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12 Pàg., Two individuals with a first-degree relationship share about 50 percent of their alleles. Parent-offspring relationships cannot be homozygous for alternative alleles (genetic exclusion)., This research was financed by the Spanish Ministry project AGL2016‐75942‐R, “Caracterización molecular de la eficiencia alimentaria y de los caracteres reproductivos en cerdo Ibérico” (IBERFIRE), and SusAn ERA‐NET project “Sustainability of pig production through improved feed efficiency” (SusPig).

Parameter for the first simulation with Easypop are in the file "TestStutterDioeciousNoStutter-n1000N100-1.txt"; The file "TestStutterDioeciousNoStutter-n1000N100-1.equ gives population genetics parameters measured at each generation of the first replicate; "TestStutterDioeciousNoStutter-n1000N100-1.gen" and "TestStutterDioeciousNoStutter-n1000N100-1.dat" are the genepop and Fstat files generated by this simulation, respectively. The file "TestStutterDioecious-n1000N100-1-10%Stuttering.xlsx", is a template for generating 10% stuttering in the first replicate of the first Easypop simulation; and the file "TestStutterDioeciousNoStutter-n1000N100-1-FstatRes.xlsx" is a template of stuttering detection (together with LD tests and other measured done by Fstat), for this first replicate of the fists simulation with 10 % stuttering. Synthetic results for dioecious and monoecious simulations are in Excel format in the supplementary file S1 "SynthesisStutteringTestSexualsSupFileS1.xlsx". Synthetic results for clonal simulations are in Excel format in the supplementary file S2 "StutteringClonesSynthesisSupFileS2.xlsx". Pooling protocols for curing simulated data are in Excel files that were compressed in a zipped file, supplementary file S3 "PoolingProtocolCureSupFileS3.zip".

Parameter for the first simulation with Easypop are in the file "TestStutterDioeciousNoStutter-n1000N100-1.txt"; The file "TestStutterDioeciousNoStutter-n1000N100-1.equ gives population genetics parameters measured at each generation of the first replicate; "TestStutterDioeciousNoStutter-n1000N100-1.gen" and "TestStutterDioeciousNoStutter-n1000N100-1.dat" are the genepop and Fstat files generated by this simulation, respectively. The file "TestStutterDioecious-n1000N100-1-10%Stuttering.xlsx", is a template for generating 10% stuttering in the first replicate of the first Easypop simulation; and the file "TestStutterDioeciousNoStutter-n1000N100-1-FstatRes.xlsx" is a template of stuttering detection (together with LD tests and other measured done by Fstat), for this first replicate of the fists simulation with 10 % stuttering. Synthetic results for dioecious and monoecious simulations are in Excel format in the supplementary file S1 "SynthesisStutteringTestSexualsSupFileS1.xlsx". Synthetic results for clonal simulations are in Excel format in the supplementary file S2 "StutteringClonesSynthesisSupFileS2.xlsx". Pooling protocols for curing simulated data are in Excel files that were compressed in a zipped file, supplementary file S3 "PoolingProtocolCureSupFileS3.zip".

Background Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. Objective The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. Methods A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. Results The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. Conclusions These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics.

The recovery of genealogy in both natural and captive populations is critical for any decision in the management of genetic resources. It allows for the estimation of genetic parameters such as heritability and genetic correlations, as well as defining an optimal mating design that maintains a large effective population size. We utilised “genotyping-by-sequencing” (GBS) in combination with bioinformatics tools developed specifically for GBS data to recover genetic relatedness, with a focus on parent-offspring relationships in a Eucalyptus nitens breeding population as well as recognition of individuals representing other Eucalyptus species and putative hybrids. We found a clear advantage on using tools specifically designed for data of highly variable sequencing quality when recovering genetic relatedness. The parent-offspring relatedness showed a significant response to data filtering from 0.05 to 0.3 when the standard approach (G1) was used, while it oscillated around 0.4 when the specifically designed method (G5) was implemented. Additionally, comparisons with commonly used tools demonstrated vulnerability of the relatedness estimates to incorrect imputation of missing data when shallow sequencing information and genetically distant individuals are present in the population. In turn, these biased imputed genotypes negatively affected the estimation of genetic relatedness between parents and offspring. Careful filtering for both genetic outliers and shallowly sequenced markers led to improvements in estimations of genetic relatedness. Alternatively, a method that avoided missing data imputation and took sequence depth into consideration improved the accuracy of parent-offspring relationship coefficients where sequencing data quality was highly variable.

