Search

22 Pags.- 3 Figs.- 4 Tabls. © 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License., Natural populations are characterized by abundant genetic diversity driven by a range of different types of mutation. The tractability of sequencing complete genomes has allowed new insights into the variable composition of genomes, summarized as a species pan-genome. These analyses demonstrate that many genes are absent from the first reference genomes, whose analysis dominated the initial years of the genomic era. Our field now turns towards understanding the functional consequence of these highly variable genomes. Here, we analysed weighted gene coexpression networks from leaf transcriptome data for drought response in the purple false brome Brachypodium distachyon and the differential expression of genes putatively involved in adaptation to this stressor. We specifically asked whether genes with variable “occupancy” in the pan-genome – genes which are either present in all studied genotypes or missing in some genotypes – show different distributions among coexpression modules. Coexpression analysis united genes expressed in drought-stressed plants into nine modules covering 72 hub genes (87 hub isoforms), and genes expressed under controlled water conditions into 13 modules, covering 190 hub genes (251 hub isoforms). We find that low occupancy pan-genes are under-represented among several modules, while other modules are over-enriched for low-occupancy pan-genes. We also provide new insight into the regulation of drought response in B. distachyon, specifically identifying one module with an apparent role in primary metabolism that is strongly responsive to drought. Our work shows the power of integrating pan-genomic analysis with transcriptomic data using factorial experiments to understand the functional genomics of environmental response., This work was supported by the USDA (NIFA-2011-67012-30663) to D.L.D., NSF (IOS-0922457) to T.E.J., the Spanish Ministry of Science and Innovation CGL2016-79790-P and PID2019-108195GB-I00, University of Zaragoza UZ2016_TEC02 grant projects to P.C., and the Joint Genome Institute FP00006675 contract to P.C. and FP00006746 to D.L.D. R.S. was funded by a Spanish Ministry of Economy and Competitiveness (Mineco) FPI PhD fellowship, Mineco and Ibercaja-CAI mobility grants and Instituto de Estudios Altoaragoneses grant. B.C.M. was funded by Fundación ARAID. P.C. and R.S. were partially funded by a European Social Fund/Spanish Aragón Government Bioflora grant.

Gramene (http://www.gramene.org), a knowledgebase founded on comparative functional analyses of genomic and pathway data for model plants and major crops, supports agricultural researchers worldwide. The resource is committed to open access and reproducible science based on the FAIR data principles. Since the last NAR update, we made nine releases; doubled the genome portal's content; expanded curated genes, pathways and expression sets; and implemented the Domain Informational Vocabulary Extraction (DIVE) algorithm for extracting gene function information from publications. The current release, #63 (October 2020), hosts 93 reference genomes—over 3.9 million genes in 122 947 families with orthologous and paralogous classifications. Plant Reactome portrays pathway networks using a combination of manual biocuration in rice (320 reference pathways) and orthology-based projections to 106 species. The Reactome platform facilitates comparison between reference and projected pathways, gene expression analyses and overlays of gene–gene interactions. Gramene integrates ontology-based protein structure–function annotation; information on genetic, epigenetic, expression, and phenotypic diversity; and gene functional annotations extracted from plant-focused journals using DIVE. We train plant researchers in biocuration of genes and pathways; host curated maize gene structures as tracks in the maize genome browser; and integrate curated rice genes and pathways in the Plant Reactome.

7 Pags.- 2 Figs. © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), RSAT (Regulatory Sequence Analysis Tools) enables the detection and the analysis of cis-regulatory elements in genomic sequences. This software suite performs (i) de novo motif discovery (including from genome-wide datasets like ChIP-seq/ATAC-seq) (ii) genomic sequences scanning with known motifs, (iii) motif analysis (quality assessment, comparisons and clustering), (iv) analysis of regulatory variations and (v) comparative genomics. RSAT comprises 50 tools. Six public Web servers (including a teaching server) are offered to meet the needs of different biological communities. RSAT philosophy and originality are: (i) a multi-modal access depending on the user needs, through web forms, command-line for local installation and programmatic web services, (ii) a support for virtually any genome (animals, bacteria, plants, totalizing over 10 000 genomes directly accessible). Since the 2018 NAR Web Software Issue, we have developed a large REST API, extended the support for additional genomes and external motif collections, enhanced some tools and Web forms, and developed a novel tool that builds or refine gene regulatory networks using motif scanning (network-interactions). The RSAT website provides extensive documentation, tutorials and published protocols. RSAT code is under open-source license and now hosted in GitHub. RSAT is available at http://www.rsat.eu/., Institut Universitaire de France (to M.T.C., N.T.T.N.); ANR [ANR-14-CE11-0006-01 to M.T.C., N.T.T.N.]; ITMO Cancer [20CM114-00 to D.T., W.S.G.]; CONACYT [11311 to A.M.R., M.P.G.]; Universidad Nacional Autónoma de México PAPIIT (UNAM) [IA203021]; Spanish State Research Agency, EUROPEAN REGIONAL DEVELOPMENT FUND (FEDER) [AGL2017-83358-R to N.K.; AEI/FEDER, UE]; Gobierno de Aragón [A08_17R, A09_20R, Phd Contract to N.K.]; PRIMA [PCI2019-103526 to B.C.M.; Programación Conjunta Internacional, Programa Estatal de l+D+i Orientada a los Retos de la Sociedad]; Erasmus+ (to N.K.); Norwegian Research Council [288404 to J.A.C.M.]; Institut Français de Bioinformatique (IFB) [ANR-11-INBS-0013]; SEP-CONACYT-ECOS-ANUIES [291235 to A.M.R., M.T.C. and D.T.]. Funding for open access charge: Institut Universitaire de France.

