Single Nucleotide Polymorphisms (SNPs), defined as single-base changes or short insertion or deletion mutations (indels), are the most abundant class of genetic variation found in eukaryotic genomes. SNPs are widespread, and present in both coding and non coding regions [[1–3]]. Until few years ago, the use of SNP markers was limited to model organisms with sequenced genomes, mostly because of the costs associated with SNP discovery. Methods for indirect SNP discovery detect heteroduplexes on the basis of mismatch-induced altered DNA characteristics [[4–8]], whereas typical direct SNP discovery strategies [9, 10] involve sequencing of locus-specific amplification (LSA) products from multiple individuals. One of the most common strategies adopted for SNP development in non-model organisms is the use of Expressed Sequence Tags (ESTs) as a resource for SNP marker detection [[11–14]]. This is a consequence of the increasing availability of EST libraries for non-model organisms. As a result, SNPs are becoming increasingly important for research on non-model organisms. SNPs offer the potential for genome wide scans of selectively neutral as well as adaptive variation [15, 16], with simple mutation models and powerful analytical methods , and with application to noninvasive analysis and historical DNA .
Most genetic surveys of natural populations focus on neutral loci. Whereas this provides valuable insights into the historical demography and evolution of populations (see ), it does not allow to understand the dynamics of genes that affect fitness along environmental gradients. Daphnia (Crustacea: Cladocera) offers a unique opportunity to study neutral and selective variation in natural populations with a known ecological background. Daphnia play a pivotal role in the ecology of standing waters, are widely used in population studies and environmental risk assessments, and are supported by a large community of ecologists, evolutionary biologists and ecotoxicologists [[19–21]]. This is in part due to their ease of culture, convenient size, short generation time and cyclic parthenogenetic reproduction, which make them very suitable for laboratory and field experiments, experimental evolution, and quantitative genetic analyses in multiple environments. Thanks to the sustained efforts of the Daphnia Genomics Consortium (http://daphnia.cgb.indiana.edu, DGC), Daphnia is also regarded as a model invertebrate in ecological genomics [21, 22]. Daphnia magna is, especially in Europe, intensively used to study stress responses to pollutants, climate change, and antagonistic interactions with predators and parasites [[23–25]]. Daphnia has also been subject to intensive population genetic study, with a strong focus on the impact of its peculiar reproduction mode, cyclical parthenogenesis, population genetic structure and among-population genetic differentiation [[26–29]]. Yet, although some knowledge has recently been acquired in the fields of functional responses to parasite infection  and proteomics , precious little is known about the complex interaction between neutral genetic variation, reflecting population genetic structure and demography, and the functional genomics underpinning phenotypic responses to environmental stressors. Among other reasons, the lack of suitable markers for functional traits has been one of the main limitations.
We developed three EST libraries using clonal lineages of D. magna exposed to standardized selection pressures, namely fish predation, exposure to parasites (Pasteuria ramosa), and exposure to pesticides (carbaryl). These environmental stressors are known to induce pronounced micro-evolutionary responses in D. magna [[23–25]]. Our EST sequences as well as EST sequences published in NCBI at the time of the analysis were mined for SNP markers targeting synonymous and non-synonymous polymorphisms. An in silico discovery tool purposely designed to mine EST sequences (Souche et al, in prep) was used for SNP discovery. The newly developed SNPs were validated by genotyping individuals from six natural populations of D. magna distributed at regional scale. The identification and characterization of genes differentially expressed in stress conditions and the validation of SNP mutations that could be linked to specific environmental stressors opens new interesting perspectives in the study of functional polymorphism in natural populations of D. magna.