dataesr/scientific-paragraphs-categorization huggingface dataset:
Discussion SNPs are considered to be the most efficient tools for comprehensive genetic studies  [32]. In Pyrus, the number of available SNPs was marginal. We developed more than 1,000 SNPs from the re-sequencing of P. communis cultivars and for the first time we included them in an array, making them easily available for further studies. These SNPs were selected based on their location within candidate genes, to ensure their usefulness for marker-trait association and for future breeding programmes. We used the apple and pear InfiniumH II 9K SNP array for the genotyping of five segregating pear populations, for a grand total of 873 individuals. The clustering of the SNPs using the GenomeStudio software depends on the minor allele frequency of the SNPs: the lower the minor allele frequency, the more samples are required to achieve accurate representation of all clusters.
Genomic Resources The pear SNPs detected by sequencing, the pear SNPs chosen for the apple and pear InfiniumH II 9K SNP array, and the GenomeStudio cluster file developed are deposited in the Genome Database for Rosaceae (www. rosaceae. org). SNPs are available in dbSNP (http://www. ncbi. nlm. nih. gov/projects/SNP/) under accessions ss527787751 to ss527789916.
Acknowledgments We thank  Dianne Hyndman  and  Rosemary Rickman  ( AgResearch Invermay, New Zealand ) and  Elisa Banchi  ( IASMA, Italy ) for providing the Illumina genotyping service. DC thanks the  Rosaceae genomics community  for kindly enabling the inclusion of pear SNPs in the apple and pear InfiniumH II 9K SNP array. We also thank the  INRA Experimental Unit (UE Horti, Angers, France)  for care of the T0036M population and the  INRA GENTYANE platform  ( UMR1095 ,  Clermont-Ferrand, France ) for SSR genotyping of this progeny.
SM is funded by the  FEM  PhD school. MS is funded by a  Massey University Doctoral Scholarship. MK is funded by the  New Zealand Ministry of Science and Innovation  grant '' Pipfruit: a juicy future '' (Contract number:  27744 ). The visiting scientist fellowship of YKK to PFR was funded by a  National Institute of Horticultural and Herbal Science grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
rubra DSM 19751 T [GenBank:KC253226] and Chromatocurvus halotolerans DSM 23344 T [GenBank: JX311416] have a sequence identity of 80. 7%, but an affiliation of both strains to the same genus would be in contradiction to phenotypic and 16S rRNA sequence data. Among all other photoheterotrophic representatives of this clade the pufLM sequence identity values are in the range between 69. 3 and 76. 6% and hence clearly below the genus level. For instance, the identity level of the pufLM genes of the two strains Ivo14 T and HTCC2080 is only 73. 6%, despite a close relationship at the 16S rRNA gene sequence level (96. 1%). The high divergence values of the pufLM genes could either indicate a rapid evolution of the photosynthetic apparatus alone or of the total genome. In order to determine representative levels of genome divergence, we have selected the housekeeping gene rpoB encoding the RNA polymerase β-subunit as an additional phylogenetic marker.
salexigens [GenBank:JX311417], H. mediterranea [GenBank:KC253225] and Chromatocurvus halotolerans [GenBank:JX311416] were determined upon retrieval by PCR amplification, while a complete rpoB gene sequence was extracted from the unpublished draft genome of H. rubra DSM 19751 T [GenBank:KC253224]. A comparison of the determined sequences with the available rpoB data set revealed that all identity values were below 85%, except between H. rubra and Chromatocurvus halotolerans, which share an rpoB gene sequence identity value of 86. 5%. This value is unusually high compared to an rpoB sequence identity value of 80. 1% between H. rubra and C. litoralis, which even share a higher 16S rRNA gene identity of 97. 0%. A phylogenetic diagram based on the partial rpoB nucleotide sequences (Figure  3B ) confirms with high bootstrap support the existence of two main lineages (NOR5-1 and NOR5-3) of BChl a-containing strains within the OM60/NOR5 clade.
Genome sequencing and phylogenetic analyses As part of the Moore Foundation Microbial Genome Sequencing Project  [64]  the genomes of Rap1red and Ivo14 T were shotgun sequenced by the J. Craig Venter Institute (JCVI). Two genomic libraries with insert sizes of 1 -4 kb and 10 -12 kb were made and sequenced from both ends to provide paired-end reads on ABI 3730xl DNA sequencers (Applied Biosystems, Foster City, CA) to approx. 8× coverage. The draft genomes of Rap1red (= NOR5-3) and Ivo14 T (= NOR5-1B T ) are deposited under GenBank accession numbers ACCX01000000 and ACCY01000000, respectively. A genome report compliant with the "Minimum Information about a Genome Sequence specification" is available from the Genomes Online Database  [65]. The genome sequences were all automatically annotated by JCVI.
