Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair

In the context of a study of wild chimpanzees, Pan troglodytes verus, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n= 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/10 μL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.     

Common species affects the utility of non‐invasive genetic monitoring of a cryptic endangered mammal: The bridled nailtail wallaby

Non‐invasive methods of monitoring wild populations (such as genotyping faeces or hair) are now widely used and advocated. The potential advantages of such methods over traditional direct monitoring (such as live capture) are that accuracy improves because sampling of non‐trappable individuals may be possible, species in difficult and remote terrain can be surveyed more efficiently, and disturbance to animals is minimal. Few studies have assessed the effects of interactions between species on remote sampling success. We test the use of non‐invasive monitoring for the cryptic, forest‐dwelling, solitary and endangered bridled nailtail wallaby (Onychogalea fraenata) that is sympatric with the ecologically similar and more common black‐striped wallaby (Macropus dorsalis). Six types of hair traps were tested for 3668 trap days, and hairs were caught with about a 10% success rate. Camera traps showed that baited hair traps targeted both wallaby species. We microscopically identified hair as bridled nailtail wallaby or black‐striped wallaby. We compared these hairs and their genotypes (using seven microsatellite loci) with known bridled nailtail wallaby hairs and genotypes derived from animal trapping. Trapped bridled nailtail wallaby hairs had characteristics that could be mistaken for black‐stripe wallaby hairs; characteristics were not diagnostic. Genetic assignment tests consistently differentiated the known bridled nailtail wallaby samples from identified black‐striped wallaby samples, however genetic overlap between most of the microsatellite markers means that they are not suitable for species identification of single samples, with the possible exception of the microsatellite locus B151. With similar trapping effort and within the same area, live‐capture mark‐recapture techniques estimated 40–60 individuals and non‐invasive methods only detected 14 genotypes. A species‐specific genetic marker would allow more efficient targeting of bridled nailtail wallaby samples and increase capture rates.     

Assessment of genotyping accuracy in a non-invasive DNA-based population survey of Asiatic black bears (Ursus thibetanus): lessons from a large-scale pilot study in Iwate prefecture,northern Japan

Non-invasive DNA genotyping using hair samples has become a common method in population surveys of Asiatic black bears (Ursus thibetanus) in Japan; however, the accuracy of the genotyping data has rarely been discussed in empirical studies. Therefore, we conducted a large-scale pilot study to examine genotyping accuracy and sought an efficient way of error-checking hair-trapping data. We collected 2,067 hair samples, successfully determined the genotypes of 1,245 samples, and identified 295 individuals. The genotyping data were further divided into 3 subsets of data according to the number of hairs used for DNA extraction in each sample (1–4, 5–9, and ≥10 hairs), and the error rates of allelic dropout and false alleles were estimated for each subset using a maximum likelihood method. The genotyping error rates in the samples with ≥10 hairs were found to be lower than those in the samples with 1–4 and 5–9 hairs. The presence of erroneous genotypes among the identified individuals was further checked using a post hoc goodness-of-fit test that determined the match between the expected and observed frequencies of individual homozygotes at 0–6 loci. The results indicated the presence of erroneous genotypes, possibly as a result of allelic dropout, in the samples. Therefore, for improved accuracy, it is recommended that samples containing ≥10 hairs should be used for genotyping and a post hoc goodness-of-fit test should be performed to exclude erroneous genotypes before proceeding with downstream analysis such as capture-mark-recapture estimation.     

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus)

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.     

Siberian Roe Deer (<Emphasis Type="Italic">Capreolus pygargus</Emphasis> Pallas, 1771) in Ukraine: Analysis of the Mitochondrial and Nuclear DNA

A molecular-genetic analysis of the nucleotide sequences of the cytochrome b gene (1140 base pairs) of the mitochondrial DNA and 17 microsatellite loci of eight samples of roe deer from the Samara forest of Dnipropetrovsk oblast (Ukraine) was carried out. For comparison, 212 corresponding mtDNA sequences of the Siberian and European roe deer and data on the variability of microsatellite markers in 49 representatives of these species were included in the study. It was noted that all the analyzed mitochondrial sequences of individuals from the Samara forest are characteristic of the Siberian roe Capreolus pygargus Pallas, 1771. Four haplotypes were described, all of which belonged to the haplogroup typical for the western part of the range of C. pygargus. A fragment analysis of the microsatellite loci of nuclear DNA confirmed the identification of the investigated group with the Siberian species.     

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis)

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as “fecal hairs”. In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I—the highest-quality DNA, grade II—high-quality DNA, and grade III—poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis. Zoo Biol 28:49–58, 2009. © 2008 Wiley-Liss, Inc.     

