest and Montastraea grows slowest.
- Should be widely distributed and ecologically significant, or be a representative of a closely related group of ecologically significant species, in order that sequence information from the genome project could be used for studies in many places and many similar species. None of the candidates have a single species which is globally distributed. There is a limitation to either the Caribbean or Indo-Pacific. However, at the generic level, all three (Acropora, Montastraea and Porites) are distributed worldwide and are the most important reef-builders. Acropora represents the largest genus with about 250 species. Porites is second with some 50 species, and Montastraea is last with about 10 species. The downside of using species-rich genera is that more taxonomic difficulties are presented.
- Existence of other relevant molecular projects, such as EST sequences. (an expressed sequence tag (EST) is a small part of the active part of a gene, made from cDNA which can be used to fish the rest of the gene out of the chromosome by matching base pairs with part of the gene. The EST can be radioactively labeled in order to locate it in a larger segment of DNA). Existence of supporting molecular projects is very important. In general, there is not much grant money to support coral molecular biology, so the community ought to stay focused. To this participant's knowledge, there were some EST projects on Acropora millepora, and another on Montastraea annularis.
- Popularity of the species in general as a model for various non-molecular research projects. Acropora is probably the most popular, M. annularis comes second and Porites seems to lag behind.
- Ultimately, the species should be reproducible in the laboratory, completing its full life cycle in less than a year, and be amenable for transgenic manipulations. The author of this model was not aware of any species of coral that would fulfill this requirement.
Based upon these model features, he concluded that there is no formally best candidate, so the choice would depend on how one would weigh the above eight considerations. He tended to put more weight into general popularity and existence of other molecular projects, so that in his view, Porites is not a good candidate. In all other respects Acropora seems better than Montastraea, except for the notion that it would be more difficult to do molecular work with Acropora.
The colonies of the mustard hill coral, Porites astreoides, are usually small, but may grow several meters in diameter. They are found in coral reefs of the tropical Atlantic (Credit: Andrew Bruckner, NOAA Fisheries)
Other participants in this discussion group responded to this model. A researcher working on a stress related syndrome of Porites lutea in the Indian Ocean would be happy to get any sequencing data, regardless of which species of coral, though he believed that the slow growing corals should be sequenced first. Another suggested the lace coral, Pocillopora damicornis, as a species that reproduces prolifically by asexual formation of planula larvae. P. damicornis has a wide distribution and is easily cultured in aquaria, where it can reproduce asexually in a year or less. P. damicornis received another endorsement from a participant for reasons that it grows rapidly in aquaria and is very hardy and adaptable to varying conditions. It has a very fine branching structure and even a modest-sized colony can be fragmented into many uniform-sized branch tips for starting replicate, genetically identical colonies for laboratory work. Very small fragments of P. damicornis, with just a few polyps, can be used to start a new colony, and attachment to new surfaces is very rapid. It has a high density of large, long polyps that are almost always well extended. The polyps are very transparent, except for their zooxanthellae, and some polyps are almost completely unobscurred by zooxanthellae which would be perfect for studies of gene expression in which genes of interest have been linked. Also, the colonies are relatively unbothered by handling and vibrations.
Another coral expert came down on the side of Porites as the best candidate for sequencing because it is an important reef builder in both the Caribbean and indo-pacific, and the third largest genus of corals. Also, the huge, massive Porites are the sources of climate records. P. lobata is the most common of the big massive ones used for climate studies and is one of the most widely spread of all the corals. Acropora is also a major reef builder in both the Caribbean and indo-Pacific. It is also the largest coral genus with some 165 species, so far. Montastraea appears not to be a good candidate because, while it is a major reef builder in the Caribbean, it is not in the Indo-Pacific, with only a few small uncommon species.
Another participant thought that the model would argue against massive growth forms as candidates in the first round of sequencing. What is important in the first round is experimental feasibility. He reluctantly stepped back from Porites and supported Pocillopora, or a robust and well-characterized Acropora. However, he agreed with his colleague that the massive Porites have the greatest colony longevity and are widely used as environmental sensors. That, plus widespread distribution and geological and ecological importance, keeps them high on the list of candidate species for sequencing. Low down on the list is Montastraea because of its paucity in the Indo-Pacific.
