Noninvasive genetic tracking of the endangered Pyrenean brown bear population

Pyrenean brown bears Ursus arctos are threatened with extinction. Management efforts to preserve this population require a comprehensive knowledge of the number and sex of the remaining individuals and their respective home ranges. This goal has been achieved using a combination of noninvasive genetic sampling of hair and faeces collected in the field and corresponding track size data. Genotypic data were collected at 24 microsatellite loci using a rigorous multiple-tubes approach to avoid genotyping errors associated with low quantities of DNA. Based on field and genetic data, the Pyrenean population was shown to be composed at least of one yearling, three adult males, and one adult female. These data indicate that extinction of the Pyrenean brown bear population is imminent without population augmentation. To preserve the remaining Pyrenean gene pool and increase genetic diversity, we suggest that managers consider population augmentation using only females. This study demonstrates that comprehensive knowledge of endangered small populations of mammals can be obtained using noninvasive genetic sampling.     

A quantitative evaluation of two methods for preserving hair samples

Hair samples are an increasingly important DNA source for wildlife studies, yet optimal storage methods and DNA degradation rates have not been rigorously evaluated. We tested amplification success rates over a one‐year storage period for DNA extracted from brown bear (Ursus arctos) hair samples preserved using silica desiccation and ?20 °C freezing. For three nuclear DNA microsatellites, success rates decreased significantly after a six‐month time point, regardless of storage method. For a 1000 bp mitochondrial fragment, a similar decrease occurred after a two‐week time point. Minimizing delays between collection and DNA extraction will maximize success rates for hair‐based noninvasive genetic sampling projects.     

Shed skin as a source of DNA for genotyping seals

Obtaining a sufficient number of DNA samples from ice‐breeding marine phocids, in a noninvasive manner, has proven difficult and has limited the ability to use molecular genetics on these species. We evaluate the ability to genotype ringed seals using a novel source of DNA, skin cells shed by the seal as it moults on sea ice. We found that shed skin samples yielded a lower quantity and purity of DNA compared to tissue samples. Nevertheless, the shed skin cells were a viable source of DNA for microsatellite analysis; we found no significant difference in allelic diversity or heterozygosities between tissue samples and shed skin cells. This source of DNA should allow the rapid collection of a large number of noninvasively collected DNA samples in ice‐breeding phocids.     

Evaluating mixed samples as a source of error in non-invasive genetic studies using microsatellites

The use of noninvasive genetic sampling (NGS) for surveying wild populations is increasing rapidly. Currently, only a limited number of studies have evaluated potential biases associated with NGS. This paper evaluates the potential errors associated with analysing mixed samples drawn from multiple animals. Most NGS studies assume that mixed samples will be identified and removed during the genotyping process. We evaluated this assumption by creating 128 mixed samples of extracted DNA from brown bear (Ursus arctos) hair samples. These mixed samples were genotyped and screened for errors at six microsatellite loci according to protocols consistent with those used in other NGS studies. Five mixed samples produced acceptable genotypes after the first screening. However, all mixed samples produced multiple alleles at one or more loci, amplified as only one of the source samples, or yielded inconsistent electropherograms by the final stage of the error-checking process. These processes could potentially reduce the number of individuals observed in NGS studies, but errors should be conservative within demographic estimates. Researchers should be aware of the potential for mixed samples and carefully design gel analysis criteria and error checking protocols to detect mixed samples.     

Reliable microsatellite genotyping of the Eurasian badger (Meles meles) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple-tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximum-likelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well-studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark-recapture models has the potential to provide more accurate results.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

Automated pneumococcal MLST using liquid-handling robotics and a capillary DNA sequencer

Multilocus sequence typing (MLST) is used by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) as a routine method for the characterization of certain bacterial pathogens. The SMPRL recently started performing MLST on strains of Streptococcus pneumoniae, and here we describe a fully automated method for MLST using a 96-well-format liquid-handling robot and a 96-capillary automated DNA sequencer.     

Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas

We characterized 79 microsatellite DNA markers, which were obtained from genomic libraries enriched for CA, GA, ATG and TAGA motif repeats, in the Pacific oyster Crassostrea gigas. For eight F₁ grandparents or great‐grandparents of mapping families, the average heterozygosity, 0.705, and average number of alleles per locus, 5.7, did not vary among motif‐repeat or motif‐complexity categories. Non‐amplifying polymerase chain reaction null alleles, which were confirmed by segregation in the mapping families, were detected at 41 (51.9%) of the 79 loci. Cross‐species amplifications from C. angulata, C. sikamea, C. ariakensis and C. virginica showed a precipitous decline with distance from the focal species C. gigas.     

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the 'acceptable' scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially 'false' scores. 'Acceptable' scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.     

