Source: UNIVERSITY OF RHODE ISLAND submitted to
SEQUENCING THE GENOME OF THE EASTERN OYSTER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005328
Grant No.
2015-67016-22942
Project No.
RI.W-2014-05870
Proposal No.
2014-05870
Multistate No.
(N/A)
Program Code
A1201
Project Start Date
Feb 1, 2015
Project End Date
Jan 31, 2019
Grant Year
2015
Project Director
Gomez-Chiarri, M.
Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881
Performing Department
Fish-Animal-Vet-Science
Non Technical Summary
The Eastern oyster, Crassostrea virginica, provides important ecological and economical services, making it the target of restoration projects and supporting a significant fishery/aquaculture industry with landings valued at more than $100 million in 2012. While traditional breeding practices have achieved production of oyster lines with fast growth and improved disease resistance, little is known about the genetic basis of traits of commercial, biological, and ecological importance. Advanced molecular breeding techniques necessitate information regarding markers and genes associated with these traits. The goal of this research is to develop tools and resources to assist in the elucidation of the genomic basis of traits of commercial, biological, evolutionary, and ecological interest in the Eastern oyster. We propose to develop these key resources and tools by performing the sequencing, assembly, and annotation of a reference genome and transcriptome for the Eastern oyster C. virginica. Genome researchers and bioinformatics experts, in collaboration with the Eastern Oyster Genome Consortium, will use state-of-the-art sequencing and assembly strategies to achieve these aims. These reference genome and transcriptomes for the Eastern oysters will aid the research community in the discovery of candidate genes and markers associated with traits of commercial, biological, and ecological importance in oysters.
Animal Health Component
25%
Research Effort Categories
Basic
0%
Applied
0%
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30408111080100%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
0811 - Shellfish;

Field Of Science
1080 - Genetics;
Goals / Objectives
The Eastern oyster, Crassostrea virginica, provides important ecological and economical services, making it the target of restoration projects and supporting a significant fishery/aquaculture industry with landings valued at more than $100 million in 2012. While traditional breeding practices have achieved production of oyster lines with fast growth and improved disease resistance, little is known about the genetic basis of traits of commercial, biological, and ecological importance. Advanced molecular breeding techniques necessitate information regarding markers and genes associated with these traits. The goal of this research is to develop tools and resources to assist in the elucidation of the genomic basis of traits of commercial, biological, and ecological interest in the Eastern oyster. The objectives of this research are to: 1) Perform the sequencing, assembly, and annotation of a reference genome for the Eastern oyster C. virginica; and 2) Perform the sequencing, assembly, and annotation of a reference transcriptome for the Eastern oyster C. virginica.
Project Methods
Our strategy for sequencing the Eastern oyster genome has been specially developed to address the challenges of assembling the genome of a highly polymorphic, medium-sized (around 600 Mbp), species with a high percentage of repetitive elements (36% in the Pacific oyster) for which there are no highly inbred lines available. First, we will carefully select the individual oyster with the lowest heterozygosity available to be used as a source of DNA for the genome sequencing. Currently, the breeding center at Rutgers University has two source populations for inbred material. One is a family produced from two-generations of sister-brother mating, and the other is an inbred line that has been maintained with small effective population sizes for over 8 generations. These two populations will be tested with a large number of genetic markers to determine which of them is the most inbred population. Another option involves developing highly inbred lines for the eastern oyster using self-fertilization and gynogenesis. Second, high levels of polymorphism can be dealt with in silico if the appropriate sequencing and assembly approach is used. Our sequencing strategy will rely on the preparation of fosmid libraries and novel sequencing technologies providing low cost and high-throughput of short-read Illumina sequencing (in order to obtain deep coverage of the genome), combined with long, accurate reads (used to close assembly scaffold gaps). A genome assembly will be created following a process similar to that used to assemble the Pacific oyster and Norway spruce genomes. Illumina sequences for each fosmid library pool will be assembled with Newbler, an overlap-layout graphical algorithm. Long sequences will be used to close existing assembly scaffold gaps for the assembled Eastern oyster genome using PBJelly. The final step in the process involves the use of the Quiver algorithm, to error correct the consensus of the PBJelly enhanced assembly. Gene annotation of the Eastern oyster genome will be accomplished with the NCBI gene annotation pipeline. Quality of the reference genome will be evaluated through standard measures such as number and size of scaffolds, consensus accuracy, integration tests, determination of how much base content has been accurately captured from de novo and modification approaches, and identification of regions in the genome assembly that present evidence of assembly discordance. Third, exhaustive sampling of RNA from oysters at different developmental stages and from different tissues from the same adult oyster used for the genome sequencing will be utilized to generate RNAseq data to ensure high quality gene models. The process starts with the identification and masking of DNA repeats with WindowMasker, performs EST, RNAseq and protein alignments, predicts genes with both evidence-informed and ab initio methods, and automatically consolidates this information into gene annotations having evidence-based quality scores. Quality control is a major issue for today's genome projects and several metrics of success are measured and reported for the user to judge the quality of each set of gene predictions. In addition, the cumulative transcriptomes derived from unique tissue types will be sequenced sufficiently for assembly and read counting purposes. Once a robust set of unique genes is obtained, a series of novel digital expression profiles will be generated that will markedly improve biological interpretation in downstream studies.

