SAES-422 Multistate Research Activity Accomplishments Report
Sections
Status: Approved
Basic Information
- Project No. and Title: WERA_OLD99 : Broodstock Management, Genetics and Breeding Programs for Molluscan Shellfish
- Period Covered: 04/01/2001 to 04/01/2002
- Date of Report: 05/17/2002
- Annual Meeting Dates: 04/13/2002 to 04/13/2002
Participants
Allen, Stan, ska@vims.edu Virginia Institute of Marine Sciences; Boudry, Pierre, pierre.boudry@ifremer.fr, IFREMER, La Tremblade; Brake, John, john.brake@hmsc.orst.edu, Hatfield Marine Science Center, Oregon State University; Camara, Mark, camara@vims.edu, Virginia Institute of Marine Sciences; Davis, Joth, jdavis32@mindspring.com, Taylor Shellfish Farms; Gaffney, Patrick, pgaffney@udel.edu, University of Delaware; Guo, Ximing, xguo@hsrl.rutgers.edu, Rutgers University; Samain, Jean-Francois, Jean.Francois.Samain@ifremer.fr, IFREMER, Brest; Hedgecock, Dennis, dehedgecock@ucdavis.edu; Bodega Marine Lab, University of California- Davis; Langdon, Chris, chris.langdon@hmsc.orst.edu, Hatfield Marine Sciences Center, Oregon State University; Li, Li, lili@hsrl.rutgers.edu, Rutgers University; Matson, Sean, sean.matson@hmsc.orst.edu, Hatfield Marine Sciences Center, Oregon State University; Meritt, Don, merit@hpl.umces.edu, Horn Point Laboratory, University of Maryland; Nicola, Jean Louis, jlnicola@ifremer.fr, IFREMER; Reece, Kimberly, kreece@vims.edu, Virginia Institute of Marine Sciences; Wikfors, Gary, gary.wikfors@noaa.gov, NOAA; Wilbur, Amy, wilbura@uncwil.edu,CMS UNCW
The meeting started at 9 AM with an introduction by Stan Allen who described the history of WCC-99 & its purpose.
Pierre Boudry reported on research of the genetics & pathology program at the IFREMER station at La Tremablade. Primary areas of research in genetics of the European & Pacific oyster are: molecular taxonomy, population genetics, phenotypic evaluation, cytogenetics & improvement of stocks by genetic selection.
Species-specific arkers have been developed for various oyster species & varieties collected from around the world. Based on their data, the mangrove oyster Crassostrea gasa is found in both Africa & South America but Crassostrea rhizophora is only found in South America. The population genetics of the European oyster has been studied & the distribution of different stocks described on the Mediterranean & Atlantic coasts. In addition, the ecophysiologies of Crassostrea gigas & Crassostrea angulata have been compared to show that the specific growth rate of C. angulata is lower than that of C. gigas. Cytogenetic studies have shown that there is correlation between aneuploidy & growth of Pacific oysters. Genomic tags are being developed for each chromosome. Some pairs of chromosomes appear to be lost more commonly than others. Pollution results in increased rates of chromosome loss. Genetic selection has resulted in improved survival of European oysters exposed to Bonamia. A large inter-institution project called MOREST has begun in France to study the heritability of growth & survival of Pacific oysters.
Jean-Francois Samain described the MOREST program, The five-year program is multidisciplinary involving 15 French laboratories & 22 teams. The five main tasks are 1) to prepare tools in all disciplines to study summer mortality, 2) to characterize the environment & identify possible causal factors, 3) to examine interactions among possible causes, including both environmental & physiological factors, 4) to identify potential pathogens using a national network of laboratories, 5) to develop long-term monitoring programs for oyster growing areas susceptible to summer mortality. Preliminary genetic selection indicated a strong genetic effect on summer mortality with family survival ranging from 17 to 97%. It was noted that mortalities do not occur until seawater temperatures rise to >19 oC & they often occur shortly before spawning.
Gary Wikfors discussed the importance of understanding feeding & nutrition of mollusks as part of the domestication process. He described an apparatus to vary the ration & frequency of feeding shellfish on algal diets. Rations of 5% whole body weight per day were optimal for growth but food conversion efficiencies were best at 2% ration per day.
Pat Gaffney described the use of various genetic markers to distinguish different populations of the Eastern oyster on the East & Gulf coasts, US. Use of 16S mitochondrial DNA sequences was useful in distinguishing Gulf from East Coast populations, with over 33 polymorphic restriction sites identified.
