Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

Chloroplast DNA variation in a hyperdiverse tropical tree community.

We investigate chloroplast DNA variation in a hyperdiverse community of tropical rainforest trees in French Guiana, focusing on patterns of intraspecific and interspecific variation. We test whether a species genetic diversity is higher when it has congeners in the community with which it can exchange genes and if shared haplotypes are more frequent in genetically diverse species, as expected in the presence of introgression.We sampled a total of 1,681 individual trees from 472 species corresponding to 198 genera and sequenced them at a noncoding chloroplast DNA fragment.Polymorphism was more frequent in species that have congeneric species in the study site than in those without congeners (30% vs. 12%). Moreover, more chloroplast haplotypes were shared with congeners in polymorphic species than in monomorphic ones (44% vs. 28%).Despite large heterogeneities caused by genus-specific behaviors in patterns of hybridization, these results suggest that the higher polymorphism in the presence of congeners is caused by local introgression rather than by incomplete lineage sorting. Our findings suggest that introgression has the potential to drive intraspecific genetic diversity in species-rich tropical forests.

Imprints of natural selection along environmental gradients in phenology-related genes of Quercus petraea.

We explored single nucleotide polymorphism (SNP) variation in candidate genes for bud burst from Quercus petraea populations sampled along gradients of latitude and altitude in Western Europe. SNP diversity was monitored for 106 candidate genes, in 758 individuals from 32 natural populations. We investigated whether SNP variation reflected the clinal pattern of bud burst observed in common garden experiments. We used different methods to detect imprints of natural selection (FST outlier, clinal variation at allelic frequencies, association tests) and compared the results obtained for the two gradients. FST outlier SNPs were found in 15 genes, 5 of which were common to both gradients. The type of selection differed between the two gradients (directional or balancing) for 3 of these 5. Clinal variations were observed for six SNPs, and one cline was conserved across both gradients. Association tests between the phenotypic or breeding values of trees and SNP genotypes identified 14 significant associations, involving 12 genes. The results of outlier detection on the basis of population differentiation or clinal variation were not very consistent with the results of association tests. The discrepancies between these approaches may reflect the different hierarchical levels of selection considered (inter- and intrapopulation selection). Finally, we obtained evidence for convergent selection (similar for gradients) and clinal variation for a few genes, suggesting that comparisons between parallel gradients could be used to screen for major candidate genes responding to natural selection in trees.

Aperture effect correction in spectroscopic full-field optical coherence tomography.

Spatially resolved spectroscopic optical coherence tomography (OCT) has been demonstrated to be a convenient tool for spectral analysis in turbid media. For a full-field OCT configuration using a Mirau objective in the visible range, we found that the effective numerical aperture varies over the field of view, leading to field-dependent spectral shifts in the reconstructed spectra. Interferograms recorded with quasi-monochromatic lights are theoretically fitted with a general Mirau interference formula, and we propose a numerical correction method for white-light spectroscopy. The method is then tested successfully for the measure of the reflectivity of a plane gold sample.

Role of waterlogging-responsive genes in shaping interspecific differentiation between two sympatric oak species.

Pedunculate (Quercus robur L.) and sessile oak (Quercus petreae Matt. Liebl.) are closely related species with a widely sympatric distribution in Europe. These two oak species are also known to display different ecological features, particularly related to their adaptation to soil waterlogging. Pedunculate oak grows in humid areas and can withstand high moisture content of the soil, whereas sessile oak requires drier soil with better drainage. The main goal of this study was to explore the role of gene expression contributing to differences in terms of waterlogging tolerance between these two species. We implemented a series of experiments aimed at evaluating whether differentially expressed genes between species are associated with their ecological preferences and underlie adaptive genetic divergence. Rooted cuttings of both species were grown in hydroponic conditions and subjected to gradual root hypoxia. White roots were sampled after 6, 12, 24 and 48 h. Real-time polymerase chain reaction (qPCR) was first used to monitor the expression of 10 known waterlogging-responsive genes, to identify discriminating sampling time points along the kinetics of hypoxia. Secondly, four subtractive suppressive hybridization libraries (sessile vs. pedunculate, pedunculate vs. sessile for early and late responses) were generated to isolate differentially expressed genes between species. A total of 2160 high-quality expressed sequence tags were obtained and annotated, and a subset of 45 genes were selected for qPCR analysis in a second independent factorial experimental design applying two stress durations per two species. Significant differences of gene expression between pedunculate and sessile oaks were detected, suggesting species-specific molecular strategies to respond to hypoxia. This study revealed that the ability of pedunculate oak to maintain glycolysis and fermentation under hypoxic conditions may help explain its tolerance to waterlogging.

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. RESULTS: We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. CONCLUSIONS: Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak.

BACKGROUND: The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity. RESULTS: We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html. CONCLUSIONS: This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.

A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study.

