Abstract Key message Mining genome-wide DNA sequences enabled the discovery of near-diagnostic markers for species assignment in four European white oaks ( Quercus petraea (Matt.) Liebl., Quercus ...pubescens Willd., Quercus pyrenaica Willd., and Quercus robur L.) despite their low interspecific differentiation. Near-diagnostic markers are almost fully fixed in one species and absent in the three others. As a result, only a handful of markers are needed for species identification, making this genetic assay a very promising operational taxonomic assignment procedure in research and forestry. Context Identifying species in the European white oak complex has been a long-standing concern in taxonomy, evolution, forest research, and management. Quercus petraea (Matt.) Liebl., Q. robur L., Q. pubescens Willd., and Q. pyrenaica Willd. are part of this species complex in western temperate Europe and hybridize in mixed stands, challenging species identification. Aims Our aim was to identify near-diagnostic single-nucleotide polymorphisms (SNPs) for each of the four species that are suitable for routine use and rapid diagnosis in research and applied forestry. Methods We first scanned existing whole-genome and target-capture data sets in a reduced number of samples (training set) to identify candidate diagnostic SNPs, i.e., genomic positions being characterized by a reference allele in one species and by the alternative allele in all other species. Allele frequencies of the candidates SNPs were then explored in a larger, range-wide sample of populations in each species (validation step). Results We found a subset of 38 SNPs (10 for Q. petraea , 7 for Q. pubescens , 9 for Q. pyrenaica , and 12 for Q. robur ) that showed near-diagnostic features across their species distribution ranges with Q. pyrenaica and Q. pubescens exhibiting the highest (0.876) and lowest (0.747) diagnosticity, respectively. Conclusions We provide a new, efficient, and reliable molecular tool for the identification of the species Q. petraea , Q. robur , Q. pubescens , and Q. pyrenaica , which can be used as a routine tool in forest research and management. This study highlights the resolution offered by whole-genome sequencing data to design near-diagnostic marker sets for taxonomic assignment, even for species complexes with relatively low differentiation.
Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, are a major cause of human bacterial enteritis. Current detection in stools is done essentially by culture on selective ...and nonselective media with filtration. These methods were compared to 2 molecular biology methods, an in-house real-time PCR and a multiplex PCR named Seeplex Diarrhea ACE Detection, and 3 immunoenzymatic methods, Premier Campy, RidaScreen Campylobacter, and ImmunoCard Stat!Campy. Out of 242 stool specimens tested, 23 (9.5%) fulfilled the positivity criteria, i.e., they were positive by one or both culture methods or, in case of a negative culture, by a positive molecular method and a positive immunoenzymatic method. The striking feature of this study is the low sensitivity of culture, in the range of 60%, in contrast to immunoenzymatic and molecular tests.
The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. ...delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼ 1900 years ago), and to the Neolithic era for bioprocesses (∼ 4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To better study and manage chestnut trees and species, we identified nuclear single nucleotide polymorphism (SNP) markers using restriction-associated DNA sequencing. Out of 343 loci tested, 68 SNP ...markers were selected that withhold stringent quality criteria such as quasi-systematic amplification across species and Mendelian segregation in both purebred and hybrid individuals. They provide sufficient power for species, hybrids and backcross characterization as well as for clonal identification, as shown by a comparison with single sequenced repeat (SSR) loci.
We describe the development of new nuclear SNP markers for the genetic timber tracking of the geographical origin of Sapelli,
Entandrophragma cylindricum
(Meliaceae). Restriction associated DNA ...sequencing (RADseq) of two reference individuals yielded 1131 putative SNPs. Among those, 131 were selected to design four MassARRAY multiplexes and screened at 178 individuals. Seventy-two loci were selected for further use in genetic tracking.
Multispecies population genetics is an emerging field that provides insight relevant to conservation biology and community ecology. However, to date, this approach is limited to species with ...available genetic resources. The use of thousands of single‐nucleotide polymorphism (SNP) markers developed from recent genotyping‐by‐sequencing (GBS) technologies is a roadmap for the study of nonmodel species, but remains cost prohibitive when several, distantly related species are involved.
We aimed to overcome this issue using a single HiSeq3000 run of restriction‐site associated DNA sequencing (RAD‐Seq) to retrieve SNP markers for 40 diverse species including plants, invertebrates, fish, and mammals. We developed a Python‐based pipeline to isolate c. 100–500 high‐quality SNP markers for each species that could be genotyped through classical PCR amplification methods. To assess the quality of these markers, we validated our approach on c. 160 of the characterized SNPs for each of 18 Neotropical fish species from the river Maroni (French Guiana, South America), using the MassARRAY iPLEX platform from Agena Bioscience (San Diego, CA, USA).