These sequences were imported into the GenDB gene annotation system  [66, 67]  and the genes were further analyzed. Despite the automatic annotations, all the gene findings in this study were based on manual gene comparison rather than automatic annotation, since in several cases the automated annotation was incorrect. In order to determine whether a gene has homologs existing in other genomes, we used the genomic BLAST tool of the NCBI  [68]  with the tblastn (search translated nucleotide database using a protein query) algorithm for searching. The Genome-To-Genome Distance Calculator  [69]  was used for genome-based species delineation as described  [70]. This system calculates DNA-DNA similarity values by comparing the genomes to obtain highscoring segment pairs (HSPs) and inferring distances from a set of three formulas (1, HSP length/total length; 2, identities/HSP length; 3, identities/total length).
Spectroscopic DNA-DNA reassociation experiments were performed according to the protocol outlined by the DSMZ Identification Service  [62]. Phylogenetic trees based on 16S rRNA, pufLM and rpoB gene sequences were reconstructed using distance matrix (neighbor-joining) and parsimony programs included in the ARB package  [71]. Maximum likelihood trees were reconstructed with the program RAxML (version 7. 2. 8) using raxmlGUI  [72]  and the GTRGAMMA option with 1000 rounds of bootstrap replicates  [73]. The dataset of aligned and almost complete 16S rRNA gene sequences was based on the ARB SILVA database release 108 (September 2011)  [74] , whereas DNA sequences of pufL, pufM and rpoB genes were obtained from GenBank and aligned using the ClustalW algorithm implemented in the ARB package. The generated alignments of pufLM and rpoB nucleotide sequences in PHYLIP format are available as Additional file 2 and Additional file 3, respectively.
Acknowledgements We thank  Ivalyo Kostadinov  and  Alexandra Meziti  for taking of samples. We are grateful to the  Genome Analytics group (HZI Braunschweig)  for providing sequence data of DSM 19751 T and to  Anne Fiebig  ( DSMZ Braunschweig ) for help with the genome assembly. The assistance of  Andrey Yurkov  ( DSMZ Braunschweig ) in performing maximum likelihood analyses is gratefully acknowledged. The excellent technical assistance of  Jörg Wulf  ( MPI Bremen ),  Nicole Mrotzek ,  Gabriele Pötter  and  Bettina Sträubler  (all  DSMZ Braunschweig ) is acknowledged. We are grateful to  Dr. J. P. Euzéby  (http:// www. bacterio. net/) for correcting the etymology of the proposed Latin name of strain Ivo14 T and to  Dr. B. T. Tindall  ( DSMZ Braunschweig ) for helpful discussions. TR was supported by the  DFG Transregio-SFB 51 Roseobacter.
A hyphen indicates that the branch was not obtained with the respective reconstruction method. Nucleotide sequence accession numbers are given in parentheses. The affiliation of strains to subclades of the OM60/NOR5 group is based on [13]. The sequence of Alcanivorax borkumensis [GenBank:Y12579] was used as outgroup (not shown). Designations given in red color indicate that the respective strains produce BChl a and/or encode genes for a photosynthetic apparatus; names in blue indicate the presence of proteorhodopsin encoding genes. Strains that were tested with specific PCR primers for the presence of pufLM and soxB genes are labeled with red and yellow circles, respectively. Closed circles indicate a positive PCR reaction and open circles a negative reaction. The bar represents an estimated sequence divergence of 5%.
Strains and accession numbers: 1, Luminiphilus syltensis Ivo14 T [GenBank:ACCY01000000]; 2, marine gammaproteobacterium HTCC 2080 [GenBank:AAVV01000000]; 3, Congregibacter litoralis KT71 T [GenBank:AAOA01000000]; 4, Congregibacter sp. Rap1red [GenBank: ACCX01000000]; 5, gammaproteobacterium IMCC3088 [GenBank:AEIG01000000]; 6, marine gammaproteobacterium HTCC2143 [GenBank:NZ_AAVT00000000].
