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Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair
Abstract
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 microL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
... Our sexing assay relies on the identification of one homozygous genotype (female) and one heterozygous genotype (male) via a bi-allelic target. In such a design, the risk of false assignation of a male to the female genotype due to allelic dropout of the Y allele is a limitation, particularly when working with templates of low DNA content 40,41 . We carried out all mammoth PCR reactions in triplicates, to comply with the multi-tube strategy developed to control for that risk. ...
... Among the mammoth extracts themselves, the decrease in amplification success rate-as well as the inconsistency in genotype call-is only observed for the 10 mammoths with the lowest CN (i.e. less than 3 copies per reaction; see Table 1). With such low DNA templates, the stochasticity of the PCR amplification process becomes a pervasive issue due to allelic dropout, and the selection of a multi-tube strategy-three replicates per sample-is a method of choice to evaluate the accuracy of the genotypes 40,41 . ...
... As discussed above, three positive replicates are usually sufficient to identify a male specimen, despite allelic dropout. However, the reciprocal confirmation of a female genotype is somewhat trickier: up to 7 positive replicates might be necessary to reach a 99% confidence in the assignment of a homozygous genotype 40,41 . Because of the rarity of the mammoth DNA, we could not spare more than three reactions per specimen. ...
After a long-standing debate, African elephants are now considered by the IUCN as two distinct species: savannah elephants (Loxodonta africana), listed as endangered on the IUCN Red List of Threatened Species, and forest elephants (Loxodonta cyclotis), critically endangered. Both are severely threatened by forest loss, fragmentation and degradation due to agriculture expansion, as well as by illegal ivory trade. Although the two species have different habitat preferences, their range overlaps in some ecotones; despite an ancient separation between these two species, hybrids have been reported in five locations. The main hybrid hotspot is located on the Democratic Republic of Congo-Uganda border and still remains understudied. Using 15 microsatellites, we investigated this hybridization zone by determining the species and hybrid status of 177 fecal samples collected in the area of Sebitoli, at the extreme North of Kibale National Park. Surprisingly for a forest area, no pure forest elephants were detected. Out of the 91 individuals sampled, a very large proportion (81.3%) were hybrid individuals mainly from a second generation or more. Only 18.7% of pure savannah elephants were detected, all originating from the DRC-Uganda border. Further analyses are necessary to assess the age of this hybridization zone. Our results emphasize that hybrids and savannah elephants can successfully range in forested area. They also show that forest elephants are rare even in their native habitat. In the current context of high threat faced by African elephant species, it is crucial to strengthen conservation efforts for these species before it is too late.
... Our sexing assay relies on the identification of one homozygous genotype (female) and one heterozygous genotype (male) via a bi-allelic target. In such a design, the risk of false assignation of a male to the female genotype due to allelic dropout of the Y allele is a limitation, particularly when working with templates of low DNA content (Gagneux et al., 1997;Taberlet et al., 1996). We carried out all mammoth PCR reactions in triplicates, to comply with the multitube strategy developed to control for that risk. ...
... Among the mammoth extracts themselves, the decrease in amplification success rateas well as the inconsistency in genotype callis only observed for the 10 mammoths with the lowest CN (i.e. less than 3 copies per reaction; see Table 1). With such low DNA templates, the stochasticity of the PCR amplification process becomes a pervasive issue due to allelic dropout, and the selection of a multi-tube strategythree replicates per sampleis a method of choice to evaluate the accuracy of the genotypes (Gagneux et al., 1997;Taberlet et al., 1996). ...
... As discussed above, three positive replicates are usually sufficient to identify a male specimen, despite allelic dropout. However, the reciprocal confirmation of a female genotype is somewhat trickier: up to 7 positive replicates might be necessary to reach a 99% confidence in the assignment of a homozygous genotype (Gagneux et al., 1997;Taberlet et al., 1996). ...
Le conflit humains-faune sauvage est très fréquent en Afrique, et est amené à s’intensifier du fait de l’expansion de l’agriculture, et de la réduction et la fragmentation de l’habitat. Les pertes liées à la consommation ou dégradation des cultures créent de l’insécurité alimentaire, ce qui accroit la pauvreté et entrave le soutien local aux efforts de conservation. Les éléphants sont souvent considérés comme les animaux responsables de la majorité des conflits. S’appuyant sur une approche pluridisciplinaire associant génétique, morphologie, sciences sociales, et eco-éthologie, notre étude vise (1) à mieux caractériser les acteurs et les interactions dans une zone située à la lisière d’une aire protégée, où vivent les deux espèces d’éléphants, et où le conflit est exacerbé par la proximité des cultures humaines et de l’habitat des éléphants ; et (2) à proposer des mesures efficaces et non violentes adaptées au contexte local. Les perspectives sont de soumettre des recommandations pour la gestion du conflit humain-faune sauvage, afin de contribuer à une meilleure protection des champs et par conséquent à l’amélioration des conditions de vie des communautés locales, ainsi qu’à la réduction des pressions de braconnage sur la faune sauvage du parc. Nous avons tout d’abord montré que les deux espèces d’éléphants d’Afrique ainsi que des individus hybrides sont présents dans la zone de Sebitoli, au nord du parc national de Kibale en Ouganda. L’étude de leur comportement en forêt et lors des incursions dans les champs montre qu’ils vivent en groupes mixtes ne permettant pas de caractériser des incursions typiques d’éléphants de forêt et d’autres typiques des éléphants de savane. Plutôt que d’adapter les recommandations de mesures de protection des cultures contre les incursions selon les espèces d’éléphants, il apparait indispensable que les mesures soient adaptées au contexte géographique, foncier, économique et social de chaque village, en tenant compte du comportement nocturne des éléphants mais aussi des autres espèces participant au conflit entre humains et faune sauvage, notamment des espèces menacées et protégées comme les chimpanzés. La diminution des interactions négatives entre humains et faune sauvage est indispensable à une cohabitation pacifique. Cela nécessite de comprendre les besoins de chaque parti et repose sur un équilibre entre développement humain et conservation de la biodiversité.
... Our sexing assay relies on the identification of one homozygous genotype (female) and one heterozygous genotype (male) via a bi-allelic target. In such a design, the risk of false assignation of a male to the female genotype due to allelic dropout of the Y allele is a limitation, particularly when working with templates of low DNA content 40,41 . We carried out all mammoth PCR reactions in triplicates, to comply with the multi-tube strategy developed to control for that risk. ...
... Among the mammoth extracts themselves, the decrease in amplification success rate-as well as the inconsistency in genotype call-is only observed for the 10 mammoths with the lowest CN (i.e. less than 3 copies per reaction; see Table 1). With such low DNA templates, the stochasticity of the PCR amplification process becomes a pervasive issue due to allelic dropout, and the selection of a multi-tube strategy-three replicates per sample-is a method of choice to evaluate the accuracy of the genotypes 40,41 . ...
... As discussed above, three positive replicates are usually sufficient to identify a male specimen, despite allelic dropout. However, the reciprocal confirmation of a female genotype is somewhat trickier: up to 7 positive replicates might be necessary to reach a 99% confidence in the assignment of a homozygous genotype 40,41 . Because of the rarity of the mammoth DNA, we could not spare more than three reactions per specimen. ...
It is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure. Molecular sexing methods developed in the last two decades have often shown limited efficiency, particularly in terms of sensitivity and specificity, due to the degradation of DNA in these samples. These limitations have also prevented their use with ancient DNA samples of elephants or mammoths. Here we propose a novel TaqMan-MGB qPCR assay to address these difficulties. We designed it specifically to allow the characterization of the genetic sex for highly degraded samples of all elephantine taxa (elephants and mammoths). In vitro experiments demonstrated a high level of sensitivity and low contamination risks. We applied this assay in two actual case studies where it consistently recovered the right genotype for specimens of known sex a priori. In the context of a modern conservation survey of African elephants, it allowed determining the sex for over 99% of fecal samples. In a paleogenetic analysis of woolly mammoths, it produced a robust hypothesis of the sex for over 65% of the specimens out of three PCR replicates. This simple, rapid, and cost-effective procedure makes it readily applicable to large sample sizes.
... Previous studies addressing the reliability of microsatellite genotyping considered DNA extracts from plucked or shed hair, feces, or urine in the context of noninvasive sampling of mammals (Taberlet et al. 1996, Gagneux et al. 1997, Goossens et al. 1998, Valiere and Taberlet 2000, Morin et al. 2001. Allele dropout was the most serious problem, with rates as high as 31% in samples extracted from a single shed hair (Gagneux et al. 1997). ...
... Previous studies addressing the reliability of microsatellite genotyping considered DNA extracts from plucked or shed hair, feces, or urine in the context of noninvasive sampling of mammals (Taberlet et al. 1996, Gagneux et al. 1997, Goossens et al. 1998, Valiere and Taberlet 2000, Morin et al. 2001. Allele dropout was the most serious problem, with rates as high as 31% in samples extracted from a single shed hair (Gagneux et al. 1997). The amplifi cation of artifact alleles was less frequent (<5%; Goossens et al. 1998) and also more readily detected on the basis of unexpected allele sizes or multiple peaks per locus. ...
... Although negative PCRs are a nuisance and increase the eff ort required to obtain data, allele dropout presents a more severe problem because it may lead to the scoring of false homozygotes (Taberlet et al. 1996, Gagneux et al. 1997, Gerloff et al. 1999, Taberlet and Luikart 1999. A defi ciency of heterozygotes at one or more loci in a population that is otherwise in Hardy-Weinberg equilibrium should be taken as a warning signal that a proportion of heterozygous genotypes may have been missed because of amplifi cation errors (Zierdt et al. 1996). ...
We address the problem of microsatellite genotyping errors associated with polymerase chain reaction (PCR) amplification from degraded and dilute template DNA and provide suggestions for improving the accuracy of genotype data in studies using older museum specimens as a source of DNA. In the course of a population genetics study of African indigobirds (Vidua spp.), we used replicate PCR to evaluate genotyping reliability for nine microsatellite loci in relation to PCR fragment length and DNA template concentration (DNA extracted from the calamus of one vs. two wing feathers). Complete amplification failure and the dropout of one allele from heterozygous genotypes were the predominant problems encountered. For samples with heterozygous genotypes, allele dropout occurred in 19.2 and 12.1% of PCR using extracts derived from one and two feathers, respectively. The amplification of artifact bands was less frequent (affecting 4.9 and 1% of positive PCR reactions with one- and two-feather extracts, respectively). Those results indicate that multiple replicates per sample and locus are required to obtain accurate genotype data from museum feather samples. Although higher DNA concentration improved success, PCR fragment size had a much stronger influence on the success and repeatability of microsatellite amplification, which suggests that the accuracy and efficiency of genotyping can be improved most easily by designing primers that amplify smaller DNA fragments.