Pat described his work on sequencing nuclear DNA of the Eastern oyster. About 1446 sequences have been described & there is evidence of a non-autonomous DNA transposon (CvA). He also reported that only 50% of the flanking regions of microsatellites of the Eastern oyster are unique, resulting in possible confusion in amplification of microsatellite markers.
Ximing Guo Numerous genetic projects are being undertaken, including 1) breeding & evaluation of NE stocks of Eastern oysters, 2) aneuploidy & triploidy in oysters, 3) production of triploid & tetraploid Eastern oysters & hard clams, 4) genetic expression analysis of oyster responses to Dermo & MSX infections, 5) gene mapping of Eastern & Pacific oysters, 6) physical mapping of chromosomes (FISH).
About 42,000 tetraploid Eastern oysters have been successfully produced. High mortality of the larvae & spat meant that large numbers of broodstock were needed to produce sufficient larvae. Tetraploid Eastern oysters were not stable & 16% showed "mosaic" ploidy after three months, a rate that was greater than for triploids.
An AFLP map has been produced for the Eastern oyster with 170 markers identified. A second map has been produced for the Pacific oyster with 145 markers.
Suppression subtraction libraries of expressed genes for Eastern & Pacific oysters exposed to Dermo & MSX infection have been produced. A wide range of gene expression responses have been detected & are currently being analyzed.
Ami Wilbur described her work on comparing different oyster stocks planted at various sites on the East coast. Stocks were identified using two 16S mtDNA loci. Four % of Eastern oyster seed sampled from "wild" oyster beds likely originated as seed produced from planted Louisiana oysters at a site in North Carolina.
Ami reported on work to determine the effectiveness of enhancement programs for Bay scallops. Research in Florida indicated that three years of scallop enhancement had no detectable effect in contributing to the recovery of scallop populations in that state. However, in Chincoteague Bay, efforts by Virginia agencies were more effective. The closed nature of the bay may have contributed to the positive results of the enhancement program.
Don Meritt described plans for a new shellfish hatchery at the Horn Point Laboratories, University of Maryland, which is planned for completion next spring. The hatchery will cost an estimated $25 million & will receive a flow-through seawater supply. The hatchery will also have a quarantine lab that will allow importation of non-native species.
Stan Allen reported on the results of the Williamsberg conference held in October, 2001, at which the aquaculture potential of triploid Suminoe oysters Crassostrea ariakensis was discussed. He recounted the history of the study of non-native species at VIMS leading up to the current industry trials with about 60,000 out-planted triploid oysters. Growth & survival results indicate that the performance of Suminoe oysters at test sites is much better than that of Eastern oysters.
A conclusion of the science panel at the Williamsburg Conference was that triploid aquaculture of Suminoe oysters would inevitably lead to the introduction of diploids either through illegal introductions, ploidy reversions, lapses in biosecurity or harvest loss. Any scenario for triploid introductions should include consideration of the fate of diploid introductions & possible effects of competition with Eastern oysters, return of the Chesapeake to a benthic-dominated system & fouling of yachts, marine structures & intake pipes. The conference recommended that more work should be undertaken to study the ecological consequences of diploid introductions. In June 2002, a committee of the National Academy of Sciences will undertake a review of the issue.
Mark Camara discussed some concepts of breeding programs. He noted that one needs to develop fast-growing lines for aquaculture using high-tech procedures, while for restoration purposes one can adopt a low-tech approach. The pros & cons of adopting a "walk-back" approach to genetic selection were discussed.
Kim Reese described research that focused on developing markers to discriminate between hatchery & natural stocks as well the identification of genes useful in selecting for disease resistance, growth rate & final size at harvest. Her lab is focusing on developing microsatellite markers using primers that amplify only at a single locus. She reported a high incidence of null alleles for many microsatellites but found that dinucleotide microsatellites were associated with fewer null alleles that tetranucleotide microsatellites. Using RFLP analysis, Kim reported that the DERBY line of disease-resistant Eastern oysters was highly inbred and, therefore, has limited use in restoration. CROSBreed lines were less inbred & showed more promise for use in restocking.