Progress 02/01/15 to 01/31/19

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in using comparative genomic tools to identify the genetic basis of a multitude of traits of biological, ecological and economical importance in bivalve shellfish and apply those tools for the management of wild and cultured populations of shellfish. These include groups involved in bivalve shellfish restoration, such as the Nature Conservancy, hatchery managers that provide seed for public (restoration) and commercial aquaculture, shellfish breeding programs, members of the East Coast Shellfish Breeding Consortium, and the Oyster Genome Consortium, researchers at the USDA Agricultural Research Services Laboratory in Shellfish Genetics, and a wide group of researchers using bivalve shellfish as models systems for the study of evolution and adaptation to environmental stress. A majority of the members of this community are associated with professional associations such as the National Shellfisheries Association, International Society for Genetics in Aquaculture, International Society for Aquatic Genomics, International Society of Fish and Shellfish Immunology, and are participants of the NRSP-8 National Animal Genome Research Program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The assembly has been shared with the research community and is now being used in a wide variety of research in invertebrate biology. Two hands-on training "Comparative Genomics in Shellfish" workshops were held in March 2017 and 2018 at the annual meetings of the National Shellfisheries Association with support from this award and an award from USDA NRSP8. These workshops were attended by graduate students, postdoctoral fellows, and a combination of junior and senior faculty. In these workshops, more than 30 members of the bivalve shellfish research community discussed how to best use genomics resources, including the eastern oyster genome, to answer key questions in invertebrate biology. Participants also shared tools and approaches for comparative genomics analysis. An additional workshop was held in October 2018 in coordination with the East Coast Shellfish Breeding Consortium, focused specifically on the applications of genomics to breeding. This Breeding and Genomics in Bivalves workshop was attended by hatchery managers and shellfish breeders. These workshops led to the design of a collaborative resequencing experiment and several large collaborative proposals submitted to the USDA AFRI program in 2017 (not funded), NSF EDGE (pending), the Atlantic States Marine Fisheries Commission in 2019 (pending), and the NIH Comparative Genomics program (to be submitted June 2019). Moreover, a group of graduate students (6) and postdoctoral fellows (3) from a variety of institutions are collaboratively working with junior (6) and senior (7) faculty on the analysis of the resequencing data gathered in this project, and will meet in May 2019 for another workshop on Epi(Genomics) in bivalves, in which we plan to write a series of collaborative manuscripts describing the major findings derived from the analysis of the eastern oyster genome. This collaborative project has offered excellent opportunities for training in bioinformatics, genome analysis, population genomics and evolution, environmental genomics, and genomically-informed breeding. At this point, at least 6 graduate students and 5 undergraduate students directly associated with the project have used or are using the eastern oyster assembly in their research. Some of these graduate students have received further training in bioinformatics tools so they can act as instructors in workshops and courses. How have the results been disseminated to communities of interest?This project has been disseminated through: 1) publication of two review articles and one general announcement in journals widely accessed by members of the bivalve shellfish research community; 2) announcements through relevant listservers, such as the mollwera listserver and Aquaculture Genomics newsletter; and 3) presentations and roundtable discussions at the Plant and Animal Genomics meeting in San Diego, January 2016, 2017, 2018, and 2019; 4) presentations at meetings attended by the shellfish industry, including the National Shellfisheries Association and the Northeast Regional Aquaculture Conference and Exposition; and 5) Presentationa at the Aquaculture Genomics, Genetics and Breeding Workshop, Auburn, Alabama, March 2016, two workshops at the 2017 and 2018 annual meetings of the National Shellfisheries Association, and two additional workshops in October 2018 and May 2019 at the University of Rhode Island. The workshop in October 2018 was specially designed to target shellfish breeders and hatchery managers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research generated a high-quality chromosome-level assembly of the eastern oyster genome, as well as several transcriptomes to be used in gene annotation. The annotated genome and transcriptomes have been deposited in NCBI and have been widely used by the research community for a series of applications. Some examples of these applications are provided in this final report. Oysters are known for their high polymorphism (1 SNP every 20 bp), a major challenge for genome assembly. To address this