BACKGROUND: Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut). RESULTS: A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher. CONCLUSION: We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

In vitro vs in silico detected SNPs for the development of a genotyping array: what can we learn from a non-model species?

BACKGROUND: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size ( approximately 23.8 Gb/C). METHODOLOGY/PRINCIPAL FINDINGS: A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). CONCLUSIONS/SIGNIFICANCE: This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.

Controlled modification of single colloidal CdSe/ZnS nanocrystal fluorescence through interactions with a gold surface.

Single colloidal CdSe/ZnS nanocrystals are deposited at various distances from a gold film in order to improve their performance as single photon sources. Photon antibunching is demonstrated and the experimental curves are accurately fitted by theoretical equations. Emission lifetime and intensity are measured and found in excellent agreement with theoretical values. The various effects of a neighbouring gold film are discussed : interferences of the excitation beam, interferences of the fluorescence light, opening of plasmon and lossy-surface-wave modes, modification of the radiation pattern leading to a modified objective collection efficiency. At 80 nm from the gold film, when using an objective with 0.75 numerical aperture, about a 2.4-fold increase of the detected intensity is evidenced.

Contribution of surface state characterization to studies of works of art.

This paper has two purposes. The first one underlines that qualitative and quantitative studies of surface states lead to relevant information for analyzing works of art, with lots of potential for art history, restorers, and curators. The discrimination between different artistic techniques and the influence of a varnish on the leveling of paint surfaces are presented. The second purpose is the comparison between different nondestructive optical topographic methods, i.e., goniophotometry, optical coherence topography, and confocal microscopy, according to their accuracy, their discriminatory ability, their practicability inside a museum, and the size limits of the studied objects.

Determination of the absorption and scattering coefficients of pigments: application to the identification of the components of pigment mixtures.

In most scattering media, the scattering centers have random shapes and sizes, preventing any theoretical approach for the determination of their absorption and scattering coefficients. Three different experimental protocols are here selected and briefly described. The obtained values are compared and then validated from a comparison with the most rigorous method. The Kubelka-Munk method, using a paint layer applied to both white and black backgrounds, is chosen to build a database of these coefficients for reference pigments. Using this database, the identification of the components of a pigment mixture using spectral measurements is realized and validated.

Skin color modeling using the radiative transfer equation solved by the auxiliary function method: inverse problem.

In a previous article [J. Opt. Soc. Am. A 24, 2196 (2007)] we have modeled skin color using the radiative transfer equation, solved by the auxiliary function method. Three main parameters have been determined as being predominant in the diversity of skin color: the concentrations of melanosomes and of red blood cells and the oxygen saturation of blood. From the reflectance spectrum measured on real Caucasian skin, these parameters are now evaluated by minimizing the standard deviation on the adjusted wavelength range between the experimental spectrum and simulated spectra gathered in a database.

Molecular and phenotypic profiling from the base to the crown in maritime pine wood-forming tissue.

Environmental, developmental and genetic factors affect variation in wood properties at the chemical, anatomical and physical levels. Here, the phenotypic variation observed along the tree stem was explored and the hypothesis tested that this variation could be the result of the differential expression of genes/proteins during wood formation. Differentiating xylem samples of maritime pine (Pinus pinaster) were collected from the top (crown wood, CW) to the bottom (base wood, BW) of adult trees. These samples were characterized by Fourier transform infrared spectroscopy (FTIR) and analytical pyrolysis. Two main groups of samples, corresponding to CW and BW, could be distinguished from cell wall chemical composition. A genomic approach, combining large-scale production of expressed sequence tags (ESTs), gene expression profiling and quantitative proteomics analysis, allowed identification of 262 unigenes (out of 3512) and 231 proteins (out of 1372 spots) that were differentially expressed along the stem. A good relationship was found between functional categories from transcriptomic and proteomic data. A good fit between the molecular mechanisms involved in CW-BW formation and these two types of wood phenotypic differences was also observed. This work provides a list of candidate genes for wood properties that will be tested in forward genetics.

Skin color modeling using the radiative transfer equation solved by the auxiliary function method.

The auxiliary function method is an efficient technique for solving the radiative tranfer equation without adding any assumption and was applied until now only for theoretical stratified media. The first application (to our knowledge) of the method to a real case, the human skin, is presented. This makes it possible to validate the method by comparing model results with experimental reflectance spectra of real skin. An excellent agreement is obtained for a multilayer model of the skin made of 22 sublayers and taking into account the anisotropic phase function of the scatterers. Thus there is the opportunity to develop interest in such models by quantitatively evaluating the influence of the parameters commonly used in the literature that modify skin color, such as the concentration of the scatterers and the thickness of each sublayer.

Nondestructive varnish identification by ultraviolet fluorescence spectroscopy.