Temporal shifts of DNA‐microsatellite allele profiles in roe deer (Capreolus capreolus L.) within three decades

DNA‐microsatellite polymorphism (four loci) was studied in 56 male roe deer (Capreolus capreolus) from a 900‐ha hunting territory in the Vosges du Nord Mountains (France), culled over 34 years (1956–1990). Changed allele frequencies at two loci within this period, and increased allelic diversity, were traced to a phase of reduced population density and subsequent immigration. Decadic population samples collected within 900‐ha were distinguished by higher genetic variability measures than were certain geographical samples across Central Europe (4–900 km). On average, the decadic cohorts were distinguished by a gene diversity index of G_ST = 0.0286, and a genetic distance of D = 0.0938, which reflect 54% (G_ST) and 69% (D) of the respective geographic (350 km) differentiation indices of roe deer in Central Europe. The importance of demography and population ecology effects for microevolution in a large mammal is demonstrated, as is the risk of artefact by composing population samples of deer over several years. Population genetic screening should cover various demes of roe deer from the same general region, and be based on many unlinked polymorphic loci, to minimize the distorting effects of genetic dynamics at the small spatial scale.     

Biochemical and DNA markers yield strikingly different results regarding variability and differentiation of roe deer (Capreolus capreolus, Artiodactyla: Cervidae) populations from northern Germany

Three mainland and two island roe deer ( Capreolus capreolus ) populations with a total sample size of 105 individuals from Schleswig–Holstein, northern Germany, were analysed with regard to genetic variability within and differentiation among populations as revealed by eight allozyme loci known to be polymorphic in roe deer, eight microsatellite loci and 404 bp of the mitochondrial control region. Surprisingly, the allozymes were completely monomorphic, but microsatellite and control region variability were high. Hypotheses as to demographic reasons for the variability patterns found, including bottlenecks, founder effects and translocations, are put forward. There were no statistically significant differences between the island and the mainland populations in terms of genetic variability as measured by expected heterozygosity, inbreeding coefficient and allelic richness. The correlations of the various variability indices were not statistically significant after Bonferroni correction. Nevertheless, there was a clear tendency for differentiation indices to yield concordant results for microsatellite and mitochondrial markers.     

A methodological approach for non-invasive sampling for population size estimates in wild boars (Sus scrofa)

Composite microsatellite genotypes were determined at five loci from 35 tissue-sampled wild boars and used as reference genotypes to estimate both allelic drop-out rate and false allele rate in comparison to genotypes from scats and hair strands of the same animals. These rates allow to assess the genotyping reliability when only non-invasively collected material is available. Polymerase chain reaction (PCR) amplification from scats was often corrupted by inhibitors and worked poorly, whereas genotyping success in hair samples was high. Body region of hair origin had no influence on PCR suitability, whereas the type of hair had. We recommend the use of bristles. PCR conditions were optimized for single-hair (bristle) genotyping.     

Isolation and characterization of polymorphic microsatellite loci in the raccoon (Procyon lotor)

We report the isolation and characterization of 17 polymorphic microsatellite loci in the North American raccoon (Procyon lotor). These loci exhibit high levels of allelic diversity, with between four and 13 alleles per locus, and heterozygosity, with observed values of 0.500-1.000 in a sample of 20 individuals. All genotypes conformed to Hardy-Weinberg expectations and there were no instances of linkage disequilibrium detected.     

Remotely plucked hair genotyping: a reliable and non-invasive method for censusing pine marten (Martes martes,L. 1758) populations

We investigated the feasibility of using genetic techniques to census pine marten (Martes martes) populations by genotyping non-invasively collected samples (plucked hair and scats), with particular reference to the genetically depauperate Irish population. Novel real-time polymerase chain reaction methods were developed for species and sex identification, targeting short DNA sequences. Background genetic variation at 17 microsatellite loci was very low in the Irish population, with an average of 2.29 alleles per locus and expected heterozygosity of 0.35. Despite such low polymorphism, a panel of eight loci with a sibling probability of identity of 0.011 reliably identified individual pine marten and their gender, as determined by reference to genotypes of live trapped individuals. With high nuclear DNA amplification success rates (93.8%) and low genotyping error rates (1.8%), plucked hairs may represent a more reliable and cost-effective DNA source than scats for monitoring populations of this elusive carnivore, and similar taxa such as the sympatric stone marten Martes foina.

     

Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes

Determining population sizes can be difficult, but is essential for conservation. By counting distinct microsatellite genotypes, DNA from noninvasive samples (hair, faeces) allows estimation of population size. Problems arise because genotypes from noninvasive samples are error-prone, but genotyping errors can be reduced by multiple polymerase chain reaction (PCR). For faecal genotypes from wolves in Yellowstone National Park, error rates varied substantially among samples, often above the 'worst-case threshold' suggested by simulation. Consequently, a substantial proportion of multilocus genotypes held one or more errors, despite multiple PCR. These genotyping errors created several genotypes per individual and caused overestimation (up to 5.5-fold) of population size. We propose a 'matching approach' to eliminate this overestimation bias.     