challenge, we sequenced the most highly inbred oyster available to us. An oyster from a family produced by gynogenesis of an already inbred female oyster (XGHG1) bred at small effective population sizes (Ne = 10 - 30) for over 12 generations was used for the genome sequencing. Genotyping with a small panel of microsatellite loci showed that this family experienced an approximately 50% reduction in the percentage of polymorphic loci compared with their perspective parents, with about 6 times lower heterozygosity than wild oysters collected from Delaware Bay. High quality genomic DNA from this oyster was sequenced using single molecule real time sequencing (~87x coverage). All reads were assembled using FALCON to a total size of 684Mb with an N50 contig length of 1.9Mb. The size of this genome assembly is within the range of the predicted genome size (578-675Mb). Total repetitive elements were estimated to be ~36% using WindowMasker. A majority of sequences (>99%) were scaffolded into the known number of 10 chromosomes using a HiC proximity map, and then aligned to an eastern oyster genetic linkage map to confirm correct association. The genome assembly and associated transcriptomes have been deposited in NCBI (C_virginica-3.0 - accession number GCA_002022765.4). Gene annotation using the automated NCBI pipeline predicts the presence of 34,596 protein coding genes and 4,230 non-coding. Larval and adult transcriptomes have been used to support the annotation of genes involved in several processes and pathways, such as epigenetics (e.g. chromatin-associated proteins), development (e.g. homeobox), immunity, apoptosis, and responses to environmental stress. With additional funding from USDA NRSP-8, the Eastern Oyster Genome Consortium has also re-sequenced 92 eastern oysters at 30X coverage from several cultured (disease-resistant lines) and wild populations from different biogeographic regions and ecotypes representative of the wide diversity of environments in which oysters are grown in the US, from the Gulf of Mexico to Maine. This re-sequencing project, still in process, is allowing the consortium to: a) characterize the overall genetic diversity in eastern oyster populations, b) understand genetic polymorphism across the geographic range; c) determine the effect of selection on this genetic diversity; and d) provide a foundation to studies investigating the mechanisms used by oysters to adapt to a diversity of environments. The resequencing project has identified over 30 million SNPs in the eastern oyster. These SNPs have been filtered and thinned using a variety of criteria, resulting in panels of 10, 50, and 200K SNPs that were used analyzed using principal component analysis. The 50 and 200K panels provide enough differentiation between populations and lines to be used in determination of population structure. These SNPs will be used in the future by the consortium to design genotyping platforms for genomically-informed breeding and other industry-related applications, such as parentage analysis. Ongoing analysis of the resequencing data include evaluation of linkage disequilibrium, identifications of outlier SNPs, environmental associations, copy number variation, expected versus observed CpGs, and annotation and curation of chromatin-associated sequences. Highlights from this analysis include observations of exceptional levels of variation in the eastern oyster genome, including lineage-expansions of gene families for genes involved in processes such as metabolism, apoptosis, and response to environmental challenge, as well as large levels of copy number variation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Guo X, Li C, Wang H. 2018. Diversity and evolution of living oysters. J. Shellfish Res., 37(4):755-771. https://doi.org/10.2983/035.037.0407
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Gerdol, M., Gomez-Chiarri, M., Castillo, M.G., Figueras, A., Fiorito, G., Moreira, R., Novoa, B., Pallavicini, A., Ponte, G., Roumbedakis, K., Venier, P., Vasta, G.R., 2018. Immunity in Molluscs: Recognition and Effector Mechanisms, with a Focus on Bivalvia, in: Cooper, E.L. (Ed.), Advances in Comparative Immunology. Springer International Publishing, Cham, pp. 225341. https://doi.org/10.1007/978-3-319-76768-0_11
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Gonzalez-Romero R, Suarez-Ulloa V, Rodriguez-Casariego J, Garcia-Souto D, Diaz G, Smith A, Pasantes JJ, Rand G, Eirin-Lopez JM. 2017. Effects of Florida Red Tides on histone variant expression and DNA methylation in the Eastern oyster Crassostrea virginica. Aquatic Toxicology 186, 196204. https://doi.org/10.1016/j.aquatox.2017.03.006
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Jones HR, Johnson KM, and Kelly MW. Synergistic effects of temperature and salinity on the gene expression and physiology of Crassostrea virginica. Integrative and Comparative Biology (in review)
