Effects of storage type and time on DNA amplification success in tropical ungulate faeces

Abstract

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium-sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.

Factors affecting genotyping success in giant panda fecal samples

“…As in other studies (Murphy et al, 2002; Santini et al, 2007; Soto-Calderon et al, 2009), we found that the storage time had a negative effect on the microsatellites amplification success, suggesting that the preservation medium could only retard but not stop the degradation of fecal DNA by endonucleases. We recommended that fecal DNA should be extracted as soon as the samples are collected in order to obtain high DNA quality, to increase the accuracy of microsatellite genotyping, and to decrease the associated cost.…”

“…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). Moreover, it was reported that the optimal storage types were dependent on the storage period (Murphy et al, 2002; Soto-Calderon et al, 2009), which could be the reason for the varied degradation rate of fecal DNA in different preservation media. Such observations necessitate the assessment of the storage type for each new fecal study.…”

Reliable genotyping of the koala (Phascolarctos cinereus) usingDNAisolated from a single faecal pellet

“…Obtaining DNA isolates of sufficient quantity and quality from a single scat negates the need to isolate DNA from multiple pellets, as is sometimes required (e.g. Soto-Calderon et al 2009), thus minimizing the chance of sample contamination between different individuals. Screening DNA isolates for total DNA concentration allows inferior isolates (<0.25 ng/lL) to be discarded prior to genotyping and samples with sufficient amounts of DNA to be analysed using the minimum number of required replicates, thereby minimizing costs.…”

“…DNA isolated from faecal samples, however, may be associated with increased rates of amplification failure and genotyping error, in particular allelic dropout and false alleles (Bonin et al 2004;Pompanon et al 2005). Genotyping reliability may also be affected by scat storage (Frantzen et al 1998;Piggott & Taylor 2003a;Soto-Calderon et al 2009), sampling (Lampa et al 2008;Stenglein et al 2010) and isolation methods (Wehausen et al 2004).…”

Cross‐species amplification of bovid microsatellites in central African duikers (genusCephalophus) and other sympatric artiodactyls

“…2007). In a recent study, Soto‐Calderón et al. (2009) were able to amplify and successfully genotype unknown faecal samples from African forest artiodactyls using six published bovid microsatellite loci.…”