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.     

Estimating mountain goat abundance using DNA from fecal pellets

Non-invasive collection of tissue samples to obtain DNA for microsatellite genotyping required to estimate population size has been used for many wildlife species but rarely for ungulates. We estimated mountain goat (Oreamnos americanus) population size on a mountain complex in southwestern British Columbia by identification of individuals using DNA obtained from fecal pellet and hair samples collected during 3 sampling sessions. We identified 55 individuals from 170 samples that were successfully genotyped, and estimated a population of 77 mountain goats (SE = 7.4). Mean capture probability was 0.38 (SE = 0.037) per session. Our technique provides one of the first statistically rigorous estimates of abundance of an ungulate species using DNA derived primarily from fecal pellets. Our technique enables managers to obtain minimum counts or population estimates of ungulates in areas of low sightability that can be used for conservation and management. © 2011 The Wildlife Society.     

River otter population size estimation using noninvasive latrine surveys

Across much of North America, river otter (Lontra canadensis) populations were extirpated or greatly reduced by the early 20th century. More recently, reintroductions have resulted in restored populations and the recommencement of managed trapping. Perhaps the best example of these river otter reintroductions occurred in Missouri, regarded as one of the most successful carnivore recovery programs in history. However, abundance estimates for river otter populations are difficult to obtain and often contentious when used to underpin management activities. We assessed the value of latrine site monitoring as a mechanism for quantifying river otter abundance. Analyses of fecal DNA to identify individual animals may result in an improved population estimate and have been used for a variety of mammal species. We optimized laboratory protocols, redesigned existing microsatellite primers, and calculated genotyping error rates to enhance genotyping success for a large quantity of river otter scat samples. We also developed a method for molecular sexing. We then extracted DNA from 1,421 scat samples and anal sac secretions (anal jelly) collected during latrine site counts along 22–34-km stretches representing 8–77% of 8 rivers in southern Missouri in 2009. Error rates were low for the redesigned microsatellites. We obtained genotypes at 7–10 microsatellite loci for 24% of samples, observing highest success for anal jelly samples (71%) and lowest for fresh samples (collected within 1 day of defecation). We identified 63 otters (41 M, 22 F) in the 8 rivers, ranging from 2 to 14 otters per river. Analyses using program CAPWIRE resulted in population estimates similar to the minimum genotyping estimate. Density estimates averaged 0.24 otters/km. We used linear regression to develop and contrast models predicting population size based on latrine site and scat count indices, which are easily collected in the field. Population size was best predicted by a combination of scats per latrine and latrines per kilometer. Our results provide methodological approaches to guide wildlife managers seeking to initiate similar river otter fecal genotyping studies, as well as to estimate and monitor river otter population sizes. © 2011 The Wildlife Society.     

Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

Highly variable microsatellite loci for studies of introduced populations of the small Indian mongoose (Herpestes javanicus)

During their introduction, non‐native species typically undergo founder events that reduce genetic variation. To allow a high‐resolution genetic investigation of introduced populations of the small Indian mongoose (Herpestes javanicus), we developed primers for nine variable microsatellite loci. Their applicability was assessed in 10 mongooses from the large Fijian population, which originated from a single pair from Calcutta, India. The number of alleles ranged from two to five per locus, possibly as a result of preservation of initial variability and in situ mutations during the rapid population expansion after introduction.     

Low Genotyping Error Rates and Noninvasive Sampling in Bighorn Sheep

Abstract Noninvasive DNA sampling allows studies of natural populations without disturbing the target animals. Unfortunately, high genotyping error rates often make noninvasive studies difficult. We report low error rates (0.0–7.5%/locus) when genotyping 18 microsatellite loci in only 4 multiplex polymerase chain reaction amplifications using fecal DNA from bighorn sheep (Ovis canadensis). The average locus-specific error rates varied significantly between the 2 populations (0.13% vs. 1.6%; P < 0.001), as did multi-locus genotype error rates (2.3% vs. 14.1%; P < 0.007). This illustrates the importance of quantifying error rates in each study population (and for each season and sample preservation method) before initiating a noninvasive study. Our error rates are among the lowest reported for fecal samples collected noninvasively in the field. This and other recent studies suggest that noninvasive fecal samples can be used in species with pellet-form feces for nearly any study (e.g., of population structure, gene flow, dispersal, parentage, and even genome-wide studies to detect local adaptation) that previously required high-quality blood or tissue samples.     

