for assistance in obtaining samples. Deutsche Forschungsgemeinschaft (Schi277/10) and the Human Frontier Science Program (HFSP RGP0221/2001-M) are gratefully acknowledged. AGC thanks NSF Grant EAR-9814845 for support of this project at an early stage.
Simple sequence repeats (SSRs, or microsatellites) have been constantly gaining importance as single-locus DNA markers in population genetics and behavioural ecology. We tested a PCR-based strategy for finding microsatellite loci in anonymous genomes, which avoids genomic library construction and screening, and the need for larger amounts of DNA. In the first step, parts of a genome are randomly amplified with arbitrary 10mer primers using RAPD fingerprinting. Labelled SSR-oligonucleotides serve as probes to detect complementary sequences in RAPD products by means of Southern analyses. Subsequently, positive RAPD fragments of suitable size are cloned and sequenced. Using GA and GT probes, we applied this approach to waterfleas (Daphnia) and revealed 37 hybridization signals in 20 RAPD profiles. Thirteen positive RAPD fragments from three Daphnia species and two hybrid 'species' were cloned and sequenced. In all cases simple sequence repeats were detected. We characterized seven perfect repeat loci, which were found to be polymorphic within and between species.
We describe a strategy that identifies molecular biomarkers and links the study of abiotic stress to evolutionary history. By utilizing the moon jellyfish Aurelia spp. as a model, we identified genes differentially regulated in response to the chemical stressor tributyltin by means of complementary DNA subtraction analyses. Expression of 3 out of 25 identified candidate genes, one oxidative stress gene, one heat shock (hsp70) gene, and one GTP-binding gene, was quantified under laboratory conditions and in field tests using semiquantitative reverse transcriptase polymerase chain reaction. Differential expression patterns were found following exposure to tributyltin and temperature treatments. The findings suggest that the identified genes are involved in response to chemical as well as heat- induced stress and may serve as biomarkers for monitoring marine habitats. Gene regulatory patterns combined with phylogenetic inferences of the hsp70 gene support a possible role of ecologically driven divergence within the genus Aurelia. We show that added information on genetic variability can raise the predictive power of molecular biomarkers in studies of individual stress response.
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