RRL SNP
Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library
Background: To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection,  a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be  identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP)  discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies  of paralogous sequences and therefore resulted in a high rate of false positive SNP identification.  Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming  methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP  discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to  generate data from a reduced representation library constructed with genomic DNA pooled from 96  unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture  (NCCCWA) broodstock population.    Results: The reduced representation library consisted of 440 bp fragments resulting from complete  digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average  6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends).  Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627  independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three  analyses were genotyped on individual fish to determine the validation rate of putative SNPs among  analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome,  examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map.  Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully  incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated  markers were associated with rainbow trout transcripts.  Conclusion: The use of reduced representation libraries and pyrosequencing technology proved to be  an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however,  modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent  genome duplication would be desirable.