... faeces, hair) can also show a high frequency of erroneous genotypes because of multiple causes such as low quantity and quality of DNA, pollution, and the presence of PCR inhibitors (Pompanon et al. 2005). Genotyping errors occurring at a high rate of 37% per locus has been reported (Gagneux et al. 1997) for microsatellites genotyped from single shed hair in wild chimpanzees. ...
... Simulation of SSRs from noninvasive sampling I simulated a number of L unlinked microsatellites, each having k alleles in a uniform frequency distribution. Due to factors such as the presence of inhibitors, pollutions, and the low quality and quantity of DNA extracted from noninvasive samples (such as faeces), a high dropout rate (ε 1 ) and a high other error rate (ε 2 ) are possible (Gagneux et al. 1997;Pompanon et al. 2005). Because dropouts were considered in NGS data, I focused on false alleles by keeping a constant and low dropout rate of ε 2 = 0.01 and by considering a wide range of ε 2 values (0.01~0.64) for each locus. ...
Marker genotype data could suffer from a high rate of errors such as false alleles and allelic dropouts (null alleles) in situations such as SNPs from low-coverage next-generation sequencing and microsatellites from noninvasive samples. Use of such data without accounting for mistyping properly could lead to inaccurate or incorrect inferences of family relationships such as parentage and sibship. This study shows that markers with a high error rate are still informative. Simply discarding them could cause a substantial loss of precious information, and is impractical in situations where virtually all markers (e.g. SNPs from low-coverage next-generation sequencing, microsatellites from noninvasive samples) suffer from a similarly high error rate. This study also shows that some previous error models are valid for markers of low error rates, but fail for markers of high error rates. It proposes an improved error model and demonstrates, using simulated and empirical data of a high error rate (say, >0.5), that it leads to more accurate sibship and parentage inferences than previous models. It suggests that, in reality, markers of high error rates should be used rather than discarded in pedigree reconstruction, so long as the error rates can be estimated and used properly in the analyses.
... Thus, the true representation of overall allele dropout is 0.9% (6/643 homozygous samples). For comparison, Gandhi et al. found 2 out of 1891 homozygous samples had allele dropout using a different commercial NGS assay [8]. Additionally, Yin et al. found allele dropout occurred at 0.35% of all buccal swab samples [9]. ...
... Allelic dropout occurs either due to sequence independent factors or allele-specific sequence variations. Poor sample quality [8,9], low DNA input concentration [10], PCR conditions [3], DNA polymerase fidelity [11][12][13], polymerase-blocking secondary structures [14] or the presence of inhibitors [15] may cause an allele to be incompletely amplified. Alleles with low expression levels may also present a potential problem [16], and the seldom-quantified ''human error" contributes hugely to genotyping errors [17]. ...
The initial discovery of cell-free DNA (cfDNA) in 1948 by Mandel and Metais has led to numerous investigations evaluating the role of cfDNA in various disease states. cfDNA has been characterized in various patient populations with similar results. cfDNA are typically 150 bp of double-stranded DNA that are thought to be released from nucleosomes during apoptosis and necrosis. They are found in circulation as monomers, dimers, and trimers. Different specimen types yield significantly different amounts cfDNA. While serum yields the highest amount of cfDNA, it contains the most genomic DNA contamination compared to Streck and plasma specimen types. The utility of cfDNA as a biomarker was advanced by the completion of the Human Genome Project and enabled interrogation of tumor markers in cancer patients. While tumor genetics may have been the initial application of cfDNA, the most successful application of cfDNA as a clinical biomarker is noninvasive prenatal testing (NIPT). CfDNA has become the gold-standard for NIPT testing, allowing for high sensitivity while maintaining specificity for aneuploidy. Because prenatal testing is essentially mixed genome analysis, application of cfDNA analysis to solid organ transplantation is a clear diagnostic target. There have been several studies examining the role of cfDNA in solid organ transplantation. These studies identified cfDNA as a surrogate marker for rejection with a high level of concordance with biopsies. While the data thus far are promising, there is still a need for more prospective studies to determine the clinical utility of cfDNA in solid organ transplant rejection.
... Hence, these SSR markers were used for further analysis. However, the DNA isolated from wood/bark samples apparently had inconsistent quality and quantity, which possibly will inf luence PCR amplification and increase the chances of null alleles/allele dropout (Gagneux et al. 1997). Therefore, GenePop 4.7.x. ...
Illegal tree felling is one of the crucial problems in forestry worldwide. It is essential to develop adequate forensic techniques that can verify the origin of timber sourced at logging concessions. Teak wood—one of the most important timber species in India—is often illegally logged and also imported from other countries without certification. In our study, the best combination of Simple Sequence Repeat (SSR) markers identified was utilized for match testing the genetic profiles of seized and evidence woods. A genetic reference database was also developed to assign unknown wood samples to their source populations. Our results found a perfect match between the genetic profiles of seized wood and their respective evidence wood which ascertains the utility of genetic profiles as an efficient forensic tool. Further, efficiency of assignment tools based on different approaches such as distance-based, model-based and machine learning were tested. Mycorrhiza, a machine learning algorithm, was identified as the best assignment tool. However, only broad provenance-level assignment was possible due to the genetic admixture in a few natural teak populations. Mycorrhiza was then used to identify the origin of two plantations in India. The assignment test predicted that both the plantations had similar origin, having sourced seeds from multiple natural populations (through clonal seed orchards and seed production areas) and local populations in different ratio. The SSR markers and assignment tool from this study can be used as an effective forensic tool to curb illegal felling and to verify integrity of timber supply chains in India. Also, provenance-based assignment guarantees usage of these markers in a global scale. However, a collective global effort to develop and deposit a robust reference database in a common repository is a prerequisite to strengthen timber traceability worldwide.
... These data correlate with studies that have shown that longer alleles of heterozygous loci tend to have higher levels of allelic dropout in scat samples as a result of degraded DNA (Gerloff et al., 1995;Goossens et al., 1998). When used for population structure and genetic diversity, this marker will greatly underestimate the level of genetic diversity (Gagneux et al., 1997;Pompanon et al., 2005;Waits & Leberg, 2000). Loci with shorter amplicons have higher amplification and lower error rates and are therefore more ideal for scat samples (Broquet et al., 2006;Frantzen et al., 1998). ...
The black‐footed cat ( Felis nigripes ) is endemic to the arid regions of southern Africa. One of the world's smallest wild felids, the species occurs at low densities and is secretive and elusive, which makes ecological studies difficult. Genetic data could provide key information such as estimates on population size, sex ratios, and genetic diversity. In this study, we test if microsatellite loci can be successfully amplified from scat samples that could be noninvasively collected from the field. Using 21 blood and scat samples collected from the same individuals, we statistically tested whether nine microsatellites previously designed for use in domestic cats can be used to identify individual black‐footed cats. Genotypes recovered from blood and scat samples were compared to assess loss of heterozygosity, allele dropout, and false alleles resulting from DNA degradation or PCR inhibitors present in scat samples. The microsatellite markers were also used to identify individuals from scats collected in the field that were not linked to any blood samples. All nine microsatellites used in this study were amplified successfully and were polymorphic. Microsatellite loci were found to have sufficient discriminatory power to distinguish individuals and identify clones. In conclusion, these molecular markers can be used to monitor populations of wild black‐footed cats noninvasively. The genetic data will be able to contribute important information that may be used to guide future conservation initiatives.
... Thus, on average, 1 Ϫ (1 Ϫ 1/25.4) 2 ϭ 7.7% of theoretically identical genotypes will mismatch. Mistyping was largely due to scoring and data-inputting errors, although four cases were consistent with the dropout of a single allele during PCR amplification (Gagneux et al. 1997;Goossens et al. 1998). ...
Although mammalian mating systems are classically characterized in terms of male competition and polygyny, it is becoming increasingly apparent that alternative male strategies and female choice may play important roles. For example, females who mate with males from a dominant dynasty risk producing inbred offspring. Many pinnipeds are highly polygynous, but in some species alternative male strategies such as aquatic mating appear to be important, even when behavioral observations suggest strong polygyny. Here, we analyze male reproductive success in the Antarctic fur seal Arctocephalus gazella, an otariid described behaviorally as being highly polygynous, by combining a microsatellite paternity analysis spanning seven consecutive breeding seasons with detailed behavioral data on both sexes. Territorial males fathered 59% of 660 pups analyzed from our study colony. Male reproductive skew was considerable, with a quarter of all paternities assigned to just 12 top individuals on a beach where mean annual pup production was 635. Most males were successful for only a single season, but those able to return over successive years enjoyed rapidly increasing success with each additional season of tenure. We found no evidence of alternative male reproductive tactics such as aquatic or sneaky terrestrial mating. However, paternity was strongly influenced by maternal status. Females observed on the beach without a pup were significantly less likely to conceive to a sampled territorial male than equivalent females that did pup. In addition, their pups carried combinations of paternal alleles that were less likely to be found on the study beach and exhibited lower levels of shared paternity. Thus, from a territorial male's perspective, not all females offer equal opportunities for fertilization.
... Although our initiative was successful, extracting DNA from eggshells is challenging as it is expected to get a lower concentration than that may yield from muscle or blood [40]. The low concentration and poor template quality may lead to amplification failure [41,42]. However, using DNA from eggshells is convenient, especially, to identify the nests because it does not require the individual ...
Alien invasive species are posing conservation challenges worldwide. Pet trade, one of the many ways, is worsening the situation. Especially, pet turtles have been released into nature due to their longer life span and peoples' religious and traditional beliefs. In addition, unwanted and undesired pets are also released. While information on the successful local establishment and subsequent dispersal into new habitats is required to designate an inva-sive and ecosystem-disturbing species, alien freshwater turtle nests have always been hard to find and identify in nature. Because one should identify nests by the eggs, which do not always guide properly, as adults abandon the sites quickly. We thought the recent advancement in DNA technology may help improve the situation. We studied Pseudemys peninsu-laris, one of the most traded freshwater turtle pet species, which has already been reported from a wide range of wild areas in South Korea. Yet, it is not designated as ecosystem-disturbing species due to a lack of adequate information on their local reproduction and establishment. We conducted surveys and found two nests in Jeonpyeongje Neighborhood Park, Maewol-dong, Seo-gu, Gwangju. We developed the methodology for extracting DNA from the eggshells and successfully identified the nests by phylogenetic analysis and verified through egg characteristics and morphological features of artificially hatched juveniles. This was the first successful initiative to extract DNA from freshwater turtle eggshells. We believe it will help future researchers identify the alien invasive turtle nests and develop their control and management policies. In addition, our study also included comparative descriptions and schematic diagrams of the eggs of eight freshwater turtles, including a native and three ecosystem disturbing species, from South Korea. We urged an immediate designation of P. peninsularis as an ecosystem-disturbing species considering its local establishment, distribution range, and potential negative impact on native ecosystems.
... The amount of DNA in the sample can also cause the lack of detection, because using the same DNA extract yields a PCR product in some loci but not in others [5,6]. ...