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Robledo, J.A.F., Yadavalli, R., Allam, B., Pales-Espinosa, E., Gerdol, M., Greco, S., Stevick, R.J., G�mez- Chiarri, M., Zhang, Y., Heil, C.A. and Tracy, A.N. (2018). From the raw bar to the bench: Bivalves as models for human health. Developmental & Comparative Immunology 92, 260-282. DOI: https://doi.org/10.1016/j.dci.2018.11.020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Downey-Wall AM, Ford B, Lotterhos K. 2019. Evaluating gene expression responses of the Eastern oyster, Crassostrea virginica, under ocean acidification. Northeast Aquaculture Conference and Exposition, ACE. Boston, MA. Jan 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Guo X. 2018. Molecular adaptations of bivalve molluscs revealed by genomics analyses. Presented at 110th Annual Meeting of National Shellfisheries Association, March 18  22, 2018, Seattle, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Guo X. 2019. Telomere sequences in shrimp and bivalves. Presented at Aquaculture 2019, March 7-11, 2019, New Orleans, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Guo X. 2019. Progress in genetic improvement of eastern oysters. Presented at the Northeast Aquaculture Conference & Exposition and the Milford Aquaculture Seminar in Boston, Massachusetts, January 9-11, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hare MP, Kutsumi Y. A genomic test for hybridization between selectively bred eastern oyster aquaculture strains and wild New York populations. National Shellfisheries Association, Seattle, WA. March 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johnson KM, La Peyre JF, Casas SM, and Kelly MW. 2019. The Influences of Environment and Dermo Infection on DNA Methylation in the Eastern Oyster Crassostrea virginica. Presented at: The Society for Integrative and Comparative Biology. Integrative and Comparative Biology, Volume 59, Issue Supplement_1, March 2019, Pages e1e260, https://doi.org/10.1093/icb/icz003
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Proestou, D.A., Markey Lundgren, K., Small, J.M., Allen, S.K. Transcriptomic responses to Dermo disease in susceptible and resistant eastern oysters.� National Shellfisheries Association Meeting.� Seattle, WA, March 18-22, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Proestou, D.A., Sullivan, M., Markey Lundgren, K. Varied transcriptomic responses to Dermo disease within an eastern oyster breeding population. Plant and Animal Genome Conference. San Diego, CA, January 12-16, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wang Z, Guo X. 2018. Developing an effective transfection protocol for gene editing in the Pacific oyster, Crassostrea gigas. Presented at 110th Annual Meeting of National Shellfisheries Association, March 18  22, 2018, Seattle, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wang, Z. and X. Guo. 2019. Gene editing with CRISPR/Cas9 in the eastern oyster Crassostrea virginica. Presented at Aquaculture 2019, March 7-11, 2019, New Orleans, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Whiteside, W. and X. Guo. 2019. Identification of genes potentially associated with shell color in the eastern oyster Crassostrea virginica. Presented at Aquaculture 2019, March 7-11, 2019, New Orleans, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Roberts EM, Proestou DP, Wikfors G, Ben-Horin T, Markey-Lundgren K, Gomez-Chiarri M. 2019. Investigating the Role of Apoptosis in Disease Resistance to Dermo in the Eastern Oyster, Crassostrea virginica. Northeastern Aquaculture Conference and Exposition, Boston MA, Jan 2019. Oral presentation. Awarded student travel grant from association.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Roberts EM, Gomez-Chiarri M. 2018. Differential Expression of Apoptosis Pathway Gene Families in Response to Immune Challenge in Magallana gigas and Crassostrea virginica. 110th Annual Meeting of National Shellfisheries Association, March 18  22, 2018, Seattle, USA. Awarded student travel grant from association.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Modak T. 2018. Eastern oyster larval transcriptomes in response to probiotic and pathogenic bacteria. Ph.D. Dissertation, University of Rhode Island, December 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Roberts EM, Gomez-Chiarri, M. 2018. Differential Expression of Apoptosis Pathway Gene Families in Response to Immune Challenge in Magallana gigas and Crassostrea virginica. Plant and Animal Genomics Conference, Aquaculture Species Group Workshop. Oral Presentation. San Diego, CA, USA. Jan, 2018. Awarded student travel grant from NRSP8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Stevick RJ, Post AF, G�mez-Chiarri M. Trends in Oyster-Associated Microbial Transcriptomes. Presented at: National Shellfisheries Association, Annual Meeting, Seattle, WA, March 18-22, 2018. Awarded student travel grant from association.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Modak T, Gomez-Chiarri M. 2019. Immunological response of American oyster, Crassostrea virginica, larvae to pathogen Vibrio coralliilyticus RE22 and probiotics Bacillus pumilus RI06-95 and Phaeobacter inhibens S4. Presented at: International Society Developmental and Comparative Immunology, Santa Fe, NM, June 2018. Awarded student travel award from society.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Modak T, Nelson DN, Rowley DR, Gomez-Chiarri M. 2018. Immunological response of Crassostrea virginica larvae to probiotic Bacillus pumilus RI06-95. Presented at: National Shellfisheries Association, Annual Meeting, Seattle, WA, March 18-22, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Modak T, Nelson DN, Rowley DR, Gomez-Chiarri M. 2019. Effect of probiotics and pathogen on Crassostrea virginica larval immunity. Northeastern Aquaculture Conference and Exposition, Boston MA, Jan 2019. Oral presentation. Awarded student travel grant from association.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Modak T, Gomez-Chiarri M, Schwartz R. Exceptional copy number variation within and among eastern oyster (Crassostrea virginica) populations. Presented at: Evolution 2019. Providence, RI, June 2019