Light spectra influence barley development, causing a diverse range of responses among cultivars that are poorly understood. Here, we exposed three barley genotypes with different light sensitivities to two light sources: fluorescent bulbs, over-representing green and red wavebands, and metal halide lamps, with a more balanced spectrum. We used RNA sequencing to identify the main genes and pathways involved in the different responses, and RT-qPCR to validate the expression values. Different grades of sensitivity to light spectra were associated with transcriptional reprogramming, plastid signals, and photosynthesis. The genotypes were especially divergent in the expression of genes regulated by transcription factors from MADS-box, WRKY, and NAC families, and in specific photoreceptors such as phytochromes and cryptochromes. Variations in light spectra also affected the expression of circadian clock, flowering time, and frost tolerance genes, among others, resembling plant responses to temperature. The relation between PPD-H1, HvVRN1, and HvFT1 expression might explain genotypic differences. Light-sensitive genotypes experienced a partial reversion of the vernalization process and senescence-related stress under the less favorable light quality conditions. The observed light-quality sensitivities reveal a complex mechanism of adaptation to regions with specific light quality features and/or possible regulation of light spectra in plant development during early spring.HighlightDevelopment genes were affected by light quality in the barley varieties tested. Different grades of sensitivity were related to the expression of transcription factors, senescence, light signaling and cold-regulated genes.

24 Pags.- 6 Figs.- 1 Tabl. © 2021 The Authors., Unraveling the evolution of plant polyploids is a challenge when their diploid progenitor species are extinct or unknown or when genome sequences of known progenitors are unavailable. Existing subgenome identification methods cannot adequately infer the homeologous genomes that are present in the allopolyploids if they do not take into account the potential existence of unknown progenitors. We addressed this challenge in the widely distributed dysploid grass genus Brachypodium, which is a model genus for temperate cereals and biofuel grasses. We used a transcriptome-based phylogeny and newly designed subgenome detection algorithms coupled with a comparative chromosome barcoding analysis. Our phylogenomic subgenome detection pipeline was validated in Triticum allopolyploids, which have known progenitor genomes, and then used to infer the identities of three subgenomes derived from extant diploid species and four subgenomes derived from unknown diploid progenitors (ghost subgenomes) in six Brachypodium polyploids (B. mexicanum, B. boissieri, B. retusum, B. phoenicoides, B. rupestre and B. hybridum), of which five contain undescribed homeologous subgenomes. The existence of the seven Brachypodium progenitor genomes in the polyploids was confirmed by their karyotypic barcode profiles. Comparative phylogenomics of nuclear versus plastid trees allowed us to formulate hypothetical homoploid hybridizations and allo- and autopolyploidization scenarios that could have generated the six Brachypodium polyploids., This work was supported by the Spanish Ministries of Economy and Competitivity (Mineco) and Science and Innovation(MICINN) CGL2016-79790-P and PID2019-108195GB-I00 and University of Zaragoza UZ2016_TEC02 grant projects and funding fromHarvard University. The work conducted by the US DOE Joint Genome Institute was supported by the Office of Science of the US Department of Energy (DOE) under Contracts no. DE-AC02-05CH11231 and FP00006675. RS was funded by a Mineco FPI PhDfellowship, Mineco and Ibercaja-CAI Mobility Grants and Instituto deEstudios Altoaragoneses Grant. BCM was funded by FundacionARAID. PC and RS were also funded by a European Social Fund/Ara-gon Government Bioflora Research Grants A01-17R and A01-20R.The work that was conducted at the University of Silesia in Katowicewas supported by the Research Excellence Initiative program.

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of interfaces to genomic data across the tree of life, including reference genome sequence, gene models, transcriptional data, genetic variation and comparative analysis. Data may be accessed via our website, online tools platform and programmatic interfaces, with updates made four times per year (in synchrony with Ensembl). Here, we provide an overview of Ensembl Genomes, with a focus on recent developments. These include the continued growth, more robust and reproducible sets of orthologues and paralogues, and enriched views of gene expression and gene function in plants. Finally, we report on our continued deeper integration with the Ensembl project, which forms a key part of our future strategy for dealing with the increasing quantity of available genome-scale data across the tree of life.

17 Pags.- 3 Tabls.- 7 Figs. © Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence., The identification of functional elements encoded in plant genomes is necessary to understand gene regulation. Although much attention has been paid to model species like Arabidopsis (Arabidopsis thaliana), little is known about regulatory motifs in other plants. Here, we describe a bottom-up approach for de novo motif discovery using peach (Prunus persica) as an example. These predictions require pre-computed gene clusters grouped by their expression similarity. After optimizing the boundaries of proximal promoter regions, two motif discovery algorithms from RSAT::Plants (http://plants.rsat.eu) were tested (oligo and dyad analysis). Overall, 18 out of 45 co-expressed modules were enriched in motifs typical of well-known transcription factor (TF) families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, and WRKY) and a few uncharacterized motifs. Our results indicate that small modules and promoter window of [–500 bp, +200 bp] relative to the transcription start site (TSS) maximize the number of motifs found and reduce low-complexity signals in peach. The distribution of discovered regulatory sites was unbalanced, as they accumulated around the TSS. This approach was benchmarked by testing two different expression-based clustering algorithms (network-based and hierarchical) and, as control, genes grouped for harboring ChIPseq peaks of the same Arabidopsis TF. The method was also verified on maize (Zea mays), a species with a large genome. In summary, this article presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to other species. A Docker software container is released to facilitate the reproduction of these analyses., This work was partly funded by the Spanish Ministry of Economy and Competitiveness grants AGL2014-52063R, AGL2017-83358-R (MCIU/AEI/FEDER/UE); and the Government of Aragón with grants A44, A08_17R and A09_17R, which were co-financed with FEDER funds. N.K. was funded by project AGL2014-52063R, two months of Erasmus plus traineeship at Aix-Marseille University (AMU; Lab.Technological Advances for Genomics and Clinics (TAGC)) and at present a PhD contract awarded by the Government of Aragón.

The COVID-19 pandemic has seen unprecedented use of SARS-CoV-2 genome sequencing for epidemiological tracking and identification of emerging variants. Understanding the potential impact of these variants on the infectivity of the virus and the efficacy of emerging therapeutics and vaccines has become a cornerstone of the fight against the disease. To support the maximal use of genomic information for SARS-CoV-2 research, we launched the Ensembl COVID-19 browser, incorporating a new Ensembl gene set, multiple variant sets (including novel variation calls), and annotation from several relevant resources integrated into the reference SARS-CoV-2 assembly. This work included key adaptations of existing Ensembl genome annotation methods to model ribosomal slippage, stringent filters to elucidate the highest confidence variants and utilisation of our comparative genomics pipelines on viruses for the first time. Since May 2020, the content has been regularly updated and tools such as the Ensembl Variant Effect Predictor have been integrated. The Ensembl COVID-19 browser is freely available at https://covid-19.ensembl.org.

16 Pags.- 8 Figs. This article is licensed under a Creative Commons Attribution 4.0 International License., Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution., The work conducted by the US DOE Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract no. DE-AC02-05CH11231 and User Agreement No. FP00006675 between the University of Zaragoza (User) and the DOE (Community Science Program 503504 proposal); S.J. was supported by the DOE Office of Science Graduate Student Research Program; P.C., B.C.-M., A.D.P., and R.S. were supported by a Spanish Ministry of Economy and Competitiveness grant CGL2016-79790-P; P.C., A.D.P., and R.S. were supported by a Bioflora A01-17 grant co-funded by the Spanish Aragon Government and the European Social Fund. R.H., E.W., and J.L. were supported by the National Science Centre Poland (grants DEC-2012/04/A/NZ3/00572 and DEC-2014/14/M/NZ2/00519). J.H.D. and C.N. were supported by Leverhulme Trust (grant no. 10754). A.C.E. and A.D. were supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013)/ERC grant agreement no [GA335691 “Effectomics”], the Austrian Science Fund (FWF): [P27429-B22, P27818-B22, I 3033-B22], and the Austrian Academy of Science (OEAW).

Sunflower germplasm collections are valuable resources for broadening the genetic base of commercial hybrids and ameliorate the risk of climate events. Nowadays, the most studied worldwide sunflower pre-breeding collections belong to INTA (Argentina), INRA (France), and USDA-UBC (United States of America&ndash, Canada). In this work, we assess the amount and distribution of genetic diversity (GD) available within and between these collections to estimate the distribution pattern of global diversity. A mixed genotyping strategy was implemented, by combining proprietary genotyping-by-sequencing data with public whole-genome-sequencing data, to generate an integrative 11,834-common single nucleotide polymorphism matrix including the three breeding collections. In general, the GD estimates obtained were moderate. An analysis of molecular variance provided evidence of population structure between breeding collections. However, the optimal number of subpopulations, studied via discriminant analysis of principal components (K = 12), the bayesian STRUCTURE algorithm (K = 6) and distance-based methods (K = 9) remains unclear, since no single unifying characteristic is apparent for any of the inferred groups. Different overall patterns of linkage disequilibrium (LD) were observed across chromosomes, with Chr10, Chr17, Chr5, and Chr2 showing the highest LD. This work represents the largest and most comprehensive inter-breeding collection analysis of genomic diversity for cultivated sunflower conducted to date.

51 Pags.- 4 Tabls.- 6 Figs. © 2019 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. The definitive version is available at: https://onlinelibrary.wiley.com/journal/1365294x, Landraces are local populations of crop plants adapted to a particular environment. Extant landraces are surviving genetic archives, keeping signatures of the selection processes experienced by them until settling in their current niches. This study intends to establish relationships between genetic diversity of barley (Hordeum vulgare L.) landraces collected in Spain and the climate of their collection sites. A high‐resolution climatic data set (5 × 5 km spatial, 1‐day temporal grid) was computed from over 2,000 temperature and 7,000 precipitation stations across peninsular Spain. This data set, spanning the period 1981–2010, was used to derive agroclimatic variables meaningful for cereal production at the collection sites of 135 barley landraces. Variables summarize temperature, precipitation, evapotranspiration, potential vernalization and frost probability at different times of the year and time scales (season and month). SNP genotyping of the landraces was carried out combining Illumina Infinium assays and genotyping‐by‐sequencing, yielding 9,920 biallelic markers (7,479 with position on the barley reference genome). The association of these SNPs with agroclimatic variables was analysed at two levels of genetic diversity, with and without taking into account population structure. The whole data sets and analysis pipelines are documented and available at https://eead-csic-compbio.github.io/barley-agroclimatic-association. We found differential adaptation of the germplasm groups identified to be dominated by reactions to cold temperature and late‐season frost occurrence, as well as to water availability. Several significant associations pointing at specific adaptations to agroclimatic features related to temperature and water availability were observed, and candidate genes underlying some of the main regions are proposed., This work was supported by the FACCE ERA-NET Plus project ClimBar (618105) and by Spanish Agencia Estatal de Investigación projects CGL2014-52135-C3-3-R, AGL2013-48756-R, AGL2016-80967-R and RFP2015-00006-00-00. BCM was funded by Fundación ARAID.

13 Pags.- 6 Figs. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder, While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution., The work conducted by the US DOE Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract no. DE-AC02-05CH11231. D.P. W. and R.A. were funded in part by the National Science Foundation (grant no. IOS–1258126), and the Great Lakes Bioenergy Research Center (Department of Energy Biological and Environmental Research Office of Science grant no. DE– FCO2–07ER64494). TEJ and DLDM were supported by NSF PGRP grant IOS-0922457. P.C. and B.C.M. were funded by Spanish MINECO (CGL2012-39953-C02-01 and CGL2016-79790-P). B.C.M. was partially funded by DGA—Obra Social La Caixa (grant number GA-LC-059-2011) and Spanish MINECO (AGL2013-48756-R, CSIC13-4E-2490). PC was partially funded by Spanish Aragon Government-European Social Fund (Bioflora).

10 Págs., 4 Figs., 2 Tabls., 1 add. file (17 Págs.) with 8 Figs., 2 Tabls. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Background - Intrinsically disordered proteins, found in all living organisms, are essential for basic cellular functions and complement the function of ordered proteins. It has been shown that protein disorder is linked to the G + C content of the genome. Furthermore, rec ent investigations have suggested that the evolutionary dynamics of the plant nucleus adds disordered segments to open reading frames alike, and these segments are not necessarily conserved among orthologous genes. Results - In the present work the distribution of intrinsically disordered proteins along the chromosomes of several representative plants was analyzed. The reported results support a non-random distribution of disordered proteins along the chromosomes of Arabidopsis thaliana and Oryza sativa, two model eudicot and monocot plant species, respectively. In fact, for most chromosomes positive correlations between the frequency of disordered segments of 30+ amino acids and both recombination rates and G + C content were observed. Conclusions - These analyses demonstrate that the presence of disordered segments among plant proteins is associated with the rates of genetic recombination of their encoding genes. Altogether, these findings suggest that high recombination rates, as well as chromosomal rearrangements, could induce disordered segments in proteins during evolution., This work was supported by grants from Ministerio de Economía y Competitividad (MAT2011-23861) and Gobierno de Aragón (DGA-GC B18 to I.Y. and DGA-GC A06 to B.C.-M.). All these grants were partially funded by the EU FEDER Program. We acknowledge support from CSIC Open Access Publication Support Initiative, through its Unit of Information Resources for Research (URICI), to help cover the Open Access fee.

25 Págs., 3 Figs. The definitive version is available at: http://aem.asm.org/content/79/24/7696, GET_HOMOLOGUES is an open-source software package that builds on popular orthology-calling approaches making highly customizable and detailed pangenome analyses of microorganisms accessible to nonbioinformaticians. It can cluster homologous gene families using the bidirectional best-hit, COGtriangles, or OrthoMCL clustering algorithms. Clustering stringency can be adjusted by scanning the domain composition of proteins using the HMMER3 package, by imposing desired pairwise alignment coverage cutoffs, or by selecting only syntenic genes. The resulting homologous gene families can be made even more robust by computing consensus clusters from those generated by any combination of the clustering algorithms and filtering criteria. Auxiliary scripts make the construction, interrogation, and graphical display of core genome and pangenome sets easy to perform. Exponential and binomial mixture models can be fitted to the data to estimate theoretical core genome and pangenome sizes, and high-quality graphics can be generated. Furthermore, pangenome trees can be easily computed and basic comparative genomics performed to identify lineage-specific genes or gene family expansions. The software is designed to take advantage of modern multiprocessor personal computers as well as computer clusters to parallelize time-consuming tasks. To demonstrate some of these capabilities, we survey a set of 50 Streptococcus genomes annotated in the Orthologous Matrix (OMA) browser as a benchmark case. The package can be downloaded at http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php., Fundación ARAID, Consejo Superior de Investigaciones Científicas [grant number 200720I038]. DGAPA-PAPIIT UNAM-México [grant number IN212010], CONACyT-México [grant numbers P1-60071 and 179133].

25 Pags., 5 Tabls., 2 Figs. The definitive version, with all the supplementary materials, is available at: http://link.springer.com/journal/122, Scald, caused by Rhynchosporium commune, is one of the most prevalent barley diseases worldwide. A search for new resistance sources revealed that Spanish landrace-derived lines SBCC145 and SBCC154 showed outstanding resistance to scald. They were crossed to susceptible cultivar Beatrix to create large DH-mapping populations of 522 and 416 DH lines that were scored for disease resistance in the greenhouse using two R. commune isolates. To ascertain the pattern of resistance, parents and reference barley lines with known scald resistance were phenotyped with a panel of differential R. commune isolates. Subpopulations were genotyped with the Illumina GoldenGate 1,536 SNP Assay and a large QTL in the centromeric region of chromosome 3H, known to harbour several scald resistance genes and/or alleles, was found in both populations. Five SNP markers closest to the QTL were converted into CAPS markers. These CAPS markers, together with informative SSR markers used in other scald studies, confirmed the presence of the Rrs1 locus. The panel of differential scald isolates indicated that the allele carried by both donors was Rrs1 Rh4 . The genetic distance between Rrs1 and its flanking markers was 1.2 cM (11_0010) proximally and 0.9 cM (11_0823) distally, which corresponds to a distance of just below 9 Mbp. The number and nature of scald resistance genes on chromosome 3H are discussed. The effective Rrs1 allele found and the closely linked markers developed are already useful tools for molecular breeding programs and provide a good step towards the identification of candidate genes., This work was funded by the German BMELV and BLE through the project “Rhynchosporium secalis-resistance in barley (project 28-1-41.009-06) and PLANT KBBE II “ExpResBar” AZ 0315702C and by the Spanish Ministry of Science and Innovation (projects GEN2006-28560-E, AGL2007-63625, RFP2012-00015-00-00, and PLANT KBBE “ExpResBar”, EUI2009-04075), and co-funded by the European Regional Development Fund. CS held an I3P contract from CSIC. BCM is supported by Fundación ARAID, Zaragoza, Spain.

17 pags., 8 Figs., High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice., This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through national funds allocated to research projects [POCI/BIA-BCM/56063/2004 and PTDC/BIA-BCM/099836/2008] and PhD scholarships [SFRH/BD/31011/2006 to TS, SFRH/BD/29258/2006 to DF, SFRH/BD/74946/2010 to AC, SFRH/BD/65229/2009 to DA, SFRH/BPD/34943/2007 to TL]. NS and IA were supported by Programa Ciência 2007, financed by POPH (QREN). AS and BCM work was supported by funding from Programa Euroinvestigación 2008 [EUI2008-03612].

16 Pags.- 4 Tabls.- 5 Figs.- Supplementary material foundable online. This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission., The pan-genome of a species is defined as the union of all the genes and non-coding sequences found in all its individuals. However, constructing a pan-genome for plants with large genomes is daunting both in sequencing cost and the scale of the required computational analysis. A more affordable alternative is to focus on the genic repertoire by using transcriptomic data. Here, the software GET_HOMOLOGUES-EST was benchmarked with genomic and RNA-seq data of 19 Arabidopsis thaliana ecotypes and then applied to the analysis of transcripts from 16 Hordeum vulgare genotypes. The goal was to sample their pan-genomes and classify sequences as core, if detected in all accessions, or accessory, when absent in some of them. The resulting sequence clusters were used to simulate pan-genome growth, and to compile Average Nucleotide Identity matrices that summarize intra-species variation. Although transcripts were found to under-estimate pan-genome size by at least 10%, we concluded that clusters of expressed sequences can recapitulate phylogeny and reproduce two properties observed in A. thaliana gene models: accessory loci show lower expression and higher non-synonymous substitution rates than core genes. Finally, accessory sequences were observed to preferentially encode transposon components in both species, plus disease resistance genes in cultivated barleys, and a variety of protein domains from other families that appear frequently associated with presence/absence variation in the literature. These results demonstrate that pan-genome analyses are useful to explore germplasm diversity., This work was supported by DGA – Obra Social La Caixa (grant number GA-LC-059-2011), Spanish MINECO (AGL2013-48756-R and CSIC13-4E-2490) and CONACyTMéxico (grant 179133) and DGAPA-PAPIIT/UNAM IN211814. CC was funded by Spanish MINECO grant BES-2011-045905 linked to AGL2010-21929. BC-M was funded by Fundación ARAID. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231.

12 Pags., 8 Figs., Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments., Funding for open access charge: Programa Euroinvestigación/Plant KBBE 2008 [EUI2008-03612].

29 Pag., 5 Tabl., 4 Fig. The definitive version is available at: http://www.springer.com/life+sciences/plant+sciences/journal/122, Flowering time is an important factor in the adaptation of barley varieties to environmental conditions and maximizing yield potential (Boyd et al. 2003; Cockram et al. 2007a; Cuesta-Marcos et al. 2009), by synchronizing the plant cycle to the prevailing environmental conditions. Flowering time is a complex trait that shows an almost continuous variation in cereals. The investigation of the genetic control of flowering time in barley has benefited from the comparative use of floral pathways in Arabidopsis thaliana (Cockram et al. 2007a) and rice, via the identification of candidate genes through orthology. The variation in flowering time is mainly due to variations in genes regulated by day length (photoperiod) or long exposures to low temperature (vernalization) (Laurie et al. 1995; Trevaskis et al. 2003; Dubcovsky et al. 2005). In barley, three genes are responsible for the vernalization requirement: VrnH1 (isolated by map-based cloning in diploid wheat, Yan et al. 2003), VrnH2 (identified by positional cloning, Yan et al. 2004) and VrnH3 (identified by homology to a known gene from Arabidopsis thaliana, Yan et al. 2006). VrnH1 is induced by vernalization and promotes the transition from vegetative to reproductive development. VrnH2 is a floral repressor that delays flowering until the plants are vernalized. The VrnH3 gene seems to be orthologous to the A. thaliana floral pathway integrator FT (FLOWERING LOCUS T) gene (Yan et al. 2006; Faure et al. 2007; Turck et al. 2008; Kikuchi et al. 2009). In A. thaliana, FT expression increases in the leaves when plants are exposed to inductive day length. In barley, expression of orthologous HvFT1 (synonymous to VrnH3) is induced by long day conditions and promotes flowering (Hemming et al. 2008). The winter growth habit of barley requires the presence of a recessive VrnH1 allele, together with an active VrnH2 allele (Cockram et al. 2007b; Hemming et al. 2009). Vernalization induces VrnH1 under both short and long days, which then represses VrnH2. Distelfeld et al. (2009) reported that the interactions among the three vernalization genes generate a feedback regulatory loop that once started, leads to an irreversible induction of flowering. The function of HvFT1 has started to be unraveled only recently. There is now mounting evidence supporting the role of the FT protein in Arabidopsis (and corresponding proteins in other species) as an important part of the florigen (Corbesier et al. 2007; Tamaki et al. 2007). Kikuchi et al. (2009) presented strong evidence suggesting that HvFT1 plays a central role in promoting flowering, integrating the photoperiod and vernalization pathways. HvFT1 expression seems to be regulated by the major photoperiod response genes: PpdH1 under LD conditions and PpdH2 under SD conditions. There are evidences on the adaptive role played by VrnH1, VrnH2 and PpdH1 during the expansion of the crop, facilitating its adaptation to new agroecological niches (Cockram et al. 2007a; Jones et al. 2008). Does VrnH3-HvFT1 also have an adaptive role? We know that the phenotypic effect of HvFT1 on flowering time can be very large (Yan et al. 2006), and therefore may be an important factor for the final determination of barley flowering time. Other open questions on this gene are: to what environmental cue does VrnH3 respond, temperature or photoperiod? What effect does it have on flowering time under natural conditions? To address these questions, we analyzed the polymorphism and the phenotypic effect of this gene on a collection of Spanish barley landraces and its variation at the sequence level, and validated its effect on a segregating population., This work was supported by the Spanish Ministry of Science and Innovation (Projects AGL2007-63625 and RTA01-088-C3) and by the European Regional Development Fund. A Djemel was supported by a fellowship from IAMZ-CIHEAM. S Yahiaoui and L Ponce were supported by fellowships from AECID-Spanish Ministry of Foreign Affairs and Cooperation.

In prokaryotes, regulation of gene expression is predominantly controlled at the level of transcription. Transcription in turn is mediated by a set of DNA-binding factors called Transcription Factors (TFs). In this study, we map the complete repertoire of ~ 300 TFs of the bacterial model, Escherichia coli, onto gene expression data for a number of non-redundant experimental conditions and show that TFs are generally expressed at a lower level than other gene classes. We also demonstrate that different conditions harbor varying number of active TFs, with an average of about 15% of the total repertoire, with certain stress and drug induced conditions exhibiting as high as one-third of the collection of TFs. Our results also show that activators are more frequently expressed than repressors, indicating that activation of promoters might be a more common phenomenon than repression in bacteria. Finally, to understand the association of TFs with different conditions and to elucidate their dynamic interplay with other TFs, we develop a network-based framework to identify TFs which act as markers, those which are responsible for condition-specific transcriptional rewiring. This approach allowed us to pinpoint several marker TFs as being central in various specialized conditions like drug-induction or growth condition variations, which we discuss in light of previously reported experimental findings. Further analysis showed that a majority of identified markers effectively control the expression of their regulons and in general transcriptional programs of most conditions can be effectively rewired by a very small number of TFs. It was also found that closeness is a key centrality measure which can aid in the successful identification of marker TFs in regulatory networks. Our results suggest the utility of the network-based approaches developed in this study to be applicable for understanding other interactomic datasets., This work was supported by MRC Laboratory of Molecular Biology and Cambridge Commonwealth Trust and by a grant from Gobierno de Aragón to the research group of José María Lasa in 2010

3D-footprint is a living database, updated and curated on a weekly basis, which provides estimates of binding specificity for all protein–DNA complexes available at the Protein Data Bank. The web interface allows the user to: (i) browse DNA-binding proteins by keyword; (ii) find proteins that recognize a similar DNA motif and (iii) BLAST similar DNA-binding proteins, highlighting interface residues in the resulting alignments. Each complex in the database is dissected to draw interface graphs and footprint logos, and two complementary algorithms are employed to characterize binding specificity. Moreover, oligonucleotide sequences extracted from literature abstracts are reported in order to show the range of variant sites bound by each protein and other related proteins. Benchmark experiments, including comparisons with expert-curated databases RegulonDB and TRANSFAC, support the quality of structure-based estimates of specificity. The relevant content of the database is available for download as flat files and it is also possible to use the 3D-footprint pipeline to analyze protein coordinates input by the user. 3D-footprint is available at http://floresta.eead.csic.es/3dfootprint with demo buttons and a comprehensive tutorial that illustrates the main uses of this resource., Consejo Superior de Investigaciones Científicas [grant number 200720I038]. Funding to pay the Open Access publication charges for this article was provided by [grant A06] from Gobierno de Aragón to the research group of J.M. Lasa in 2009.

Specific protein-DNA interactions are central to a wide group of processes in the cell and have been studied both experimentally and computationally over the years. Despite the increasing collection of protein-DNA complexes, so far only a few studies have aimed at dissecting the structural characteristics of DNA binding among evolutionarily related proteins. Some questions that remain to be answered are: (a) what is the contribution of the different readout mechanisms in members of a given structural superfamily, (b) what is the degree of interface similarity among superfamily members and how this affects binding specificity, (c) how DNA-binding protein superfamilies distribute across taxa, and (d) is there a general or family-specific code for the recognition of DNA. We have recently developed a straightforward method to dissect the interface of protein-DNA complexes at the atomic level and here we apply it to study 175 proteins belonging to nine representative superfamilies. Our results indicate that evolutionarily unrelated DNA-binding domains broadly conserve specificity statistics, such as the ratio of indirect/direct readout and the frequency of atomic interactions, therefore supporting the existence of a set of recognition rules. It is also found that interface conservation follows trends that are superfamily-specific. Finally, this article identifies tendencies in the phylogenetic distribution of transcription factors, which might be related to the evolution of regulatory networks, and postulates that the modular nature of zinc finger proteins can explain its role in large genomes, as it allows for larger binding interfaces in a single protein molecule.

21 Pags.- 2 Tabls.- 5 Figs.- 1 Tabl. Suppl.- 5 Figs. Suppl., Background Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells. Results We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2. Conclusions YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as opposed to YY1, suggesting that YY2 exerts unique contributions to gene regulation. YY2 binding was not generally associated with gene promoters. However, several YY2 binding sites are linked to long noncoding RNA (lncRNA) genes and we show that the expression levels of a few of those are Yy2-dependent., This work has been supported by grant PI07119 from FIS/CarlosIII, Ministry of Health; PAMER grants, Aragon Health Sciences Institute, Spain; grant PI110/09 from Dpto de CTU (Gobierno de Aragón) and annual grants B77 from the Government of Aragon (Department of Research and Innovation)/European Social Funds. R. PérezPalacios and S. Macías-Redondo were supported by PhD fellowships B138/10 and C071/2014, respectively, from the DGA (Aragón, Spain).

28 Pags.- 8 Figs. The definitive version is available at: http://link.springer.com/bookseries/7651 and http://link.springer.com/book/10.1007/978-1-4939-6396-6., FootprintDB is a database and search engine that compiles regulatory sequences from open access libraries of curated DNA cis-elements and motifs, and their associated transcription factors (TFs). It systematically annotates the binding interfaces of the TFs by exploiting protein-DNA complexes deposited in the Protein Data Bank. Each entry in footprintDB is thus a DNA motif linked to the protein sequence of the TF(s) known to recognize it, and in most cases, the set of predicted interface residues involved in specific recognition. This chapter explains step-by-step how to search for DNA motifs and protein sequences in footprintDB and how to focus the search to a particular organism. Two real-world examples are shown where this software was used to analyze transcriptional regulation in plants. Results are described with the aim of guiding users on their interpretation, and special attention is given to the choices users might face when performing similar analyzes., This work was funded by grant Euroinvestigación EUI2008-03612 under the framework of the Transnational (Germany, France, Spain) Cooperation within the PLANT-KBBE Initiative.

1 .pdf file of 46 Pags. with text and 7 Figs. and 5 .pdfs files of Supplementary Tables. The definitve version is available at: http://gbe.oxfordjournals.org/, The advent of whole-genome sequencing has provided an unprecedented detail about the evolution and genetic significance of species-specific variations across the whole Mycobacterium tuberculosis Complex. However, little attention has been focused on understanding the functional roles of these variations in the protein coding sequences. In this work, we compare the coding sequences from 74 sequenced mycobacterial species including M. africanum, M. bovis, M. canettii, M. caprae, M. orygis, and M. tuberculosis. Results show that albeit protein variations affect all functional classes, those proteins involved in lipid and intermediary metabolism and respiration have accumulated mutations during evolution. To understand the impact of these mutations on protein functionality, we explored their implications on protein ductility/disorder, a yet unexplored feature of mycobacterial proteomes. In agreement with previous studies, we found that a Gly71Ile substitution in the PhoPR virulence system severely affects the ductility of its nearby region in M. africanum and animal-adapted species. In the same line of evidence, the SmtB transcriptional regulator shows amino acid variations specific to the Beijing lineage, which affects the flexibility of the N-terminal trans-activation domain. Furthermore, despite the fact that MTBC epitopes are evolutionary hyperconserved, we identify strain- and lineage-specific amino acid mutations affecting previously known T-cell epitopes such as EsxH and FbpA (Ag85A). Interestingly, in silico studies reveal that these variations result in differential interaction of epitopes with the main HLA haplogroups., This work was supported by Gobierno de Aragón (DGA-GC B18 and B25), the Spanish Ministry of Science and Competitiveness (BIO2014-52580P, CSIC13-4E-2490), Instituto de Salud Carlos III (PI12/01970) and the European Commission Horizon 2020 (H2020-PHC-643381). Some of these grants were partially financed by the EU FEDER Program. This work was also supported by Fundação para a Ciência e Tecnologia, Portugal (IF/00474/2014) and cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER).

14 Pags.- 5 Figs. © Crop Science Society of America. This is an open access article distributed under the CC BY-NC-ND, Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus) conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high-resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBSLRR (nucleotide-binding site–leucine-rich repeat containing protein) genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence-absence variation) in the composition of the NBS-LRR cluster. Finally, a template-guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS-LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next-generation sequencing) tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

26 Pags.- 8 Figs.- 2 Tabls. The definitive version is available at: http://link.springer.com/bookseries/7651 and http://link.springer.com/book/10.1007/978-1-4939-6396-6, In this protocol, we explain how to run ab initio motif discovery in order to gather putative transcription factor binding motifs (TFBMs) from sets of genomic regions returned by ChIP-seq experiments. The protocol starts from a set of peak coordinates (genomic regions) which can be either downloaded from ChIP-seq databases, or produced by a peak-calling software tool. We provide a concise description of the successive steps to discover motifs, cluster the motifs returned by different motif discovery algorithms, and compare them with reference motif databases. The protocol is documented with detailed notes explaining the rationale underlying the choice of options. The interpretation of the results is illustrated with an example from the model plant Arabidopsis thaliana., This work was funded in part by Fundación ARAID and by the EnseignantsChercheurs invités program of Aix-Marseille Université (to B.C.M.). C.R. was supported by the France Génomique National infrastructure, funded as part of the Investissements d’Avenir, program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09). J.C-M PhD grant is funded by the Ecole Doctorale des Sciences de la Vie et de la Santé (EDSVS), AixMarseille Université.

17 Pags.- 4 Tabls.- 2 Figs. The definitive version is available at: http://link.springer.com/bookseries/7651 and http://link.springer.com/book/10.1007/978-1-4939-6396-6, The plant-dedicated mirror of the Regulatory Sequence Analysis Tools (RSAT, http://​plants.​rsat.​eu) offers specialized options for researchers dealing with plant transcriptional regulation. The website contains whole-sequenced genomes from species regularly updated from Ensembl Plants and other sources (currently 40), and supports an array of tasks frequently required for the analysis of regulatory sequences, such as retrieving upstream sequences, motif discovery, motif comparison, and pattern matching. RSAT::Plants also integrates the footprintDB collection of DNA motifs. This protocol explains step-by-step how to discover DNA motifs in regulatory regions of clusters of co-expressed genes in plants. It also explains how to empirically control the significance of the result, and how to associate the discovered motifs with putative binding factors., This work was funded in part by Fundación ARAID and by the Enseignants-Chercheurs invités program of Aix-Marseille Université (to BCM). CR was supported by the France Génomique National infrastructure, funded as part of the Investissements d”Avenir, program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09).

Intron boundaries were extracted from genomic data and mapped onto single-domain human and murine protein structures taken from the Protein Data Bank. A first analysis of this set of proteins shows that intron boundaries prefer to be in non-regular secondary structure elements, while avoiding alpha-helices and beta-strands. This fact alone suggests an evolutionary model in which introns are constrained by protein structure, particularly by tertiary structure contacts. In addition, in silico recombination experiments of a subset of these proteins together with their homologues, including those in different species, show that introns have a tendency to occur away from artificial crossover hot spots. Altogether, these findings support a model in which genes can preferentially harbour introns in less constrained regions of the protein fold they code for. In the light of these findings, we discuss some implications for protein modelling and design.

22 pags.- 4 Figs., The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences., Part of this work was supported by the French National Research Agency (CERES, Grant ANR-BLAN-1238) and the PLANT-KBBE Initiative (STREG, ANR-08-KBBE-011-01). http:// www.agence-nationale-recherche.fr/. The work of Z.K., F.S. and A.S. was supported by the STREG project. W.X. was supported by a fellowship from the China Scholarship Council (CSC).