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology. ispartof: PLOS ONE vol:15 issue:4 ispartof: location:United States status: published

Background A reliable method to detect gene polymorphisms must be established to eliminate genotyping errors due to false PCR amplification. In the previous study, we have developed AS-PCR (Allele Specific-Polymerase Chain Reaction) to detect HER2 Ile655Val gene polymorphism with good specificity and sensitivity, yet it produces some errors. This study is aimed to eliminate the source of genotyping errors mainly by betaine treatment and PCR program modification. Methods Genotyping errors produced by AS-PCR was qualitatively and quantitatively evaluated using two genomic DNA that each contained AA genotype and GG genotype of HER2 Gene. Betaine treatment or PCR program modification was tested to eliminate the occurrence of genotyping errors during AS-PCR amplification. Results The types of genotyping errors exhibited by HER2 Ile655Val AS-PCR are diverse, ranging from LDO (Locus Drop Out), preferential amplification to ADO (Allele Drop Out). The rate of genotyping errors was from 10% to 50% depending on the amount and ratio of DNA template and the annealing temperature of PCR. In the mixed DNA template model, the betaine treatment has shown to reduce ADO only in preferentially amplified GG genotype amplicon. Alternatively, reducing the template of the heterozygous DNA by half ( -0.5 ng of DNA template) for such case has effectively recovered the AS-PCR from ADO. Furthermore, increasing the denaturation temperature to 96 °C with an annealing time of 40 s at the first 10 cycles of AS-PCR has succeeded in eliminating severe preferential amplification of AA genotype amplicon by preventing the DNA template with GG genotype from forming into a G-quadruplex structure. The guideline offered in this study has been successfully applied for clinical samples of breast cancer that show preferential amplification. Conclusion Betaine and the modifying AS-PCR program can reduce significantly genotyping errors making AS-PCR for HER2 Ile655Val detection more reliable to be used as a molecular tool for genotyping purpose.

This work was supported by CGL2015-64164-R project from the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund. Research was partly conducted during a research visit of JJRA to St Andrews University, hosted by OEG and funded by PRX14/00611 mobility grant from the Spanish Ministry of Education, Culture and Sports. OEG was supported by MASTS (the Marine Alliance for Science and Technology for Scotland). 1 . Microsatellite-based genetic assignment is used broadly to monitor contemporary effective dispersal among populations. The need to investigate the robustness of this method to common genotyping errors was emphasized more than a decade ago, but it remains unaddressed. 2 . We evaluate here for the first time the effect of mistaken and null alleles on estimates of contemporary seed and pollen migration rates obtained with genetic assignment methods. We also introduce a novel Bayesian approach to jointly estimate seed and pollen migration rates, genotyping error rates and null allele frequencies, not requiring independent reference or duplicate genotypic data. 3 . Unaccounted-for mistaken alleles caused positive bias and increased the root mean square error (RMSE) of pollen migration rate estimates, whereas seed migration rate estimates were weakly sensitive to mistyping. Jointly estimating mistyping rates minimized the bias and RMSE they introduce on pollen migration estimates, while yielding seed migration rate estimates with similar or slightly larger bias and RMSE than those obtained when ignoring mistyping. 4 . Ignoring genotyping errors can be especially problematic when there is no actual migration, because it can lead to the wrong conclusion that there is statistically significant exchange of pollen and/or seeds among populations that are actually isolated. 5 . Unaccounted-for null alleles are problematic when among-population pollen dispersal is present, leading to underestimation of pollen migration rates and overestimation of seed migration rates. Jointly estimating null allele frequencies minimized these two biases, reduced the RMSE of seed migration rate estimates and produced relatively small changes in the RMSE of pollen dispersal estimates. 6 . Synthesis. Disregarding genotyping errors and null alleles can produce biased and less accurate estimates of the rates at which present-day plant populations are exchanging seed and pollen. An approach is proposed here to minimize the effect of genotyping problems on contemporary migration rate estimates, which should help avoiding erroneous migration inference, monitoring and management, especially when dealing with low migration rates and their associated uncertainty. Postprint

International audience; In the search for genetic associations with complex traits, population isolates offer the advantage of reduced genetic and environmental heterogeneity. In addition, cost-efficient next-generation association approaches have been proposed in these populations where only a sub-sample of representative individuals is sequenced and then genotypes are imputed into the rest of the population. Gene mapping in such populations thus requires high quality genetic imputation and preliminary phasing. To identify an effective study-design, we compare by simulation a range of phasing and imputation software and strategies. We simulated 1,115,604 variants on chromosome 10 for 477 members of the large complex pedigree of Campora, a village within the established isolate of Cilento in southern Italy. We assessed the phasing performance of IBD-based software ALPHAPHASE and SLRP, LD-based software SHAPEIT2, SHAPEIT3, and BEAGLE, and new software EAGLE which combines both methodologies. For imputation we compared IMPUTE2, IMPUTE4, MINIMAC3, BEAGLE, and new software PBWT. Genotyping errors and missing genotypes were simulated to observe their effects on the performance of each software. Highly accurate phased data were achieved by all software with SHAPEIT2, SHAPEIT3, and EAGLE2 providing the most accurate results. MINIMAC3, IMPUTE4, and IMPUTE2 all performed strongly as imputation software and our study highlights the considerable gain in imputation accuracy provided by a genome sequenced reference panel specific to the population isolate.

