Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. See also the Perspective by Massana
ISSN: 0962-1083

hal-01545321
https://hal.archives-ouvertes.fr/hal-01545321
DOI : 10.1111/mec.13160

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly , Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deino-coccus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.
ISSN: 2045-8827

Droits : http://creativecommons.org/licenses/by/
hal-01560945
https://hal-amu.archives-ouvertes.fr/hal-01560945
https://hal-amu.archives-ouvertes.fr/hal-01560945/document
https://hal-amu.archives-ouvertes.fr/hal-01560945/file/Theodorakopoulos-2013.pdf
DOI : 10.1002/mbo3.119

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : In order to analyze the large-scale structure of the genome of Norway spruce (Picea abies Karst.), a pseudo-testcross genetic linkage map was built using markers of six different types, belonging to the low (amplified fragment length polymorphisms, simple sequence repeats) or high (sequence-specific amplified polymorphisms, inter-retrotransposon amplified polymorphisms) copy-number fraction of the genome, and including expressed region-derived markers (expressed sequence tag polymorphisms). Twenty seven and 23 linkage groups of at least four markers were obtained for the female and the male parent maps, respectively. A subset of these linkage groups coalesced into 13 bi-parental linkage groups through markers shared between the two maps. This map was used to investigate the frequency of each marker type over chromosomes and the distribution of marker types relative to each other, using autocorrelation techniques. Our results show that, while the composition of chromosomes is homogeneous, low- and high-copy-number markers tend to occupy separate regions of the linkage groups, and that expressed sequences are preferentially associated with microsatellites and separated from retrotransposons. These results indicate that the spatial structure of Norway spruce chromosomes is not homogeneous
ISSN: 1614-2942

hal-01032033
https://hal.archives-ouvertes.fr/hal-01032033
DOI : 10.1007/s11295-005-0012-2

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : International audience
Microsatellites are widely used in population genetics to uncover recent evolutionary events. They are typically genotyped using capillary sequencer, which capacity is usually limited to 9, at most 12 loci for each run, and which analysis is a tedious task that is performed by hand. With the rise of next-generation sequencing (NGS), a much larger number of loci and individuals are available from sequencing: for example, on a single run of a GS Junior, 28 loci from 96 individuals are sequenced with a 30X cover. We have developed an algorithm to automatically and efficiently genotype microsatellites from a collection of reads sorted by individual (e.g. specific PCR amplifications of a locus or a collection of reads that encompass a locus of interest). As the sequencing and the PCR amplification introduce artefactual insertions or deletions, the set of reads from a single microsatellite allele shows several length variants. The algorithm infers, without alignment, the true unknown allele(s) of each individual from the observed distributions of microsatellites length of all individuals. MicNeSs, a python implementation of the algorithm, can be used to genotype any microsatellite locus from any organism and has been tested on 454 pyrosequencing data of several loci from fruit flies (a model species) and red deers (a nonmodel species). Without any parallelization, it automatically genotypes 22 loci from 441 individuals in 11 hours on a standard computer. The comparison of MicNeSs inferences to the standard method shows an excellent agreement, with some differences illustrating the pros and cons of both methods.
ISSN: 1755-098X

hal-01544754
https://hal.archives-ouvertes.fr/hal-01544754
DOI : 10.1111/1755-0998.12467

