2012 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. This is due to the fact that 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, Oregon, 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. This will encompass 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.
This cooperative research has been concluded and the completed objectives are detailed in this final report. This research directly contributed to objective 3 of the in-house project: Identify genetic markers for economically important traits in (Pacific) oysters to enable maker-assisted selection.
In order to take advantage of collaborating research with Rutgers University on identifying markers for disease resistance to Juvenile Oyster Disease, we compared partial DNA sequences of 60 individuals from 2 families that showed differential susceptibility to Juvenile Oyster Disease. The resistant family showed about 20% mortality in response to bacterial challenge while the susceptible family showed about 80% mortality. DNA libraries were made from individuals prior and after challenge to Juvenile Oyster Disease in order to evaluate the impact of bacterial challenge on susceptibility or resistance to disease.
DNA libraries from resistant and susceptible individuals were sequenced using next generation sequencing leading to a huge database of sequencing data. The best assembly of the DNA expression data included about 172,000 unique sequences. We have identified thousands of single nucleotide polymorphisms (SNPs), small differences in the DNA sequences of genes involved in immunity and other physiological processes. This separates a critical first step in the development of genetic markers intended to assist breeding programs produce disease-resistant broodstock.
The high coverage of the sequencing combined with the number of individuals sequenced (60), provided an excellent representation of the SNPs that may have utility as molecular markers. During the performance of this research, a new genotyping technique called RAD-Tag became available. A meeting with other oyster geneticists led to the decision to focus on the development of RAD-Tags as a genotyping method. We are now in the position of adding the SNPs identified in this research to the genetic map where disease resistance markers can be developed.