Location: Cool and Cold Water Aquaculture Research
Title: Single nucleotide polymorphism discovery in rainbow trout Authors
Submitted to: Aquaculture America Conference
Publication Type: Abstract Only
Publication Acceptance Date: February 5, 2010
Publication Date: March 1, 2010
Citation: Rexroad Iii, C.E., Castano, C., Smith, T.P., Vallejo, R.L., Jianbo, Y., Wiedmann, R.T. 2010. Single nucleotide plymorphism discovery in rainbow trout. Aquaculture America Conference. Technical Abstract: To enhance capabilities for genetic analyses in rainbow trout, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be developed. However, the evolutionarily recent whole genome duplication event complicates the use of standard approaches in the discovery of genetic markers. For instance, strategies which aim to identify single nucleotide polymorphisms (SNPs) from expressed sequence tag data encounter a high frequency of assemblies including paralogous sequences and therefore result in a high rate of false positive SNP identification. In an effort to identify and validate a large set of SNPs, 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. The reduced representation library consisted of 440bp 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 288 individual DNA samples 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, 36% of the sequences from the validated markers were associated with rainbow trout transcripts. In a subsequent effort to increase the frequency of validated SNPs, a second data set was generated by sequencing a reduced representation library created from the doubled haploid Swanson fish. Doubled haploids are the result of doubling the maternal or paternal genome; therefore they do not retain allelic variation. Any putative SNPs are the result of assemblies of paralogous sequences; determination of assembly and discovery parameters which rule out these false positives will increase the frequency of SNP validation from the pooled library data set.