The identification of the chemical nature of varnish is essential for art restorers in order to choose a suitable solvent during its removal. Until today, such identification has been performed using chemical analysis after sampling. An innovative technique is presented here, using ultraviolet (UV) fluorescence spectroscopy. The method is nondestructive, workable in situ, and leads to results in real time. It is based on the comparison between the emission spectrum of an unknown varnish with those of fresh, artificially aged, or old reference resins and varnishes, for different monochromatic excitation wavelengths. The resin and the nature of the varnish as spirit, oil, or mixed can thus be identified. Various examples are presented on home-made samples applied on fluorescent backgrounds and on real works of art.

The proteome of maritime pine wood forming tissue.

Wood is one of our most important natural resources. Surprisingly, we know hardly anything about the details of the process of wood formation. The aim of this work was to describe the main proteins expressed in wood forming tissue of a conifer species (Pinus pinaster Ait.). Using high resolution 2-DE with linear pH gradient ranging from 4 to 7, a total of 1039 spots were detected. Out of the 240 spots analyzed by MS/MS, 67.9% were identified, 16.7% presented no homology in the databases, and 15.4% corresponded to protein mixtures. Out of the 57 spots analyzed by MALDI-MS, only 15.8% were identified. Most of the 175 identified proteins play a role in either defense (19.4%), carbohydrates (16.6%) and amino acid (14.9%) metabolisms, genes and proteins expression (13.1%), cytoskeleton (8%), cell wall biosynthesis (5.7%), secondary (5.1%) and primary (4%) metabolisms. A summary of the identified proteins, their putative functions, and behavior in different types of wood are presented. This information was introduced into the PROTICdb database and is accessible at http://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php. Finally, the average protein amount was compared with their respective transcript abundance as quantified through EST counting in a cDNA-library constructed with mRNA extracted from wood forming tissue.

Automated SNP detection in expressed sequence tags: statistical considerations and application to maritime pine sequences.

We developed an automated pipeline for the detection of single nucleotide polymorphisms (SNPs) in expressed sequence tag (EST) data sets, by combining three DNA sequence analysis programs: Phred, Phrap and PolyBayes. This application requires access to the individual electrophoregram traces. First, a reference set of 65 SNPs was obtained from the sequencing of 30 gametes in 13 maritime pine (Pinus pinaster Ait.) gene fragments (6671 bp), resulting in a frequency of 1 SNP every 102.6 bp. Second, parameters of the three programs were optimized in order to retrieve as many true SNPs, while keeping the rate of false positive as low as possible. Overall, the efficiency of detection of true SNPs was 83.1%. However, this rate varied largely as a function of the rare SNP allele frequency: down to 41% for rare SNP alleles (frequency < 10%), up to 98% for allele frequencies above 10%. Third, the detection method was applied to the 18498 assembled maritime pine (Pinus pinaster Ait.) ESTs, allowing to identify a total of 1400 candidate SNPs, in contigs containing between 4 and 20 sequence reads. These genetic resources, described for the first time in a forest tree species, were made available at http://www.pierroton.inra/genetics/Pinesnps. We also derived an analytical expression for the SNP detection probability as a function of the SNP allele frequency, the number of haploid genomes used to generate the EST sequence database, and the sample size of the contigs considered for SNP detection. The frequency of the SNP allele was shown to be the main factor influencing the probability of SNP detection.

Identification and characterization of water-stress-responsive genes in hydroponically grown maritime pine (Pinus pinaster) seedlings.

Growth, development and productivity of long-lived organisms such as forest trees are continuously challenged by abiotic stresses, and may also be greatly affected by predicted climatic change. As a first step toward creating stress-resistant maritime pine (Pinus pinaster Ait.) varieties by marker-assisted breeding, we describe the identification and characterization of water-stress-responsive genes in hydroponically grown seedlings that were well watered (-0.08 MPa) or subjected to water deprivation (-0.45 MPa) by the addition of polyethylene glycol. The cDNA amplified fragment-length polymorphism (cDNA-AFLP) technique was used to identify genes regulated by water deprivation. Approximately 4000 transcript-derived fragments (TDFs) were screened, of which 28 increased and 20 decreased in seedlings subjected to water deprivation. Of these 48 TDFs, 62.6% corresponded to proteins of known function, which indicate the main mechanisms involved in the osmotic stress response (photosynthesis, carbohydrate metabolism, cell wall synthesis and plant defense). We found that 16.6% of the 48 TDFs were similar to Arabidopsis thaliana (L.) Heynh gene products, 10.4% were similar to Pinus taeda L. expressed sequence tags (ESTs) and 10.4% did not match any sequences in the public databases. The relative abundance of these transcripts was quantitatively analyzed by reverse northern of both needle and root tissues, confirming the effectiveness of the cDNA-AFLP technique in detecting differentially expressed genes. The identification and characterization of water-stress-responsive genes provide new insights into the nature of the machinery involved in the response to water deprivation in a forest tree.