Dennis Hedgecock described the approach of Lynx Therapeutics for measuring genetic expression of oysters using massively parallel analysis. The method is based on the use of millions of microbeads each providing a unique 20 base pair signature sequence. A total of about 3 million sequences can be analyzed at a time. Dennis used the method to compare expressed genes in inbred & outbred lines (MBP families 1.35 & 1.51). About 2 to 3% of the expressed genes were non-additive (i.e. their expression was either greater or less than the average of the two parental inbred lines). Of these non-additive genes, about one third were over-dominant & one third were under-dominant. A total of 120 were significantly non-additive in both hybrids & are candidate genes of interest in explaining hybrid vigor in these lines. The 20 base-pair sequences will be used to produce longer 250 base-pair sequences for GenBank searches. The non-additively expressed genes of interest will be evaluated as QTLs for traits of interest.
Dennis also described progress in mapping the genome of Pacific oysters. Optimal PCR methods have been developed for 190 microsatellite primers & 79 markers have been mapped. A high proportion (49/96) of microsatellites are associated with null alleles indicting a high level (0.0123) of polymorphism - a rate that is ten times that reported for humans. Dennis estimated that this level of polymorphism in oysters would result in a SNP every 80 base pairs.
Joth Davis reported on the results of the WRAC project for cross-breeding inbred lines of Pacific oysters. Taylor Farms Inc. has established a bivalve research & testing center with a capacity for 50 x 100 liter tanks supplied with 0.2 micron-filtered, UV-treated seawater. The nursery has 96 upwellers for growth of juveniles. The grow-out system consists of bag suspended from long-lines. Large differences in the growth of seed from various hybrid lines were observed.
Chris Langdon described progress for the Molluscan Broodstock Program (MBP). Analysis of yields of Pacific oysters derived from selected broodstock showed a realized heritability of 9.4% for improvement in yield compared with yields of non-selected "wild" oysters. Both growth & survival affect yield, with the relative proportion differing among sites & cohorts. In Tomales Bay, 79% of the variation in yield among families was due to differences in survival.
John Brake discussed methods to describe shell & mantle color as well as cup shape of Pacific oysters. These parameters will be added to descriptions of the various MBP families on the MBP web site. Future experiments are planned to determine the heritability of shell & mantle color as well as shell shape.
Sean Matson described the results of a commercial-scale test of MBP seed. The performance of seed derived from crossing MBP broodstock was compared with that of seed from a standard commercial cross. The seed were produced on a commercial-scale at Taylor‘s hatchery & planted either on-cultch oysters in Samish Bay or as singles in bags in Totten Inlet. Microsatellite analysis of the seed at planting indicated a contamination level of about 8%. The parentage of the contaminants was unknown, indicating that contamination likely occurred in the hatchery or nursery.
At harvest, MBP-derived oysters were heavier with a lower percentage of extra smalls & debris (empty shells) compared with controls. In support of this finding, the volume ratio of harvested oysters to discard material was 5 to 1 for MBP oysters but only 2:1 for control oysters. There was a likely mix-up of oyster meats during shucking as meat weight data for the MBP & control oysters were not correlated with whole weights - a very unlikely result.
Stan Allen volunteered to remain Chairperson for a second year with Chris Langdon as Secretary. The venue for the next meeting was not determined. Adjourned at 5.30 pm.
Pierre Boudry reported on research of the genetics & pathology program at the IFREMER station at La Tremablade. Primary areas of research in genetics of the European & Pacific oyster are: molecular taxonomy, population genetics, phenotypic evaluation, cytogenetics & improvement of stocks by genetic selection.
Species-specific arkers have been developed for various oyster species & varieties collected from around the world. Based on their data, the mangrove oyster Crassostrea gasa is found in both Africa & South America but Crassostrea rhizophora is only found in South America. The population genetics of the European oyster has been studied & the distribution of different stocks described on the Mediterranean & Atlantic coasts. In addition, the ecophysiologies of Crassostrea gigas & Crassostrea angulata have been compared to show that the specific growth rate of C. angulata is lower than that of C. gigas. Cytogenetic studies have shown that there is correlation between aneuploidy & growth of Pacific oysters. Genomic tags are being developed for each chromosome. Some pairs of chromosomes appear to be lost more commonly than others. Pollution results in increased rates of chromosome loss. Genetic selection has resulted in improved survival of European oysters exposed to Bonamia. A large inter-institution project called MOREST has begun in France to study the heritability of growth & survival of Pacific oysters.