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699
Spontaneous gene flow between wild and cultivated chicory, Cichorium intybus L., may have implications for the genetic structure and evolution of populations and varieties. One aspect of this crop-wild gene flow is the dispersal of transgenes from genetically modified varieties, e.g. gene flow from GM chicory to natural chicory could have unwanted consequences. With the purpose to identify and quantify crop-wild gene flow in chicory, we analysed introgression in 19 wild chicory populations and 16 accessions of chicory varieties and landraces distributed across Northern, Central and Mediterranean Europe. The analysis used 281 AFLP markers and 75 SSAP markers giving a total of 356 polymorphic markers. Results from model based assignments with the program STRUCTURE indicated many incidents of recent gene flow. Gene flow was observed both between cultivars and wild populations, between landraces and wild populations, between different wild populations as well as between cultivars. Population structure visualized by distance-based clustering showed a North-South geographical structuring of the wild populations, and a general grouping of the cultivars corresponding to known origin. The results indicated, however, that the structuring between the two groups of wild and cultivated types was weak. As crop and wild recipients are genetically close and genes are transferred between the two types rather frequently, focus on mitigating crop-wild gene flow should be increased, before transgenic varieties are cultivated openly.
ISSN: 0925-9864

hal-01032184
https://hal.archives-ouvertes.fr/hal-01032184
DOI : 10.1007/s10722-008-9375-1

Éditeur(s) : HAL CCSD Lippincott, Williams & Wilkins
Résumé : International audience
Purpose of review Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Recent findings Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Summary Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.
ISSN: 0951-7375

hal-01390970
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390970
DOI : 10.1097/QCO.0000000000000275
PUBMED : 27191201

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
Background: The Ras/Raf/MEK/ERK signaling pathway is involved in essential cell processes and it is abnormally activated in similar to 30 % of cancers and cognitive disorders. Two ERK isoforms have been described, ERK1 and ERK2; ERK2 being regarded by many as essential due to the embryonic lethality of ERK2 knock-out mice, whereas mice lacking ERK1 are viable and fertile. The controversial question of why we have two ERKs and whether they have differential functions or display functional redundancy has not yet been resolved. Results: To investigate this question we used a novel approach based on comparing the evolution of ERK isoforms' sequences and protein expression across vertebrates. We gathered and cloned erk1 and erk2 coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. For the first time, we measured each isoforms' relative protein level in phylogenetically distant animals using anti-phospho antibodies targeting active ERKs. We demonstrate that squamates (lizards, snakes and geckos), despite having both genes, do not express ERK2 protein whereas other tetrapods either do not express ERK1 protein or have lost the erk1 gene. To demonstrate the unexpected squamates' lack of ERK2 expression, we targeted each ERK isoform in lizard primary fibroblasts by specific siRNA-mediated knockdown. We also found that undetectable expression of ERK2 in lizard is compensated by a greater strength of lizard's erk1 promoter. Finally, phylogenetic analysis revealed that ERK1 amino acids sequences evolve faster than ERK2' s likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant. Conclusions: ERK isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least 400 Mya. Our results demonstrate that tetrapods can live by expressing either one or both ERK isoforms, supporting the notion that ERK1/2 act interchangeably. Substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate ERKs further suggesting functional redundancy. We suggest that future ERK research should shift towards understanding the role and regulation of total ERK quantity, especially in light of newly described erk2 gene amplification identified in tumors.
ISSN: 1471-2148

hal-01546165
https://hal.archives-ouvertes.fr/hal-01546165
DOI : 10.1186/s12862-015-0450-x