The isolation of microsatellite loci in the Mediterranean fruitfly Ceratitis capitata (Diptera: Tephritidae) using a biotin/streptavidin enrichment technique

The Medfly (Ceratitis capitata) is a polyphagous dipteran pest which has spread from North Africa to the countries of the Mediterranean Basin and has also invaded tropical and subtropical regions throughout the world. Colonizing populations typically possess low levels of genetic variability. Microsatellites provide an effective means of investigating the population structure of such genetically depauperate populations, however, microsatellite markers traditionally require a long phase of development in new taxa. We used a biotin/streptavidin capture technique to isolate microsatellites directly from C. capitata genomic DNA and we describe here the identification of seven polymorphic microsatellite markers in C. capitata.     

Plant microsatellite genotyping with 4-color fluorescent detection using multiple-tailed primers

We extended the concept of fluorescent microsatellite genotyping with a single-universal tailed primer to the simultaneous use of three different tailed primers to allow multiplexed 4-color detection for medium throughput genotyping of plant species. The method was tested on Eucalyptus DNA samples using three forward primer sequences of human microsatellite markers labeled with different fluorescent dyes. The robustness of the method was tested for the simultaneous detection and genetic analysis of microsatellites in a genetic mapping experiment. This method allows reliable and cost-effective genotyping with the same level of multiplexing attained in regular microsatellite fluorescent detection assays. Besides the enhanced quality of the genotypic data provided by the fluorescent detection method when compared to colorimetric ones, the economy brought about by this method becomes greater with an increasing number of microsatellite markers. This method has been particularly useful for genotyping populations of several tropical tree species addressing community-wide population genetics and conservation questions.     

Genetic integrity and individual identification-based population size estimate of the endangered long-tailed goral,Naemorhedus caudatus from Seoraksan National Park in South Korea,based on a non-invasive genetic approach

ABSTRACT

The long-tailed goral (also called the Amur goral) Naemorhedus caudatus (subfamily Caprinae), a vulnerable and protected species designated by IUCN and CITES, has sharply been declining in the population size and is now becoming critically endangered in South Korea. This species has been conserved as a natural monument by the Korean Cultural Heritage Administration since 1968. In this study, using 78 fecal DNA samples with a non-invasive genetic approach, we assessed the genetic integrity and individual identification-based population size for the goral population from Seoraksan National Park representing the largest wild population in Korea. Using the successfully isolated 38 fecal DNA, phylogeographic and population genetic analyses were performed with mitochondrial DNA control region (CR) sequences and nine microsatellite loci. We found seven CR haplotypes, of which five were unique to the Seoraksan population, considering previously determined haplotypes in Korean populations. The Seoraksan population showed higher haplotype diversity (0.777?±?0.062) and mean number of alleles (4.67?±?1.563) relative to southern populations in Korea reported from previous studies, with no signal of a population bottleneck. Microsatellite-based individual identification estimate based on probability of identity (PID) indicated a population size of ≥30 in this population. Altogether, we suggest that for future management efforts of this species in the Seoraksan National Park, conserving its genetic integrity as an ‘endemic’ lineage, and curbing a decrease in its number through mitigating habitat destruction might be key to secure the population for the long term.     

Species identification and genetic structure of threatened seahorses in Gran Canaria Island (Spain) using mitochondrial and microsatellite markers

A non-invasive DNA analysis of seahorse populations was carried out after extensive underwater surveys in Gran Canaria Island (Spain). In this geographical area, the presence of two species, Hippocampus hippocampus and H. guttulatus, has been previously reported. Sequencing of 16S ribosomal DNA (16S rDNA) was used for specific identification of live seahorses sampled in situ, as a previous step to evaluate genetic structure based on ten microsatellite markers. Phylogenetic analyses revealed the presence of a single species, H. hippocampus, in the seahorse communities found at Gran Canaria. No evidences of H. guttulatus or interspecific hybrids were found based on 16S rDNA and microsatellite data. The nuclear markers revealed low genetic diversity and lack of population structure across populations of Gran Canaria Island, with evidence of small population sizes. This study provides critical information to support conservation strategies of Gran Canaria seahorses.     

Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.     

Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.     

Development of polymorphic microsatellite loci for North American river otters (Lontra canadensis) and amplification in related Mustelids

In order to complement ecological research on native and reintroduced populations of the North American river otter (Lontra canadensis), we developed 10 polymorphic microsatellite loci that would permit us to evaluate population genetic structure and conduct fecal DNA analyses. The number of alleles per locus ranged from 3 to 14 and observed heterozygosity ranged from 0.23 to 0.68. Preliminary screening revealed that primers were polymorphic in a subset of other Mustelids.     