In this study, parental assignment was studied in the 10th generation of a pedigreed selected Nile tilapia ( Oreochromis niloticus ) population (GIFT) and their offspring, by comparing two types of molecular markers, microsatellites and SNPs, using an exclusion-based (Vitassign) and a likelihood-based (Cervus) method. For the experiment, G10 parents were divided in 4 groups (cohorts) and allowed to produce offspring by natural group mating. In total 173 offspring were tested against 238 parents, using either 12 microsatellites (PIC = 0.639; exclusion power 68.0%) or 122 SNPs (PIC = 0.341; exclusion power 99.9%). In this study, more than half of the candidate parents were either full- or half-sibs with other parents. Furthermore, 13.8% of the parents died before being sampled for DNA. When offspring were assigned to parents in the same cohort, using Vitassign, for microsatellites, allowing up to 2 mismatches, 37.6% offspring got unique assignments, 45.1% got multiple assignments, and 17.3% were not assigned; for SNPs with up to 15 mismatches allowed, 83.8% offspring got unique assignments while 13.9% got multiple assignments. Only 2.3% were not assigned. Using Cervus, for microsatellites, the mean ‘strict’ (> 95% CF) assignment rate across the 4 cohorts was 18%, the ‘relax’ (80–95% CF) assignment rate was 43%, and 39% were not assigned; for SNPs, 39% ‘strict’ assignments were obtained (mean across 4 cohorts); the remaining offspring were not assigned. In general assignment rates were higher when cohort offspring were assigned to all parents combined, irrespective of method (Vitassign or Cervus) or marker used. However, consistency of assignments between microsatellites and SNPs was low: 28% with Vitassign and 16% with Cervus. Consistency of assignments between Cervus and Vitassign was high with SNPs (65%), but was low with microsatellites (31%). We conclude that missing parents and relatedness among candidate parents resulted in low assignment rates. Furthermore, low exclusion power of the microsatellite set resulted in low assignment rates and multiple parent pair assignments irrespective of method used. Exclusion methods and likelihood-based methods can be equally good for parental assignments, providing that good marker sets with high exclusion power are available.

High-density linkage maps are important tools for genome biology and evolutionary genetics by quantifying the extent of recombination, linkage disequilibrium, and chromosomal rearrangements across chromosomes, sexes, and populations. They provide one of the best ways to validate and refine de novo genome assemblies, with the power to identify errors in assemblies increasing with marker density. However, assembly of high-density linkage maps is still challenging due to software limitations. We describe Lep-MAP2, a software for ultradense genome-wide linkage map construction. Lep-MAP2 can handle various family structures and can account for achiasmatic meiosis to gain linkage map accuracy. Simulations show that Lep-MAP2 outperforms other available mapping software both in computational efficiency and accuracy. When applied to two large F2-generation recombinant crosses between two nine-spined stickleback (Pungitius pungitius) populations, it produced two high-density (∼6 markers/cM) linkage maps containing 18,691 and 20,054 single nucleotide polymorphisms. The two maps showed a high degree of synteny, but female maps were 1.5-2 times longer than male maps in all linkage groups, suggesting genome-wide recombination suppression in males. Comparison with the genome sequence of the three-spined stickleback (Gasterosteus aculeatus) revealed a high degree of interspecific synteny with a low frequency (

The impact of erroneous genotypes having passed standard quality control (QC) can be severe in genome-wide association studies, genotype imputation, and estimation of heritability and prediction of genetic risk based on single nucleotide polymorphisms (SNP). To detect such genotyping errors, a simple two-locus QC method, based on the difference in test statistic of association between single SNPs and pairs of SNPs, was developed and applied. The proposed approach could detect many problematic SNPs with statistical significance even when standard single SNP QC analyses fail to detect them in real data. Depending on the data set used, the number of erroneous SNPs that were not filtered out by standard single SNP QC but detected by the proposed approach varied from a few hundred to thousands. Using simulated data, it was shown that the proposed method was powerful and performed better than other tested existing methods. The power of the proposed approach to detect erroneous genotypes was similar to 80% for a 3% error rate per SNP. This novel QC approach is easy to implement and computationally efficient, and can lead to a better quality of genotypes for subsequent genotype-phenotype investigations. Genet. Epidemiol. 34:854-862, 2010. (C) 2010 Wiley-Liss, Inc. Refereed/Peer-reviewed

Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

Coherent ecological networks (EN) composed of core areas linked by ecological corridors are being developed worldwide with the goal of promoting landscape connectivity and biodiversity conservation. However, empirical assessment of the performance of EN designs is critical to evaluate the utility of these networks to mitigate effects of habitat loss and fragmentation. Landscape genetics provides a particularly valuable framework to address the question of functional connectivity by providing a direct means to investigate the effects of landscape structure on gene flow. The goals of this study are (1) to evaluate the landscape features that drive gene flow of an EN target species (European pine marten), and (2) evaluate the optimality of a regional EN design in providing connectivity for this species within the Basque Country (North Spain). Using partial Mantel tests in a reciprocal causal modeling framework we competed 59 alternative models, including isolation by distance and the regional EN. Our analysis indicated that the regional EN was among the most supported resistance models for the pine marten, but was not the best supported model. Gene flow of pine marten in northern Spain is facilitated by natural vegetation, and is resisted by anthropogenic landcover types and roads. Our results suggest that the regional EN design being implemented in the Basque Country will effectively facilitate gene flow of forest dwelling species at regional scale. This study has been funded by the Basque Government through the Research group on "Systematics, Biogeography and Population Dynamics'' (Ref. IT317-10; GIC10/76; IT575/13) and by the University of the Basque Country (UPV-EHU) and the Department of Environment, Territorial Planning, Agriculture and Fisheries (Basque Government) through IKT S.A under the University-Enterprise research program (Ref. UE07/02). Ruiz-Gonzalez holds a Post doc fellowship awarded by the Dept. of Education Universities and Research of the Basque Government (Ref. DKR-2012-64). Several samples analysed in this study have been obtained in the framework of different carnivore surveys funded by regional or national administrations (Spanish Ministry of Environment, Regional Governments of Navarre and Aragon, Alava Provincial Council). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background Runs of homozygosity are long, uninterrupted stretches of homozygous genotypes that enable reliable estimation of levels of inbreeding (i.e., autozygosity) based on high-throughput, chip-based single nucleotide polymorphism (SNP) genotypes. While the theoretical definition of runs of homozygosity is straightforward, their empirical identification depends on the type of SNP chip used to obtain the data and on a number of factors, including the number of heterozygous calls allowed to account for genotyping errors. We analyzed how SNP chip density and genotyping errors affect estimates of autozygosity based on runs of homozygosity in three cattle populations, using genotype data from an SNP chip with 777 972 SNPs and a 50 k chip. Results Data from the 50 k chip led to overestimation of the number of runs of homozygosity that are shorter than 4 Mb, since the analysis could not identify heterozygous SNPs that were present on the denser chip. Conversely, data from the denser chip led to underestimation of the number of runs of homozygosity that were longer than 8 Mb, unless the presence of a small number of heterozygous SNP genotypes was allowed within a run of homozygosity. Conclusions We have shown that SNP chip density and genotyping errors introduce patterns of bias in the estimation of autozygosity based on runs of homozygosity. SNP chips with 50 000 to 60 000 markers are frequently available for livestock species and their information leads to a conservative prediction of autozygosity from runs of homozygosity longer than 4 Mb. Not allowing heterozygous SNP genotypes to be present in a homozygosity run, as has been advocated for human populations, is not adequate for livestock populations because they have much higher levels of autozygosity and therefore longer runs of homozygosity. When allowing a small number of heterozygous calls, current software does not differentiate between situations where these calls are adjacent and therefore indicative of an actual break of the run versus those where they are scattered across the length of the homozygous segment. Simple graphical tests that are used in this paper are a current, yet tedious solution.

Microsatellite markers are important tools in population, conservation and forensic studies and are frequently used for species delineation, the detection of hybridization and introgression. Therefore, marker sets that amplify variable DNA regions in two species are required; however, cross-species amplification is often difficult, as genotyping errors such as null alleles may occur. To estimate the level of potential misidentifications based on genotyping errors, we compared the occurrence of parental alleles in laboratory and natural Daphnia hybrids (Daphnia longispina group). We tested a set of 12 microsatellite loci with regard to their suitability for unambiguous species and hybrid class identification using F(1) hybrids bred in the laboratory. Further, a large set of 44 natural populations of D. cucullata, D. galeata and D. longispina (1715 individuals) as well as their interspecific hybrids were genotyped to validate the discriminatory power of different marker combinations. Species delineation using microsatellite multilocus genotypes produced reliable results for all three studied species using assignment tests. Daphnia galeata × cucullata hybrid detection was limited due to three loci exhibiting D. cucullata-specific null alleles, which most likely are caused by differences in primer-binding sites of parental species. Overall, discriminatory power in hybrid detection was improved when a subset of markers was identified that amplifies equally well in both species.

Background Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. Results Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Conclusions A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Several options are available to the scientific community for genetic map construction but few are simple to install and use. Available programs either lack intuitive interface or are commercial, expensive for many laboratories. We present MapDisto, a free, user-friendly and powerful program for constructing genetic maps from experimental segregating populations. MapDisto is freely available at http://mapdisto.free.fr/DL/.Current version: 1.7.5.

Single nucleotide polymorphisms (SNPs) which represent the most widespread source of sequence variation in genomes, are becoming a routine application in several fields such as forensics, ecology and conservation genetics. Their use, requiring short amplifications, may allow a more efficient genotyping of degraded DNA. We provide the first application of SNP genotyping in an Italian non-invasive genetic monitoring project of the wolf. We compared three different techniques for genotyping SNPs: pyrosequencing, SNaPshot? and TaqMan? Probe Assay in Real-Time PCR. We successively genotyped nine SNPs using the TaqMan Probe Assay in 51 Italian wolves, 57 domestic dogs, 15 wolf x dog hybrids and 313 wolf scats collected in the northern Apennines. The obtained results were used to estimate genetic variability and PCR error rates in SNP genotyping protocols compared to standard microsatellite analysis. We evaluated the cost, laboratory effort and reliability of these different markers and discuss the possible future use of VeraCode, SNPlex and Fluidigm EP1 system in wild population monitoring.

13 pages; International audience; We report 22 new polymorphic microsatellites for the Ivory gull (Pagophila eburnea), and we describe how they can be efficiently co-amplified using multiplexed polymerase chain reactions. In addition, we report DNA concentration, amplification success, rates of genotyping errors and the number of genotyping repetitions required to obtain reliable data with three types of noninvasive or nondestructive samples: shed feathers collected in colonies, feathers plucked from living individuals and buccal swabs. In two populations from Greenland (n = 21) and Russia (Severnaya Zemlya Archipelago, n = 21), the number of alleles per locus varied between 2 and 17, and expected heterozygosity per population ranged from 0.18 to 0.92. Twenty of the markers conformed to Hardy-Weinberg and linkage equilibrium expectations. Most markers were easily amplified and highly reliable when analysed from buccal swabs and plucked feathers, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. Although DNA amplification success using single shed feathers was generally high, the genotypes obtained from this type of samples were prone to error and thus need to be amplified several times. The set of microsatellite markers described here together with multiplex amplification conditions and genotyping error rates will be useful for population genetic studies of the Ivory gull.

