Conditions determining PCR success in otter landscape genetics

Non-invasive sampling for genetic analysis using DNA from feces is a useful technique to monitor animal populations. But despite being a very promising tool, it still has some limitations and can give some problems such as low success rates of genotyped samples, contamination concerns and high microsatellite genotyping error rates. To optimize success, it is important to follow specific steps, including a stringent sample selection in the field, proper storage conditions, and choice of the optimal DNA extraction method for the study species. Furthermore, the PCR success rate could potentially be influenced by factors prior to collection, such as temperature and the diet of the species, as well as by sample storage time. Here, we investigated the effect of temperature at time of collection, average maximum temperature one week prior to collection and storage time on the amplification success rate of microsatellite DNA extracted from XX fecal samples of the Eurasian otter (Lutra lutra) in the Netherlands. To do this, we considered two cases: firstly, we performed a logistic regression to assess whether temperature at time of collection and/or storage time could be used to predict the genotyping success rate for one microsatellite locus, that is used as a primary selection tool for sample quality. Secondly, we considered PCR success rate as the number of successfully scored loci out of the total 13 loci used in the landscape genetic study of Dutch otters and assessed the effect of temperature at time of collection, average maximum temperatures one week prior to collection and storage time. Results showed that when considering one locus only, the amplification success rate was not influenced by temperature. However, when considering all the loci temperature at time of collection and temperature one week prior positively influenced the PCR success rate, meaning that as the temperature raised, the success rate increased. This result was contrasting most of the literature, indeed, as it would be expected, high temperatures should cause a more rapid degradation of DNA caused by higher activity of hydrolytic enzymes in feces. Probably there were outside factors (e.g. the otters’ diet) that we did not control and that were confounded with temperature requiring the need for further investigation. Storage time had no effect on DNA amplification success rate in any of our tests. Our results suggest that DNA amplification success rate is influenced by many factors. Before starting a non-invasive study, it is fundamental to take into account several factors and to study to what extent each factor influences the amplification success in order to maximize the DNA amplification success rate and to reduce genotyping error rates.