Rapid multiplex PCR based species identification of wild tigers using non-invasive samples

Conservation and management of rare and elusive species requires accurate data on presence or absence. In such cases, molecular genetics based species identification approaches can prove invaluable, especially in conjuncture with non-invasive DNA sampling. However, non-invasive sources yield DNA in low concentration that is degraded, which could result in false negatives for species identification. In this paper, we developed a set of primers for PCR-based species identification of tigers. Our results reveal high rates (upto 90%) of species identification for both fresh (less than 48 h) and old (between 7 days and 3 months) fecal samples from the field. Experiments reveal that multiplex PCR (amplifying more than one genomic region) results in an increase in conclusive species identification (and a consequent decrease in the number of false negatives) from 55% to 89% for old fecal samples. We demonstrate that this increased success is because we experimentally overcome the problems of low DNA template quantity (using the multiplex PCR kit, increases species identification from 55% to 72%) and low template DNA quality (two sets of primers increase the species identification success from 72% to 89%). We recommend that multiplex PCR based methods be used (in conjuncture with species specific primers) for other rare and elusive species since such methods will potentially significantly decrease error in species identification.     

Development of novel real‐time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan^® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.     

A centralized model for creating shared,standardized, microsatellite data that simplifies inter-laboratory collaboration

We demonstrate an efficient model for standardizing microsatellite DNA data among laboratories studying Oncorhynchus mykiss. Eight laboratories standardized 13 microsatellite loci following allele nomenclature of a central laboratory (average inter-laboratory genotyping concordance >98%). Following this central model, we have currently standardized 298 alleles from throughout the species native range. Although we focus here on O. mykiss, our experiences and recommendation apply equally to other broadly distributed species that may benefit from multi-laboratory collaborative data collection.     

A refined panel of 42 microsatellite loci to universally genotype catarrhine primates

1. Microsatellite genotyping is an important genetic method for a number of research questions in biology. Given that the traditional fragment length analysis using polyacrylamide gel or capillary electrophoresis has several drawbacks, microsatellite genotyping‐by‐sequencing (GBS) has arisen as a promising alternative. Although GBS mitigates many of the problems of fragment length analysis, issues with allelic dropout and null alleles often remain due to mismatches in primer binding sites and unnecessarily long PCR products. This is also true for GBS in catarrhine primates where cross‐species amplification of loci (often human derived) is common.
2. We therefore redesigned primers for 45 microsatellite loci based on 17 available catarrhine reference genomes. Next, we tested them in singleplex and different multiplex settings in a panel of species representing all major lineages of Catarrhini and further validated them in wild Guinea baboons (Papio papio) using fecal samples.
3. The final panel of 42 microsatellite loci can efficiently be amplified with primers distributed into three amplification pools.
4. With our microsatellite panel, we provide a tool to universally genotype catarrhine primates via GBS from different sample sources in a cost‐ and time‐efficient way, with higher resolution, and comparability among laboratories and species.

     

Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.     

Microsatellites for eastern Australian Banksia species

We developed eight primer pairs for Banksia microsatellite markers (five using DNA from Banksia oblongifolia and three from Banksia robur) in order to study the processes of speciation within hybridizing B. oblongifolia, B. robur and Banksia paludosa complex. We genotyped four populations of B. oblongifolia and B. robur, and three of B. paludosa. Numbers of alleles ranged from 1 to 13 across the three species and observed average heterozygosities ranged from 0.000 to 0.833. At least four loci completely discriminated B. robur from B. oblongifolia and three discriminated B. paludosa from B. oblongifolia. Seven of these primers amplified DNA from at least two of three other local species.     

Evaluation of Comparative DNA Amplification Fingerprinting for Rapid Species Identification within the Genus Clusia

Understanding the taxonomiy of the tropical genus Clusia (Fam. Clusiaceae, Ord. Theales) has been hampered by the difficulties inherent in studying tropical dioecious, succulent, arborescent, epi- or hemiepiphytic taxa. Species identification by morphological traits often requires the terminal inflorescences and/or the succulent capsular fruits. To allow species differentiation based exclusively on vegetative tissue, a frequent necessity during ecological field studies, a procedure has been developed for rapid isolation of genomic DNA from Clusia leaf tissue followed by DNA amplification fingerprinting with a set of single arbitrary oligomer primers (23–27 mers). Fingerprints obtained with independent DNA preparations from one individual as well as DNA preparations from several individuals of the same species were identical for the major amplification products, although minor bands were somewhat variable. Polymorphic fingerprints have been obtained with 3 different primers for 3 Clusia species (C. minor L., C. alata Pl. & Tr., C. multiflora H. B. K.), and the related Oedematopus obovatus Spruce ex. PL (Clusiaceae). The interspecific Randomly Amplified Polymorphic Markers (RAPDs) thus obtained allow a rapid identification of vegetative tissue samples collected in the field, and will assist in a revision of the controversial taxonomy of the genus Clusia.