Background Several thousand human genome epidemiology association studies are published every year investigating the relationship between common genetic variants and diverse phenotypes. Transparent reporting of study methods and results allows readers to better assess the validity of study findings. Here, we document reporting practices of human genome epidemiology studies. Methods Articles were randomly selected from a continuously updated database of human genome epidemiology association studies to be representative of genetic epidemiology literature. The main analysis evaluated 315 articles published in 2001–2003. For a comparative update, we evaluated 28 more recent articles published in 2006, focusing on issues that were poorly reported in 2001–2003. Results During both time periods, most studies comprised relatively small study populations and examined one or more genetic variants within a single gene. Articles were inconsistent in reporting the data needed to assess selection bias and the methods used to minimize misclassification (of the genotype, outcome, and environmental exposure) or to identify population stratification. Statistical power, the use of unrelated study participants, and the use of replicate samples were reported more often in articles published during 2006 when compared with the earlier sample. Conclusion We conclude that many items needed to assess error and bias in human genome epidemiology association studies are not consistently reported. Although some improvements were seen over time, reporting guidelines and online supplemental material may help enhance the transparency of this literature.

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

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.

Times Cited: 2; International audience; In noninvasive studies, the intersample variance in DNA quality and quantity is large, and produces multilocus genotypes of highly variable quality. Here we propose a standardized method for testing the reliability of the genotyping procedure when using the multiple-tube approach. The quality indexes generated will allow reliable comparisons among samples, loci, studies, and field and/or laboratory protocols. These indexes represent a powerful tool for the quality management of noninvasive studies.

Strategic management of brushtail possum populations in New Zealand is presently dependent on the use of a standardized trap-catch procedure for monitoring population trends. Where this has been used in the first few months after control, calculated rates of increase often far exceed known reproductive and dispersal rates, suggesting that trapping-based population indices immediately following control are biased low. We are investigating the problem by genotyping DNA extracted from possum fecal pellets and using matching genotypes in the ear tissue of trapped possums as a measure of trappability. We have used quantitative polymerase chain reaction (PCR) to determine the threshold of possum DNA required from fecal samples in order to obtain an accurate genotype (>99%) from field samples. This has enabled the removal of “allelic dropout” as a source of error in obtaining accurate genotypes from such noninvasive DNA samples. Validation tests were conducted on fecal samples collected from caged (i.e., identifiable) possums, and fecal pellets and ear tissue from trapped possums in the field. The tests confirmed that the sample collection and preservation procedures used resulted in accurate identification of possums from both types of sample material, although fecal pellets older than about 7 days were unlikely to yield sufficient DNA for amplification and genotyping. Genotyping of a large quantity of sample material collected before control, and at 1, 4, and 9 months after control, in 2 replicated field trials is proceeding and revealing information on the trappability of possums that survive control, and the contribution of immigration to the populations after control.

We evaluated the feasibility of using faeces as a non-invasively collected DNA source for the genetic study of an endangered bird population (capercaillie; Tetrao urogallus). We used a multitube approach, and for our panel of 11 microsatellites genotyping reliability was estimated at 98% with five repetitions. Experiments showed that free DNases in faecal material were the major cause of DNA degradation. Our results demonstrate that using avian faeces as a source of DNA, reliable microsatellite genotyping can be obtained with a reasonable number of PCR replicates.

Overexploitation and subsequent collapse of major worldwide fisheries has made it clear that marine stocks are not inexhaustible. Unfortunately, the perception remains that marine fishes are resilient to large population reductions, as even a commercially ‘collapsed’ stock will still consist of millions of individuals. Coupled with this notion is the idea that fisheries can, therefore, have little effect on the genetic diversity of stocks. We used DNA from archived otoliths collected between 1924 and 1972 together with 2002 juvenile's tissue to estimate effective population size (Ne) in plaice (Pleuronectes platessa).Ne was estimated at 20 000 in the North Sea and 2000 in Iceland. These values are five orders of magnitude smaller than the estimated census size for the two locations. Populations examined between 1924 and 1960 were in Hardy–Weinberg equilibrium, whereas populations examined after approximately 1970 were not. Extensive testing was performed to rule out genotyping artefacts and Wahlund effects. The significant heterozygote deficiencies found from 1970 onward were attributed to inbreeding. The emergence of inbreeding between 1950 and 1970 coincides with the increase in fishing mortality after World War II. Although the biological mechanisms remain speculative, our demonstration of inbreeding signals the need for understanding the social and mating behaviour in commercially important fishes.

