Background: Microsatellites (MSs) are DNA markers with high analytical power, which are widely used in population genetics, genetic mapping, and forensic studies. Currently available software solutions for high-throughput MS design (i) have shortcomings in detecting and distinguishing imperfect and perfect MSs, (ii) lack often necessary interactive design steps, and (iii) do not allow for the development of primers for multiplex amplifications. We present a set of new tools implemented as extensions to the STADEN package, which provides the backbone functionality for flexible sequence analysis workflows. The possibility to assemble overlapping reads into unique contigs (provided by the base functionality of the STADEN package) is important to avoid developing redundant markers, a feature missing from most other similar tools.
Climate warming involves not only a rise of air temperature means, but also more frequent heat waves in many regions on earth, and is predicted to intensify physiological stress especially in extremely changeable habitats like the intertidal. We investigated the heat-shock response (HSR) and enzymatic antioxidant defense levels of Patagonian shallow-water limpets, adapted to distinct tidal exposure conditions in the sub-and intertidal. Limpets were sampled in the temperate Northern Patagonia and the subpolar Magellan region. Expression levels of two Hsp70 genes and activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in submerged and 2-and 12-h air-exposed specimens. Air-exposed Patagonian limpets showed a tiered HSR increasing from South to North on the latitudinal gradient and from high to low shore levels on a tidal gradient. SOD activities in the Magellan region correlated with the tidal rhythm and were higher after 2 and 12 h when the tide was low at the experimental site compared to the 6 h value taken at high tide. This pattern was observed in intertidal and subtidal specimens, although subtidal individuals are little affected by tides. Our study shows that long-term thermal adaptation shapes the HSR in limpets, while the oxidative stress response is linked to the tidal rhythm. Close to the warm border of their distribution range, energy expenses to cope with stress might become overwhelming and represent one cause why the limpets are unable to colonize the shallow intertidal zone.
BackgroundRising temperatures and other environmental factors influenced by global climate change can cause increased physiological stress for many species and lead to range shifts or regional population extinctions. To advance the understanding of species’ response to change and establish links between individual and ecosystem adaptations, physiological reactions have to be compared between populations living in different environments. Although changes in expression of stress genes are relatively easy to quantify, methods for reliable comparison of the data remain a contentious issue. Using normalization algorithms and further methodological considerations, we compare cellular stress response gene expression levels measured by RT-qPCR after air exposure experiments among different subpopulations of three species of the intertidal limpet Nacella.ResultsReference gene assessment algorithms reveal that stable reference genes can differ among investigated populations and / or treatment groups. Normalized expression values point to differential defense strategies to air exposure in the investigated populations, which either employ a pronounced cellular stress response in the inducible Hsp70 forms, or exhibit a comparatively high constitutive expression of Hsps (heat shock proteins) while showing only little response in terms of Hsp induction.ConclusionsThis study serves as a case study to explore the methodological prerequisites of physiological stress response comparisons among ecologically and phylogenetically different organisms. To improve the reliability of gene expression data and compare the stress responses of subpopulations under potential genetic divergence, reference gene stability algorithms are valuable and necessary tools. As the Hsp70 isoforms have been shown to play different roles in the acute stress responses and increased constitutive defenses of populations in their different habitats, these comparative studies can yield insight into physiological strategies of adaptation to environmental stress and provide hints for the prudent use of the cellular stress response as a biomarker to study environmental stress and stress adaptation of populations under changing environmental conditions.
10In this study we provide eight polymorphic microsatellite markers for the two South 11American patellogastropods Nacella magellanica and N. deaurata.
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