A few north Appalachian populations are the source of European black locust.

The role of evolution in biological invasion studies is often overlooked. In order to evaluate the evolutionary mechanisms behind invasiveness, it is crucial to identify the source populations of the introduction. Studies in population genetics were carried out on Robinia pseudoacacia L., a North American tree which is now one of the worst invasive tree species in Europe. We realized large-scale sampling in both the invasive and native ranges: 63 populations were sampled and 818 individuals were genotyped using 113 SNPs. We identified clonal genotypes in each population and analyzed between and within range population structure, and then, we compared genetic diversity between ranges, enlarging the number of SNPs to mitigate the ascertainment bias. First, we demonstrated that European black locust was introduced from just a limited number of populations located in the Appalachian Mountains, which is in agreement with the historical documents briefly reviewed in this study. Within America, population structure reflected the effects of long-term processes, whereas in Europe it was largely impacted by human activities. Second, we showed that there is a genetic bottleneck between the ranges with a decrease in allelic richness and total number of alleles in Europe. Lastly, we found more clonality within European populations. Black locust became invasive in Europe despite being introduced from a reduced part of its native distribution. Our results suggest that human activity, such as breeding programs in Europe and the seed trade throughout the introduced range, had a major role in promoting invasion; therefore, the introduction of the missing American genetic cluster to Europe should be avoided.

A nuclear DNA barcode for eastern North American oaks and application to a study of hybridization in an Arboretum setting.

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.

Dealing with paralogy in RADseq data: in silico detection and single nucleotide polymorphism validation in Robinia pseudoacacia L.

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.

Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level.

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.

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii.

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.

New methods for detection of campylobacters in stool samples in comparison to culture.

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.