Jean-Francois Samain described the MOREST program, The five-year program is multidisciplinary involving 15 French laboratories & 22 teams. The five main tasks are 1) to prepare tools in all disciplines to study summer mortality, 2) to characterize the environment & identify possible causal factors, 3) to examine interactions among possible causes, including both environmental & physiological factors, 4) to identify potential pathogens using a national network of laboratories, 5) to develop long-term monitoring programs for oyster growing areas susceptible to summer mortality. Preliminary genetic selection indicated a strong genetic effect on summer mortality with family survival ranging from 17 to 97%. It was noted that mortalities do not occur until seawater temperatures rise to >19 oC & they often occur shortly before spawning.
Gary Wikfors discussed the importance of understanding feeding & nutrition of mollusks as part of the domestication process. He described an apparatus to vary the ration & frequency of feeding shellfish on algal diets. Rations of 5% whole body weight per day were optimal for growth but food conversion efficiencies were best at 2% ration per day.
Pat Gaffney described the use of various genetic markers to distinguish different populations of the Eastern oyster on the East & Gulf coasts, US. Use of 16S mitochondrial DNA sequences was useful in distinguishing Gulf from East Coast populations, with over 33 polymorphic restriction sites identified.
Pat described his work on sequencing nuclear DNA of the Eastern oyster. About 1446 sequences have been described & there is evidence of a non-autonomous DNA transposon (CvA). He also reported that only 50% of the flanking regions of microsatellites of the Eastern oyster are unique, resulting in possible confusion in amplification of microsatellite markers.
Ximing Guo Numerous genetic projects are being undertaken, including 1) breeding & evaluation of NE stocks of Eastern oysters, 2) aneuploidy & triploidy in oysters, 3) production of triploid & tetraploid Eastern oysters & hard clams, 4) genetic expression analysis of oyster responses to Dermo & MSX infections, 5) gene mapping of Eastern & Pacific oysters, 6) physical mapping of chromosomes (FISH).
About 42,000 tetraploid Eastern oysters have been successfully produced. High mortality of the larvae & spat meant that large numbers of broodstock were needed to produce sufficient larvae. Tetraploid Eastern oysters were not stable & 16% showed "mosaic" ploidy after three months, a rate that was greater than for triploids.
An AFLP map has been produced for the Eastern oyster with 170 markers identified. A second map has been produced for the Pacific oyster with 145 markers.
Suppression subtraction libraries of expressed genes for Eastern & Pacific oysters exposed to Dermo & MSX infection have been produced. A wide range of gene expression responses have been detected & are currently being analyzed.
Ami Wilbur described her work on comparing different oyster stocks planted at various sites on the East coast. Stocks were identified using two 16S mtDNA loci. Four % of Eastern oyster seed sampled from "wild" oyster beds likely originated as seed produced from planted Louisiana oysters at a site in North Carolina.
Ami reported on work to determine the effectiveness of enhancement programs for Bay scallops. Research in Florida indicated that three years of scallop enhancement had no detectable effect in contributing to the recovery of scallop populations in that state. However, in Chincoteague Bay, efforts by Virginia agencies were more effective. The closed nature of the bay may have contributed to the positive results of the enhancement program.
Don Meritt described plans for a new shellfish hatchery at the Horn Point Laboratories, University of Maryland, which is planned for completion next spring. The hatchery will cost an estimated $25 million & will receive a flow-through seawater supply. The hatchery will also have a quarantine lab that will allow importation of non-native species.
Stan Allen reported on the results of the Williamsberg conference held in October, 2001, at which the aquaculture potential of triploid Suminoe oysters Crassostrea ariakensis was discussed. He recounted the history of the study of non-native species at VIMS leading up to the current industry trials with about 60,000 out-planted triploid oysters. Growth & survival results indicate that the performance of Suminoe oysters at test sites is much better than that of Eastern oysters.
A conclusion of the science panel at the Williamsburg Conference was that triploid aquaculture of Suminoe oysters would inevitably lead to the introduction of diploids either through illegal introductions, ploidy reversions, lapses in biosecurity or harvest loss. Any scenario for triploid introductions should include consideration of the fate of diploid introductions & possible effects of competition with Eastern oysters, return of the Chesapeake to a benthic-dominated system & fouling of yachts, marine structures & intake pipes. The conference recommended that more work should be undertaken to study the ecological consequences of diploid introductions. In June 2002, a committee of the National Academy of Sciences will undertake a review of the issue.