This thesis deals with the application of molecular tools, combined with field data, in wildlife management, in conservation and in understanding species' biology and behavior. We used the brown bear (Ursus arctos) as a model species and the Scandinavian bear population as a case study. The first part of this thesis is a methodological part, in which we developed or reviewed technical aspects in molecular biology and parentage analysis; the second part is devoted to the application of molecular genetics to estimate population sizes and to understand mating systems. Noninvasive methods are gaining widespread use in genetic studies as they do not require the handling or disturbance of the study animal. However, DNA recovered from noninvasive samples, such as hairs or feces, is usually degraded and/or in small quantities, leading to genotyping errors and resulting in the identification of incorrect genotypes. This is a major concern, especially for small or endangered populations, as it can lead to biases in population size estimates. With the aim of increasing the quality and quantity of the desired DNA template, and to avoid the need for numerous replicates, we devised a two-step polymerase chain reaction (PCR) method. This “multiplex pre-amplification method” was tested on different species and compared with a conventional PCR approach. It significantly improved microsatellite amplification and decreased error rates for fecal DNA in limiting conditions. To more specifically amplify DNA from noninvasive samples of brown bears, we also redesigned microsatellite primers and one sex-specific primer and combined a semi-nested PCR with the multiplex pre-amplification method. These new approaches could be transposed to other species where conventional PCR methods experience low success due to limiting DNA concentration and/or quality.Genotyping errors remain a taboo subject in population genetics studies, in spite of their occurrence in most datasets and the negative consequences they may cause in the interpretation of the results. We considered four case studies representing a large variety of population genetics investigations, to track genotyping errors and identify their causes. In these datasets the estimated genotyping error rate ranged from 0.8% to 2.6%, depending on the study organism and the marker used. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs (Amplified Fragment Length Polymorphism), but in both cases, human factors were non-negligible error generators. We present suggestions to limit and quantify genotyping errors at each step of the genotyping process and recommend the systematic reporting of the error rate in population genetics studies.Parentage analyses using multilocus genotypes are widely used to assess reproductive success, mating patterns, kinship and fitness in natural populations. Several approaches, based on maximum likelihood estimations and /or Bayesian inference, have been recently developed, but they often remain theoretical and difficult for biologists to apply. However, there is a clear lack of parentage assignment softwares that are able to consider several generations of individuals and that allow the determination of both parents without any prior assumptions. We developed the software PARENTE to conduct parentage inference using molecular data from diploid codominant markers. Based on the principle of genetic compatibility, PARENTE looks for maternity, paternity or simultaneously for both potential parents, using multilocus genotypes and birth and death dates of individuals (if available). It also calculates the probability of successfully allocating an individual offspring to its parents.Estimates of population size and population density are essential for successful management and conservation of species. However, few attempts have been made to evaluate the accuracy of the estimates obtained. Using the protocols developed for amplifying fecal DNA, we first compared four census methods based on noninvasive genetic methods. Two methods used rarefaction indices and two were based on capture-mark-recapture (CMR) estimators. A total of 1904 fecal samples were collected over 2 consecutive years in a 49,000-km² study area in south-central Sweden. Population size estimates ranged from 378 to 572 bears in 2001 and 273 to 433 bears in 2002, depending on the method used. Based on a calculated minimum population size from radio-telemetry data, we concluded that the estimate from the best model in program MARK, a CMR estimator, was the most accurate. This model included heterogeneity and temporal variation in detection probabilities, which appeared to be present in our samples. Second, we evaluated the reliability of three traditional field methods in comparison with the best performing noninvasive genetic method in a smaller study area (7,328 km²). All three field methods tended to underestimate population size; the genetic method using the MARK estimator seemed to perform the best. We concluded that approximately 550 (482-648) bears were present in the 49,000-km² study area and 223 (188-282) bears were present in the 7,328-km² study area during 2001 and 2002. We suggest that the brown bear has reached a threshold density in the core area and currently expands on the edge of this area. A cost/benefit analysis showed that the noninvasive genetic method was less expensive than the most reliable field method and it is preferable from an ethical point of view. In conclusion, we recommend the use of noninvasive genetic methods, using the MARK estimator, to estimate population size over large areas. We also point out the importance of an adequate and well-distributed sampling effort and advise calibration with independent estimates in case of biased sampling, if possible. Future studies should aim at collecting 2.5 to 3 times the number of fecal samples as the “assumed” number of animals. These studies also confirmed that the present management of the Scandinavian bears has been successful and that this population is in a good conservation status. The knowledge of mating systems is important for understanding the evolution of sexual selection. We studied two major aspects of the brown bear mating system, namely the mating strategies employed by both sexes in relation to sexually selected infanticide (SSI) and female mate selection. Infanticide, the killing of dependent young, can be considered as sexually selected and adaptive for males, if the following three requirements are fulfilled: i) infanticide shortens the time to the mother's next estrus, ii) the perpetrator is not the father of the killed infants, and iii) perpetrators sire the female's next litter. However, this is not of benefit for females and they may have evolved counterstrategies in order to defend their infants against infanticidal males. We documented eight cases of infanticide in the field. From genetic samples collected at the sites and from observations, we verified that all requirements for SSI were fulfilled, suggesting that SSI may be an adaptive male mating strategy in this nonsocial carnivore. Contrary to social species, where mostly immigrant males kill young, mainly resident adult males were infanticidal in Scandinavian brown bears. This implies that they are able to differentiate their own progeny from unrelated cubs, perhaps by recognizing the females they mated with. Moreover, we genetically documented a minimum of 14.5% multiple paternities (28% for litters with 3 young or more). Female promiscuity to confuse paternity may therefore be an adaptive counterstrategy to avoid SSI. Further, we assessed on which criteria female brown bears chose their reproductive partner(s). We hypothesized that females may be faced with a dilemma: either select a high quality partner based on morphological or genetic criteria, as suggested by theories of mate choice, or rather mate with future potentially infanticidal males, i.e. the geographically closest males. We tested whether different male traits influenced paternity determination and found that females significantly selected the geographically closest males, but also the more heterozygous, largest and oldest males. We suggest that female brown bears might mate with the closest males as a counter-strategy to infanticide and exercise a post-copulatory cryptic choice, based on morphological traits such as body size or dominance, reflecting male genetic quality., Cette thèse traite de l'application de l'outil moléculaire pour la gestion, la conservation et la compréhension de la biologie et du comportement des espèces animales. Nous avons étudié l'ours brun (Ursus arctos) en tant qu'espèce modèle et la population d'ours bruns de Scandinavie en tant que cas d'étude. La première partie de cette thèse est une partie méthodologique, dans laquelle nous avons développé des aspects techniques en biologie moléculaire et en analyse de parenté. La seconde partie concerne l'application de ces outils moléculaires pour estimer les tailles de population et comprendre les systèmes d'appariement. Les méthodes non invasives sont de plus en plus utilisées en génétique des populations car elles ne nécessitent pas la manipulation ni le dérangement de l'animal étudié et sont particulièrement recommendabls pour l'étude des populations en danger d'extinction. Cependant, l'ADN extrait de ce type d'échantillons, tels que poils ou fèces, est en général dégradé et/ou en faible quantité, ce qui peut conduire à des erreurs de génotypage. Dans le but d'accroître la qualité et quantité de l'extrait d'ADN, nous avons mis au point une métode PCR (polymerase chain reaction) en deux étapes (“multiplex pre-amplification”). Cette méthode a été testée sur différentes espèces et, en comparaison avec une approche PCR conventionnelle, a permis d'améliorer l'amplification d'ADN et de diminuer le taux d'erreur. Pour amplifier plus spécifiquement l'ADN à partir d'échantillons non invasifs d'ours brun, nous avons également défini de nouvelles amorces microsatellites ainsi qu'un marqueur de sexe spécifique, et combiné une PCR en nid avec la méthode “multiplex pre-amplification”. Ces nouvelles approches peuvent être transposées à d'autres espèces pour lesquelles les méthodes conventionnelles ne sont pas appropriées à cause d'une faible quantité/qualité d'ADN. Les erreurs de génotypage sont un sujet « tabou » dans les études de génétique des populations, malgré leur incidence dans la plupart des jeux de données et le biais qu'elles peuvent causer dans l'interprétation des résultats. Nous avons considéré quatre cas d'étude représentant une large variété d'investigations en génétique des populations, pour détecter les erreurs de génotypage et identifier leurs causes. Dans ces jeux de données, le taux d'erreur estimé variait de 0.8% à 2.6% , selon l'organisme étudié et le marqueur utilisé. Les sources d'erreur principales étaient les pertes d'allèles pour les microsatellites et les différences d'intensité de pics pour les AFLP (Amplified Fragment Length Polymorphism), ainsi que des erreurs d'origine humaine dans les deux cas. Nous présentons des suggestions pour limiter et quantifier les erreurs de génotypage à chaque étape du processus et recommandons le report systématique du taux d'erreur dans les études de génétique des populations. Les analyses de parenté basées sur les génotypes multilocus sont largement utilisées pour estimer les succès reproducteurs, les appariements et la fitness dans les populations naturelles. Les approches proposées sont basées sur des estimations du maximum de vraisemblance ou des inférences Bayésiennes et restent en général assez théoriques et difficiles à appliquer pour les biologistes. Il existe un réel manque de logiciels capables de considérer plusieurs générations d'individus et permettant la détermination des deux parents sans hypothèse à priori. Le logiciel PARENTE, que nous avons développé, détermine les maternités, paternités ou les deux parents simultanément, basé sur la compatibilité des génotypes multilocus (marqueurs diploïdes codominants) et des dates de naissance et de mort des individus (si disponibles). Ce logiciel calcule également la probabilité de parenté à partir des fréquences alléliques, du taux d'échantillonnage de la population et du taux d'erreur de génotypage. Les estimations de taille de population sont essentielles pour la bonne gestion et conservation des espèces. Cependant, de manière générale, peu d'études évaluent la précision des estimations obtenues. Nous avons, dans un premier temps, comparé quatre estimateurs de taille de population, basés sur des méthodes génétiques non invasives. Deux méthodes utilisaient des indices de raréfaction et deux étaient basées sur des estimateurs de capture-marquage-recapture (CMR). Au total, 1904 fèces d'ours bruns ont été collectés sur deux années consécutives sur le terrain (49 000-km2 en Suède centrale). Les estimations variaient de 378 à 572 ours en 2001 et de 273 à 433 ours en 2002, selon l‘estimateur utilisé. La détermination d'une taille de population minimale obtenue à partir de données de radio-télémétrie nous a permis de conclure que l'estimation donnée par une des méthodes de CMR était la plus précise. Cet estimateur incluait une hétérogénéité et une variation temporelle dans les probabilités de détection, ce qui paraissait réaliste dans notre échantillonnage. Deuxièmement, nous avons évalué la fiabilité de trois méthodes de terrain traditionnelles en comparaison avec la méthode génétique la plus performante, dans une aire d'étude plus réduite (7 328-km2). Les trois méthodes de terrain tendaient à sous-estimer la taille de population ; la méthode génétique paraissait être la plus exacte. Nous avons conclu qu'environ 550 (482-648) ours étaient présents dans l'aire de 49 000-km2 et 223 (188-282) ours étaient présents dans l'aire de 7 328-km2. Nous suggérons que la population d'ours a atteint une densité de saturation dans l'aire centrale et disperse à présent sur les bords de cette aire centrale. Une analyse en termes de coûts/bénéfices a démontré que la méthode génétique était moins onéreuse que la méthode de terrain la plus fiable. De plus, elle est préférable d'un point de vue éthique. En conclusion, nous recommandons l'utilisation de méthodes génétiques basées sur un principe de CMR, pour estimer les tailles de population sur de larges aires. Nous insistons sur l'importance d'un effort d'échantillonnage adéquat et, en cas d'échantillonnage biaisé, nous conseillons le calibrage avec des estimations indépendantes, si possible. Nous recommandons La collecte d'un nombre d'échantillons supérieur de 2,5 à 3 fois le nombre « présumé » d'animaux. Ces études ont également confirmé que la gestion actuelle de la population d'ours a été bénéfique et que cette population est actuellement dans un bon statut de conservation.La connaissance des systèmes d'appariement est importante dans la compréhension de la sélection naturelle. Nous avons étudié deux aspects majeurs du système d'appariement de l'ours brun : les stratégies d'appariement employées par les deux sexes en relation avec l'infanticide sexuellement sélectionné (SSI) et la sélection du partenaire par la femelle. L'infanticide, le meurtre de jeunes non sevrés, peut être considéré comme sexuellement sélectionné si les trois conditions suivantes sont réunies : i) l'infanticide réduit le délai du prochain oestrus de la femelle ; ii) le mâle commettant l'infanticide n'est pas le père des jeunes tués ; iii) le mâle commettant l'infanticide produit la portée suivante de la femelle. Nous avons documenté huit cas d'infanticide sur le terrain. A partir d'observations et d'échantillons collectés sur sites, nous avons vérifié que les trois conditions pour le SSI étaient vérifiées. Cela suggère que le SSI pourrait être une stratégie adaptative pour le mâle chez ce carnivore non social. Contrairement aux espèces sociales où les mâles immigrants tuent les jeunes, la plupart des mâles commettant l'infanticide étaient résidents chez les ours scandinaves. Ceci implique qu'ils sont capables de différencier leurs propres jeunes des jeunes non apparentés, probablement en reconnaissant les femelles avec lesquelles ils se sont accouplés l'année précédente. De plus, nous avons démontré génétiquement un minimum de 14.5% de paternités multiples (28% pour les portées de 3 jeunes ou plus). La promiscuité des femelles, dans le but de confondre les paternités, pourrait donc être une contre-stratégie adaptative pour éviter le SSI. D'autre part, nous avons évalué sur quels critères les femelles ours bruns sélectionnaient leur partenaire reproductif. Nous avons émis l'hypothèse que les femelles pourraient faire face à un dilemme: soit choisir un partenaire de bonne qualité d'un point de vue phénotypique, comme suggéré par les théories de choix du partenaire, soit s'accoupler avec des mâles susceptibles de commettre l'infanticide l'année suivante, c'est à dire les plus proches géographiquement. Nous avons conclu que les femelles sélectionnaient significativement les mâles les plus proches mais aussi les plus hétérozygotes, les plus gros et les plus âgés. Nous suggérons que les femelles ours s'accouplent avec les mâles les plus proches comme contre-stratégie au SSI et exercent un choix post-copulatoire du partenaire reproducteur, basé sur des critères morphologiques tels qu'une large taille corporelle, ou sur des critères de statut de dominance, reflétant la qualité génétique du mâle.