Null alleles are alleles that are recessive to codominant markers without any effect on the phenotype. In SSR assays, there are several reasons for the lack of amplification at a locus: the primer does not bind well, longer fragments do not amplify due to imperfections in the PCR reaction, or the amount of DNA in the sample is insufficient. In microsatellite studies, null alleles are mostly used in pedigree analysis and population genetics calculations such as diversity estimation. Null alleles in pedigree analysis can cause rejection of the true parent; if not recognized while in population genetics they distort the results in underestimating diversity. In this review, the effects caused by null-alleles in viticultural research and its possible solutions were summarized.
... The third cause of null alleles may be due to inconsistent quality or the low quantity of DNA templates. Some loci are relatively easy to amplify, yet others cannot be amplified within the same DNA preparation 45 . When a null allele is present, the www.nature.com/scientificreports/ ...
Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10–12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.
... For non-invasive genetic tracking of wildlife, a multipletubes approach is widely used, whereby a sample is analysed several times to derive a consensus result from these single analyses. There are several designs for a multiple-tubes approach, which vary in the number of DNA isolations and repeated PCRs (Caniglia et al. 2012;Gagneux et al. 1997;Lucchini et al. 2002). The classical approach includes up to seven PCRs from one DNA isolate (Taberlet et al. 1996). ...
Contamination and degradation are known challenges for reliable genotyping, since they can cause, among other problems, false microsatellite profiles. In this study we described a method to decrease the proportion of false microsatellite profiles from fish scale samples of endangered allis shads (Alosa alosa) from a reintroduction program, where cross-contamination with DNA from other individuals and potentially degradation of samples occurred. To maximize the portion of reliably measurable results, we modified and combined two known approaches—thresholds used in forensic DNA analyses and a multiple-tubes approach. This combined approach increased reliable microsatellite profiles compared with single approaches. The forensic thresholds and the multiple-tubes approach increased the measurable results from 55 to 67% and 75%, respectively, whereas the combined approach accomplished an increase to 90%. This illustrates the potential of the combined approach for other studies with comparable problems or sample material.
... In this case, the null alleles occur when an allele fails to amplify via PCR and heterozygotes might falsely be scored as homozygote. Null alleles can possible be either caused by mutations in the flanking regions of a microsatellite sequence (Chapuis & Estoup 2007;Dakin & Avise 2004), or associated with shorter alleles outperforming the longer alleles, usually due to lower DNA quality or quantity (Gagneux et al. 1997;Wattier et al. 1998). ...
Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii. Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations. Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations. Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.
... The estimated null allele frequencies were not significant at all loci (nf < 0.25). Null alleles are caused by either mutation within the primer region or large allele drop-out (Callen et al. 1993;Gagneux et al. 1997). Null alleles at low frequencies (nf < 0.25) have minor effect on the estimates of population genetic parameters (Dakin and Avise 2004). ...
The Freshwater bivalve, Lamellidens marginalis is native to Southeast Asia and one of
the most widely used mussels for pearl production in the region. However, no studies
have been carried out on stock characterization of L. marginalis due to lack of
molecular markers. In the present study, genome of L. marginalis was sequenced at
low coverage using Illumina paired-end approach. A total of 268,403 contigs with a
size range of 258 to 13,308 bp were assembled from 2.5 million high quality reads. The
contigs were mined and 26, 877 simple sequence repeats (SSRs) were identified using
MIcroSAtellite identification tool (MISA) software. Among SSRs, di-nucleotide repeats
(15,633 total) were more than the tri- (6,306), tetra- (4,768), penta- (146) and hexanucleotide
(24 ) repeats. Eighteen loci representing di- (8 total), tri- (7) and tetra- (3)
nucleotide repeats were characterized using 30 individuals of L. marginalis. All loci
showed moderate to high Polymorphic Information Content (PIC) values and no
linkage disequilibria between any pair of loci. Fourteen microsatellites were amplified in
the closely related species Lamellidens corrianus. These polymorphic microsatellite
markers will be useful for genetic selection programmes, as well as population and
conservation genetics of L. marginalis and L. corrianus.
... The software arranged the samples of clutch and metamorphs to clusters with a probability of sibship ranging between 0 and 1. Clusters with a probability higher than 0.8 were used for further analysis and defined to represent offspring of a matriline. Some clusters were grouped without clutch sample, which could be due to allelic dropouts that may occur due to the low DNA concentrations we used (Gagneux, Boesch, & Woodruff, 1997). To compare variance in phenotypic traits of offspring within and between matrilines, we only used clusters comprising at least six full-sibs for further analysis. ...
In meiner Dissertation untersuche ich das Paarungs- und Fortpflanzungsverhalten des Europäischen Grasfrosches (Rana temporaria) in einem evolutionären Kontext. Mein Ziel ist es zu verstehen, welche Mechanismen zur Bildung von Paaren führen, ob die Partnerwahl die Paarungsmuster erklärt, die wir beobachten können, und ob es evolutive Vorteile gibt, die sich aus der Paarung mit einem bestimmten Partner ergeben. Die Suche nach und die Konkurrenz um Paarungspartner führt zur Entwicklung verschiedener Paarungssysteme, Strategien und Taktiken, um den Reproduktionserfolg während der gesamten Lebensdauer zu erhöhen. Das Paarungsverhalten wird durch natürliche und sexuelle Selektion beeinflusst, wobei beide in unterschiedliche Richtungen wirken können. Für die meisten Individuen ist das Überleben unerlässlich, um sich so oft wie möglich zu reproduzieren, und dadurch die reproduktive Gesamtfitness zu erhöhen. Andererseits könnte ein auffälliges Verhalten bei der Fortpflanzung das Prädationsrisiko erhöhen. Der Akt der Paarung selbst kann bereits mit Risiken verbunden sein, welche sich auf die Überlebensraten auswirken können. Durch sexuelle Selektion könnten bestimmte sekundäre Geschlechtsmerkmale begünstigt werden, entweder aufgrund von Vorteilen im Wettbewerb innerhalb eines Geschlechts (intrasexuell), oder aufgrund spezifischer Präferenzen zwischen den Geschlechtern (intersexuelle Selektion). Damit sich die Partnerwahl entwickeln kann, muss der gewählte Paarungspartner Vorteile aufweisen, von denen der wählende Partner profitiert, denn die Wahl ist mit energetischen Kosten und zeitlichem Aufwand verbunden. Als Frühlaicher muss der Europäische Grasfrosch mit einem eingeschränktem Paarungszeitraum umgehen. Die Männchen konkurrieren um den Zugang zu Weibchen und es wird angenommen, dass sich Weibchen während der Paarung und Reproduktion passiv verhalten, da der hohe "Männchen-Überschuss" keine Wahl zulassen würde. Aus evolutionärer Sicht sollten Weibchen jedoch das wählerische Geschlecht sein und entscheiden mit wem sie sich paaren, da sie mehr Energie in die Eierproduktion investieren.
... The molecular origin of null alleles (substitution and indel mutations) resulting from polymorphism in the annealing region has been assessed directly by sequencing the annealing sites of microsatellite locus primers for both null and visible alleles (Callen et al. 1993). Other possible causes of microsatellite null alleles include the preferential amplification of short alleles (due to inconsistent DNA template quality or quantity) or slippage during PCR amplification (Gagneux et al. 1997;Shinde et al. 2003). These technical problems associated with amplification will not be considered here. ...
Correspondance : chapuimp@supagro.inra.fr
... Therefore, sources of genotyping error (Taberlet 1996, Hoffman andAmos 2005) were considered when developing protocols for determining whether microsatellite data supported cannibalism. For example, allelic dropout, or the failure of one allele of a heterozygous individual to be amplified via PCR, can lead to incorrect genotyping of that individual as a homozygote (Gagneux et al. 1997, Soulsbury et al. 2007, and null alleles, or alleles that do not amplify by PCR, can lead to blank or incorrectly identified genotypes (Shaw et al. 1999, Van Oosterhout et al. 2004. Another source of genotyping error stems from PCR artifacts (i.e., stutters) in which amplification products are generated that can be misinterpreted as true alleles (Taberlet 1996, Goossens et al. 1998, Bradley and Vigilant 2002 Cannibalism was considered to have occurred when at least two different alleles were observed between a lionfish and its prey across the four loci tested. ...
Species invasions have been increasing in frequency and severity in recent decades, furthering the need for research on ecological impacts from invasions and means to mitigate them. The spread of Indo-Pacific lionfish (Pterois spp.) into the northern Gulf of Mexico (nGOM) causes concern, given potential negative impacts on native fish and invertebrate communities.
To characterize trophic impacts of lionfish on native reef fish communities of the nGOM, I report a comprehensive assessment of feeding ecology using traditional diet, stable isotope, and DNA barcoding analyses. Results indicate lionfish are generalist mesopredators that become more piscivorous at larger sizes. However, lionfish exhibit a more varied diet at artificial reefs, where they forage on open substrates away from reef structure. DNA barcoding of unidentified fish prey significantly increased diet resolution and exposed potential cannibalism occurrence. Cannibalism was later confirmed using microsatellite genotyping, and increased in frequency through time, mirroring increases in lionfish density.
Next, I estimated age, growth, and condition of lionfish sampled over five years of invasion, with the objective to test for density-dependent effects. Significant declines in mean size-at-age and condition as a function of lionfish density indicated density-dependent effects that were likely due to inter- and intra-specific competition. The increase in these effects through time likely explains the plateauing of nGOM populations in latter years of study.
Finally, I conducted a 2-year experiment to examine the effectiveness and ecological benefits of lionfish removals. Removals reduced lionfish densities, but juveniles and adults quickly recruited to cleared reefs, thus removal efforts were insufficient to achieve native reef fish recovery. This work has important implications for invasive lionfish population dynamics and carrying capacity in the nGOM, and data herein will be used to parameterize models estimating the removal effort necessary to mitigate their impacts effectively.
... We then genotyped 1 sample/individual to 20 loci (21 including sex) to allow sufficient power to assign parentage and identify full siblings. To eliminate genotypes created through genotyping error (Gagneux et al. 1997, Goossens et al. 1998, Taberlet et al. 1999, Paetkau 2003, we further scrutinized 20-locus genotypes for close mismatches. We reanalyzed the mismatching markers of all pairs of samples that mismatched at 1, 2, or 3 loci to confirm the genotype or resolve errors (Paetkau 2003, Kendall et al. 2009). ...
Population fragmentation is stressing wildlife species worldwide. In populations with minimal genetic structure across potential fractures, detecting fragmentation can be challenging. Here we apply a relatively unused approach, genetic pedigree analysis, to detect fragmentation in the American black bear (Ursus americanus) across 2 highway corridors that are bordered by large, contiguous populations. We compared our results with movements detected through Global Positioning System (GPS) telemetry of collared bears between 2005 and 2010. We used 20-locus microsatellite genotypes to identify 104 first-order relatives (parent–offspring or full siblings) within 383 black bears, sampled between 2002 and 2012. We compared numbers of pairs of immediate relatives found on either side of 2 highways—U.S. Highway 2 in northwestern Montana, USA, and BC Highway 3 in southeastern British Columbia, Canada—with an expected rate, the mean across 22 lines parallel to each highway at 1-km intervals. We found that over similar geographic scales, dispersal was lower across the transportation corridors than adjacent areas without a highway corridor. The observed number of migrants across Highway 2 was 3, well below the confidence interval of the expected number of 15.1 migrants/available bears (95% CI = 12.2–18.0). Highway 3 had 6 migrants, compared with the expected 13.1 bears (95% CI = 10.8–15.5). None of 16 black bears wearing GPS radiocollars for 1 year crossed Highway 2, yet 6 of 18 crossed Highway 3. These results suggest that even though 33% of radiocollared black bears crossed Highway 3, there appeared to be less dispersal across the transportation corridors than across other regions in the study area. Pedigree and telemetry results were more closely aligned in the Highway 2 system, with both methods suggesting more intense fragmentation than we found along Highway 3. Our results identified pedigree analysis as another tool for investigating population fragmentation, particularly in situations where genetic differentiation is too weak to determine migration rates using individual-based methods, such as population assignment.
... To address issues associated with low quality DNA, particularly allelic dropout (Gagneux, Boesch, and Woodruff 1997), we applied a multiple-tubes PCR approach. ...
The overarching goal of my thesis is to characterize the relationship between kinship and
social behaviour in a species with a cooperative, multilevel social structure – the sperm
whale. To do so, I use a combination of genetic, behavioural and acoustic data collected
during a longitudinal study of sperm whale social units, in the eastern Caribbean. Social
units are a stable and basal component of sperm whale social structure. Associations
between social units occur within large cultural groups, called vocal clans. To deal with
degraded DNA from non-invasive sampling, I develop a protocol that maximizes
genotyping success with degraded DNA, while quantifying and minimizing error rates.
Using microsatellite loci and mitochondrial DNA haplotypes, I evaluate kinship among
sperm whales, and I examine its relationship to social association, alloparental care and
vocal repertoires. First, I characterize the extent and pattern of kinship in and among
sperm whale social units, and test whether association is predicted by kinship. I
document that social units have a clear matrilineal basis, but do not appear to be strictly
matrilineal. My findings also indicate paternal relatedness between social units. Within
units, I find individuals associate more with their closer relatives, but this is not the case
among units. Second, I investigate calf care in relation to kinship. I demonstrate that
behavioural observations are not always sufficient for assigning maternity, and that
alloparental care is considerable in some cases and correlates positively with maternal
kinship. Exceptions to the general pattern, however, demonstrate that, in addition to kinselection, other factors influence alloparental care, perhaps including reciprocity, group
augmentation or gaining maternal experience. Lastly, I examine acoustic repertoires of
individuals and social units, in the context of kinship and social association. Variation in
vocal repertoires was not explained by close kinship or social bonds. This supports the
prevailing hypothesis that these vocalizations are culturally transmitted, and not
determined genetically. Further, this suggests that vocal learning occurs broadly within
clans, rather than preferentially from close kin or close social associates, or that biases in
vocal learning at lower levels of social structure are diffused by clan-level processes.
Also, by observing an absence of signals of kinship in vocalizations, my results suggest
that a different mechanism, perhaps familiarity, regulates kin-selection among sperm
whales. In conclusion, kinship clearly influences social unit composition, association
preferences and alloparental care among sperm whales. However, I also reveal variability
in social behaviour that is unexplained by kinship, which highlights the complexity of
drivers behind social structure, cooperation and communication in this cultural, highly
social and large-brained species.
... Karena sifat kompetitif pada proses PCR, alel DNA yang berukuran lebih kecil memiliki peluang lebih besar berhasil diamplifikasi daripada alel yang lebih panjang. Selain itu menurut Gagneux et al., 1997;Garcia et al., 1998 null alel lebih disebabkan karena kualitas atau kuantitas DNA hasil ekstraksi. ...
Ikan tongkol lisong dan krai merupakan salah satu jenis tuna yang berperan nyata untuk usaha perikanan tangkap di Indonesia. Pengelolaan sumberdaya ikan tersebut harus selalu dapat dilakukan untuk menjaga tingkat pemanfaatannya supaya tidak lebih tangkap. Kajian keragaman genetik merupakan salah satu teknik dalam pengelolaan pemanfaatan sumberdaya perikanan dengan cara mengetahui tingkat keragaman genetik pada suatu struktur populasi. Kajian keragaman genetik ini diharapkan dapat menjadi basis kajian stok dan opsi dalam pengelolaan sumberdaya perikanan tongkol agar pemanfaatannya dapat dilakukan secara berkelanjutan. Awal mula analisis keragaman genetik dilakukan dengan memperbanyak DNA secara in vitro menggunakan teknik PCR (Polymerase Chain Reaction). Keberhasilan proses PCR dipengaruhi oleh beberapa faktor seperti suhu dan waktu penempelan oligonukleotida primer. Berdasarkan hal tersebut, penelitian ini bertujuan untuk mengetahui suhu dan waktu optimal pada primer Aro2-38. Sampel penelitian diperoleh dari hasil tangkapan pukat cincin yang didaratkan di PPN Palabuhanratu, Jawa Barat. Optimasi PCR menggunakan 12 suhu dan 2 waktu penempelan yang berbeda yaitu : 520C; 52,80C; 540C; 55,50C; 57,20C; 59,10C; 60,90C; 62,80C; 64,50C; 65,90C; 67,20C dan 680C, dan suhu penempelan 30 dan 15 detik. Hasil analisis menunjukkan bahwa produk PCR optimum (menghasilkan pita alel DNA) pada ikan tongkol krai berhasil waktu penempelan 30 detik dengan rentang suhu 52-540C. Sedangkan pada sampel ikan tongkol lisong, produk PCR yang optimum muncul pada waktu penempelan 15 dan 30 detik, dengan rentang suhu 52-60,90C.Frigate and bullet tuna constitute one of tuna species that plays a significant role in Indonesian fishing business. Management of fisheries resources must always be done to maintain the level of utilization so that it is not excessive. Genetic study is one of techniques in managing fisheries resource utilization by knowing the level of genetic diversity in a population structure. This genetic diversity study is expected to be the basis and option in the management of tuna fishing resources so that their utilization can be carried out sustainably. Genetic diversity analysis is start by multiplying fish DNA using PCR (Polymerase Chain Reaction) technique. The success of the PCR process is influenced by several factors such as temperature and time of primary oligonucleotide attachment. Based on this, this study aims to determine the optimal temperature and time in primers Aro2-38. The research sample was obtained from the catch of purse seine landed in PPN Palabuhanratu, West Java. PCR optimization uses 12 temperatures and 2 different annealing times: 520C; 52.80C; 54ÚC; 55,50C; 57.20C; 59.10C; 60.90C; 62.80C; 64,50C; 65,90C; 67.20C and 680C, and the annealing times are 30 and 15 seconds. The results of the analysis showed that the optimum PCR product (producing DNA allele bands) on the cretaceous tuna was successfully pasted for 30 seconds with a temperature range of 52-540C. Whereas in the sample of tuna lisong, the optimum PCR product appeared at the time of attachment of 15 and 30 seconds, with a temperature range of 52-60.90C.
... These errors can result from degradation due to environmental exposure (Murphy et al. 2007;Brinkman et al. 2010;Panasci et al. 2011) and the presence of inhibitory compounds in scat (i.e., complex polysaccharides, bile salts, lipids, urate) that can interfere with sample extraction and PCR (Schrader et al. 2012). Genotyping errors have the potential to bias the conclusions of individual-based analyses, such as population size estimates (Waits and Leberg 2000) and parentage analyses (Gagneux et al. 1997), and can also influence populationlevel analyses (Pompanon et al. 2005). Therefore, it is important to conduct a pilot study to determine if reliable and informative DNA fingerprints can be obtained from G. agassizii scat. ...
Noninvasive fecal genotyping can be a useful tool for population monitoring of elusive species. We tested extraction protocols on scat samples from the threatened Mojave Desert tortoise, Gopherus agassizii, to evaluate whether scat-based mark–recapture and population genetic monitoring studies are feasible. We extracted DNA from G. agassizii scat samples collected in California and Nevada using several extraction protocols and evaluated the reliability of resulting genotypes using quality scores, maximum likelihood reliability estimates, and paired scat and blood genotypes from the same individuals. Finally, we assessed probabilities of identity and sibship, and locus amplification quality, and calculated genotyping error rates for 19 microsatellite loci to determine the best set of loci to use with G. agassizii scat extractions. We found that genotype quality depended more on the sample quality than on the extraction method, and that the Qiagen DNeasy Plant Mini extraction kit is an efficient method for extracting tortoise DNA from tortoise scat. We identified 6 G. agassizii microsatellite loci that can be used to generate a unique molecular tag for individual tortoises. We characterized the reliability of an additional 13 microsatellite loci for use in population genetic analyses where additional power at the expense of some increase in error may be advantageous. As proof of concept, with very low error rates, we matched 3 opportunistically collected scat samples to blood genotypes from animals captured during population surveys within the study area and discovered at least 3 new individuals, even after 2 yrs of extensive survey work. These results suggest that genotyping of field-collected scat can complement existing methods used in long-term demographic and movement studies of G. agassizii and other, closely related, tortoise species.
... 本 模型使用 R 2 表示纯合子阳性 PCR 的正确率, 2 ˆ R 在许 多研究中小于 0.98 [20,39] . [3,18] , 但也有研究发现它们的 扩增效率不存在差异 [40] . 另一方面, 如果 2 个等位基 因的长度相差过大, 导致它们的扩增效率差别很大, 那么方程(1)将转换为(推导过程见附录 1): ...
... Small fragment alleles exert significant amplification advantage due to the loss of large fragments. The fitness of DNA quality may also be a possible reason for null alleles corresponding to different microsatellite markers [29]. ...
A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P < 0.05). All 45 primers were not significant with regard to linkage disequilibrium (P > 0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r = − 0.741, P = 0.022), whereas the positive correlation with the number of the samples (r = 0.847, P = 0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or ≥ 15 (P = 0.7472 ~ 0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.
... The software arranged the samples of clutch and metamorphs to clusters with a probability of sibship ranging between 0 and 1. Clusters with a probability higher than 0.8 were used for further analysis and defined to represent offspring of a matriline. Some clusters were grouped without clutch sample, which could be due to allelic dropouts that may occur due to the low DNA concentrations we used (Gagneux, Boesch, & Woodruff, 1997). To compare variance in phenotypic traits of offspring within and between matrilines, we only used clusters comprising at least six full-sibs for further analysis. ...