Mark Camara discussed some concepts of breeding programs. He noted that one needs to develop fast-growing lines for aquaculture using high-tech procedures, while for restoration purposes one can adopt a low-tech approach. The pros & cons of adopting a "walk-back" approach to genetic selection were discussed.
Kim Reese described research that focused on developing markers to discriminate between hatchery & natural stocks as well the identification of genes useful in selecting for disease resistance, growth rate & final size at harvest. Her lab is focusing on developing microsatellite markers using primers that amplify only at a single locus. She reported a high incidence of null alleles for many microsatellites but found that dinucleotide microsatellites were associated with fewer null alleles that tetranucleotide microsatellites. Using RFLP analysis, Kim reported that the DERBY line of disease-resistant Eastern oysters was highly inbred and, therefore, has limited use in restoration. CROSBreed lines were less inbred & showed more promise for use in restocking.
Dennis Hedgecock described the approach of Lynx Therapeutics for measuring genetic expression of oysters using massively parallel analysis. The method is based on the use of millions of microbeads each providing a unique 20 base pair signature sequence. A total of about 3 million sequences can be analyzed at a time. Dennis used the method to compare expressed genes in inbred & outbred lines (MBP families 1.35 & 1.51). About 2 to 3% of the expressed genes were non-additive (i.e. their expression was either greater or less than the average of the two parental inbred lines). Of these non-additive genes, about one third were over-dominant & one third were under-dominant. A total of 120 were significantly non-additive in both hybrids & are candidate genes of interest in explaining hybrid vigor in these lines. The 20 base-pair sequences will be used to produce longer 250 base-pair sequences for GenBank searches. The non-additively expressed genes of interest will be evaluated as QTLs for traits of interest.
Dennis also described progress in mapping the genome of Pacific oysters. Optimal PCR methods have been developed for 190 microsatellite primers & 79 markers have been mapped. A high proportion (49/96) of microsatellites are associated with null alleles indicting a high level (0.0123) of polymorphism - a rate that is ten times that reported for humans. Dennis estimated that this level of polymorphism in oysters would result in a SNP every 80 base pairs.
Joth Davis reported on the results of the WRAC project for cross-breeding inbred lines of Pacific oysters. Taylor Farms Inc. has established a bivalve research & testing center with a capacity for 50 x 100 liter tanks supplied with 0.2 micron-filtered, UV-treated seawater. The nursery has 96 upwellers for growth of juveniles. The grow-out system consists of bag suspended from long-lines. Large differences in the growth of seed from various hybrid lines were observed.
Chris Langdon described progress for the Molluscan Broodstock Program (MBP). Analysis of yields of Pacific oysters derived from selected broodstock showed a realized heritability of 9.4% for improvement in yield compared with yields of non-selected "wild" oysters. Both growth & survival affect yield, with the relative proportion differing among sites & cohorts. In Tomales Bay, 79% of the variation in yield among families was due to differences in survival.
John Brake discussed methods to describe shell & mantle color as well as cup shape of Pacific oysters. These parameters will be added to descriptions of the various MBP families on the MBP web site. Future experiments are planned to determine the heritability of shell & mantle color as well as shell shape.
Sean Matson described the results of a commercial-scale test of MBP seed. The performance of seed derived from crossing MBP broodstock was compared with that of seed from a standard commercial cross. The seed were produced on a commercial-scale at Taylor‘s hatchery & planted either on-cultch oysters in Samish Bay or as singles in bags in Totten Inlet. Microsatellite analysis of the seed at planting indicated a contamination level of about 8%. The parentage of the contaminants was unknown, indicating that contamination likely occurred in the hatchery or nursery.
At harvest, MBP-derived oysters were heavier with a lower percentage of extra smalls & debris (empty shells) compared with controls. In support of this finding, the volume ratio of harvested oysters to discard material was 5 to 1 for MBP oysters but only 2:1 for control oysters. There was a likely mix-up of oyster meats during shucking as meat weight data for the MBP & control oysters were not correlated with whole weights - a very unlikely result.
Stan Allen volunteered to remain Chairperson for a second year with Chris Langdon as Secretary. The venue for the next meeting was not determined. Adjourned at 5.30 pm.
[Minutes]
Accomplishments
Principal accomplishments of this group include major work on the West Coast in tetraploidy research, primarily coming out of the MBP & USDA Western Regional Aquaculture Consortium funded projects. Triploid & tetraploid technology has moved from research to application, supported, in part, by USDA-SBIR funding. MBP select stocks are being propagated commercially. The WRAC project has one commercial test of hybrid oyster yield in the field; however, more are planned. There is vigorous industry support for these activities.
Work parallel to that of the West Coast concerns the Eastern Oyster & includes investigations of aneuploidy, triploidy & tetraploidy & some important disease investigations related to Dermo & MSX. FISH is being used to map the chromosomes of the Eastern Oyster.
There have been important key papers published relating to the causes & uses of hybrid vigor & the development of microsatellite DNA markers for confirming parentage in experiments & for gene-mapping. A number of the members of WCC-099 also participate in the Regional project NE-186, Genetic Maps of Aquaculture Species. A genetic linkage map of the Pacific Oyster based on microsatellite technology has been developed & there has been analysis of gene expression in hybrid Pacific Oysters by massively parallel signature sequencing.
There have been important research efforts from members of this group to identify & conserve the Kumamoto oyster. Work has also taken place on the nutrition of mollusks in relationship to the domestication process.
Work parallel to that of the West Coast concerns the Eastern Oyster & includes investigations of aneuploidy, triploidy & tetraploidy & some important disease investigations related to Dermo & MSX. FISH is being used to map the chromosomes of the Eastern Oyster.
There have been important key papers published relating to the causes & uses of hybrid vigor & the development of microsatellite DNA markers for confirming parentage in experiments & for gene-mapping. A number of the members of WCC-099 also participate in the Regional project NE-186, Genetic Maps of Aquaculture Species. A genetic linkage map of the Pacific Oyster based on microsatellite technology has been developed & there has been analysis of gene expression in hybrid Pacific Oysters by massively parallel signature sequencing.
There have been important research efforts from members of this group to identify & conserve the Kumamoto oyster. Work has also taken place on the nutrition of mollusks in relationship to the domestication process.
Impacts
- Triploid & tetraploid technology has moved rapidly from research to application supported by various funding sources, e.g., USDA-SBIR, to members of this project. MBP select stocks are being reared on farms & are being propagated commercially by two west coast firms. West Coast industry is contributing the essential facilities & manpower to support the projects. Their support is a clear indication of their confidence in USDA-supported research for hybrid vigor & a commitment to crossbreeding.
Publications
Launey, Sophie; Hedgecock, Dennis. High genetic load in the Pacific oyster Crassostrea gigas. 2001. Genetics 159: 255-265.
Hallerman, E., M. Leffler, S. Mills, and S.K. Allen, Jr. 2002. Aquaculture of triploid Crassostrea ariakensis in Chesapeake Bay: A Symposium Report. Md/Va Sea Grant Publication, UM-sg-ts-2002-01/VSG-02-03, 20 pp.
Sophie Hubert, Gang Li, Dennis Hedgecock. 2002. Development of a genetic linkage map, using microsatellite markers in the Pacific oyster, Crassostrea gigas. http://www.intl-pag.org/pag/10/abstracts/PAGX_W16.html
Hedgecock, D., Lin, J.-Z. DeCola, S., Haudenschild, C., Meyer, E., Manahan D.T., Bowen, B. 2002. Analysis of gene expression in hybrid Pacific oysters by massively parallel signature sequencing.
http://www.intl-pag.org/pag/10/abstracts/PAGX_W15.html
Hallerman, E., M. Leffler, S. Mills, and S.K. Allen, Jr. 2002. Aquaculture of triploid Crassostrea ariakensis in Chesapeake Bay: A Symposium Report. Md/Va Sea Grant Publication, UM-sg-ts-2002-01/VSG-02-03, 20 pp.
Sophie Hubert, Gang Li, Dennis Hedgecock. 2002. Development of a genetic linkage map, using microsatellite markers in the Pacific oyster, Crassostrea gigas. http://www.intl-pag.org/pag/10/abstracts/PAGX_W16.html
Hedgecock, D., Lin, J.-Z. DeCola, S., Haudenschild, C., Meyer, E., Manahan D.T., Bowen, B. 2002. Analysis of gene expression in hybrid Pacific oysters by massively parallel signature sequencing.
http://www.intl-pag.org/pag/10/abstracts/PAGX_W15.html