Progress 02/01/17 to 01/31/18

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in using comparative genomic tools to identify the genetic basis of a multitude of traits of biological, ecological and economical importance in bivalve shellfish and apply those tools for the management of wild and cultured populations of shellfish. These include groups involved in bivalve shellfish restoration, such as the Nature Conservancy, hatchery managers that provide seed for public (restoration) and commercial aquaculture, shellfish breeding programs, members of the East Coast Shellfish Breeding Consortium, the Molluscan Broodstock Program, and the Oyster Genome Consortium, researchers at the USDA Agricultural Research Services Laboratory in Shellfish Genetics, and a wide group of researchers using shellfish as models systems for the study of evolution and adaptation to environmental stress. A majority of the members of this community are associated with professional associations such as the National Shellfisheries Association, International Society for Genetics in Aquaculture, International Society for Aquatic Genomics, and the International Society of Fish and Shellfish Immunology, and are participants of the NRSP-8 National Animal Genome Research Program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A hands-on training "Comparative Genomics in Shellfish" workshop was held in March 2017 at the annual meeting of the National Shellfisheries Association with support from this award and an award from USDA NRSP8. In this workshop, more than 30 members of the bivalve shellfish research community discussed how to best use genomics resources to answer key questions in invertebrate biology. Participants also shared tools and approaches for comparative genomics analysis. This workshop led to the design of a community resequencing experiment (in progress) and a large collaborative proposal submitted to the USDA AFRI program (not funded). The assembly has been shared with the research community and is now being used in a wide variety of research in invertebrate biology. Another workshop is planned for March 2018 in Seattle, Washington, as part of the annual meeting of the National Shellfisheries Association. How have the results been disseminated to communities of interest?This project has been disseminated through: 1) publication of two review article and one general announcement in journals widely accessed by members of the bivalve shellfish research community; 2) announcements through relevant listservers, such as the mollwera listserver and Aquaculture Genomics newsletter; and 3) presentations and roundtable discussions at the Plant and Animal Genomics meeting in San Diego, January 2016, 2017, 2018, the Aquaculture Genomics, Genetics and Breeding Workshop, Auburn, Alabama, March 2016, and two workshops at the 2017 and 2018 annual meetings of the National Shellfisheries Association. What do you plan to do during the next reporting period to accomplish the goals?Quality of the reference genome will be evaluated through standard measures such as number and size of scaffolds, consensus accuracy, integration tests, determination of how much base content has been accurately captured from de novo and modification approaches, and identification of regions in the genome assembly that present evidence of assembly discordance. Sampling of RNA from oysters at different developmental stages and from different tissues from the same adult oyster used for the genome sequencing has been done to generate RNAseq data, which is currently being analyzed. A series of novel digital expression profiles will be generated that will markedly improve biological interpretation in downstream studies. Moreover, the Eastern Oyster Genome Consortium decided during the 2017 Comparative Genomics workshop to use remaining funds in the project, complemented with funds provided by the NRPS-8, to embark on a community resequencing project. We are now in the process of sequencing the genome of samples of oysters from several cultured (disease-resistant lines) and wild populations from different biogeographic regions and ecotypes representative of the wide diversity of environments in which oysters are grown in the US, from the Gulf of Mexico to Maine. This re-sequencing project will: a) allow to characterize the overall genetic diversity in eastern oyster populations, b) understand genetic polymorphism across the geographic range; c) determine the effect of selection on this genetic diversity; and d) provide a foundation to studies investigating the mechanisms used by oysters to adapt to a diversity of environments. The consortium will held in March 2018 another hands-on workshop targeting the development of a publication showcasing the results from the analysis of the genome by the community.