Successful reproduction is an important determinant of the fitness of an individual and of the dynamics of populations. Offspring of the European common frog (Rana temporaria) exhibit a high degree of variability in metamorphic traits. However, environmental factors alone cannot explain this phenotypic variability, and the influence of genetic factors remains to be determined. Here, we tested whether the maternal genotype influences developmental time, body size, and body condition of offspring in a forest pond in Germany. We collected fertilized eggs from all 57 clutches deposited in the pond. We used multilocus genotypes based on seven microsatellite loci to assign metamorphosed offspring to mothers and to determine the number of fathers for a single matriline. We tested the influence of genetic effects in the same environment by comparing variability of metamorphic traits within and between full‐sib offspring grouped to matrilines and tested whether multiple paternity increases the variability of metamorphic traits in a single matriline. The variability in size and body condition was higher within matrilines than between them, which indicates that these traits are more strongly influenced by environmental effects, which are counteracting underlying genetic effects. The developmental time varied considerably between matrilines and variability increased with the effective number of fathers, suggesting an additive genetic effect of multiple paternity. Our results show that metamorphic traits are shaped by environmental as well as genetic effects. We show the variability of metamorphic traits in an natural ecosystem, by assigning anuran metamorphs to their respective matrilines, using multi‐locus microsatellite data. We looked at multiple paternity within matrilines and how it affects metamorphic trait variability in the respective offspring.
... To address issues associated with low-quality DNA, particularly allelic dropout [43], we applied a multiple-tubes PCR approach. This allowed us to determine rates of genotyping errors and improve confidence in genotypes. ...
Sperm whales have a multi-level social structure based upon long-term, cooperative social units. What role kinship plays in structuring this society is poorly understood. We combined extensive association data (518 days, during 2005–2016) and genetic data (18 microsatellites and 346 bp mitochondrial DNA (mtDNA) control region sequences) for 65 individuals from 12 social units from the Eastern Caribbean to examine patterns of kinship and social behaviour. Social units were clearly matrilineally based, evidenced by greater relatedness within social units (mean r = 0.14) than between them (mean r = 0.00) and uniform mtDNA haplotypes within social units. Additionally, most individuals (82.5%) had a first-degree relative in their social unit, while we found no first-degree relatives between social units. Generally and within social units, individuals associated more with their closer relatives (matrix correlations: 0.18–0.25). However, excepting a highly related pair of social units that merged over the study period, associations between social units were not correlated with kinship (p > 0.1). These results are the first to robustly demonstrate kinship's contribution to social unit composition and association preferences, though they also reveal variability in association preferences that is unexplained by kinship. Comparisons with other matrilineal species highlight the range of possible matrilineal societies and how they can vary between and even within species.
... Therefore, sources of genotyping error (Taberlet et al. 1996;Hoffman and Amos 2005) were considered when developing protocols for determining whether microsatellite data supported cannibalism. For example, allelic dropout, or the failure of one allele of a heterozygous individual to be amplified via PCR, can lead to incorrect genotyping of that individual as a homozygote (Gagneux et al. 1997;Soulsbury et al. 2007), and null alleles, or alleles that do not amplify by PCR, can lead to blank or incorrectly identified genotypes (Shaw et al. 1999;Van Oosterhout et al. 2004). Another source of genotyping error stems from PCR artifacts (i.e., stutters) in which amplification products are generated that can be misinterpreted as true alleles (Taberlet et al. 1996;Goossens et al. 1998;Bradley and Vigilant 2002). ...
DNA barcoding is used in a variety of ecological applications to identify organisms, including partially digested prey items from diet samples. That particular application can enhance the ability to characterize diet and predator–prey dynamics but is problematic when genetic sequences of prey match those of consumer species (i.e., self-DNA). Such a result may indicate cannibalism, but false positives can result from contamination of degraded prey samples with consumer DNA. Here, nuclear-encoded microsatellite markers were used to genotype invasive lionfish, Pterois volitans, consumers and their prey (n = 80 pairs) previously barcoded as lionfish. Cannibalism was confirmed when samples exhibited two or more different alleles between lionfish and prey DNA across multiple microsatellite loci. This occurred in 26.2% of all samples and in 42% of samples for which the data were considered conclusive. These estimates should be considered conservative given rigorous assignment criteria and low allelic diversity in invasive lionfish populations. The highest incidence of cannibalism corresponded to larger sized consumers from areas with high lionfish densities, suggesting cannibalism in northern Gulf of Mexico lionfish is size- and density-dependent. Cannibalism has the potential to influence population dynamics of lionfish which lack native western Atlantic predators. These results also have important implications for interpreting DNA barcoding analysis of diet in other predatory species where cannibalism may be underreported.
... Genotyping success of hair by Poole et al. (2011) was 32%, similar to my results which suggests this rate may only be increased by a significant improvement in extraction or amplification of DNA. Presumably the poor success for goat hair is due to the degraded nature of the DNA in sloughed hair (Gagneux et al. 1997) Using pellets has the advantage of sampling all animals in the population and avoids the potential biases associated with the differential timing of shedding. Pellets of juveniles can be sorted to focus sampling on adults or to assign age to juveniles for analysis. ...
I tested the use of non-invasive sampling and genetic identification methods for estimating
mountain goat (Oreamnos americanus) abundance. I collected hair and fecal pellets by walking
transects in goat habitat during July 2005 in a 140 km2 study area. Microsatellite genotyping was used
to identify 61 individual goats which were detected 75 times across 3 sampling sessions. There were
adequate detections to estimate population size, albeit with low precision, despite low genotyping
success (31%). Seventy adult goats were seen in the study area during an aerial census during July
2004 and 45 adults were seen during February 2005. A mark-recapture model was used to estimate the
adult population size as 123 goats during summer 2005 although precision was poor (95% CI 90-190).
The DNA-based population estimate suggested sightability of >50% during the summer aerial census
and <50% during the winter census, assuming the same number of goats were present during all three
periods. DNA sampling was more expensive than aerial surveys. The cost of a single DNA-based
sampling session and an aerial census or survey was similar in this study area. DNA-based sampling
for long-term population monitoring will likely be more similar in annual expense to an aerial census
than a population estimate generated during a single year. The amount of helicopter access required for
field sampling greatly affects the cost of genetic sampling. Genetic analysis was also a large part of the
cost but recent improvements in genotyping success have reduced genetic costs substantially.
... Co-purifying contaminants such as complex polysaccharides, polyphenolic substances from plant tissues, and potent PCR inhibitors could inhibit subsequent utilization of DNA for genetic analyses (13)(14)(15). A harsh cell lysis step in the extraction method yield severely fragmented DNA, which often results in poor amplification, false alleles, and allele dropout (16)(17)(18). Although some measures such as inclusion of polyvinylpyrrolidone in PCR amplification (19,20), increasing sampling frequency (21), and repeated extraction and PCR analysis (22,18) partially correct these problems, they involve additional time and expenses of analysis. ...
A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.
... As a result, researchers continue to recommend a suite of laboratory and analytical guidelines aimed at uncovering and reducing sources of bias and imprecision known to be associated with non-invasively collected samples (Kwok 1990, Woods et al. 1999, Paetkau 2003 regardless of the purpose of the study. These include study-specifi c selection and design of appropriate markers, application of particular quality control procedures such as 'multiple tubes' approaches (Taberlet et al. 1996, Gagneux et al. 1997, culling of low quality samples using initial screening and analysis techniques and/or comparison of results generated from different tissue sources from the same individual (Paetkau 2003), and application of reliability testing (e.g., Miller et al. 2002). The use of buccal swabs for collecting epithelial cell DNA less invasively and less disruptively than collecting blood has become routine in clinical human research, following demonstration of reliability based on analyses of paired (blood, swab) samples (Richards et al. 1992, Dickinson et al. 2001, Abraham et al. 2012, and commercial kits are available. ...
We evaluated the efficacy of using swabs to collect cells from the epidermis of octopus as a non-invasive DNA source for classical genetic studies, and demonstrated value of the technique by incorporating it into an effort to determine, within a day, the lineage of captured, live Enteroctopus (E. dofleini or a cryptic lineage). The cryptic lineage was targeted for captive behavioral and morphological studies, while once genetically identified, the non-target lineage could be more rapidly released back to the wild. We used commercially available sterile foamtipped swabs and a high-salt preservation buffer to collect and store paired swab and muscle (arm tip) tissue sampled from live Enteroctopus collected from Prince William Sound, Alaska. We performed a one-day extraction of DNA from epithelial swab samples and amplification of two diagnostic microsatellite loci to determine the lineage of each of the 21 individuals. Following this rapid lineage assessment, which allowed us to release non-target individuals within a day of laboratory work, we compared paired swab and muscle tissue samples from each individual to assess quantity of DNA yields and consistency of genotyping results, followed by assessment of locus-by-locus reliability of DNA extracts from swabs. Epithelial swabs yielded, on average, lower quantities of DNA (170.32 ± 74.72 (SD) ng/μL) relative to DNA obtained from tissues collected using invasive or destructive techniques (310.95 ± 147.37 (SD) ng/μL. We observed some decrease in yields of DNA from extractions of swab samples conducted 19 and 31 months after initial extractions when samples were stored at room temperature in lysis buffer. All extractions yielded quantities of DNA sufficient to amplify and score all loci, which included fragment data from 10 microsatellite loci (nine polymorphic loci and monomorphic locus EdoμA106), and nucleotide sequence data from a 528 base pair portion of the nuclear octopine dehydrogenase gene. All results from genotyping and sequencing using paired swab and muscle tissue extracts were concordant, and experimental reliability levels for multilocus genotypes generated from swab samples exceeded 97%. This technique is useful for studies in which invasive sampling is not optimal, and in remote field situations since samples can be stored at ambient temperatures for at least 31 months. The use of epithelial swabs is thus a noninvasive technique appropriate for sampling genetic material from live octopuses for use in classical genetic studies as well as supporting experimental and behavioral studies.
... Null allele frequencies ranged from 0.00 to 0.33 (Table 1). Null alleles may be due to individuals missing genotype data for loci, the low number of alleles present, or the sample size (Gagneux et al. 1997, Garcia de Leon et al. 1998, Kwok et al. 1990). Eight individuals from Marco Island were missing data for a single locus (GP26, GP19, or GP102), 1 individual from Marco Island was missing data from 2 loci (GP96 and GP61), and 2 individuals from RBNERR were missing data for locus GP102. ...
Gopherus polyphemus (Gopher Tortoise) is a prominent species found in pine flatwoods, upland scrub, and coastal dunes of the southeast United States. Geoclimatic and anthropogenic sources of habitat changes have fragmented Gopher Tortoises into isolated populations that may reduce gene flow, promote inbreeding, and ultimately impact population viability. Rapid urbanization along the southwest Gulf Coast of Florida has degraded habitat, and fragmented insular and coastal populations. To assess the diversity in these vestige populations, we assessed the genetic structure of Gopher Tortoises on heavily developed Marco Island (n = 61) and the adjacent mainland within Rookery Bay National Estuarine Research Reserve (RBNERR) (n = 23) in Collier County, FL. Using microsatellite markers, we determined that the Marco Island tortoises are genetically distinct from the RBNERR tortoises. We identified unique alleles and reduced allelic richness in both populations, suggesting isolation has reduced gene flow. We therefore encourage careful management of the Marco Island Gopher Tortoises to maintain the uniqueness of the population while preventing further loss of diversity.
