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.

Ventilator modes and settings during non-invasive ventilation: effects on respiratory events and implications for their identification.

Compared with invasive ventilation, non-invasive ventilation (NIV) has two unique characteristics: the non-hermetic nature of the system and the fact that the ventilator-lung assembly cannot be considered as a single-compartment model because of the presence of variable resistance represented by the upper airway. When NIV is initiated, the ventilator settings are determined empirically based on a clinical evaluation and diurnal blood gas variations. However, NIV is predominantly applied during sleep. Consequently, to assess overnight patient-machine 'agreement' and efficacy of ventilation, more specific and sophisticated monitoring is needed. The effectiveness of NIV might therefore be more correctly assessed by sleep studies than by daytime assessment. The most available and simple monitoring can be done from flow and pressure curves from the mask or the ventilator circuit. Examination of these tracings can give useful information to evaluate if the settings chosen by the operator were the right ones for that patient. However, as NIV allows a large range of ventilatory parameters and settings, it is mandatory to have information about this to better understand patient-ventilator interaction. Ventilatory modality, mode of triggering, pressurisation slope, use or not of positive end expiratory pressure and type of exhalation as well as ventilator performances may all have physiological consequences. Leaks and upper airway resistance variations may, in turn, modify these patterns. This article discusses the equipment available for NIV, analyses the effect of different ventilator modes and settings and of exhalation and connecting circuits on ventilatory traces and gives the background necessary to understand their impact on nocturnal monitoring of NIV.

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.

"Contrasting patterns of selection at Pinus pinaster Ait. Drought stress candidate genes as revealed by genetic differentiation analyses".

The importance of natural selection for shaping adaptive trait differentiation among natural populations of allogamous tree species has long been recognized. Determining the molecular basis of local adaptation remains largely unresolved, and the respective roles of selection and demography in shaping population structure are actively debated. Using a multilocus scan that aims to detect outliers from simulated neutral expectations, we analyzed patterns of nucleotide diversity and genetic differentiation at 11 polymorphic candidate genes for drought stress tolerance in phenotypically contrasted Pinus pinaster Ait. populations across its geographical range. We compared 3 coalescent-based methods: 2 frequentist-like, including 1 approach specifically developed for biallelic single nucleotide polymorphisms (SNPs) here and 1 Bayesian. Five genes showed outlier patterns that were robust across methods at the haplotype level for 2 of them. Two genes presented higher F(ST) values than expected (PR-AGP4 and erd3), suggesting that they could have been affected by the action of diversifying selection among populations. In contrast, 3 genes presented lower F(ST) values than expected (dhn-1, dhn2, and lp3-1), which could represent signatures of homogenizing selection among populations. A smaller proportion of outliers were detected at the SNP level suggesting the potential functional significance of particular combinations of sites in drought-response candidate genes. The Bayesian method appeared robust to low sample sizes, flexible to assumptions regarding migration rates, and powerful for detecting selection at the haplotype level, but the frequentist-like method adapted to SNPs was more efficient for the identification of outlier SNPs showing low differentiation. Population-specific effects estimated in the Bayesian method also revealed populations with lower immigration rates, which could have led to favorable situations for local adaptation. Outlier patterns are discussed in relation to the different genes' putative involvement in drought tolerance responses, from published results in transcriptomics and association mapping in P. pinaster and other related species. These genes clearly constitute relevant candidates for future association studies in P. pinaster.

Transcriptome analysis of bud burst in sessile oak (Quercus petraea).

Expression patterns of hundreds of transcripts in apical buds were monitored during bud flushing in sessile oak (Quercus petraea), in order to identify genes differentially expressed between the quiescent and active stage of bud development. Different transcriptomic techniques combining the construction of suppression subtractive hybridization (SSH) libraries and the monitoring of gene expression using macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were performed to dissect bud burst, with a special emphasis on the onset of the process. We generated 801 expressed sequence tags (ESTs) derived from six developmental stages of bud burst. Macroarray experiment revealed a total of 233 unique transcripts exhibiting differential expression during the process, and a putative function was assigned to 65% of them. Cell rescue/defense-, metabolism-, protein synthesis-, cell cycle- and transcription-related transcripts were among the most regulated genes. Macroarray and real-time RT-PCR showed that several genes exhibited contrasted expressions between quiescent and swelling buds, such as a putative homologue of the transcription factor DAG2 (Dof Affecting Germination 2), previously reported to be involved in the control of seed germination in Arabidopsis thaliana. These differentially expressed genes constitute relevant candidates for signaling pathway of bud burst in trees.

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.

Long-term ventilation in restrictive ventilatory disorders.

Long-term ventilation (LTV) had its beginning with restrictive ventilatory disorders. Today, LTV is predominately represented by noninvasive ventilation (NIV). Invasive ventilation was used for a long time but is now usually restricted for selected patients in which NIV fails.