Owl pellets as a source of DNA for genetic studies of small mammals

Owl pellets contain a good skeletal record of the small mammals consumed, and correspond to the undigested portions of prey which are regurgitated. These pellets are easy to find at the roosting site of owls. As it has been demonstrated that amplifiable DNA can be isolated from ancient bone remains, the possibility of using owl pellets as a source of DNA for small mammal genetics studies via the polymerase chain reaction has been investigated. The main uncertainties when isolating DNA from such a material are firstly the possibility that the extracted DNA would be too degraded during the digestion in the stomach of the owl, and secondly that extensive cross-contaminations could occur among the different prey consumed. The results obtained clearly demonstrate that cross-contamination does not occur, and that mitochondrial and nuclear DNA can be amplified using skulls of small mammals found in owl pellets as a source of DNA. The relative efficiency of two methods of DNA extraction is estimated and discussed. Thus, owl pellets represent a non-invasive sampling technique which provides a valuable source of DNA for studying population genetics of small mammals.     

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping

ABSTRACT: BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman TM and Illumina Beadchip TM genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek(R)) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by GenProbe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ug, range 0.2-52 ug) than from blood (mean 210 ug, range 58-577 ug) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.     

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.     

Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.)

DNA microsatellites are ubiquitously present in eukaryotic genomes [30] and represent a vast source of highly informative markers [30, 33, 34, 2]. We describe in this article a (GGC)_n microsatellite which is widely distributed in eukaryotic genomes. Using polymerase chain reaction (PCR) techniques and DNA sequencing, we demonstrated for the first time in plant species that a (GGC)_n microsatellite locus is moderately polymorphic. Six alleles are present at this locus in rice and length polymorphisms are caused by variation in the number of tandem GGC repeats. By scoring a backcross mapping population, we were able to demonstrate that this locus is stably inherited and does not link to any known RFLP markers on the rice RFLP map. Our results suggest that DNA microsatellites should be useful in plants for construction of genetic linkage maps, extension of the existing genetic linkage maps, linkage analysis of disease and pest resistance genes, and the study of population genetics.     

Silent blood chimaerism in a mare confirmed by DNA marker analysis of hair bulbs

Microsatellite DNA markers in a mare's hair bulbs not concordant with markers in her blood confirmed the hypothesis of chimaerism which had been proposed to explain the apparent parentage exclusion of the mare from her suckling foal. Parentage analysis for this foal based on genetic markers not originating from blood cells of its dam supported a parentage verification conclusion.     

Applying genomic data in wildlife monitoring: Development guidelines for genotyping degraded samples with reduced single nucleotide polymorphism panels

The genomic era has led to an unprecedented increase in the availability of genome‐wide data for a broad range of taxa. Wildlife management strives to make use of these vast resources to enable refined genetic assessments that enhance biodiversity conservation. However, as new genomic platforms emerge, problems remain in adapting the usually complex approaches for genotyping of noninvasively collected wildlife samples. Here, we provide practical guidelines for the standardized development of reduced single nucleotide polymorphism (SNP) panels applicable for microfluidic genotyping of degraded DNA samples, such as faeces or hairs. We demonstrate how microfluidic SNP panels can be optimized to efficiently monitor European wildcat (Felis silvestris S.) populations. We show how panels can be set up in a modular fashion to accommodate informative markers for relevant population genetics questions, such as individual identification, hybridization assessment and the detection of population structure. We discuss various aspects regarding the implementation of reduced SNP panels and provide a framework that will allow both molecular ecologists and practitioners to help bridge the gap between genomics and applied wildlife conservation.     

Application of Quantitative Real-Time Polymerase Chain Reaction for Noninvasive Genetic Monitoring

ABSTRACT Noninvasive genetic monitoring of animal populations has become a widely used method in animal conservation and wildlife management due to its known advantages in sample availability of endangered or elusive species. A variety of methods have been suggested to overcome the difficulties of collecting reliable genetic data despite poor DNA quality and quantity of samples. We used quantitative real-time polymerase chain reaction (qPCR) to quantify DNA contents and preselect extracts suitable for microsatellite genotyping of noninvasive samples from 2 carnivore species, wolf (Canis lupus) and Eurasian otter (Lutra lutra). We tested 2 concentration thresholds for DNA extracts containing either 5 pg/μL or 25 pg/μL at minimum and evaluated the effect of excluding samples from genotyping falling below either of these DNA concentrations. Depending on species and threshold concentration applied, we reduced the genotyping effort by 21% to 47% and genotyping errors by 7% to 45%, yet we could still detect 82% to 99% of available genotypes. Thus, qPCR may potentially reduce genotyping effort and enhance data reliability in noninvasive genetic studies. Genetic laboratories working on noninvasive population genetic studies could transfer this approach to other species, streamline genetic analyses and, thus, more efficiently provide wildlife managers with reliable genetic data of wild populations.     

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at –20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (146 bp) and a nuclear DNA(nDNA) locus (200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26–88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.     

smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samples

Aims: To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices. Methods and Results: A real‐time PCR (RTi‐PCR) for the quantitative and species‐specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false‐negative results. The smcL‐IAC RTi‐PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R² > 0·9989) and PCR efficiency (E > 0·984) over a 6‐log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL‐IAC RTi‐PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid. Conclusions: This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. Significance and Impact of the Study: We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments.