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United States Department of Agriculture

Agricultural Research Service

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Research Project: HIGH-THROUGHPUT SCREENING OF SNPS IN THE EASTERN OYSTER

Location: Forage Seed and Cereal Research

2010 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. Documents SCA with U of RI.


3.Progress Report

The new SCA agreement has been finalized, research plans have been finalized, the postdoctoral researcher position to implement the plan has been advertised, and candidates are currently being interviewed. In the coming year, 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 US. 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.

The ADODR monitored the cooperator’s performance on this extramural agreement primarily through email correspondence and telephone calls.


Last Modified: 8/30/2014
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