Impacts
What was accomplished under these goals? Oysters are known for their high polymorphism (1 SNP every 20 bp), a major challenge for genome assembly. To address this challenge, we have sequenced the most highly inbred oyster available to us. An oyster from a family produced by gynogenesis of an already inbred female oyster (XGHG1) bred at small effective population sizes (Ne = 10 - 30) for over 12 generations was used for the genome sequencing. Genotyping with a small panel of microsatellite loci showed that this family experienced an approximately 50% reduction in the percentage of polymorphic loci compared with their perspective parents, with about 6 times lower heterozygosity than wild oysters collected from Delaware Bay. High quality genomic DNA from this oyster was sequenced using single molecule real time sequencing (~87x coverage). All reads were assembled using FALCON to a total size of 684Mb with an N50 contig length of 1.9Mb. The size of this genome assembly is within the range of the predicted genome size (578-675Mb). Total repetitive elements were estimated to be ~36% using WindowMasker. A majority of sequences (>99%) were scaffolded into the known number of 10 chromosomes using a HiC proximity map, and then aligned to an eastern oyster genetic linkage map to confirm correct association. The genome assembly and associated transcriptomes have been deposited in NCBI (C_virginica-3.0 - accession number GCA_002022765.4). Gene annotation using the automated NCBI pipeline predicts the presence of 34,596 protein coding genes and 4,230 non-coding.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Abdelrahman, H., ElHady, M., Alcivar-Warren, A., Allen, S., Al-Tobasei, R., Bao, L., Beck, B., Blackburn, H., Bosworth, B., Buchanan, J., Chappell, J., Daniels, W., Dong, S., Dunham, R., Durland, E., Elaswad, A., Gomez-Chiarri, M., Gosh, K., Guo, X., Hackett, P., Hanson, T., Hedgecock, D., Howard, T., Holland, L., Jackson, M., Jin, Y., Kahlil, K., Kocher, T., Leeds, T., Li, N., Lindsey, L., Liu, S., Liu, Z., Martin, K., Novriadi, R., Odin, R., Palti, Y., Peatman, E., Proestou, D., Qin, G., Reading, B., Rexroad, C., Roberts, S., Salem, M., Severin, A., Shi, H., Shoemaker, C., Stiles, S., Tan, S., Tang, K.F.J., Thongda, W., Tiersch, T., Tomasso, J., Prabowo, W.T., Vallejo, R., van der Steen, H., Vo, K., Waldbieser, G., Wang, H., Wang, X., Xiang, J., Yang, Y., Yant, R., Yuan, Z., Zeng, Q., Zhou, T., 2017. Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research. BMC Genomics 18, 191. doi:10.1186/s12864-017-3557-1
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: Gerdol M, Gomez-Chiarri M, Castillo MG, Figueras A, Fiorito G, Moreira R, Novoa B, Pallavicini A, Ponte G, Roumbedakis K, Venier P, Vasta GR. Immunity in Molluscs: Recognition and Effector Mechanisms, with a Focus on Bivalvia. In: Comparative Immunology. Ed: Cooper E. Springer Link. (in press)


Progress 02/01/16 to 01/31/17

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in using comparative genomic tools to identify the genetic basis of a multitude of traits of biological, ecological and economical importance in bivalve shellfish and apply those tools for the management of wild and cultured populations of shellfish. These include groups involved in bivalve shellfish restoration, such as the Nature Conservancy, hatchery managers that provide seed for public (restoration) and commercial aquaculture, shellfish breeding programs, members of the East Coast Shellfish Breeding Consortium, the Molluscan Broodstock Program, and the Oyster Genome Consortium, researchers at the USDA Agricultural Research Services Laboratory in Shellfish Genetics, and a wide group of researchers using shellfish as models systems for the study of evolution and adaptation to environmental stress. A majority of the members of this community are associated with professional associations such as the National Shellfisheries Association, International Society for Genetics in Aquaculture, International Society for Aquatic Genomics, and the International Society of Fish and Shellfish Immunology, and are participants of the NRSP-8 National Animal Genome Research Program. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?It is estimated that the draft genome will be available on March 2017. A hands-on training workshop will occur the last week of March 2017 with support from this award and an award from NRSP8. In this workshop, members of the research community will acquire training on how to access the genome and demonstrate how to exploit this and other genomics resources to answer key questions in invertebrate biology. Members of the community will be allowed to access the tools, first through password access to those members that request it, and then through open access to the whole research community after the tools have been tested and validated by the oyster research community. How have the results been disseminated to communities of interest?This project has been disseminated through: 1) publication of one review article and one general announcement in journals widely accessed by members of the bivalve shellfish research community; 2) announcements through relevant listservers, such as the mollwera listserver and Aquaculture Genomics newsletter; and 3) presentations and roundtable discussions at the Plant and Animal Genomics meeting in San Diego, January 2016 and 2017 and the Aquaculture Genomics, Genetics and Breeding Workshop, Auburn, Alabama, March 2016. What do you plan to do during the next reporting period to accomplish the goals?Quality of the reference genome will be evaluated through standard measures such as number and size of scaffolds, consensus accuracy, integration tests, determination of how much base content has been accurately captured from de novo and modification approaches, and identification of regions in the genome assembly that present evidence of assembly discordance. Gene annotation will be accomplished with the NCBI gene annotation pipeline. Exhaustive sampling of RNA from oysters at different developmental stages and from different tissues from the same adult oyster used for the genome sequencing will be utilized to generate RNAseq data to ensure high quality gene models. In addition, the cumulative transcriptomes derived from unique tissue types will be sequenced sufficiently for assembly and read counting purposes. Once a robust set of unique genes is obtained, a series of novel digital expression profiles will be generated that will markedly improve biological interpretation in downstream studies. In addition to the workshop scheduled for March 2017, we will have another hands-on workshop targeting the development of a publication showcasing the results from the analysis of the genome by the community.