A run of the pipeline applying stringent filtering parameters enabled the successful design of between 130 and 3,492 SNP markers for 30 of the 40 study species. Relaxing pipeline parameters allows for an increase in the number of detected SNPs. Across the 18 species from French Guiana, an average of 85% of markers were successfully amplified, polymorphic, and scored in ≥90% of individuals (c. 200 individuals per species). The great majority (>98%) of these markers were at Hardy–Weinberg equilibrium in each sampling site from the river Maroni.
This SNP discovery was performed at the cost of c. $US110 for each of the 40 species. Genotyping was performed at the cost of c. $US6000 for each of the 18 fish species with an average of 200 individuals per species. This strategy was found cost‐and‐time efficient to develop hundreds of SNP markers for a large range of nonmodel species, which can be used to investigate ecological and evolutionary questions that do not require whole‐genome coverage.
Résumé
La génétique des populations comparée entre plusieurs espèces, constitue un axe émergent dans l'appréhension de questions relatives à la biologie de la conservation et à l’écologie des communautés. Cependant, cette approche reste à ce jour restreinte aux espèces pour lesquelles des ressources génétiques sont disponibles. L'utilisation de plusieurs milliers de marqueurs de type SNP (single‐nucleotide polymorphism) développés via les technologies de génotypage‐par‐séquençage présente un avantage certain pour l’étude d'espèces non‐modèles, mais garde un coût prohibitif lorsque plusieurs espèces sont impliquées.
Nous nous proposons de répondre à ces considérations par le développement de marqueurs SNP pour 40 espèces de divers taxa incluant plantes, invertébrés, poisons et mammifères à partir du séquençage de marqueurs RAD‐sequencing sur un seul run de HiSeq3000. Nous présentons un pipeline informatique ayant pour but d'isoler, pour chaque espèce, 100 à 500 SNPs de haute qualité, c'est‐à‐dire pouvant être génotypés de façon robuste via des techniques classiques d'amplification par PCR. Pour évaluer la qualité de ces marqueurs, nous validons notre approche sur un sous‐échantillonnage de c. 160 des SNPs spécifiquement caractérisés pour 18 espèces de poissons néotropicaux provenant du bassin versant du Maroni (Guyane française, Amérique du Sud). Nous utilisons pour cela les équipements proposés par une plateforme de type MassARRAY iPLEX, et développés par Agena Bioscience (San Diego, CA, USA).
L'utilisation de paramètres stringents au cours d'un run du pipeline a permis d'isoler entre 130 et 3492 SNPs pour 30 des 40 espèces focales. Des paramètres plus tolérants augmentent le nombre de SNPs isolés. Parmi les 18 espèces de Guyane française, en moyenne 85% des marqueurs ont été amplifiés avec succès chez au moins 90% des individus génotypés (c. 200 par espèce). La grande majorité (>98%) de ces marqueurs étaient à l’équilibre de Hardy‐Weinberg dans chacun des sites d’échantillonnage du bassin du Maroni.
Le coût de ce développement de marqueurs SNP avoisinait 110$ pour chacune des 40 espèces. La procédure de génotypage consécutive avoisinait 6000$ pour chacune des 18 espèces de Guyane française avec en moyenne 200 individus par espèce. La stratégie proposée s'avère d'une grande efficacité pour développer jusqu’à plusieurs centaines de marqueurs SNPs pour une large gamme d'espèces non‐modèles, ouvrant des perspectives d’étude de questions écologiques ou évolutives qui ne nécessiteraient toutefois pas d'approche génomique à très haute densité de marqueurs.
A set of single nucleotide polymorphism (SNP) markers was developed from the transcriptome of the solitary ascidian
Halocynthia papillosa
. 1399 putative SNPs with coverage larger than 100 reads were ...identified, and a selected set of 320 SNPs was tested using a MassARRAY System on 95 samples from the NW Mediterranean. A total of 175 SNPs were successfully genotyped, polymorphic and in Hardy-Weinberg equilibrium over all samples, with significant linkage desequilibrium in only 8 pairs of SNPs. The newly developed loci will be a valuable tool for population genetics studies of this endemic ascidian species of the Mediterranean Sea.
