Progress 01/01/09 to 12/31/12
Outputs
OUTPUTS: Activities: We developed over 4000 SNP candidates from new EST sequences generated both in collaboration with the U.S. DOE Joint Genome Institute (~140,000 EST sequences in GenBank) and by in-house RNA-Seq experiments. We collaborated with scientists at the Institute of Oceanology Chinese Academy of Sciences (IOCAS), to align SNP-containing ESTs to the oyster genome assembled at the Beijing Genome Institute (BGI) and to identify intron-exon boundaries. This enabled creation of a high-quality, 1536-plex GoldenGate bead assay, which was used to genotype 384 samples, representing five mapping families, various conspecific stocks of the Pacific oyster (Japan, New Zealand, France), four closely related species (C. sikamea, C. angulata, C. ariakensis, and C. hongkongensis), and replicate samples for estimation of genotyping error (zero errors detected in 11,645 trials). Most of the assays were successful (65% of markers scored in all 384 samples; 98% of markers scored in at least half of the samples), though not all SNPs proved to polymorphic and null alleles were surprisingly abundant for exonic SNPs (e.g. 85 markers in one F2 family had a null allele in one or both parents). Across all five mapping families, 1,025 SNPs were placed on at least one linkage map, a 10-fold increase in marker density over previous microsatellite-based maps. Dr. Patrick Gaffney, the University of Delaware subcontractor on the award, developed protocols for moderate-throughput screening of BAC pools, using a liquid handling robot, and applied these to assign mapped SNPs (n = 322 to date) to BAC clones. Dr. Ximing Guo, the Rutgers University subcontractor on the award, used 53 BAC clones for FISH and successfully mapped 40, covering all 10 chromosomes. Dr. Guos lab has done double hybridizations of mapped clones to assign linkage groups to chromosomes; morphological analysis to name each chromosome has been completed. Events: We held a video-conferencing workshop for project participants and students in August 2009. We had a workshop with colleagues at BGI/IOCAS in November 2010 to coordinate research with the international oyster genome project. We organized a genomics session at the National Shellfisheries Association annual meeting in 2011; Hedgecock delivered invited plenary addresses at the 2009 NRSP-8 meeting, the PAG XVII Conference, and at the 2012 NSA national meeting. Products: We produced 2nd-generation linkage maps with a tenfold increase in marker density. We developed HRM assays for 54 mapped SNP markers to verify parentage efficiently in a commercial breeding program. Because Illumina dropped the GoldenGate assay during the course of this project, we initiated research into SNP genotyping-by-synthesis. Dissemination: SNP genotypes for French disease-resistant and wild stocks were communicated to colleagues in France, who had donated DNA for the GoldenGate assays. PARTICIPANTS: Individuals: As PD, Dennis Hedgecock communicated with the international oyster research community, including the IOCAS and BGI, was PI on the linked, DOE Joint Genome Institute project (CSP 06-SE-02) that supplied EST sequences for this project, recruited and worked with Dr. Manoj Samanta as a consulting bioinformatician for the project, annotated the JGI EST sequences, identified SNP candidates, and supervised the construction of the GoldenGate multiplex assay and its application to samples from mapping families, diverse Pacific oyster stocks, and congeneric species. Hedgecock performed linkage analyses, prepared linkage maps, and identified sets of equally spaced SNPs for HRM marker development. He supervised a full-time technician, Grace Shin, who helped with all wet lab aspects of the project, a visiting postdoctoral scholar, Dr. Alberto Arias Perez, who developed genotyping-by-synthesis protocols, and a visiting Chinese graduate student, Xiujun Sun, who helped to develop and validate high-resolution melt (HRM) assays of SNPs mapped by this project. Project Co-PDs were Patrick Gaffney, University of Delaware, and Ximing Guo, Rutgers University. Gaffney, with the help of Ocean University of China Ph.D. candidate Zhongming Huo, assigned 322 mapped SNPs to BAC clones, by PCR tests of BAC superpools and pools, providing correspondences between the physical and linkage maps for the Pacific oyster. Gaffney annotated BAC-clone sequences that were produced by the JGI project and made these available to the IOCAS-BGI sequencing project to check genome assembly. Guo produced a comprehensive cytogenetic map for the Pacific oyster, identified the correspondence of linkage groups and chromosomes, and was chief liaison between the PDs and the IOCAS-BGI sequencing project. Guo supervised a graduate student, S. Wang, who assisted in making the integrated genetic and cytogenetic maps. Taylor Shellfish Farms, an industry partner in the project, reared stocks for research and provided matching support for the development of HRM assays of SNPs for efficient confirmation of parentage and pedigree in commercial broodstocks. Collaborators at Oregon State University and the ARS (Chris Langdon and Mark Camara) and IFREMER in France (Pierre Boudry, Sylvie Lapegue) provided DNA samples representing geographic populations of the Pacific oyster. TARGET AUDIENCES: A primary target audience for this project was the IOCAS-BGI genome sequencing project, as our goal was to provide mapping information that could be used for genome assembly. Although delays experienced by our project put us behind the release of the first draft genome, the information generated in this project will be useful (and necessary) in producing a second draft of the genome assembly. BAC sequences were supplied to BGI for comparison to the genome assembly. Another target audience for our project was the U.S. West Coast oyster aquaculture industry, in particular Taylor Shellfish Farms (TSF), Shelton, WA, which has been a long-term partner in oyster genetic research and is actively applying outcomes to an in-house breeding program. Specifically, we worked with TSF to develop the HRM assays of SNPs and to use these markers to continue our long-standing practice of genotyping all prospective broodstock to confirm pedigree. PROJECT MODIFICATIONS: The project was beset by various technical delays beyond the PDs control. For example, we did not receive the EST sequences from JGI until August, 2009, eight months after the project began. Then, though a list of SNP candidates and DNA samples were delivered to the USC Keck School of Medicine Genome Center in June 2011, we did not receive the final GoldenGate results until July 2012, just five months before termination.