A total of 1,444 bases were identified. The sequence was compared with available sequences in GenBank using a BLAST search  [36]. The strain exhibited 96% nu-cleotide sequence similarities with Halopiger xanaduensis  [3]. These values were lower than the 98. 7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization  [37]. A phylogenetic tree (Figure  1 ) was constructed using the neighbor-joining method with the MEGA 5 program package  [38]  after multiple alignments of the data using MUSCLE  [39]. Evolutionary distances were calculated using the Tamura-Nei model  [40]. , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence).
These evidence codes are from the Gene Ontology project  [35]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. Standards in Genomic Sciences Sequences were aligned using MUSCLE, and phylogenetic inferences obtained using the MEGA software. Numbers at the nodes are from a bootstrap analysis done using 1,000 replicates to generate a majority consensus tree. Methanospirillum hungatei was used as outgroup. Phenotypic characterization was carried out according to the recommended minimal standards for the description of new taxa in the order Halobacteriales  [41]. Under optimal growth conditions on SG agar medium and after incubation for 15-20 days at 55°C, colonies were salmon pigmented, circular with a diameter of 1-2 mm.
Mass spectrometric measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2000 to 20,000 DA. The acceleration voltage was 20 kV. The time of acquisition was between 30 seconds and 1 minute per spot. Spectra were collected as a sum of 240 shots across a spot. Preprocessing and identification steps were performed using the manufacturer's parameters. The IIH3 T spectrum (Figure  4 ) was imported into the MALDI BioTyper software (version 2. 0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the spectra of Haloferax mediterranei, Natrinema gari, Natrinema pallidum, Haloterrigena thermotolerans, Haloterrigena. sp, Halogeometricum. sp, Haloarcua. sp and Halopiger. sp used as reference data in the BioTyper database (Figure  5 ).
A score enabled the identification, or not, from the tested species: a score > 2. 3 with a validly published species enabled the identification at the species level, a score > 1. 7 but < 2 enabled the identification at the genus level; and a score < 1. 7 did not enable any identification. For strain IIH3 T , none of the obtained scores was > 1, thus suggesting that our isolate was not a member of a known species. We added the spectrum from strain IIH3 T to our database for future reference. Figure  5  shows the MALDI-TOF MS spectrum differences between H. goleamassiliensis and other Archaea.
Genome project history The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the genus Halopiger, and as part of a study of archaeal diversity in hypersaline lakes of Algeria. It is the second genome of a Halopiger species and the first se-quenced genome of H. goleamassiliensis sp. nov. The EMBL accession number is CBMB010000001-CBMB010000011 and it consists of 3 scaffolds (HG315690-HG315692). A summary of the project information (PRJEB1780) and its association with MIGS version 2. 0 recommendations  [27]  is shown in Table  3.
Genome annotation Open Reading Frames (ORFs) were predicted using prodigal with default parameters  [43]. ORFs spanning a sequencing gap region were excluded. Assessment of protein function was obtained by comparing the predicted protein sequences with sequences in the GenBank  [44]  and the Clusters of Orthologous Groups (COG) databases using BLASTP. RNAmmer  [45]  and tRNAscan-SE 1. 21  [46]  were used for identifying the rRNAs and tRNAs, respectively. SignalP  [47]  and TMHMM  [48]  were used to predict signal peptides and transmembrane helices, respectively. For alignment lengths greater than 80 amino acids, ORFans were identified if their BLASTP E-value was lower than 1e-03. An E-value of 1e-05 was used if alignment lengths were smaller than 80 amino acids. DNA Plotter  [49]  was used for visualization of genomic features and Artemis  [50]  was used for data management.
The mean level of nucleotide sequence similarity was estimated at the genome level between H. goleamassiliensis and 5 other members of the Halobacteriaceae family (Table  6 ), by BLASTN comparison of orthologous ORFs in pairwise genomes. Orthologous proteins were detected using the Proteinortho software using the following parameters: e-value 1e-05, 30% identi-ty, 50% coverage and 50% of algebraic connectivity  [51].
Cells are susceptible to bacitracin, novobiocin, streptomycin, and sulfamethoxazole but resistant to ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, kanamycin, nalidixic acid, penicillin G, rifampicin, tetracycline, and vancomycin. The G+C content of the DNA is 66. 06%. The 16S rRNA and genome sequences are deposited in GenBank and EMBL under accession numbers KC430940 and CBMB010000001-CBMB0100000-11, respectively. The type strain IIH3 T (=CSUR P3036 = DSM 27562) was isolated from an evaporitic sediment in El Golea Lake, Algeria.