... Genotyping errors can be defined as differences between two or more molecular genotypes obtained independently from the same sample. These happen mainly when an allele fails to amplify (allelic dropout) (Gagneux et al. 1997;Navid et al. 1992;Taberlet et al. 1996;Walsh et al. 1992), or because false alleles are amplified due to errors that happen during PCR (Bradley and Vigilant 2002;Taberlet et al. 1996). These problems can lead to errors in the identification of individuals (Paetkau 2003;Taberlet and Luikart 1999) and distortions in population size estimates (Creel et al. 2003;McKelvey and Schwartz 2004). ...
We analyze the way deer populations should be estimated taking into account terrain characteristics, and deer species behaviour.
... Finally, three individuals were deleted from the dataset, because of the deficient amplifying and scoring problems with more loci. Effective purification of DNA controlled the quality of extraction (Gagneux et al. 1997) and the optimization of PCR reactions was important to reduce null alleles due to technician deficiencies in the amplification process (Flores-Rentería and Krohn 2013). True null alleles (mutations in the primer range) and determination of allele dosage are problematic points in the investigation of polyploid species because the intensity of peaks alone is not enough to estimate them. ...
Aims:
Understanding the role of genetics in biological invasions has become an important aspect for modern plant ecology. Many studies suggest that increased ploidy level benefits the success of an invasive species, but the basis for this phenomenon is not fully understood. In its native, North American range, Solidago gigantea has 3 geo-cytotypes comprising di-, tetra- and hexaploid populations, while in Europe, where it is highly invasive, S. gigantea stands are composed primarily of tetraploid individuals. Our study investigates whether North American hexaploids can induce a greater risk of invasion, due to their higher performance in a non-native range, as compared to the existing tetraploids of that range.
Methods:
We performed greenhouse and common garden experiments along with microsatellite analyses to test whether differences in chromosome number and origin of the species mean superior fitness in the introduced range.
Important Findings:
Genetic diversity was significantly higher in the native hexaploid populations (AR = 6.04; He = 0.7794), rather than the non-native tetraploid populations (AR = 4.83; He = 0.6869). Furthermore, differentiation between geo-cytotypes was moderate (ρST = 0.1838), which was also confirmed by their clear segregation in PCA and structure analyses, proving their different genetic structure. In contrast to genetic diversity, the non-native tetraploid geo-cytotype performed better in the common garden experiment, implying that higher genetic diversity does not always mean better success. Our results suggest that native hexaploids do not present a greater risk, as assessed by their performance in the introduced range, when compared to the non-native tetraploids, as was suggested by previous studies. Nevertheless their introduction is still undesirable due to their different genetic structure, which, through hybridization, could give a new drive to the invasion of S. gigantea.
... Finally, three individuals were deleted from the dataset, because of the deficient amplifying and scoring problems with more loci. Effective purification of DNA controlled the quality of extraction ( Gagneux et al. 1997) and the optimization of PCR reactions was important to reduce null alleles due to technician deficiencies in the amplification process (Flores-Rentería and Krohn 2013 genotypes of polyploids only if the individual is homozygote or has the exact number of alleles as its ploidy level. In other cases, we could not make use of any allele dosage information (Dufresne et al. 2014). ...
Giant goldenrod (Solidago gigantea) significantly reduces species diversity by forming dense monocultures in its invaded range. Solidago is one of the most aggressive invasive neophytes in Central Europe. The plant occupies mainly wet habitats but can also take over semi-dry and disturbed areas. It spreads long-distance by seed dispersal and locally by rhizomes. Therefore, controlling this species raises difficult issues, and so far there have been no generally applicable methods to combat this problem.
We surveyed the effectiveness of different control methods against Solidago that are commonly applied at the Hungarian national parks (grazing, mowing, flooding, and their combinations) in different habitat types. We also set up a three years controlled experiment (in 50×50 cm plots) in a common garden, with six different control methods (mowing once or twice a year, selective herbicide treatment, and their combinations). Linear mixed models were used for statistical analyses.
Our results show that all methods applied at the national parks had positive effects on species diversity (df=11; F=75.519; P<0.001) compared to the untreated controls. However, grazing had the largest positive effect (t=-16.849; P<0.001).
In our experiment every treatment decreased Solidago stem number significantly (df=6; F=4.713; P=0.00347). Herbicide application had completely eliminated Solidago by the second year, but it also decreased species diversity the most. Mowing also had a negative effect on Solidago stem number in the second year, but species diversity remained similar to the control plots.
Based on our results, Solidago invasion can be controlled by most methods; however, the actual site characteristics and integrity of the native community should be considered when selecting the most applicable one.
The influence of bottom-up food resources and top-down mortality risk underlies the demographic trajectory of wildlife populations. For species of conservation concern, understanding the factors driving population dynamics is crucial to effective management and, ultimately, conservation. In southeastern British Columbia, Canada, populations of the mostly omnivorous grizzly bear (Ursus arctos) are fragmented into a mosaic of small isolated or larger partially connected sub-populations. They obtain most of their energy from vegetative resources that are also influenced by human activities. Roads and associated motorized human access shape availability of food resources but also displace bears and facilitate human-caused mortality. Effective grizzly bear management requires an understanding of the relationship between habitat quality and mortality risk. We integrated analyses of bottom-up and top-down demographic parameters to understand and inform a comprehensive and efficient management paradigm across the region. Black huckle-berry (Vaccinium membranaceum) is the key high-energy food for grizzly bears in much of southeastern British Columbia. Little is known about where and why huckleberries grow into patches that are useful for grizzly bears (i.e., densely clustered fruiting shrubs that provide efficient access to high energy food) and how Wildlife Monographs. 2023;e1078. wileyonlinelibrary.com/journal/wmon |
Genetic monitoring using non-invasive samples provides a complement or alternative to traditional population monitoring methods. However, Next Generation Sequencing approaches to monitoring typically require high quality DNA and the use of non-invasive samples (e.g. scat) is often challenged by poor DNA quality and contamination by non-target species. One promising solution is a highly multiplexed sequencing approach called Genotyping-in-thousands by sequencing (GT-seq), which can enable cost-efficient genomics-based monitoring for populations based on non-invasively collected samples. Here, we develop and validate a GT-seq panel of 324 single nucleotide polymorphisms (SNPs) optimized for genotyping of polar bears based on DNA from non-invasively collected fecal samples. We demonstrate 1) successful GT-seq genotyping of DNA from a range of sample sources, including successful genotyping (>50% loci) of 62.9% of non-invasively collected fecal samples determined to contain polar bear DNA, and 2) that we can reliably differentiate individuals, ascertain sex, assess relatedness, and resolve population structure of Canadian polar bear subpopulations based on a GT-seq panel of 324 SNPs. Our GT-seq data reveal spatial-genetic patterns similar to previous polar bear studies but at lesser cost per sample and through use of non-invasively collected samples, indicating the potential of this approach for population monitoring. This GT-seq panel provides the foundation for a non-invasive toolkit for polar bear monitoring and can contribute to community-based programs – a framework which may serve as a model for wildlife conservation and management for species worldwide.
Likelihood methods have been developed to partition individuals in a sample into full-sib and half-sib families using genetic marker data without parental information. They invariably make the critical assumption that marker data are free of genotyping errors and mutations and are thus completely reliable in inferring sibships. Unfortunately, however, this assumption is rarely tenable for virtually all kinds of genetic markers in practical use and, if violated, can severely bias sibship estimates as shown by simulations in this article. I propose a new likelihood method with simple and robust models of typing error incorporated into it. Simulations show that the new method can be used to infer full- and half-sibships accurately from marker data with a high error rate and to identify typing errors at each locus in each reconstructed sib family. The new method also improves previous ones by adopting a fresh iterative procedure for updating allele frequencies with reconstructed sibships taken into account, by allowing for the use of parental information, and by using efficient algorithms for calculating the likelihood function and searching for the maximum-likelihood configuration. It is tested extensively on simulated data with a varying number of marker loci, different rates of typing errors, and various sample sizes and family structures and applied to two empirical data sets to demonstrate its usefulness.
Meer as een vaar in ’n krokodilbroeisel verhoog die effektiewe populasiegrootte en lei tot ’n stadiger verlies van genetiese variasie as gevolg van inteling en lukraak genetiese swerwing. Meer as een vaar kan ook die variasie met betrekking tot eienskappe wat van kommersiële belang is tussen krokodille uit dieselfde broeisel verklaar. Vrugvliese kan ’n nie-ingrypende bron van DNS verskaf waarmee die genotipe van Nylkrokodilbroeilinge (Crocodylus niloticus) bepaal kan word. Die doel van hierdie studie was om vas te stel hoe doeltreffend die genotipe van Nylkrokodilbroeilinge uit die vrugvliese wat in uitgebroeide eiers agterbly bepaal kan word en of ’n broeisel uit ’n kommunale teeldam op ’n kommersiële plaas meer as een vaar kan hê. Elf mikrosatellietloki is gebruik om die DNS-profiele van 4–6 (gemiddeld 4.4) vrugvliesmonsters (VVMe) van elk van 25 broeisels uit dieselfde teeldam op ’n kommersiële Nylkrokodilplaas te bepaal. DNS het op al 11 loki in 95 van die 110 individue vermeerder, op 1–10 loki in 13 en op geen lokus nie in twee. Drie tot 20 allele is per lokus gevind. Afsonderlike beoordeling van loki het getoon dat 13 broeisels minstens twee vaars gehad het. Met ’n multilokusprogram (Colony) is afgelei dat 19 broeisels minstens twee vaars gehad het, en dat poliandrie en poliginie algemeen was. Verdere navorsing is nodig om die nuttigheid van vrugvliese as ’n bron van DNS vir nesse uit die natuur te bepaal en om, deur meer VVMe per broeisel te gebruik, die mate van poliandrie en poliginie op Nylkrokodilplase en in die natuur meer presies te bepaal.
Gorillas are one of our closest living relatives, the largest of all living primates, and teeter on the brink of extinction. These fascinating animals are the focus of this in-depth and comprehensive examination of gorilla biology. Gorilla Biology combines recent research in morphology, genetics and behavioural ecology to reveal the complexity and diversity of gorilla populations. The first section focuses on morphological and molecular variation and underscores the importance of understanding diverse biological patterns at all levels in testing evolutionary and adaptive hypotheses and elucidating subspecies and species diversification. Following are discussions of the ecological constraints that influence gorilla social organization and highlight their surprising flexibility. The book ends with discussions of the conservation status of gorillas and the many and increasing threats to their continued survival. Giving insight into the evolutionary biology of these unique primates, this book will be essential reading for primatologists, anthropologists and evolutionary biologists.