Another participant in this discussion didn’t agree that a coral that is primarily useful for fossil/paleoclimate studies would be one of the best choices for a genomics project. He thought that the coral community should figure out what would be the coral equivalent of a laboratory rat or fruit fly. A species should be selected that is most amenable to laboratory manipulations and studies on living organisms; or, a target organism should be found that will provide the most useful leads on the reagents needed to do field studies on mRNA’s or proteins isolated from specimens in the wild. He further opined that if no such hermatypic coral exists, the community might be better served by picking another model cnidarian that is more conducive to laboratory manipulation.
Stylophora is an ecologically important genus of Indo-pacific corals. The species are branching forms with common names that include club finger coral, brush coral, and finger coral. (Credit: Andrew Bruckner, NOAA Fisheries)
A participant with experience in sequencing DNA from many Caribbean corals joined in the discussion. A consideration, also mentioned earlier by another researcher in selecting a scleractinian coral species, is the availability of zooxanthellae-free tissues. DNA from the zooxanthellae is often amplified (and subsequently sequenced) in addition to the coral DNA, unless the tissue is free from zooxanthellae or the primers are specific to cnidarians. The symbiotic dinoflagellates have a genome size which is estimated to be 100 times larger than the coral genome, further complicating the effort. Spermatozoa from broadcasting species were suggested. This investigator’s preference for a candidate is Porites for a number of reasons: microsatellites for P. astreoides have already been developed; there are several representatives in the Caribbean and Indo-Pacific (microsatellites for Montastraea cavernosa have also been developed, but it would be more useful for molecular biologists to select a species from a genus with a wide distribution), thus the Porites genome can be used as a model for efficiently developing genetic markers for several species of Porites; it is easy to conduct molecular analyses on Porites (high amplification and sequencing success); and as brooders that release larvae multiple times throughout the year, molecular biologists can take advantage of breeding experiments without having to hope for good weather conditions on the couple of evenings of mass spawning. The participant concluded her remarks with the observation that regardless of what species is selected, the sequencing information would be extremely useful for those interested in genetic structure and gene flow of coral species. Mitochondrial markers used for population genetics on other organisms cannot be used in corals due to the slow rate of evolution in the mitochondrial genome. Therefore, other markers need to be developed, and having a model genome available to develop them will save the research community time and money.
Other participants presented their cases, pro and con, for the model species. One suggested that P. damicornis and the cauliflower coral, Stylophora pistillata, are already coral “guinea pigs” and are widely distributed, important reef-builders with large literature bases. Another added that S. pistillata has been used extensively in experiments on metabolism and zooxanthellae, and and was even referred to as the ‘coral lab rat’ in the 9th International Coral Reef Symposium. It is widespread in the Indo-Pacific, though not as widespread as P. damicornis. However, the genus is not present in the Caribbean. In the Caribbean, the acroporids and Montastraea sp seem to be the logical choices. In fact they were already selected by the Coral Health and Disease Consortium (CHDC) as candidates for coral ‘lab rats’ in culture. Their life histories are also more representative of the majority of corals.
Another argued that Porites made the most sense as the best candidate based on the criteria presented. Pocillopora is not as durable in shipping and handling and is susceptible to Vibrio (a bacterium) infections. This participant also added a new candidate genus to the discussion, the petaloid coral, Psammocora. It is widespread globally, easy to grow and to ship, and has a post-transit recovery capacity at least as good as Porites. Another participant offered that the ecological significance of the species really doesn’t matter. What matters is the “phylogenetic pedigree” and a body of work in the offering on gene-environmental processes. These considerations put Porites, Acropora and Pocillopora at the head of the list, in roughly that order, with one of the prominent faviid genera next. He suggested that preliminary trials be done on the top-ranking species before committing to any one, since many of the genetic/methodological criteria for the model may not be known yet for either Porites or Acropora. He concluded however, that a Porites species would probably top the list, as these are widespread and phylogenetically and ecologically important, whether the massive ones, for which we have climate records and can relate genotypes to historical conditions, or the branching ones, which satisfy more of the ‘lab rat’ criteria. Porites also has interesting syndromes in the field that would make genetic studies interesting. For example, the pink-line syndrome, abundance of growth tumors, permanent white patches, mucus sheaths of mysterious function, broad temperature acclimation range, long lifespan, etc. Perhaps, he suggests, the yellow finger porites, Porites cylindrica (or other branching Porites), might do better satisfying the distribution and lab rat requirements, having an interesting evolutionary/ecological history as well as being workable, thus satisfying other criteria. In second place would come one of the widely distributed Acropora head/cushion species, with relatively broad environmental tolerance, or Pocillopora damicornis, the “lab rat par excellence.”