Influence of pH on the Biology of M. Oryzae The wild type Guy11 strain was grown on solid malt medium and then transferred to the same medium buffered to pH 5, 6, 7 or 8. The radial growth rates were measured and very similar results were obtained from pH 5 (0. 660. 1 cm/day) to pH 8 (0. 6360. 1 cm/day). These results were unchanged when yeast extract or potato extract replaced malt in the culture medium (data not shown). Following such growth at pH 5 or at pH 8 for 14 days, counting of the collected conidia revealed a 5-fold higher production at pH 8 than at pH 5 (Fig. 1a ). This was not due to a delay in conidiation at pH 5 since a similar pattern could be observed at 21 days. Germination of these conidia, conversely, decreased with pH, and this effect could be observed from pH 8 to pH 3 (Fig.
M. Oryzae Modulates the pH of its Environment The wild type Guy11 strain was grown in different liquid media and pH was monitored over time. When a complex medium containing yeast extract (TNK-YE) was used, a rise of about three pH units was observed in 96 hours (from pH 5. 6 to pH 8. 2) and a parallel accumulation of ammonia could be measured (Fig. 2a ). When rice or barley extracts were provided as nutrients, alkalinization was also observed as the pH of the medium increased from 6. 5 to 7. 5 in 72 hours (data not shown). Lastly, an increase in pH was also observed during conidia-derived infection of both barley and rice detached leaves; the pH rose by about two units and reached pH 7. 5 between 72 and 144 hours postinoculation (Fig. 2b ).
Conservation of the pH-signaling Pathway Genes in M. Oryzae and Gene Expression Analysis The A. nidulans PalA, PalB, PalC, PalF, PalH, PalI and PacC protein sequences were used to perform BlastP searches of the Broad Institute M. oryzae protein database (http://www. broad. mit. edu). All the homologs in M. oryzae were easily identified due to strong conservation of each of the protein sequence between the two fungi (Table  1 ). The PACC gene homolog, hereafter named MoPACC, clusters with other fungal PACC genes in coherence with the expected fungal phylogeny (Fig. S1 ). The genome-based predicted MoPACC open reading frame was confirmed by reverse transcription of MoPACC mRNA, amplification and cloning of the cDNA and sequence analysis (data not shown). Expression of MoPACC and all the MoPAL genes was then measured.
Using PCR, these results were confirmed by the lack of detection of the MoPACC gene in all T1-T7 strains, its detection in T11, the amplification of the integrated cassette's left and right junctions in T1-T7, and the absence of these fragments in T11 (Fig. 4ce ). Finally, amplification of the HPH gene was positive in all transformants except T11 (data not shown). Transformants T1, T2 and T3 were selected for the rest of the study together with strain C2 (ectopic integration of a HPH gene-only DNA cassette). Similar results were collected for the T1, T2 and T3 strains, and those obtained in the T2 strain are presented below. Figure  3. Expression of the M. oryzae pH-signaling related genes. Following 15 minutes (grey) or 2 hours (black) transfer of the wild type Guy11 strain from non-buffered liquid medium to fresh medium buffered to pH 5 or pH 8, total mRNA was extracted and quantitative PCR analysis was carried out using primers specific of the MoPACC and all the six MoPAL genes (Table  S1 ).
Impact of MoPACC Deletion on Fungal Growth, Alkalinization and Expression of the MoPAL Genes The parental, control (C2) and deletion (T2) strains were inoculated onto solid medium either buffered to pH 5 or to pH 8 and their radial growth was monitored over time. Growths of the parental and control strains were identical at pH 5 and pH 8 (data not shown). Growth of the deletion strain was identical to that of the parental strain at pH 5, but dropped by 50% at pH 8 (Fig. 5a ). We then used three buffer systems allowing pH overlaps to generate solid media ranging from pH 5. 5 to pH 7. 9, and we monitored growth rate. As shown in figure  5b , the growth rates of both the parental and control strains changed only slightly over the whole pH range tested. On the contrary, that of the deletion strain gradually and continuously decreased with increasing pH.
868%). Virulence was not delayed since identical results were observed after longer infection time (14 more days). The lesions looked identical on the leaves infected by The M. oryzae PAL and PACC genes are presented. Introns were searched using the Softberry software and both their number (bold) and positions (start-end) are given. The length of the corresponding predicted proteins and the identity of these proteins to their A. nidulans counterparts is provided. doi:10. 1371/journal. pone. 0069236. t001 the parent and mutant strains and, interestingly, conidia were similarly visible on the lesions caused by these two strains (data not shown). Lastly, when the rice Maratelli and CO-39 cultivars were used (more resistant towards M. oryzae), difference in virulence could no longer be observed between the parent and mutant strains.