Impacts
Of 1536 exonic SNPs typed in our Illumina GoldenGate assay, 1025 were subsequently mapped in one or more of five families. Six of 10 linkage groups have over 100 SNP markers each, while linkage groups 2 and 9 have only 11 and 30 SNPs, respectively. The average linkage map spans ~550 centimorgans, suggesting a marker density close to 1 per centimorgan. Besides their utility in quantitative trait locus mapping, these second-generation linkage maps reveal assembly errors in the oyster genome scaffolds recently reported by Zhang et al. (2012): 143 genome scaffolds contain two or more of the 672 exonic SNPs mapped with consistency across two or more Pacific oyster families; SNPs in 63 of these scaffolds, unexpectedly map to two or more different linkage groups. A manuscript on the second-generation linkage maps is in preparation. To facilitate population genetic analyses and parentage assignment in commercial and research broodstocks, we developed high-resolution melt (HRM) assays for short amplicons containing 54 of the mapped SNPs. Application of HRM assays to parent-offspring sets of samples revealed substantial heritable variation beyond that expected from the focal SNP; sequencing of variant haplotypes revealed the bases of variant melting profiles. A second manuscript is being prepared to describe the inheritance of HRM variants. Together with our industry partner, we have applied HRM of SNPs to parentage assignment in their breeding program. At the 54 HRM SNPs, North American and Japanese populations show slight but significant divergence, while U.S. and British Columbia populations are essentially homogeneous; a third manuscript is being prepared to describe these results. Finally, given decreasing cost of short-read sequencing, we initiated research into SNP genotyping-by-sequencing (GBS), which is likely to be more affordable than assays of fixed SNPs. We identified ApoI and BamH1 as restriction enzymes capable of reducing genome complexity and demonstrated that at least 48 barcoding libraries (individuals) multiplexed per lane of sequencing produces sufficient depth of coverage for reliable calling of SNPs. Annotation of aligned BAC contigs, capturing allelic variation in an F1 hybrid cross between two inbred lines, revealed extensive polymorphism in the form of SNPs and indels, the latter including length variation in simple sequence repeats, transposable element insertions and structural variants; a manuscript is in preparation. A manuscript describing the first cytogenetic map of the Pacific oyster with all ten chromosomes identified and linked to genetic maps is in preparation.
Publications
- Hedgecock, D. 2009. Genomics on the half shell: Challenges and opportunities for the Pacific oyster, a non-model orphan in the post-genomic era. Plant & Animal Genomes XVII Conference, NRSP-8 invited plenary, http://www.intlpag.org/2013/index.php/archives/pg-i-to-pag-xix-archiv es. Gaffney, P.M., Cunningham, C., Jenny, M., Hedgecock, D., and Guo, X.M. 2011. BAC haplotypes show extensive sequence polymorphism in the Pacific oyster genome. J. Shellfish Res. 30: 507-507. Hedgecock, D., Gaffney, P.M., Guo, X.M., Shin, G., Gracey, A., Qi, H.G., Li, L., Zhang, G.F., and Samanta M.P. 2011. GIGASNP: Integrating genetic, physical and cytogenetic maps of the Pacific oyster Crassostrea gigas. J. Shellfish Res. 30: 515-515. Wang, S., Gaffney, P.M., Hedgecock, D., Bao, Z.M., and Guo, X.M. 2011. Construction of a cytogenetic map for the Pacific oyster (Crassostrea gigas). J. Shellfish Res. 30: 561-561. Zhang, G.F., Guo, X.M., Li, L., Xu, F., Wang, X.T., Qi, H.G., Zhang, L.L., Que, H.Y., Wu, H.G., Wang, S.H., Hedgecock, D., Gaffney, P.M., Luo, R.B., Fang, X.D., and Wang, J. 2011. The oyster genome project: An update on assembly and annotation. J. Shellfish Res. 30:567-567. Hedgecock, D. 2012. Shellfish biology in the genomic and post-genomic eras. J. Shellfish Res. 31:295-295. (Invited plenary talk, National Shellfisheries Association annual meeting.) Zhang, G., Fang, X., Guo, X., Li, L., Luo, R., Xu, F., Yang, P., Zhang, L., Wang, X., Qi, H. Xiong, Z., Que, H., Xie, Y., Holland, P. W. H., Paps, J., Zhu, Y., Wu, F., Chen, Y., Wang, J., Peng, C., Meng, J., Yang, L., Liu, J., Wen, B., Zhang, N., Huang, Z., Zhu, Q., Feng, Y., Mount, A., Hedgecock, D., Xu, Z., Liu, Y., Domazet-Loso, T., Du, Y., Sun, X., Zhang, S., Liu, B., Cheng, P., Jiang, X., Li, J., Fan, D., Wang, W., Fu, W., Wang, T., Wang, B., Zhang, J., Peng, Z., Li, Y., Li, N., Wang, J., Chen, M., He, Y., Tan, F., Song, X., Zheng, Q., Huang, R., Yang, H., Du, X., Chen, L., Yang, M., Gaffney, P. M., Wang, S., Luo, L., She, Z., Ming, Y., Huang, W., Zhang, S., Huang, B., Zhang, Y., Qu, T., Ni, P., Miao, G., Wang, J., Wang, Q., Steinberg, C. E. W., Wang, H., Li, N., Qian, L., Zhang, G., Liu, X., Li, Y., Yin, Y., and Wang, J. 2012. The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490: 49-54.
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