One participant thought that the proposition that one coral species can be a representative ‘lab rat’ for physiological, etc., studies of corals is flawed by not considering the evolutionary history of living corals. Corals are probably not a monophyletic group and the different families differ significantly in their physiology, ecology, and genetics. Results from one species cannot be safely extrapolated to responses of species from different groups. However, even though there is no single species of coral that is representative of corals in general, a start has to begin somewhere with a single species, and do others as resources may allow. Furthermore, in terms of selection factors, ease of culture, shipping survivability and other practical considerations may be more important in terms of getting as much generic benefit from the genetic results than are the ecological or regional importances of the species.
The starlet sea anemone, Nematostella vectensis, is a non-tropical cnidarian (not a coral) that occurs in shallow North American and western European coastal waters. It is becoming an increasingly important model system for the study of development, evolution, reproductive biology, genomics, and ecology. (Credit: Biology Department, Technische Universitat Darmstadt)
A non-U.S. participant, only recently aware of this discussion forum, thought that there were some things that should be more widely known than they appeared to be. It seemed to him that there appears to be misapprehension that a stony coral genome would be the first cnidarian to be sequenced. He thought it more likely that a proposal for sequencing the genome of the starlet sea anemone, Nematostella vectensis, will be successful, having strong support from the evo-devo community (in 1999, developmental biology and evolutionary studies merged when evolutionary developmental biology, or "evo-devo," was granted its own division in the Society for Integrative and Comparative Biology (SICB). Evolutionary biologists seek to understand how organisms evolve and change their shape and form. The roots of these changes are found in the developmental mechanisms that control body shape and form. Developmental biologists try to understand how alterations in gene expression and function lead to changes in body shape and pattern). Further, it seemed essential that the approximate size of the genome of the coral candidate be known. Genome size is a function of both the number of chromosomes and the size of the chromosomes. This participant, in reference to the model criteria put forward, pointed out the requirement or desirability of technology and tools for the coral species selected. In this light, he discussed the advantages of Acropora millepora. Acropora is the second best represented cnidarian (behind Hydra) in the genomic databases. Its genome appears to be at the low end of cnidarian genome sizes. Also, most of the molecular tools are there for A. millepora (genome libraries in lambda and cosmid vectors; cDNA libraries for six different embryonic and larval stages, as well as adult colonies; an extensive EST data set; microarrays featuring 3000 EST’s of known sequences are available. In addition, in situ hybridization technology works with A. millepora, whereas he doesn’t believe this method has been established for any other coral. Therefore, as far as the molecular basics being in place, Acropora is a much more advanced system than is any other coral. He is sure that the evo-devo community would strongly support a proposal to sequence the genome of A. millepora.