DNA barcoding has proved difficult in a number of woody plant genera, including the ecologically important oak genus Quercus. In this study, we utilized restrictionsite‐associated DNA sequencing ...(RAD‐seq) to develop an economical single nucleotide polymorphism (SNP) DNA barcoding system that suffices to distinguish eight common, sympatric eastern North American white oak species. Two de novo clustering pipelines, PyRAD and Stacks, were used in combination with postclustering bioinformatic tools to generate a list of 291 potential SNPs, 80 of which were included in a barcoding toolkit that is easily implemented using MassARRAY mass spectrometry technology. As a proof‐of‐concept, we used the genotyping toolkit to infer potential hybridization between North American white oaks transplanted outside of their native range (Q. michauxii, Q. montana, Q muehlenbergii/Q. prinoides, and Q. stellata) into a horticultural collection surrounded by natural forests of locally native trees (Q. alba and Q. macrocarpa) in the living collection at The Morton Arboretum (Lisle, IL, USA). Phylogenetic and clustering analyses suggested low rates of hybridization between cultivated and native species, with the exception of one Q. michauxii mother tree, the acorns of which exhibited high admixture from either Q. alba or Q. stellata and Q. macrocarpa, and a hybrid between Q. stellata that appears to have backcrossed almost exclusively to Q. alba. Together, RAD‐seq and MassARRAY technologies allow for efficient development and implementation of a multispecies barcode for one of the more challenging forest tree genera.
Studying hybridization among forest trees in multispecies hybrid zones typically requires large numbers of nuclear markers. In this article, we develop an economical DNA barcoding system for the white oaks of northeastern North America and use it to study hybridization in a botanical garden setting. Remarkably, and contrary to expectation, the progeny of oaks introduced to the garden appear mostly not to be hybrids, suggesting that botanical gardens may be able to limit hybridization between introduced and native species through planning and clustering of trees in single‐species groups.
Although many yeasts are useful for food production and beverage, some species may cause spoilage with important economic loss. This is the case of Dekkera/Brettanomyces bruxellensis, a contaminant ...species that is mainly associated with fermented beverages (wine, beer, cider and traditional drinks). To better control Brettanomyces spoilage, rapid and reliable genotyping methods are necessary to determine the origins of the spoilage, to assess the effectiveness of preventive treatments and to develop new control strategies.
Despite several previously published typing methods, ranging from classical molecular methods (RAPD, AFLP, REA-PFGE, mtDNA restriction analysis) to more engineered technologies (infrared spectroscopy), there is still a lack of a rapid, reliable and universal genotyping approach. In this work, we developed eight polymorphic microsatellites markers for the Brettanomyces/Dekkera bruxellensis species. Microsatellite typing was applied to the genetic analysis of wine and beer isolates from Europe, Australia and South Africa. Our results suggest that B. bruxellensis is a highly disseminated species, with some strains isolated from different continents being closely related at the genetic level. We also focused on strains isolated from two Bordeaux wineries on different substrates (grapes, red wines) and for different vintages (over half a century). We showed that all B. bruxellensis strains within a cellar are strongly related at the genetic level, suggesting that one clonal population may cause spoilage over decades. The microsatellite tool now paves the way for future population genetics research of the B. bruxellensis species.
•We developed 8 microsatellite markers for Brettanomyces bruxellensis, a spoilage wine yeast.•Microsatellite typing shows that B. bruxellensis is a highly disseminated species.•Recent and older B. bruxellensis isolates from two Bordeaux wineries were genotyped.•B. bruxellensis strains within a cellar are strongly related at the genetic level.•Our results suggest that a clonal population may cause spoilage over decades.
The RADseq technology allows researchers to efficiently develop thousands of polymorphic loci across multiple individuals with little or no prior information on the genome. However, many questions ...remain about the biases inherent to this technology. Notably, sequence misalignments arising from paralogy may affect the development of single nucleotide polymorphism (SNP) markers and the estimation of genetic diversity. We evaluated the impact of putative paralog loci on genetic diversity estimation during the development of SNPs from a RADseq dataset for the nonmodel tree species Robinia pseudoacacia L. We sequenced nine genotypes and analyzed the frequency of putative paralogous RAD loci as a function of both the depth of coverage and the mismatch threshold allowed between loci. Putative paralogy was detected in a very variable number of loci, from 1% to more than 20%, with the depth of coverage having a major influence on the result. Putative paralogy artificially increased the observed degree of polymorphism and resulting estimates of diversity. The choice of the depth of coverage also affected diversity estimation and SNP validation: A low threshold decreased the chances of detecting minor alleles while a high threshold increased allelic dropout. SNP validation was better for the low threshold (4×) than for the high threshold (18×) we tested. Using the strategy developed here, we were able to validate more than 80% of the SNPs tested by means of individual genotyping, resulting in a readily usable set of 330 SNPs, suitable for use in population genetics applications.
Paralogy can impact the estimate of genetic diversity and single nucleotide polymorphism (SNP) development from RADseq data. We explored putative paralogy in a dataset obtained on Robinia pseudoacacia L. Thanks to a filtering approach, we validated a set of SNPs useful for genotyping in the species.
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