First set of microsatellite markers for genetic characterization of the Eurasian beaver (Castor fiber) based on tissue and hair samples

Noninvasive genetic techniques have become indispensible tools in wildlife conservation and management. Here, we report the development of the first set of microsatellite markers for the Eurasian beaver (Castor fiber). All 15 loci show considerable variation within the sampled region in southwestern Germany, with number of alleles ranging from two to six alleles per locus. A comparison between tissue and hair samples revealed that amplification success was only slightly lower for hair samples, making their use in noninvasive monitoring feasible. Despite some evidence for false alleles and allelic dropout, 77% of all loci were genotyped successfully among all hair samples and loci tested. The developed markers will be used for subspecies differentiation and reconstruction of dispersal routes, following reintroductions in Central Europe.     

Reliable genotyping of samples with very low DNA quantities using PCR.

Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.     

Identification of European (Capreolus capreolus L.) and Siberian (C. pygargus Pall.) roe deer hybrids by microsatellite marker analysis

An analysis of 130 tissue specimens of the European (Capreolus capreolus) and the Siberian (C. pygargus) roe deer from nine regions of Russia and Ukraine using microsatellite loci analysis was conducted, aimed at the identification of hybrid animals. An optimized complex of 21 microsatellite loci was selected for the determination of species specificity and the search for interspecies hybrids. Hybrid animals were revealed in the Moscow and Volgograd regions. Their ratio in the total sample obtained in the European part of Russia was 11.9%.     

Determination of the optimal tissue source and number of microsatellites for detection of zygotic origin of cattle twins

Abstract

Twenty pairs of cattle twins were genotyped for 3 to 12 microsatellites each using semen, blood, milk and hair roots. Chimaerism was recognized in 19 pairs by discrepancies in microsatellite analysis from milk and blood as opposed to semen and hair, or by detection of more than 2 alleles per genotype from milk and blood. Chimaerism of 2, 3 or 4 alleles was demonstrated in genotypes of twins from blood as compared to 1 or 2 alleles only from hair. The appearance of predominant bands in genotypes from blood or milk representing alleles of only one of the co‐twins was not consistent among the different microsatellites for the same twins. No evidence for germ cell chimaerism was found in semen of dizygotic male twins although our PCR system could detect cell mixes as small as a 1:100 ratio. Genotyping from either hair or semen for 4 microsatellites are sufficient to confirm zygotic origin of twins at .98 accuracy. Researchers should be aware of the possibility of erroneous genotyping when analyzing DNA from twins derived from blood or milk and the potential of chimaerism as an experimental model to study different immunological characteristics of cattle co‐twins.     

Landscape connectivity influences gene flow in a roe deer population inhabiting a fragmented landscape: an individual-based approach

Changes in agricultural practices and forest fragmentation can have a dramatic effect on landscape connectivity and the dispersal of animals, potentially reducing gene flow within populations. In this study, we assessed the influence of woodland connectivity on gene flow in a traditionally forest-dwelling species--the European roe deer--in a fragmented landscape. From a sample of 648 roe deer spatially referenced within a study area of 55 x 40 km, interindividual genetic distances were calculated from genotypes at 12 polymorphic microsatellite loci. We calculated two geographical distances between each pair of individuals: the Euclidean distance (straight line) and the 'least cost distance' (the trajectory that maximizes the use of wooded corridors). We tested the correlation between genetic pairwise distances and the two types of geographical pairwise distance using Mantel tests. The correlation was better using the least cost distance, which takes into account the distribution of wooded patches, especially for females (the correlation was stronger but not significant for males). These results suggest that in a fragmented woodland area roe deer dispersal is strongly linked to wooded structures and hence that gene flow within the roe deer population is influenced by the connectivity of the landscape.     

Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.     

Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.     

A New Method for Noninvasive Genetic Sampling of Saliva in Ecological Research

Noninvasive samples for genetic analyses have become essential to address ecological questions. Popular noninvasive samples such as faeces contain degraded DNA which may compromise genotyping success. Saliva is an excellent alternative DNA source but scarcity of suitable collection methods makes its use anecdotal in field ecological studies. We develop a noninvasive method of collection that combines baits and porous materials able to capture saliva. We report its potential in optimal conditions, using confined dogs and collecting saliva early after deposition. DNA concentration in saliva extracts was generally high (mean 14 ng μl^-1). We correctly identified individuals in 78% of samples conservatively using ten microsatellite loci, and 90% of samples using only eight loci. Consensus genotypes closely matched reference genotypes obtained from hair DNA (99% of identification successes and 91% of failures). Mean genotyping effort needed for identification using ten loci was 2.2 replicates. Genotyping errors occurred at a very low frequency (allelic dropout: 2.3%; false alleles: 1.5%). Individual identification success increased with duration of substrate handling inside dog’s mouth and the volume of saliva collected. Low identification success was associated with baits rich in DNA-oxidant polyphenols and DNA concentrations <1 ng μl^-1. The procedure performed at least as well as other noninvasive methods, and could advantageously allow detection of socially low-ranked individuals underrepresented in sources of DNA that are involved in marking behaviour (faeces or urine). Once adapted and refined, there is promise for this technique to allow potentially high rates of individual identification in ecological field studies requiring noninvasive sampling of wild vertebrates.