Background Schistosomiasis japonica remains a major public health problem in China. Integrating molecular analyses, such as population genetic analyses, of the parasite into the on-going surveillance programs is helpful in exploring the factors causing the persistence and/or spread of Schistosoma japonicum. However, genotyping errors can seriously affect the results of such studies, unless accounted for in the analyses. Methods We assessed the genotyping errors (missing alleles or false alleles) of seven S. japonicum microsatellites, using a pedigree data approach for schistosome miracidia, which were stored on Whatman FTA cards. Results Among 107 schistosome miracidia successfully genotyped, resulting in a total of 715 loci calls, a total of 31 genotyping errors were observed with 25.2 % of the miracidia having at least one error. The error rate per locus differed among loci, which ranged from 0 to 9.8 %, with the mean error rate 4.3 % over loci. With the parentage analysis software Cervus, the assignment power with these seven markers was estimated to be 89.5 % for one parent and 99.9 % for a parent pair. One locus was inferred to have a high number of null alleles and a second with a high mistyping rate. Conclusion To the authors’ knowledge, this is the first time that S. japonicum pedigrees have been used in an assessment of genotyping errors of microsatellite markers. The observed locus-specific error rate will benefit downstream epidemiological or ecological analyses of S. japonicum with the markers. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1074-0) contains supplementary material, which is available to authorized users.