Another coral scientist offered that the proposed sequencing for Porites lobata would be a real plus for coral biology. He chided the respondents for nominating their own favorite species instead of the proposed species. In the first go-round they should get squarely behind their colleagues and help them promote an idea, realizing that success with the first species will help everyone move forward. Too much sniping in the community leads to confusion in the funding agencies, which end up funding other disciplines. Another coral scientist also urged that all pull together and support the Porites sequencing proposal, noting however, that the postings have been worthwhile and very valid points on alternate species have been brought up. These discussions also pointed out that those that work in genomics need to better convey the power, potential, and applications of the technologies to researchers in other scientific disciplines, as well managers and policy makers. However, she was concerned that the discussion was beginning to lose sight of the real goal in the effort to have a coral genome sequenced: to generate vital coral genome sequence data, and make it widely available to the research community via the public domain. It would be a great advantage for the coral community to have a sequenced coral genome added to the short list of other fully sequenced eukaryotic (organisms with cells having a distinct nucleus with nDNA, and intracellular membranes) genomes. This includes all protists, fungi, plants and animals) genomes. This effort also satisfies a major mandate of the CDHC National Research Plan. The completion of a genomic sequence will have many positive effects on the field of coral research and others, many of which cannot be foreseen. There was a short deadline, ergo, a narrow window of opportunity to respond as a unified research community to say that a coral genome needs to be sequenced, and the community will use these resources to move the field of coral research and conservation management forward.
Lastly, the scientist that sparked this discussion by asking for letters of support to a funding agency for sequencing the genome of Porites lobata, said that for the past couple of years, his group has been evaluating criteria for a ‘coral lab rat’, an organism representative of scleractinians to be used as a model for molecular genetics, cell biology, biochemistry, lipid chemistry, sterol/polyphenol chemistry, environmental/physiological monitoring, ecotoxicology, stress physiology, coral immunity, coral oncology, coral endocrinology and coral neurophysiology. He had asked a diverse group of coral biologists to nominate and justify a coral candidate, but the responses were slow. He stated that a laboratory biologist, like himself, needs a ‘lab rat’ for other laboratories to repeat his experiments or take the work further. From his point of view, the most important criterion for a coral lab rat is accessibility. The point is not to sequence the genome from a Caribbean or Pacific species, as has been mentioned several times in this exchange. Everyone must have relative ease in obtaining genetically identifiable laboratory strains which means that the first strain will come from a single colony and mass cultured. Some group must have the facilities to rear this coral in abundance and be able to distribute it to any laboratory in the world. The coral must also be hardy enough to survive the trip. Porites meets the criterion of survivalship. Few species besides Gonistrea or Pavona has the resilience of Porites.
Another issue is the amenability of many of the tools of molecular, cellular and physiological biology applied to that species. He has had considerable experience with Acropora and Montastraea, and he ruled out both of them. The ‘lab rat’ also has to be a good species for applying cell culture techniques. He states that acroporids aren’t bad and others have had success with Pocillopora, Porites and Oculina. Oculina varicosa, or any of its sibling species, would be a good candidate for a ‘lab rat’. However it is not a major reef builder and doesn’t handle shipping very well. From a laboratory technique perspective, Porites exhibits the least amount of technique artifact and can be shipped easily with high success of survival after shipping. In terms of physiological ecology, Porites and Acropora are found worldwide, but Acropora is not as resilient as Porites, and is the first to “crash” during an environmental event, such as an oil spill or unusually high sea surface temperature. The candidate species must be vigorous enough to survive after the environmental insult.
The sequenced genome is a platform that may be used to extend basic research into areas of coral biology where it has been so difficult to go in the past, or a platform to develop new technologies to allow us to see further. Basic science questions can be greatly aided by knowing the sequence of the coral genome. For example, is this coral immunocompetent or endocrine modulated? We need to know the genes that contribute to these systems to explore their individual and combined behaviors. Cnidarians have the most primitive of the metazoan nervous systems. Are their neuropeptides different from higher organisms, and why? Corals often develop areas of abnormal growth referred to as hyperplasms or neoplasms. These areas contain tissues which are radically different from normal polyps. Can our understanding of cancer in mammals be aided by our understanding and the future discoveries of how corals get cancer? The technologies that can be developed from a sequenced coral genome are only limited by imagination and determination.
As of May, 2004, the funding agency looks favorably on the proposal to sequence the genome of a coral, but a decision has yet to be made on which species, though it probably has come down to Porites or Acropora. Once that decision has been made, the selected coral species will be prioritized among the other candidates species for sequencing, and a final decision on funding for this cycle will be made.