Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms.

The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.

Current trends in microsatellite genotyping.

Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost-effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next-generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non-model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well-established strategy including standardized high-throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

Plasticity of maritime pine (Pinus pinaster) wood-forming tissues during a growing season.

The seasonal effect is the most significant external source of variation affecting vascular cambial activity and the development of newly divided cells, and hence wood properties. Here, the effect of edapho-climatic conditions on the phenotypic and molecular plasticity of differentiating secondary xylem during a growing season was investigated. Wood-forming tissues of maritime pine (Pinus pinaster) were collected from the beginning to the end of the growing season in 2003. Data from examination of fibre morphology, Fourier-transform infrared spectroscopy (FTIR), analytical pyrolysis, and gas chromatography/mass spectrometry (GC/MS) were combined to characterize the samples. Strong variation was observed in response to changes in edapho-climatic conditions. A genomic approach was used to identify genes differentially expressed during this growing season. Out of 3512 studied genes, 19% showed a significant seasonal effect. These genes were clustered into five distinct groups, the largest two representing genes over-expressed in the early- or late-wood-forming tissues, respectively. The other three clusters were characterized by responses to specific edapho-climatic conditions. This work provides new insights into the plasticity of the molecular machinery involved in wood formation, and reveals candidate genes potentially responsible for the phenotypic differences found between early- and late-wood.

Cross-species transferability and mapping of genomic and cDNA SSRs in pines.

Two unigene datasets of Pinus taeda and Pinus pinaster were screened to detect di-, tri- and tetranucleotide repeated motifs using the SSRIT script. A total of 419 simple sequence repeats (SSRs) were identified, from which only 12.8% overlapped between the two sets. The position of the SSRs within their coding sequences were predicted using FrameD. Trinucleotides appeared to be the most abundant repeated motif (63 and 51% in P. taeda and P. pinaster, respectively) and tended to be found within translated regions (76% in both species), whereas dinucleotide repeats were preferentially found within the 5'- and 3'-untranslated regions (75 and 65%, respectively). Fifty-three primer pairs amplifying a single PCR fragment in the source species (mainly P. taeda), were tested for amplification in six other pine species. The amplification rate with other pine species was high and corresponded with the phylogenetic distance between species, varying from 64.6% in P. canariensis to 94.2% in P. radiata. Genomic SSRs were found to be less transferable; 58 of the 107 primer pairs (i.e. 54%) derived from P. radiata amplified a single fragment in P. pinaster. Nine cDNA-SSRs were located to their chromosomes in two P. pinaster linkage maps. The level of polymorphism of these cDNA-SSRs was compared to that of previously and newly developed genomic-SSRs. Overall, genomic SSRs tend to perform better in terms of heterozygosity and number of alleles. This study suggests that useful SSR markers can be developed from pine ESTs.

Correction of selenium deficiency in hemodialyzed patients.

Selenium is an essential trace element important for glutathione peroxidase activity. Selenium deficiency has been found in association with skeletal and cardiac myopathy and may increase the risk for cardiovascular diseases and for cancer. We studied 39 hemodialysis patients and 15 control subjects. Plasma selenium, plasma glutathione peroxidase activity and erythrocyte glutathione peroxidase activity were lower than in controls (38 +/- 14 vs. 88 +/- 17 micrograms/liter (P less than 0.01); 153 +/- 32 vs. 334 +/- 41 IU/liter (P less than 0.01), 19 +/- 4 vs. 26 +/- 4 IU/g Hb (P less than 0.01), respectively). Plasma selenium and plasma glutathione peroxidase activity were strongly correlated with duration of dialysis. There was no correlation between plasma selenium and protein or calorie intakes. Plasma selenium was lower in patients dialyzed with highly permeable membranes (P less than 0.01). The total muscle mass, assessed by anthropometry, was lower in the patients who had the lowest plasma selenium (P less than 0.01) and plasma glutathione peroxidase activity (P less than 0.05). Interventricular septum hypertrophy, documented by echocardiography, was greater in patients with the lowest plasma selenium and plasma glutathione peroxidase activity (P less than 0.01). Twenty hemodialysis patients had oral supplementation of 500 micrograms/day of sodium selenite for three months, and then, 200 micrograms/day for the next three months. Plasma selenium increased as early as the first week and reached a plateau similar to the control levels after three weeks. Plasma glutathione peroxidase activity increased after two months but remained below controls. Erythrocyte glutathione peroxidase activity reached a higher value than controls after one month.(ABSTRACT TRUNCATED AT 250 WORDS)