Location: Forage Seed and Cereal Research2011 Annual Report
1a. Objectives (from AD-416)
The objective of this cooperative research project is to perform a large-scale, genome-wide screening to identify single nucleotide polymorphisms (SNPs) markers for the eastern oyster, Crassostrea virginica, using next generation sequencing approaches. The SNP markers are a critical resource for efforts to conduct marker-assisted selection with this species.
1b. Approach (from AD-416)
The identification and characterization of both QTL and polymorphisms in functional genes that control complex traits of economic importance for commercial-scale oyster culture requires a high density genetic map and thus a large number of genetic markers. While microsatellite and amplified fragment length polymorphism (AFLP) markers have been used for these purposes, single nucleotide polymorphism (SNP) markers have recently become the marker of choice because they are co-dominant, unambiguous to score, amenable to high-throughput analysis, and if derived from expressed sequence tags, located within functional genes. Deep sequencing of reduced representation libraries using next generation sequencing has been highly successful for detecting high numbers of valid single nucleotide polymorphisms (SNPs) in other species. In order to apply this approach to the Eastern oyster, Crassostrea virginica, the cooperator will hire a postdoctoral fellow who, in collaboration with the East Coast Shellfish Breeding Consortium and the ARS Shellfish Genetics Program in Newport, OR, will screen the C. virginica genome for large numbers of SNPs and develop high-throughput protocols for future QTL mapping and functional genomics research. The postdoctoral fellow will construct reduced representation libraries from pools of 40 individuals from several oyster lines and stocks encompassing the diverse germplasm available as well as the wide range of environmental conditions and disease challenges experienced by the cultured oyster industry on the East coast of the U.S. Libraries will be made using standard procedures and following recommendations by the manufacturer of the high-throughput, next-generation sequencing platform of choice. This technology will be selected among competing technologies available at the start of the project based on a compromise between costs and the expected length of reads in order to maximize both depth and coverage. SNPs will be identified using MAQ software. Several filtering criteria will be used to eliminate the least reliable SNPs, including thresholds for sequence quality and predictions of minor allele frequencies. Collaborators from the East Coast Shellfish Breeding Consortium will participate in the experimental design by helping choose the lines to be used in the construction of reduced representation libraries and providing the genomic DNA from the selected oyster lines. This research will provide a large number of SNPs that will be preliminarily annotated using searches against a variety of existing DNA sequence databases, and subsequently validated and mapped in future research by genotyping of reference oyster lines maintained by the East Coast Shellfish Breeding Consortium. Validated SNPs will be used in the future to construct a high-density SNP chip that will greatly reduce the effort necessary for the construction of a linkage map and the identification of quantitative trait loci for disease resistance in oysters as well as other important production traits.
3. Progress Report
Scientists at ARS, Corvallis, OR, have constructed libraries of expressed genes from a pool of 60 oysters from families that are resistant or susceptible to Juvenile Oyster Disease and Dermo disease. Libraries were made from individuals prior and after challenge to Juvenile Oyster Disease, in order to evaluate the impact of bacterial challenge on allele frequency (i.e. associations of SNPs with susceptibility or resistance to disease). ARS, Corvallis, Scientists have identified more than 320,000 SNPs in the susceptible family and more than 230,000 in a resistant family, using a stringent selection criteria of 35% minor allele frequency and a minimum coverage of 10 sequences. In collaboration with researchers from the Oyster Genome Consortium, we have adopted a procedure to screen for SNPs based on the experience of a project on SNP mining in a related species, the Pacific oyster. These criteria include minimum size of the read (100 bp) and distance to exon/intron junction of more than 50 bp (intron/exon junctions show more variation compared to other areas of the genome). These criteria increase the chance that primers for SNP screening will amplify. We are currently waiting for the Beijing Genome Institute to map our reads to the Pacific oyster genome so we can identify intron/exon junctions. In the coming year, collaborators from the East Coast Shellfish Breeding Consortium will participate in the experimental design by providing the genomic DNA from selected reference oyster families and wild oysters for SNP validation. The project was monitored through site visits, meetings, email correspondence and review of data.