Validation of single nucleotide polymorphism (SNP) markers from an immune Expressed Sequence Tag (EST) turbot, Scophthalmus maximus, database

An expressed sequence tag (EST) turbot database from immune tissues was tracked to identify and validate single nucleotide polymorphism (SNP) markers suitable for map construction and population analysis in turbot (Scophthalmus maximus). By means of a stringent bioinformatic tracking strategy, 341 contigs containing true SNPs were identified among the 1827 existing in the database. The number of true SNPs per contig ranged between one and 20 for most contigs analyzed. However, some contigs showed higher numbers (many of them presumed artifactual SNPs), as a result of the bioinformatics workflow used for database construction and SNP searching. Two hundred and four contigs containing between one and nine SNPs or representing genes of special relevance were included for validation. Seventy-seven out of 166 SNPs that were successfully amplified resulted polymorphic and were combined in multiplexes for high-throughput genotyping. A total of 47 transitions, 28 transversions and two indels were detected among polymorphic SNPs, C/T being the commonest (34) and G/T the least common (4) substitutions observed. This represented a transition/transversion ratio of 1.885, similar to that observed in other fish studies. Minimum Allele Frequencies of the 77 polymorphic SNPs ranged from 0.031 to 0.500 (mean: 0.228 +/- 0.015) and unbiased expected heterozygosity (He) from 0.063 to 0.529 (mean: 0.329 +/- 0.016). Only two SNP loci deviated from Hardy-Weinberg expectations and no pair of loci deviated from random genotypic associations after correction for multiple tests. Analysis of segregation in eight large turbot families showed that only four loci deviated from Mendelian segregation after Bonferroni correction. The same analysis detected the presence of null alleles at two SNPs in accordance with the high frequency of null alleles estimated at these loci from population data. Fifteen contigs containing polymorphic SNPs (19.5%) could not be annotated after screening public databases with AutoFACT. Among those annotated, 43 (55.8%) were located in the 3' untranslated region and 19 (24.7%) in the coding region, nine representing synonymous and 10 non-synonymous substitutions. Most of these genes appeared related to immune response and constitute candidates for functional changes in the correspondent proteins leading to putative phenotypic changes. These SNPs are the first validated in Scophthalmus maximus, a species of high aquaculture relevance, and provide new genetic tools for genetic mapping and QTL identification, and for population structure and parentage analysis. (C) 2011 Elsevier B.V. All rights reserved.