Bioinformatics and Genes Studied Blast and protein domains searches were performed at the Broad Institute (M. oryzae database: http://www. broad. mit. edu), EBI (Interproscan: http://www. ebi. ac. uk/interproscan) and NCBI (Blast -nr: http://www. ncbi. nlm. nih. gov/blast/). Alignments were performed with ClustalW and displayed using Genedoc. Phylogenetic analyses were performed using Mega 3. 1. The resulting tree was drawn using MEGA version 3. 1. Numbers at each node indicates percentage of 1000 bootstrap replicates. The fungal species used in the study were Fusarium oxysporoum, F. verticillioides, F. graminearum, Trichoderma reesei, Neurospora crassa, Chaetonium globosum, Botrytis cinerea, Sclerotinia sclerotiorum, Phaeosphaeria nodorum, Aspergillus nidulans, A. fumigatus, A. niger, Yarrowia lipolytica, Candida albicans, Ashbya gossypii, Sacharomyces cerevisiae and Ustilago maydis.
This work was supported by the  CNRS , the  University Lyon 1 and Bayer CropScience. P. Landraud was supported by a grant from the  French ministry of Industry and Bayer CropScience. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Part of the experiments were funded by  Bayer CropScience. The authors are affiliated to UMR 5240 Microbiologie, Adaptation et Pathogenie; UniversiteLyon 1, CNRS.
Ethics This study was approved by the appropriate biomedical research ethics committee in February 2009 (Bichat-Claude Bernard Hospital, Paris, France; #IRB0006477; n° 08-071). No incentives to participation were offered. Oral informed consent was obtained from at least one of the parents or legal guardian; hereafter, the term "parents" is used for both. The study was registered on Clinicaltrial. gov (NCT00948779) on July 28, 2009. The education sessions were delivered to the parents accompanying the children.
Primary data analysis At least 136 patients were required in each group to detect a 15% difference in the proportions of satisfied parents between the two groups (80% versus 65%) with 80% power and a twotailed α value of 0. 05. The value in the control group was estimated from the literature and the value in the intervention group was the smallest improvement deemed relevant  [18]. We did not compute the sample size needed to detect a significant difference regarding attitudes. We expected to be able to contact 90% of the families and we consequently planned to include 150 patients in each arm. Statistical analyses were performed using SAS ® (v9. 12, SAS Institute Inc. , Cary, NC, USA). Qualitative variables were described as frequencies (percentages) and quantitative variables as means (SD) when distribution was normal and as medians (quartiles 1 and 3) otherwise.
Acknowledgements We thank  Dr Guillaume Hebert ,  Dr Zinedine Haouari ,  Dr Anne-Sophie Lelong , all the pharmacists, and all the physicians at the pediatric emergency department of the  Robert Debre Teaching Hospital  for their contribution to the study. We also thank  Adyla Yacoubi  and  Damir Mohamed  of the clinical epidemiology unit,  Robert Debre Teaching Hospital  and  Yannick Vacher  from the  "Délégation Interrégionale à la Recherche Clinique d'Ile de France". We are indebted to the children and their parents for participating in the study.
Molecular Data Standard and touchdown polymerase chain reaction (PCR) protocols and fungal-specific primer sets (Table  S2 ) were used to amplify and sequence four gene regions: the internal transcribed spacer ribosomal RNA gene (ITS), the 28S large subunit ribosomal RNA gene (LSU), elongation factor (EF1a), and the second largest subunit of RNA polymerase II (RPB2). EF1a and RPB2 could not be amplified for many Tuber species. To address this problem we designed a new set primers with enhanced specificity for the Tuberaceae: EF1a Tuber_f (59 AGC GTG AGC GTG GTA TCA C 39 -forward), EF1a Tuber_r (59 GAG ACG TTC TTG ACG TTG AAG 39 -reverse), and RPB2 Tuber_f (59 Y AAY CTG ACY TTR GCY GTY AA 39) paired with the reverse primer RPB2_Tuber_r (59 CR GTT TCC TGY TCA ATC TCA-39). Sequences produced for this study have been deposited in GenBank under the accession numbers JQ925626-Figure  1.