USE OF MOLLUSC SHELLS FOR DNA-BASED MOLECULAR ANALYSES

Genetic approaches are increasingly being considered for investigations into the systematics and the conservation biology of molluscs. Here, we investigate the potential of using shell material for DNA-based analyses, using the example of endangered freshwater pearl mussels (Margaritifera margaritifera). Tissue and shells from 15 dead specimens were sampled. Their shells were split into four-quarters and re-exposed to original stream water for zero (unexposed), 1, 3 and 6 months. The influences of exposure time and resulting shell degradation, different grinding procedures and different DNA extraction methods on the quantity and quality of extractable DNA were assessed, using tissue samples as a reference. A combination of short shell-exposure time, medium grinding size and phenol-chloroform extraction procedures resulted in the most robust PCR and in lowest microsatellite genotyping errors. Amplification of nine microsatellite loci was successful in 89% of samples derived from fresh shell DNA. Genotyping errors were mostly the result of false alleles (7.8%), whereas allelic dropout only occurred at a rate of 5.7%. Shell material re-exposed to stream water for >= 1 month did not produce reliable genotyping results but amplification of mitochondrial COI was still successful. Real-time quantitative PCR of microsatellite-flanking DNA regions was shown to be an effective tool for pre-assessing DNA-quantity in shell material. These results suggest that fresh shell material may be a useful source of DNA for genetic analyses in mollusc species if the increased risk of errors compared to the use of soft tissues is adequately addressed.