Evaluating preservation medium for the storage of DNA in African lion Panthera leo faecal samples

Abstract

Lion faecal samples, collected in the field between 1 hour to 1 week after defecation were preserved in three different media (ethanol, ASL buffer and Two-step storage). The aim was to determine which faecal DNA field preservation method best enhances PCR amplification success. Samples stored in ethanol showed a significantly higher amplification success of microsatellite loci than samples stored in the other two media. In contrast, amplification success of a mitochondrial locus was similar among the samples stored in the three types of media. We reviewed twelve previous studies that employed different media for the storage of faeces, although patterns of success were not fully consistent among different media, ethanol storage was scored highest in the majority of these tests [Current Zoology 60 (3): 351-358, 2014].Keywords Amplification success, Faecal sample DNA, Lion Panthera leo, PCR, Preservation medium For small and elusive wildlife populations, noninvasive DNA sampling from faecal samples is a feasible method for obtaining genetic data (Kohn and Wayne, 1997). Several studies have employed this method to estimate and monitor wildlife (Kohn et al., 1999;Ernest et al., 2000;Banks et al., 2002, Flagstad et al., 2004, Bensch et al., 2006Perez et al., 2006;Zhang et al., 2009). For elusive carnivores such as lions Panthera leo non-invasive sampling is especially suitable because faecal samples are easily encountered and recovered along trails where they defecate to mark territory boundaries (Macdonald, 1980). However, studies that employ non-invasive sampling are confronted with pitfalls such as low quantity and quality DNA which can result in low genotyping success and frequent genotyping errors (Taberlet et al., 1997).The amount and quality of DNA initially present in faeces samples at the time of collection may change during the period of storage and will determine the amount and quality of DNA that can be extracted and used in downstream analyses. It is therefore important to transfer samples into a storage condition that best preserves the DNA prior to the laboratory analysis. The choice of a good preservation medium should be of prime importance for every genetic study, especially when collected samples cannot be processed immediately at the collection site. The identification of effective preservation methods can enhance PCR amplification success rate, increase genotyping accuracy and increase the feasibility of using faecal DNA for genetic studies.Faecal DNA has so far mainly been used in studies of mammals and less often for studies of birds (Idaghdour et al., 2003;Regnaut et al., 2006). In mammals, DNA has been recovered from faeces that were preserved dried (Höss et al., 1992;Kohn et al., 1995;Foran et al., 1997;Taberlet et al., 1997), in 70% ethanol (Höss, 1992;Kohn et al., 1995), in 99%-100% ethanol (Gerloff et al., 1995) and frozen at -20°C (Reed et al., 1997).Several studies have evaluated the relative genotyping success from two or more preservation media (Wasser et al., 1997;Frantzen et...

Peer Review #2 of "Factors affecting genotyping success in giant panda fecal samples (v0.1)"

“…Similar to our results, EtOH scored the highest in the study on brown bear (Murphy et al 2002), Eurasian badger (Frantz et al 2003), lion (Tende et al 2014), or was equally high as the other methods in the study on primate scats (Whittier et al 1999), gorilla (Roeder et al 2004), and coyote (Panasci et al 2011), although the preservation methods evaluated in these studies were different. These findings revealed that EtOH was a good preservation buffer for fecal samples, irrespective of the diet of the species and this method could be used in either tropical or temperate weather (Roeder et al 2004;Tende et al 2014).…”

“…This renders the conclusion of genetic studies unreliable and reduces the confidence in inferring such results for formulating management and conservation strategies (Pompanon et al 2005). Several investigations suggest that careful choice of microsatellite loci and the method used for feces preservation could enhance the genotyping success and feasibility of the use of fecal samples in such studies (Broquet et al 2007;Tende et al 2014).…”

“…A comparison of storage conditions has been made among oven-dried, frozen, and ethanol (EtOH) and bufferpreserved fecal samples from different species (reviewed in Tende et al 2014). The conclusions have, however, been inconsistent, even for the same preservation medium, suggesting that the optimal storage medium varies with species, environmental conditions, and other factors (Piggott & Taylor 2003).…”

A paired comparison of scat-collecting versus scat-swabbing methods for noninvasive recovery of mesocarnivore DNA from an arid environment

“…A variety of storage methods have been used and tested for efficacy, including desiccation, freezing, or storage in buffers or ethanol. In most studies, pure ethanol (i.e., 95–100%) works similarly to or better than other methods for shorter storage periods and can substantially outperform them during extended storage periods (Murphy et al , Frantz et al , Piggott and Taylor , Beja‐Pereira et al , Panasci et al , Tende et al ). However, acquiring, storing, and transporting sufficient quantities of ethanol for sample storage can be difficult in remote field conditions.…”

High‐resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile

“…Significant improvements have been reported based on fecal DNA analysis for both extraction and quantification of nucleic acids as part of genotyping and species identification (Kanthaswamy, Premasuthan, Ng, Satkoski, & Goyal, ). Molecular analysis of feces is a noninvasive method with a high degree of integrity and quality when using specific commercial kits for these complex matrices (Deshpande, Villarreal, & Mills, ; Ramón‐Laca, Soriano, Gleeson, & Godoy, ; Tende, Hansson, Ottosson, & Bensch, ). The main challenge of this study was to obtain DNA of good quality from fecal samples collected in the Atacama Desert.…”