Although the Louisiana black bear (Ursus americanus luteolus) is currently listed as threatened under the Endangered Species Act, there have been no attempts to estimate range-wide abundance. This subspecies was thought to occupy a near contiguous range across southern Mississippi, Louisiana and east Texas but is now restricted to three isolated areas in Louisiana. In 1964, Louisiana initiated a restocking program in which black bears from Minnesota were introduced into two of these areas. It is not clear how the additions affected population structure or if substantial breeding occurred between native and introduced bears. Using baited sites to snare hair samples, and microsatellite DNA analysis to distinguish individuals, we estimated abundance of two geographically isolated bear populations in south central Louisiana: Inland and Coastal. Additionally, we examined genetic variation both within and between the two populations. Mark recapture analysis of the distribution of individual captures during two primary sampling periods resulted in population estimates of 77 ± 9 for Coastal and 41 ± 6 for Inland. Genetic analysis revealed significant population differentiation (FST = 0.206) between the two populations. The apparently smaller Inland population exhibited more diversity than the Coastal, which suggests that the genetic structure of the Inland population has been influenced by the reintroduction. Both of these populations are isolated and face considerable demographic and genetic threats, thus conservation measures to protect both are warranted. However, the Coastal population is more representative of Louisiana black bears prior to reintroduction and special consideration should be given to insure its integrity.
This genetic study is based on a geographically isolated population of Hanuman langurs that live around the city of Jodhpur, north-west Rajasthan. Here, the majority of langurs live in harem troops (with a single resident male for 95% of the troop's history) and bachelor bands. Behavioural studies of these langurs suggest that the troops are matrilineal, with males being the dispersing sex. It has therefore been hypothesised that females of a troop are closely related, both through their mothers and through cohorts sharing the same father. This would explain the high levels of cooperation seen between females, such as allogrooming and home range defence. Conversely, members of all male bands, particularly the young adults who control the bands' movements, are unlikely to be related, because of the constantly changing membership and the high mortality rate suffered by the nomadic males. This study has tested these hypotheses using non-invasive techniques to obtain DNA samples from troops and bands in the population. 89 individuals of five troops and one band have been genotyped at eight polymorphic microsatellite loci. Analysis of the microsatellite data using Queller and Goodnight's RELATEDNESS and KINSHIP programs has shown that on average, troops are related by 0.17 ± 0.04, troop females by 0.14 ± 0.07, and non-adult troop members by 0.27 ± 0.07. Conversely, the relatedness of the band was only 0.05 ± 0.08. In three troops the resident male could not be excluded as the father of any non-adult, suggesting that these residents had had long term mating monopoly in these troops, whereas in the remaining two troops where takeover had recently occurred, the new residents could be excluded as fathers in all but 2/12 cases. Additionally, the population proved to be highly structured, and troops appeared outbred, an indication of female philopatry combined with polygyny. These results provide genetic evidence in support of the social organisation suggested from long-term behavioural data.
The Chimpanzees of the Taï Forest - edited by Christophe Boesch November 2019
Anthropology is considered a holistic discipline that studies humans in varied manners, ranging from their social cultural contexts, to their biological makeup both in the past as well as in the present. The complexity of the human species is well present in these various forms of expression. It is, therefore, a knowledge that draws from various disciplines, one of which has deserved special attention due to its overarching implication in human life: that is genetics. In recent years much has been written about genes, genetics and the human genome, and about ancestry and related species – particularly non-human primates.
To explore genetics in a discipline such as anthropology is to cross the boundaries between biology and culture, and build bridges between nature and culture whilst exploring its social and cultural ramifications. Within this context, the role of genetics in Anthropology is wide, ranging from evolutionary human biology, past human populations studies (henceforward referred to as bioarchaeology) and living population genetics, paleopathology and even forensic anthropology. The current chapter will explore genetics in anthropology within a four-field anthropology. It will first introduce the basic concepts of four-field anthropology, and its specificities of research, as well as basic concepts in genetics, particularly those that find their way into the anthropological discourse; secondly, it will introduce the overall methodology used to explore genetics and discuss it most significant limitations; thirdly, it will address past and present research agendas and results – providing a comprehensive, although summarized, state of the art on the subject. The conclusion will provide an overall appreciation of the role of genetics in anthropology, offering a historical perspective on the subject and future avenues for research.
The decreasing cost and increasing scope and power of emerging genomic technologies are reshaping the field of molecular ecology. However, many modern genomic approaches (e.g., RAD-seq) require large amounts of high quality template DNA. This poses a problem for an active branch of conservation biology: genetic monitoring using minimally invasive sampling (MIS) methods. Without handling or even observing an animal, MIS methods (e.g. collection of hair, skin, faeces) can provide genetic information on individuals or populations. Such samples typically yield low quality and/or quantities of DNA, restricting the type of molecular methods that can be used. Despite this limitation, genetic monitoring using MIS is an effective tool for estimating population demographic parameters and monitoring genetic diversity in natural populations. Genetic monitoring is likely to become more important in the future as many natural populations are undergoing anthropogenically-driven declines, which are unlikely to abate without intensive adaptive management efforts that often include MIS approaches. Here we profile the expanding suite of genomic methods and platforms compatible with producing genotypes from MIS, considering factors such as development costs and error rates. We evaluate how powerful new approaches will enhance our ability to investigate questions typically answered using genetic monitoring, such as estimating abundance, genetic structure and relatedness. As the field is in a period of unusually rapid transition, we also highlight the importance of legacy datasets and recommend how to address the challenges of moving between traditional and next generation genetic monitoring platforms. Finally, we consider how genetic monitoring could move beyond genotypes in the future. For example, assessing microbiomes or epigenetic markers could provide a greater understanding of the relationship between individuals and their environment.
Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.
Population-scale molecular studies of endangered and cryptic species are often limited by access to high-quality samples. The use of non-invasively collected samples or museum-preserved specimens reduces the pressure on modern populations by removing the need to capture and handle live animals. However, endogenous DNA content in such samples is low, making shotgun sequencing a financially prohibitive approach. Here, we apply a target enrichment method to retrieve mitochondrial genomes from 65 museum specimens and 56 non-invasively collected fecal samples of two endangered great ape species, Grauer's gorilla and the eastern chimpanzee. We show that the applied method is suitable for a wide range of sample types that differ in endogenous DNA content, increasing the proportion of target reads to over 300-fold. By systematically evaluating biases introduced during target enrichment of pooled museum samples we show that capture is less efficient for fragments shorter or longer than the baits, that the proportion of human contaminating reads increases post-capture although capture efficiency is lower for human compared to gorilla fragments with a gorilla-generated bait, and that the rate of jumping PCR is considerable, but can be controlled for with a double-barcoding approach. We succeed in capturing complete mitochondrial genomes from fecal samples, but observe reduced capture efficiency as sequence divergence increases between the bait and target species. As previously shown for museum specimens, we demonstrate here that mitochondrial genome capture from field-collected fecal samples is a robust, and reliable approach for population-wide studies of non-model organisms. This article is protected by copyright. All rights reserved.
Population estimates for bears (Ursidae), by a combination of noninvasive genetic sampling using hair-snares and capture-mark-recapture methods using DNA individual identification, are widely used in the world. In several areas of Japan, attempts have been undertaken to apply these noninvasive techniques. Compared with conventional methods, noninvasive methods have several advantages: 1) live-trapping of bears is unnecessary, 2) sampling is less biased than that of live-trapping, 3) sampling can cover larger geographical areas than traditional capture-mark-recapture sampling, and 4) genetic tags are permanent. However, we have to understand the structure of the methods and solve many issues before we apply this method, in order to estimate an accurate population size. In this paper, we describe procedures for these methods, including trap structures, study site selection, trap placement, hair sampling, individual identification by DNA analysis, and capture-mark-recapture models to estimate population size, and described points to notice.
It is urgent to establish practicable methods for estimating bear population size for the management and conservation of two species of bear (Ursus arctos and U. thibetanus) in Japan. Noninvasive sampling techniques and DNA-based capture-mark-recapture methods have drawn attention as population estimate measures because these methods can be more efficient and less biased than the traditional capture-mark-recapture methods with live-trapping. These methods have become increasingly common and have proven to be powerful means for estimating bear population size in many countries. However, in Japan, the methods have not been adopted effectively due to some open problems. To consider the effective application of these methods, we scrutinized previous studies conducted in Japan and other countries and reviewed problems in three processes: sampling, genotyping, and population estimation. We suggest that well-designed planning by the ecologist, geneticist and mathematical modeling specialist is most vital to the success of DNA-based capture-mark-recapture methods. We also suggest that DNA-based capture-mark-recapture methods would work more effectively if applied to large scale projects targeting the whole range of a local bear population.
The documentation of vertebrate diversity traditionally involved the collection of specimens for further study. Properly curated collections constitute treasure troves for biologists interested in systematics, biogeography, and evolutionary biology. The collection of specimens and their preservation was justified on the grounds that our understanding of nature depended upon it. This is no longer always the case, and in situations involving rare or threatened species it may be completely unjustified. Collections that were well-intended at the time can seem appalling in today’s conservation-minded world. Four examples illustrate the conflicts between the need to conduct scientific research and the need to conserve the subjects of that research:
The 1906 California Academy of Sciences expedition to the Galapagos dis¬ covered that the Pinzon island race of giant tortoises was, in fact, not extinct and accordingly collected the remaining 86 for study (Thornton, 1971).
Reported incidences of tool use and tool making for three wild chimpanzee populations increase from Mahale (12 and 3 types of use and making, respectively), Gombe (16 and 3) to Taï (19 and 6). Sticks are commonly used and prepared at all three sites. However, Taï chimpanzees seem to perform more modifications on the material before using it. They are also the only chimpanzees seen to pound objects with tools and to combine two different tool uses to get access to one food item. Tool making is the rule for abundant material (grass, twigs), but appears to be rarer for scarce, hard material (clubs, stones). Factors involved in the acquisition and the benefit of tool use are discussed along with factors affecting the frequency and complexity of tool making in chimpanzees.
Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.
Hypotheses about chimpanzee social behavior, phylogeography, and evolution were evaluated by noninvasive genotyping of free-ranging individuals from 20 African sites. Degrees of relatedness among individuals in one community were inferred from allele-sharing at eight nuclear simple sequence repeat (SSR) loci. Males are related on the order of half-siblings, and homozygosity is significantly increased at several SSR loci compared to Hardy-Weinberg expectations. These data support the kin-selection hypothesis for the evolution of cooperation among males. Sequence variation patterns at two mitochondrial loci indicate historically high long-distance gene flow and clarify the relationships among three allopatric subspecies. The unexpectedly large genetic distance between the western subspecies, Pan troglodytes verus, and the other two subspecies suggests a divergence time of about 1.58 million years. This result, if confirmed at nuclear loci and supported by eco-behavioral data, implies that P. t. verus should be elevated to full species rank.
Trinucleotide repeat mutations of normal alleles at the human androgen receptor locus were studied by typing approximately 4,300 sperm. Control experiments established that the mutation events were of germline origin. The mutation rate for 20-22 repeat alleles was similar to that shown by family analysis. Alleles with 28-31 repeats had a 4.4 times greater rate of mutation with contractions outnumbering expansions. Preliminary experiments on the trinucleotide repeat associated with myotonic dystrophy gave similar results although in one donor expansions were six times greater than contractions. Comparison of the sperm data to mutations of disease alleles in SBMA families suggests that expansions may have a different origin than contractions.
