TECHNICAL ADVANCES: Effects of genotyping protocols on success and errors in identifying individual river otters (Lontra canadensis) from their faeces

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.     

Urine as another potential source for template DNA in polymerase chain reaction (PCR)

This technical note examines the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred μl of urine yielded a template DNA for PCR. This research will increase the opportunity for scientists to examine the genetic backgrounds of their target animals by using non‐invasive sample collection in the wild. Am. J. Primatol. 48:299–304, 1999. © 1999 Wiley‐Liss, Inc.     

Non-invasive genetic sampling and individual identification

Individual identification via non-invasive sampling is of prime importance in conservation genetics and in behavioural ecology. This approach allows for genetics studies of wild animals without having to catch them, or even to observe them. The material used as a source of DNA is usually faeces, shed hairs, or shed feathers. It has been recendy shown that this material may lead to genotyping errors, mainly due to allelic dropout. In addition to these technical errors, there are problems with accurately estimating the probability of identity (PI, or the probability of two individuals having identical genotypes) because of the presence of close relatives in natural populations. As a consequence, before initiating an extensive study involving non-invasive sampling, we strongly suggest conducting a pilot study to assess both the technical difficulties and the PI for the genetic markers to be used. This pilot study could be carried out in three steps: (i) estimation of the PI using preliminary genetic data; (ii) simulations taking into account the PI and choosing the technical error rate mat is sufficiently low for assessing the scientific question; (iii) polymerase chain reaction (PCR) experiments to check if it is technically possible to achieve this error rate.     

Plucked hair samples as a source of DNA: reliability of dinucleotide microsatellite genotyping

To test whether plucked hairs are a reliable source of DNA for genotyping microsatellite loci, we carried out experiments using one, three, or 10 hairs per extract for 50 alpine marmots. For each extract, seven independent genotypings were performed for the same locus (multiple-tubes approach). Two types of genotyping errors were recorded: a false homozygote defined as the detection of only one allele of a true heterozygote, and a false allele defined as a PCR-generated allele that was not one of the alleles of the true genotype. Using DNA extracted from one, three, or 10 hairs, the overall error rate was 14.00%, 4.86%, and 0.29%, respectively. Based on our results, we conclude that 10 hairs should be used to obtain consistently reliable genotypings using the single-tube approach, and that a single plucked hair could represent a reliable source of DNA if the multiple-tubes approach is used. For future studies of dinucleotide repeat diversity using DNA extracted from one to three shed or plucked hairs, we strongly recommend initiating an appropriate pilot study to quantify the error rate and to determine the reliability of the single-tube approach.     

A test of the multiplex pre-amplification approach in microsatellite genotyping of wolverine faecal DNA

Recently, a two-step PCR approach, referred to as multiplex pre-amplification, was proposed to improve microsatellite amplification from non-invasive samples such as faecal DNA. Here, we compare this new approach to standard PCR with respect to amplification success and genotyping error rates in microsatellite analysis (18 markers) of wolverine faecal DNA (48 extracts initially shown to contain amplifiable DNA). The multiplex pre-amplification approach was clearly advantageous both in terms of successful PCR amplifications (91% vs. 80%) and allelic dropout rate (2.4% vs. 12.5%). However, dropouts were to a high extent repeated in all second-step amplifications following multiplex pre-amplification, indicative of being generated during the initial PCR. Analysing more than one PCR from the initial multiplex PCR product may thus be of limited value. We instead suggest to perform two initial multiplex PCRs and to analyse a single second-step PCR from each of them. This was tested for 22 extracts at 18 loci and proved to be an effective way to obtaining a correct genotype.     

Noninvasive population genetics: a review of sample source,diet, fragment length and microsatellite motif effects on amplification success and genotyping error rates

Noninvasive population genetics has found many applications in ecology and conservation biology. However, the technical difficulties inherent to the analysis of low quantities of DNA generally tend to limit the efficiency of this approach. The nature of samples and loci used in noninvasive population genetics are important factors that may help increasing the potential success of case studies. Here we reviewed the effects of the source of DNA (hair vs. faeces), the diet of focal species, the length of mitochondrial DNA fragments, and the length and repeat motif of nuclear microsatellite loci on genotyping success (amplification success and rate of allelic dropout). Locus-specific effects appeared to have the greatest impact, amplification success decreasing with both mitochondrial and microsatellite fragments’ length, while error rates increase with amplicons’ length. Dinucleotides showed best amplification success and lower error rates compared to longer repeat units. Genotyping success did not differ between hair- versus faeces-extracted DNA, and success in faeces-based analyses was not consistently influenced by the diet of focal species. While the great remaining variability among studies implies that other unidentified parameters are acting, results show that the careful choice of genetic markers may allow optimizing the success of noninvasive approaches.     

