Abstract
BackgroundSingle nucleotide polymorphisms (SNPs) represent the most abundant type of DNA variation in the vertebrate genome, and their applications as genetic markers in numerous studies of molecular ecology and conservation of natural populations are emerging. Recent large-scale sequencing projects in several fish species have provided a vast amount of data in public databases, which can be utilized in novel SNP discovery in salmonids. However, the suggested duplicated nature of the salmonid genome may hamper SNP characterization if the primers designed in conserved gene regions amplify multiple loci.ResultsHere we introduce a new intron-primed exon-crossing (IPEC) method in an attempt to overcome this duplication problem, and also evaluate different priming methods for SNP discovery in Atlantic salmon (Salmo salar) and other salmonids. A total of 69 loci with differing priming strategies were screened in S. salar, and 27 of these produced ~13 kb of high-quality sequence data consisting of 19 SNPs or indels (one per 680 bp). The SNP frequency and the overall nucleotide diversity (3.99 × 10-4) in S. salar was lower than reported in a majority of other organisms, which may suggest a relative young population history for Atlantic salmon. A subset of primers used in cross-species analyses revealed considerable variation in the SNP frequencies and nucleotide diversities in other salmonids.ConclusionSequencing success was significantly higher with the new IPEC primers; thus the total number of loci to screen in order to identify one potential polymorphic site was six times less with this new strategy. Given that duplication may hamper SNP discovery in some species, the IPEC method reported here is an alternative way of identifying novel polymorphisms in such cases.
Highlights
Single nucleotide polymorphisms (SNPs) represent the most abundant type of DNA variation in the vertebrate genome, and their applications as genetic markers in numerous studies of molecular ecology and conservation of natural populations are emerging
The diversification of the variety of molecular markers available has been an important development in the field of genetics over the past two decades [1], with one of the more recent additions to the 'molecular toolbox' being single nucleotide polymorphisms (SNPs): a variant of traditional DNA sequencing which potentially enables highthroughput analysis of numerous independent
Exon- vs. intron-primed SNP discovery strategies Out of a total of 47 loci for which primers were designed using the EPIC strategy, only 14 (30%) primer pairs produced PCR products suitable for direct sequencing – i.e., PCR amplification resulted in a single, strong band as visualized by agarose gel electrophoresis (Table 1a)
Summary
Single nucleotide polymorphisms (SNPs) represent the most abundant type of DNA variation in the vertebrate genome, and their applications as genetic markers in numerous studies of molecular ecology and conservation of natural populations are emerging. The increase in the range of molecular markers partly stems from the realisation that no particular marker type is ideal for all situations, and SNPs are no exception to this. Their beneficial features include having a relatively simple mutation model [2,3] and a high abundance in the genome It is clear that SNPs are an important class of molecular markers for genomics research and can potentially be applied in a wide range of studies
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