Impacts
What was accomplished under these goals? Oysters are known for their high polymorphism (1 SNP every 20 bp), a major challenge for genome assembly. To address this challenge, we are sequencing the most highly inbred oyster available to us. An oyster from a family produced by gynogenesis of an already inbred female oyster (XGHG1) bred at small effective population sizes (Ne = 10 - 30) for over 12 generations was used for the genome sequencing. Genotyping with a small panel of microsatellite loci showed that this family experienced an approximately 50% reduction in the percentage of polymorphic loci compared with their perspective parents, with about 6 times lower heterozygosity than wild oysters collected from Delaware Bay. The sequencing of the genome (50x PacBio) resulted in a de novo assembly of 819Mb in 1,310 contigs of N50 contig length of 1.59Mb. Based on the difference with the predicted genome size based on cytogenetic staining (578-675Mb), there was the concern that a significant amount of assembled heterozygous loci would be present in the assembly. Therefore, the genome was aligned to itself and estimated varied amounts of redundant sequence were defined by 97% or greater identity and different alignment length cutoffs. At a polymorphism rate of 3% (97% identity) and a 3kb alignment length cutoff, a total of 131 Mb could be redundant, 21 Mb remaining after repeat masking. Total interspersed repeats were estimated to be ~40% as measured by the RepeatModel r tool. However, large portions of non-redundant sequence internal to each contig may be removed if actual aligned bases within each scaffold or contig are removed. Therefore, sequence total for all scaffolds that are subsumed by other scaffolds were re-estimated and only ~1Mb of redundant sequence was found. A total of 88% of the 819 Mb assembly was assigned to linkage groups using a newly established sex averaged linkage map (RADseq), but marker order is not maintained, as seen in other bivalves. Thus scaffold assignments are considered chromosome aggregates. Mapping of transcriptome reads to the draft genome is in process.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: McDowell, I.C., Modak, T.H., Lane, C.E., Gomez-Chiarri, M., 2016. Multi-species protein similarity clustering reveals novel expanded immune gene families in the eastern oyster Crassostrea virginica. Fish & Shellfish Immunology. doi:10.1016/j.fsi.2016.03.157


Progress 02/01/15 to 01/31/16

Outputs
Target Audience:This research is an integral part of efforts to aid in the growth of the local and national oyster aquaculture industry, represented in the United States of America by the East Coast Shellfish Growers Association (ECSGA) and the Pacific Coast Shellfish Growers Association (PCSGA). In addition to the oyster aquaculture industry, this research is targeted to a wide range of members of the bivalve shellfish community that are interested in using comparative genomic tools to identify the genetic basis of a multitude of traits of biological, ecological and economical importance in bivalve shellfish and apply those tools for the management of wild and cultured populations of shellfish. These include groups involved in bivalve shellfish restoration, such as the Nature Conservancy, hatchery managers that provide seed for public (restoration) and commercial aquaculture, shellfish breeding programs, members of the East Coast Shellfish Breeding Consortium, the Molluscan Broodstock Program, and the Oyster Genome Consortium, researchers at the USDA Agricultural Research Services Laboratory in Shellfish Genetics, and a wide group of researchers using shellfish as models systems for the study of evolution and adaptation to environmental stress. A majority of the members of this community are associated with professional associations such as the National Shellfisheries Association, International Society for Genetics in Aquaculture, International Society for Aquatic Genomics, and the International Society of Fish and Shellfish Immunology, and are participants of the NRSP-8 National Animal Genome Research Program. Changes/Problems:The sequencing strategy was modified according to changes in the available technology and the decreasing costs of long read sequencing. Instead of relying on the creation of fosmid libraries, we are relying on long-read sequencing using the Pacific Biosciences technology. This strategy has provided promising data on a preliminary assembly. What opportunities for training and professional development has the project provided?Once the draft genome is available (target date of September 2016), this project will involve members of the research community that will participate in annotation through a series of workshops. Members of the community will be allowed to access the tools, first through password access to those members that request