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.
Using DNA amplified from shed or plucked hair follicles it is now possible to genotype individual primates at many nuclear
and mitochondrial gene loci. Sequence specific primers and the polymerase chain reaction permit the rapid production of sufficient
DNA from a single hair for numerous analyses. The direct sequencing of relatively conservative mtDNA sequences like cytochromeb is proving useful in establishing species and subspecies-level relationships. More variable sequences (e.g. the mtDNA control
region or D-loop) are useful at the population and social community levels. Paternity exclusion, pedigree relationships, and
community structure can be determined using simple sequence length polymorphisms (SSLPs) of multiple hypervariable nuclear
microsatellite or simple sequence repeat (SSR) loci. Studies involving captive and free-ranging chimpanzees, gibbons, and
macaques illustrate the resolving power of these new non-invasive molecular genetic genotyping techniques.
Variation in a 252-nucleotide segment of the cytochrome b gene from 26 gibbons is described. DNA was extracted from hair, amplified, and directly sequenced. These sequences represent seven of the nine nominal species and three of the four hylobatid subgenera. Variation was observed at 55 sites, 42 of which are phylogenetically informative. Levels of transitional and transversional divergence between the taxa are similar to those reported for homologous mtDNA sequences in other mammals. Parsimony, maximum likelihood, and bootstrap analyses (1) support some traditional phylogenetic hypotheses (monophyly of the concolor gibbons), (2) suggest previously unrecognized affinities between the lar species group and Hylobates klossi and between H. lar and H. agilis unko, and (3) show that this segment does not contain information sufficient for completely resolving gibbon relationships at the subgeneric level. The study demonstrates the great potential of noninvasive DNA sampling for phylogenetic analyses of mammals.
Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.
The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed.
The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.
Selective sensory ganglionectomy by means of retrograde suicide transport of adriamycin was performed on 3 patients with neuropathic pain in the areas of the trigeminal and intercostal nerves, producing significant pain relief, particularly from hyperalgesic pain. Adriamycin ganglionectomy is considered as a less invasive and highly selective pain treatment, which may possibly become an alternative for surgical ganglionectomy or rhizotomy.
A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. The procedure involves dividing the sample among several tubes, then amplifying and typing the contents of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded fragments in the sample is as small as 30. The procedure remains effective even in the presence of small amounts of laboratory contamination. We find that the multiple-tubes procedure is superior to the standard one-tube procedure, either when the sample is small or when laboratory contamination is a potential problem; and we recommend its use in these situations. Because the procedure is statistical, it allows the degree of certainty in the result to be quantified and may be useful in other PCR applications as well.
Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR-based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative.
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Selected References
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Weber JL, May PE. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet. 1989 Mar;44(3):388–396. [PMC free article] [PubMed]
Hunting is often considered one of the major behaviors that shaped early hominids' evolution, along with the shift toward a drier and more open habitat. We suggest that a precise comparison of the hunting behavior of a species closely related to man might help us understand which aspects of hunting could be affected by environmental conditions. The hunting behavior of wild chimpanzees is discussed, and new observations on a population living in the tropical rain forest of the Taï National Park, Ivory Coast, are presented. Some of the forest chimpanzees' hunting performances are similar to those of savanna-woodlands populations; others are different. Forest chimpanzees have a more specialized prey image, intentionally search for more adult prey, and hunt in larger groups and with a more elaborate cooperative level than savanna-woodlands chimpanzees. In addition, forest chimpanzees tend to share meat more actively and more frequently. These findings are related to some theories on aspects of hunting behavior in early hominids and discussed in order to understand some factors influencing the hunting behavior of wild chimpanzees. Finally, the hunting behavior of primates is compared with that of social carnivores.
The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.
The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.
While genotyping wild red deer (Cervus elaphus) at microsatellite loci for paternity assignment, we found three loci (MAF65, BOVIRBP and CelJP23) with segregating nonamplifying alleles. Nonamplifying alleles were detected through mismatches between known mother-offspring pairs and by significant deviations from Hardy-Weinberg equilibria. In a wide range of molecular ecology application, and especially in parentage assignment, the possible existence of undetectable alleles must be taken into account; this may be particularly important for microsatellite data.
We report the use of hypervariable simple sequence repeat (SSR) nuclear loci to study paternity in a community of wild chimpanzees (Pan troglodytes schweinfurthii) in Gombe National Park, Tanzania. All 43 living members of a habituated community were sampled and 35 were genotyped at 8 SSR loci using DNA amplified from hair. Paternity exclusions were performed for 25 chimpanzees including 10 for whom the mother was also genotyped. In each case 12-20 males were potential fathers based on their age and/or direct observation of sexual behaviour. 179 tests involving potential father/offspring combinations were performed. In four cases the data permit the probable identification of the previously undetermined father; these are the first such determinations for free-ranging chimpanzees, and the first based on non-invasive sampling. In another four cases we were able to exclude all but two to five potential fathers, and in the remaining cases we were able to exclude all living males. For molecular ecologists SSR genotype databases offer important advantages over currently popular minisatellite DNA finger-printing: they can be analysed unequivocally using traditional population genetics techniques and they can be expanded through time and space by other researchers.
Some small European populations of the brown bear (Ursus arctos) are threatened by the risk of extinction in the near future. The reinforcement of these populations with bears from other regions might provide a solution to their future survival. However, before any population transfer, the different conservation units must be identified. The phylogeographic approach has been advocated for this purpose. The different European populations were assayed for mitochondrial (mt) DNA polymorphism. A remarkable degree of concordance was found between the geographic distribution and the mtDNA haplotypes. Two clearly distinct lineages differing by more than 7% in mtDNA control region sequences were found and, furthermore, the western lineage appears to be organized into two clades which correspond to two different ancestral refugia. The potential conservation units can be deduced from these results, and a management policy can consequently be inferred. This study clearly demonstrates the relevance of the molecular phylogeographic approach to the identification of conservation units.
As an aid to the management of the Pyrenean population of the brown bear Ursus arctos, a sexing method based on the amplification of a Y chromosome specific sequence has been developed, and tested using hairs found in the field as a source of DNA. This method involves a two-step polymerase chain reaction (PCR) which allows the detection of a very small amount of DNA, probably a single SRY gene molecule. The sex can reliably be identified using about 50pg of DNA extract as template. It is possible that this approach could, with adjustments, be used to identify the sex of other species of eutherian mammals.
Twenty-three (AC)n repeat markers from chromosome 16 were typed in the parents of the 40 CEPH (Centre d'Etude du Polymorphisme Humain) families. Where parents were informative, the entire families were then typed. There were seven markers in which null alleles were demonstrated, as recognized by the apparent noninheritance, by a sib, of a parental allele. Four of these markers showed a null allele in a single sibship, while in the other three at least 30% of the CEPH sibships were shown to have a null allele segregating. One null allele was sequenced and shown to be the result of an 8-bp deletion occurring within the priming sequence for PCR amplification of the (AC)n repeats. In gene mapping or in application to diagnosis, the presence of a segregating null allele will not corrupt the linkage data but could result in loss of information. In isolated instances a segregating null allele may be interpreted as nonpaternity. The presence of a null allele may generate misleading data when individuals are haplotyped to determine the presence of linkage disequilibrium with a disease gene.
Keywords:DNA fingerprinting;microsatellite;null alleles;PCR;population generics;Ursidae
A highly variable portion of the mitochondrial DNA control region was sequenced in 63 free-living and captive gorillas including representatives of the three recognized subspecies. This region has proven useful for evaluation of relative levels of genetic variability in populations, for clarification of the subspecies identity of a wild population, and for examination of the phylogenetic relationships of the three subspecies. The eastern lowland (Gorilla gorilla graueri) and mountain gorilla (Gorilla gorilla beringei) sequences are distinct but closely related, with low variability within each subspecies. Two currently isolated populations of mountain gorillas, one in the Virungas Volcanoes region and the other in the Bwindi Forest, are indistinguishable using this mitochondrial DNA region for comparison. The subspecies identity of the Bwindi Forest group has previously been debated. Mitochondrial D-loop DNA variability within the western lowland gorillas (Gorilla gorilla gorilla) is very high. The genetic distance between the most divergent gorilla sequences is approximately as great as the distance between sequences of chimpanzees (Pan troglodytes) and bonobos (Pan paniscus).
Molecular Ecology of the Endangered Northern Hairy-nosed WombatLasiorhinus krefftiiand Application to Conservation and Management
- Taylorac
Taylor AC (1995) Molecular Ecology qf the Endangered Northern
Hairy-nosed Wombat Lasiorhinus krefftii and Application to
Conservation and Management. PhD thesis, University of New
South Wales, Sydney.
Amplification of hypervariable simple sequence repeats (microsatellites) from excremental DNA of wild bonobos
- U Gerloff
- C Sclotterer
- K Rassmann
Gerloff U, Sclotterer C, Rassmann K, et al. (1995) Amplification of
hypervariable simple sequence repeats (microsatellites) from
excremental DNA of wild bonobos (Pan paniscus). Molecular
A hypervariable microsatellite revealed by
- M Litt
- J A Luty
Litt M, Luty JA (1989) A hypervariable microsatellite revealed by
A I~t h~o ) d o g y 78
- Ltd Blackwell Science
Blackwell Science Ltd, Molecular Ecology, 6, 861-868
A I~t h~o ) d o g y
78, 547-573.
Paternity testing in chimpanzees with DNA amplification from hairs and buccal cells in wadges. A preliminary note
- H Takasaki
Takasaki H, Takenaka 0 (1991) Paternity testing in chimpanzees
with DNA amplification from hairs and buccal cells in
wadges. A preliminary note. In: Prirnatology Today: Proceedings
of the 13th Congress ofthe International Primatological Society (eds
Ehara A, Kinura T, Takenaka 0, Iwamoto M), pp. 613-616.
Elsevier, Amsterdam.
Dinucleotide repeat polymorphisins at the DlhS260, DlhS261, Dl6S265, D16S266, and Dl65267 loci
- J L Wcber
- A E Kwitek
- P E May
Wcber JL, Kwitek AE, May PE (1990) Dinucleotide repeat polymorphisins at the DlhS260, DlhS261, Dl6S265, D16S266, and
Dl65267 loci. Nuclcic Acids Rescrrrcli 18, 4034.
Woodruff's group is conducting parallel studies o f molecular evolution in three primate genera: Purr, Hylobntcs and Cdlithrix
- Phil Morin
Phil Morin. Woodruff's group is conducting parallel studies o f
molecular evolution in three primate genera: Purr, Hylobntcs and
Cdlithrix.
Lasiorhinus krefftii , PhD thesis
- A C Taylor
A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac actin gene
- Lift M.