Éditeur(s) : HAL CCSD
Résumé : International audience
Clear cell renal cell carcinoma (ccRCC) has a poor prognosis with a 50% risk of metastases. Little is known about the phenotypic and molecular profiles of metastases regarding their corresponding primary tumors. This study aimed to screen phenotypic and genotypic differences between metastases and their corresponding primary tumors. We selected four cases with available frozen material. The histological, immunohistochemical (VEGFA, CD31, SMA, Ki67, p53, PAR-3), FISH (VHL gene), next-generation sequencing (VHL and c-MET genes), multiplex ligation-dependent probe amplification, and array-(comparative genomic hybridization) CGH analyses were realized. Metastases were nodal, hepatic (synchronous), adrenal, and pulmonary (metachronous). High-grade tumor cells were significantly more frequent in metastases (p = 0.019). Metastases and high-grade zones of primary tumors shared similar characteristics compared to low-grade zones: a lower microscopic vascular density (43.5 vs 382.5 vessels/mm(2); p = 0.0027), a higher expression of VEGF (73 vs 10%, p = 0.045), Ki67 (37.6 vs 8.3%; p = 0.011), and p53 (54 vs 10.6%; p = 0.081), and a cytoplasmic and membranous PAR-3 staining. Metastases exhibited more chromosomal imbalances than primary tumors in total (18.75 ± 6.8; p = 0.044) with more genomic gains (13.5 ± 7; p = 0.013). The loss of chromosome 9 and gain of Xq were found in both primary tumors and metastases but gains of loci or chromosomes 2p, 3q, 5, 8q, 12, and 20 were only found in metastases. The VHL gene status was similar in each tumor couple. Although metastases and primary tumors share common histological features, this study highlights chromosomal differences specific to metastases which could be involved in ccRCC metastatic evolution.
Virchows Archiv

hal-01560281
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560281
DOI : 10.1007/s00428-017-2124-0
PUBMED : 28488172

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
The etiology of congenital heart defect (CHD) combines environmental and genetic factors. So far, there were studies reporting on the screening of a single gene on unselected CHD or on familial cases selected for specific CHD types. Our goal was to systematically screen a proband of familial cases of CHD on a set of genetic tests to evaluate the prevalence of disease-causing variant identification. A systematic screening of GATA4, NKX2-5, ZIC3 and Multiplex ligation-dependent probe amplification (MLPA) P311 Kit was setup on the proband of 154 families with at least two cases of non-syndromic CHD. Additionally, ELN screening was performed on families with supravalvular arterial stenosis. Twenty-two variants were found, but segregation analysis confirmed unambiguously the causality of 16 variants: GATA4 (1 x), NKX2-5 (6 x), ZIC3 (3 x), MLPA (2 x) and ELN (4 x). Therefore, this approach was able to identify the causal variant in 10.4% of familial CHD cases. This study demonstrated the existence of a de novo variant even in familial CHD cases and the impact of CHD variants on adult cardiac condition even in the absence of CHD. This study showed that the systematic screening of genetic factors is useful in familial CHD cases with up to 10.4% elucidated cases. When successful, it drastically improved genetic counseling by discovering unaffected variant carriers who are at risk of transmitting their variant and are also exposed to develop cardiac complications during adulthood thus prompting long-term cardiac follow-up. This study provides an important baseline at dawning of the next-generation sequencing era.
ISSN: 1018-4813

hal-01404211
https://hal-univ-bourgogne.archives-ouvertes.fr/hal-01404211
DOI : 10.1038/ejhg.2015.105
PUBMED : 26014430

Éditeur(s) : HAL CCSD Nakanishi Printing Co
Résumé : International audience
In Vibrio cholerae, the etiological agent of cholera, most of the virulence genes are located in two pathogenicity islands, named TCP (Toxin-Co-regulated Pilus) and CTX (Cholera ToXins). For each V. cholerae pathogenicity gene, we retrieved every primer published since 1990 and every known allele in order to perform a complete in silico survey and assess the quality of the PCR primers used for amplification of these genes. Primers with a melting temperature in the range 55-60 degrees C against any target sequence were considered valid. Our survey clearly revealed that two thirds of the published primers are not able to properly detect every genetic variant of the target genes. Moreover, the quality of primers did not improve with time. Their lifetime, i.e. the number of times they were cited in the literature, is also not a factor allowing the selection of valid primers. We were able to improve some primers or design new primers for the few cases where no valid primer was found. In conclusion, many published primers should be avoided or improved for use in molecular detection tests, in order to improve and perfect specificity and coverage. This study suggests that bioinformatic analyses are important to validate the choice of primers.
ISSN: 1342-6311

hal-01546178
https://hal.archives-ouvertes.fr/hal-01546178
DOI : 10.1264/jsme2.ME11317