A select panel of polymorphic microsatellite loci for individual identification of snow leopards (Panthera uncia)

Snow leopards (Panthera uncia) are elusive endangered carnivores found in remote mountain regions of Central Asia. New methods for identifying and counting snow leopards are needed for conservation and management efforts. To develop molecular genetic tools for individual identification of hair and faecal samples, we screened 50 microsatellite loci developed for the domestic cat (Felis catus) in 19 captive snow leopards. Forty‐eight loci were polymorphic with numbers of alleles per locus ranging from two to 11. The probability of observing matching genotypes for unrelated individuals (2.1 × 10^?11) and siblings (7.5 × 10^?5) using the 10 most polymorphic loci was low, suggesting that this panel would easily discriminate among individuals in the wild.     

赤眼蜂田间混合种群的分离、鉴定及其性比的确定

介绍了一种适用于赤眼蜂Trichogramma田间混合种群分离、鉴定及性比确定的简单而有效的方法,并据此方法成功地鉴定出4种自然寄生于小菜蛾卵的寄生蜂种类,拟澳洲赤眼蜂TrichogrammaconfusumViggiani、玉米螟赤眼蜂Trichogramma.ostriniaePangetChen、微突赤眼蜂TrichogrammaraioNagaraja及卷蛾分索赤眼蜂TrichogrammatoideabactraeNagaraja。     

Urine — a source for noninvasive genetic monitoring in wildlife

Noninvasive samples are of increasing importance to study wild populations. In this study, we investigate the applicability of urine samples as the sole source of DNA for routine noninvasive genetic monitoring of wildlife using wolves (Canis lupus) as an example. Within the scope of a long‐term wolf population survey, we collected during winter snow tracking in Bieszczady Mountains, Poland 41 urine samples considered as utilizable for genetic analyses. DNA concentration was determined by quantitative real‐time polymerase chain reaction (qPCR) and six microsatellite loci were genotyped in threefold repeated genotyping experiments to assess the reliability of genetic analyses of urine. DNA concentration of 33 urine samples was successfully quantified and of 14 samples, we obtained congruent results for all analysed loci and all repeated genotyping experiments. The gender of urine samples was identified with a Y‐chromosome‐linked marker. Considering the high discovery rate of urine in conjunction with its genotype reliability, our study confirms that urine is a valuable source in noninvasive genetic monitoring. Additionally, preselection of samples via qPCR proved to be a powerful tool contributing to a beneficial cost‐value ratio of genetic analyses by minimizing genotyping errors.     

A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome- b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.     

Locus effects and sources of error in noninvasive genotyping

In spite of more than a decade of research on noninvasive genetic sampling, the low quality and quantity of DNA in noninvasive studies continue to plague researchers. Effects of locus size on error have been documented but are still poorly understood. Further, sources of error other than allelic dropout have been described but are often not well quantified. Here we analyse the effects of locus size on allelic dropout, amplification success and error rates in noninvasive genotyping studies of three species, and quantify error other than allelic dropout.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non-maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.     

Ageing and environmental factors affect PCR success in wolf (Canis lupus) excremental DNA samples

Individual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf (Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species—about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed species-specific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost- and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.     

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

Estimation of the number of genetic markers required for individual animal identification accounting for genotyping errors

Nearly all studies that consider the power of exclusion for individual identification using genetic markers ignore the possibility of erroneous genotypes, although individual genotype error rates are approximately 1% for microsatellites. Single nucleotide polymorphisms (SNPs) have lower error rates, but because of their lower information content, more SNPs than microsatellites will be required to obtain the same power of exclusion for traceability. In this study, we accounted for genotyping mistakes by requiring at least two discrepancies to reject a match. Exclusion probabilities were computed analytically and by simulation. A microsatellite with five alleles was approximately comparable in exclusion power to 2-2.25 SNPs. At least eight SNPs were required to achieve a 99% probability of rejection for a match between two individuals, while with 25 SNPs there was a <1% chance for a match between any of five million individuals.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

Individual assignment using microsatellite DNA reveals unambiguous breed identification in the domestic dog

Modern individual clustering methods utilising hypervariable nuclear microsatellite DNA polymorphisms are being increasingly applied in the field of population genetics. This study explores the efficiency of the clustering methods in identifying the breeds of origin of 250 domestic dog (Canis familiaris) individuals based on 10 microsatellite loci. An allele sharing distance (DAS) matrix and the corresponding neighbour-joining tree of individuals revealed monophyletic assemblages that corresponded perfectly with the breeds of origin of the dogs. Individual assignment tests using a Bayesian statistical approach, an allele frequency based method, and a DCE genetic distance based method were all extremely powerful. Most strikingly, the Bayesian method provided 100% assignment success of individuals into their correct breeds of origin and 100% exclusion success of individuals from all alternate reference populations with a high level of statistical confidence (P < 0.0001). A Bayesian Markov Chain Monte Carlo clustering approach revealed clear distinction of individuals into groups according to their breeds of origin, with a near-zero level of 'genetic admixture' among breeds. The results demonstrate that an FST of 0.18, mean expected gene diversity of 0.6 across 10 loci, and approximately 50 individuals per reference population suffice to provide maximum individual assignment success in C. familiaris. This refutes the traditional view that DNA based dog breed identification is not feasible at the individual level of resolution.