gemini: software for testing the effects of genotyping errors and multitubes approach for individual identification

Improvements in the determination of individual genotypes from samples with low DNA quantity and quality are of prime importance in molecular ecology and conservation for reliable genetic individual identification (molecular tagging using microsatllites loci). Thus, errors (e.g. allelic dropout and false allele) appearing during samples genotyping must be monitored and eliminated as far as possible. The multitubes approach is a very effective but a costly and time‐consuming solution. In this paper, we present a simulation software that allows evaluation of the effect of genotyping errors on genetic identification of individuals and the effectiveness of a multitubes approach to correct these errors.     

gimlet: a computer program for analysing genetic individual identification data

Growing interest in microsatellite genotyping, combined with noninvasive genetic sampling has led to the increased production of data. New tools to analyse these data are required. gimlet is a user‐friendly software package designed to perform several simple tasks: (i) construction of consensus genotypes from repeated genotyping; (ii) estimation of genotyping error rates; (iii) identification of identical genotypes; (iv) comparison of new genotypes to a set of reference genotypes; (v) determination of the kinship; and (vi) estimation of several population parameters such as allele frequencies, heterozygosity, probability of identity, and population size.     

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

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

Molecular identification of individual North Atlantic right whales (Eubalaena glacialis) using free-floating feces

During the 1990s, North Atlantic right whales had significantly decreased reproduction and showed signs of compromised health, prompting the initiation of noninvasive fecal-based studies to investigate potential causal factors. The interpretation of these studies is enhanced when the defecator is identified, as data can then be linked to individual life history information. Fecal samples (n= 118) were either collected from single photoidentified whales, associated with several individuals by photoidentification of whales in the vicinity upon sample collection, or were collected when no whales were in the vicinity. Genetic profiles from fecal DNA comprising sex, mitochondrial haplotype, and five microsatellite loci helped assign specific samples to individual right whales based on existing genetic profiles. Profiles were informative in assigning 61 fecal samples to known individuals, 24 of which were collected when no whales were in the vicinity. Whales identified genetically were typically photographed in the same habitat area and on the same day of sample collection (n= 35/48). Twelve profiles new to the genetic database were identified, suggesting fecal sampling provides a means to obtain genetic profiles from previously unsampled individuals, which may help refine estimates of population size and habitat use patterns if annual fecal sampling continues.     

Quantification and genetic profiling of DNA isolated from free-floating feces of the North Atlantic right whale (Eubalaena glacialis)

Fecal analysis from the highly endangered North Atlantic right whale provides valuable information about health and reproductive parameters of individual animals. Genetically profiling the feces facilitates this connection when the sample originator is unknown. Although genetic analysis of feces collected in terrestrial systems has become well established, genetic studies of cetacean DNA are rare. Here, the use of free‐floating feces as a source of right whale DNA and the reliability of the genotypes produced are examined. On average, fecal extracts yielded 25 ng of DNA/mg dry weight, but less than 1% was right whale DNA. Although all samples were amplified using genus‐specific mitochondrial control region primers, the quantity of right whale DNA present was over estimated when compared to amplifications using nuclear primers. No correlation was found between the quantity of right whale DNA recovered and the duration the sample sat in the water. Composite microsatellite profiles from multiple amplifications of 28 fecal samples of known origin were consistent with profiles of the same individuals obtained from skin biopsies, however, the rate of allelic dropout varied depending on the amount of right whale DNA added. A screening and genotyping protocol for reliable genetic profiling based on fecal DNA quantification is presented.     

Multiplexed microsatellites for rapid identification and characterization of individuals and populations of Cercopithecidae

Cross-amplification of 15 human microsatellites was performed successfully in cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques and 11 other Cercopithecidae species of biomedical and conservation relevance. To allow for quick, efficient, and high-throughput genotyping to assess intra- and interspecific genetic variation, we performed three multiplex sets, each comprised of five markers from different parts of the genome (i.e., autosomes, the MHC region, and the X-chromosome). These multiplex sets are likely to reveal allelic divergence between taxa, which could be used for their discrimination. Population studies on three regional populations of M. fascicularis and one of M. mulatta revealed that most of the loci, with the exception of one monomorphic locus, displayed polymorphisms (the expected heterozygosities were 0.48-0.91 for M. fascicularis, and 0.61-0.93 for M. mulatta), which makes them useful for population genetics. For the multiplex set M1, including the nonlinked autosomal markers, low probabilities of identity were observed: P(ID) values ranged from 8 x 10(-7) to 3 x 10(-5). This multiplex set is reliable for forensic applications, such as individual identification, parentage testing, and kinship analysis, in wild and captive populations.     

The long-term genetic stability and individual specificity of the human gut microbiome

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Using naturally shed feathers for individual identification, genetic parentage analyses, and population monitoring in an endangered Eastern imperial eagle (Aquila heliaca) population from Kazakhstan

Genetic analyses on noninvasively collected samples have revolutionized how populations are monitored. Most noninvasive monitoring studies have used hair or scat for individual identification of elusive mammals, but here we utilize naturally shed feathers. The Eastern imperial eagle (EIE) is a species of conservation concern throughout Central Asia and, like most raptors, EIEs are inherently challenging to study because adults are difficult to capture and band using conventional techniques. Over 6 years, we noninvasively collected hundreds of adult feathers and directly sampled EIE chicks at a national nature reserve in Kazakhstan. All samples were genetically sexed and genotyped at a suite of microsatellite loci. Genetically profiled adult feathers identified and monitored the presence of individual eagles over time, enabling us to address a variety of issues related to the biology, demography, and conservation of EIEs. Specifically, we characterized (i) the genetic mating system, (ii) relatedness among mated pairs, (iii) chick sex ratios, and (iv) annual turnover in an adult breeding population. We show that EIEs are genetically monogamous and furthermore, there is no apparent relatedness-based system of mate choice (e.g. inbreeding avoidance). Results indicate that annual adult EIE survivorship (84%) is lower than expected for a long-lived raptor, but initial analyses suggest the current reproductive rate at our study site is sufficient to maintain a stable breeding population. The pristine habitat at our study site supports an EIE population that is probably the most demographically robust in the world; thus, our results caution that populations in marginal habitats may not be self-sustaining.     

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

ABSTRACT

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

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.