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The LUminometric Methylation Assay ( LUMA) measures global DNA methylation. LUMA depends on digestion of DNA with methyl-sensitive and methyl-insensitive restriction enzymes, followed by pyrosequencing. Until recently, LUMA has been principally used for biomedical research. Here, we use chickens as a model to investigate sample quality issues relating to LUMA and then apply the method to ecological species. First, we assessed the effect of tissue storage conditions on DNA methylation values. This is an important consideration for ecological species because samples are not always ideally preserved and LUMA is sensitive to poor DNA quality. We found that good quality LUMA data could be obtained from chicken liver and brain tissues stored at 21 °C for at least 2 and 12 h, respectively. Longer storage times introduced nonspecific peaks to pyrograms which were associated with reduced DNA methylation. Repeatedly, freezing and thawing the tissues did not affect LUMA data. Second, we measured DNA methylation in 12 species representing five animal classes: amphibians ( African and Western clawed frog), reptiles (green anole lizard), fish (yellow perch, goldfish, lake trout), mammals ( American mink, polar bear, short-beaked common dolphin, Atlantic white-sided dolphin) and birds (chicken, Japanese quail). We saw a pattern of high DNA methylation in fish (84-87%), and intermediate levels in mammals (68-72%) and birds (52-71%). This pattern corresponds well with previous measures of DNA methylation generated by HPLC. Our data represent the first Cp G methylation values to be reported in several species and provide a basis for studying patterns of epigenetic inheritance in an ecological context. [ABSTRACT FROM AUTHOR]

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003–2004 pilot study using noninvasive genetic sampling for the brown bear ( Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality ( n = 33) and hair DNA extracts of poor ( n = 32) and good ( n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses. [ABSTRACT FROM AUTHOR]

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole ( Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material. [ABSTRACT FROM AUTHOR]

Shed feathers obtained by noninvasive genetic sampling (NGS) are a valuable source of DNA for genetic studies of birds. They can be collected across a large geographical range and facilitate research on species that would otherwise be extremely difficult to study. A limitation of this approach is uncertainty concerning the quality of the extracted DNA. Here we investigate the relationship between feather type, feather condition and DNA quality (amplification success) in order to provide a simple, cost-effective method for screening samples prior to genetic analysis. We obtained 637 shed feathers of the powerful owl ( Ninox strenua) from across its range in southeastern Australia. The extracted DNA was amplified using polymerase chain reaction for a range of markers including mitochondrial DNA, ND3 and nuclear DNA, a simple sequence repeat (Nst02) and a portion of the CHD-1 gene (P2/P8). We found that feather condition significantly influenced the amplification success of all three loci, with feathers characterized as ‘good’ having greater success. Feather type was found to be of lower importance, with good quality feathers of all types consistently producing high success for all three loci. We also found that the successful amplification of multilocus genotypes was dependant on the condition of the starting material and was highly correlated with successful amplification of the sex-linked CHD-1 locus. Samples with low DNA quality have a higher probability of amplification failure and are more likely to produce incorrect genotypes; therefore, identifying samples with high DNA quality can save substantial time and cost associated with the genetic analysis of NGS. As a result, we propose a method for screening shed feathers in order to provide a subset of samples which will have a greater probability of containing high quality DNA suitable for the amplification of multilocus genotypes. [ABSTRACT FROM AUTHOR]