it, and then through open access to the whole research community after the tools have been tested and validated by the oyster research community. How have the results been disseminated to communities of interest?This project has been disseminated through: 1) publication of one review article and one general announcement in journals widely accessed by members of the bivalve shellfish research community (see publications); 2) announcements through relevant listservers, such as the mollwera listserver and Aquaculture Genomics newsletter; and 3) presentations and roundtable discussions at the Plant and Animal Genomics meeting in San Diego, January 2016 and the Aquaculture Genomics, Genetics and Breeding Workshop, Auburn, Alabama, March 2016. What do you plan to do during the next reporting period to accomplish the goals?Sequencing will continue until a target of about 50x in coverage is reached. Further assemblies of the genome will be performed and quality of the reference genome will be evaluated through standard measures such as number and size of scaffolds, consensus accuracy, integration tests, determination of how much base content has been accurately captured from de novo and modification approaches, and identification of regions in the genome assembly that present evidence of assembly discordance. Gene annotation will be accomplished with the NCBI gene annotation pipeline. Exhaustive sampling of RNA from oysters at different developmental stages and from different tissues from the same adult oyster used for the genome sequencing will be utilized to generate RNAseq data to ensure high quality gene models. In addition, the cumulative transcriptomes derived from unique tissue types will be sequenced sufficiently for assembly and read counting purposes. Once a robust set of unique genes is obtained, a series of novel digital expression profiles will be generated that will markedly improve biological interpretation in downstream studies.

Impacts
What was accomplished under these goals? Oysters are known for their high polymorphism (1 SNP every 20 bp), a major challenge for genome assembly. To address this challenge, we are sequencing the most highly inbred oyster available to us. The Guo lab (Rutgers University) has produced using other funding several inbred oyster families through: 1) gynogenesis of an already inbred female oyster (XGHG1) that has bred at small effective population sizes (Ne = 10 - 30) for over 12 generations; and 2) self-fertilization of a hermaphroditic oyster from one of Rutgers disease-resistant strain (NEH72). To confirm pedigree and identify the most inbred oyster for genome sequencing, oysters were genotyped at 12 and 22 months of age with 11 and 15 microsatellite markers, respectively. In both tests, oysters from the two inbred families were significantly more inbred than their parents and wild oysters. This restricted genotyping showed that the progeny in both families experienced an approximately 50% reduction in the percentage of polymorphic loci compared with their perspective parents: from 0.258 to 0.112 in XGHG1 and from 0.730 to 0.358 in XGNH1. XGHG1 is clearly the most inbred family. Compared with XGHG1, percent polymorphic loci is about 3 times higher in XGNH1 and about 6 times higher in wild oysters collected from Delaware Bay. An oyster from the most inbred family (RU13XGHG1#28) was selected for genome sequencing. Genomic DNA from this oyster was extracted by collaborators at the USDA ARS Shellfish Genetics Laboratory and sent to the McDonnell Genome Institute at Washington University for sequencing. A draft assembly of the PacBio sequencing data (about 25x coverage) available at the time of the submission of this report showed a contig count of 2,807 with an assembly length of809,991,539 bp, an N50 contig length of 930,591 bp, in which the largest contig is 9,207,522 bp in length. In this assembly, about 47% of the contigs are larger than 1Mbp.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: G�mez-Chiarri, M., Guo, X., Tanguy, A., He, Y., Proestou, D. The use of -omic tools in the study of disease processes in marine bivalve mollusks. Journal of Invertebrate Pathology (2015), Pathogens and Disease Processes in Marine Molluscs 131, 137154. doi:10.1016/j.jip.2015.05.007
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: G�mez-Chiarri, M., Warren, W.C., Guo, X., Proestou, D. Developing tools for the study of molluscan immunity: The sequencing of the genome of the eastern oyster, Crassostrea virginica. Fish & Shellfish Immunology, SI: Molluscan Immunity (2015) 46, 24. doi:10.1016/j.fsi.2015.05.004
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Guo X. et al. Current status of oyster genome assembly and perspectives for application of genome-based technologies. Aquaculture Genomics, Genetics and Breeding Workshop, Auburn, Alabama, March 2016