25 Pags.- 2 Tabls. The definitive version is available at: http://link.springer.com/journal/11032, The BARLEYMAP pipeline was designed to map both genomic sequences and transcripts against sequence-enriched genetic/physical frameworks, with plant breeders as the main target users. It reports the most probable genomic locations of queries after merging results from different resources so that diversity obtained from re-sequencing experiments can be exploited. In addition, the application lists surrounding annotated genes and markers, facilitating downstream analyses. Pre-computed marker datasets can also be created and browsed to facilitate searches and cross referencing. Performance is evaluated by mapping two sets of long transcripts and by locating the physical and genetic positions of four marker collections widely used for high-throughput genotyping of barley cultivars. In addition, genome positions retrieved by BARLEYMAP are compared to positions within a conventional genetic map for a population of recombinant inbred lines, yielding a gene-order accuracy of 96 %. These results reveal advantages and drawbacks of current in silico approaches for barley genomics. A web application to make use of barley data is available at http://floresta.eead.csic.es/barleymap. The pipeline can be set up for any species with similar sequence resources, for which a fully functional standalone version is available for download., This work was funded by DGA - Obra Social La Caixa [grant number GA-LC-059-2011] and by the Spanish Ministry of Science and Innovation [projects AGL2010-21929 and RTA2009-00006-C04-02]. Carlos P. Cantalapiedra is funded by [grant BES-2011-045905 linked to project AGL2010-21929]. Ridha Boudiar was supported by a Master’s fellowship from IAMZ-CIHEAM.

29 Pags.- 4 Tabls.- 1 Fig. The definitive version is available at: http://link.springer.com/journal/11032, This study is a retrospective analysis of an elite cross from the Spanish National Barley Breeding Program. This was the most successful cross produced in the breeding program in the past 20 years. The progeny from this cross has been investigated at two points in the program, before and after selection, through the analysis of allelic frequencies at a number of genetic loci with molecular markers. Shifts in allelic frequencies after selection allowed the identification of genomic regions with selection footprints likely due to the breeding process. The cross was replicated in three different years, and therefore, the three progenies represent different selection histories but, in all cases, were preferentially selected compared to the lines from other crosses used in the program. The progenies were sampled at two generations, before conscious selection (F2) and after six generations of selection (F8). The F2 plants were genotyped with microsatellites, whereas 31 F8 lines were surveyed for SNP and presence/absence variation polymorphisms using a genotyping-by-sequencing system (DArTseq). The DArTseq markers were aligned to the barley physical map, and, after curation, over 3,000 were still available for the analysis. Overall, 15 genomic regions in the F8 lines had allele frequencies beyond chosen thresholds, indicating selection, eight toward parent Orria and seven toward Plaisant. These selection footprints partially validated QTLs detected through classical linkage mapping in a RIL population of the same cross. These validated selection footprints convey useful information for barley breeding, either through marker-assisted selection or through genomic selection., This work was supported by the Spanish Ministry of Science and Innovation (MICINN), who funded this work with the scholarship BES‐2008‐009623 (EM), and the projects AGL2010‐21929, RTA03‐028‐C4, RTA2006‐00020‐C04 and RTA2009‐00006‐C04, and by grant A06 from the Aragon Government. Genotype by sequencing was funded by DGA ‐ Obra Social La Caixa [grant number GA‐LC‐059‐2011]. WTBT acknowledges funding by the Scottish Government’s Rural and Environment Science and Analytical Services Division.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited., [Background] Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes., [Results] Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions., [Conclusions] The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes., The work of Z.K., G.H., A.S., F.S., C.B., and L.B. was supported by the STREG project. W.X.’s work was supported by the China Scholarship Council (CSC). The authors would like to acknowledge the support of Jasmin Huebner and Nina Schmidt in the generation and analysis of transgenic A. thaliana.

35 Pags.- 5 Figs. The definitive version is available at: http://link.springer.com/book/10.1007/978-1-4939-1720-4, GET_HOMOLOGUES is an open-source software package written in Perl and R to define robust core- and pan-genomes by computing consensus clusters of orthologous gene families from whole-genome sequences using the bidirectional best-hit, COGtriangles, and OrthoMCL clustering algorithms. The granularity of the clusters can be fine-tuned by a user-configurable filtering strategy based on a combination of blastp pairwise alignment parameters, hmmscan-based scanning of Pfam domain composition of the proteins in each cluster, and a partial synteny criterion. We present detailed protocols to fit exponential and binomial mixture models to estimate core- and pan-genome sizes, compute pan-genome trees from the pan-genome matrix using a parsimony criterion, analyze and graphically represent the pan-genome structure, and identify lineage-specific gene families for the 12 complete pIncA/C plasmids currently available in NCBI’s RefSeq. The software package, license, and detailed user manual can be downloaded for free for academic use from two mirrors: http://www.eead.csic.es/compbio/soft/gethoms.php and http://maya.ccg.unam.mx/soft/gethoms.php.

This chapter presents a practical overview and case study of the usage and capabilities of the primers4clades version 1.0 web server [Cont reras-Moreira et al., 2009], a software tool useful to desi gn degenerate PCR primers for gene-targeted analysis of metagenomic DNA (see also Chapter 28, Vol. I) and multilocus sequence analysis. Although direct shotgun sequencing of community DNA is now feasible [Venter et al., 2004], polymerase chain reaction (PCR) remains the most widely used technology to gain molecular markers for studies in molecular ecology and systematics.

Primers4clades is an easy-to-use web server that implements a fully automatic PCR primer design pipeline for cross-species amplification of novel sequences from metagenomic DNA, or from uncharacterized organisms, belonging to user-specified phylogenetic clades or taxa. The server takes a set of non-aligned protein coding genes, with or without introns, aligns them and computes a neighbor-joining tree, which is displayed on screen for easy selection of species or sequence clusters to design lineage-specific PCR primers. Primers4clades implements an extended CODEHOP primer design strategy based on both DNA and protein multiple sequence alignments. It evaluates several thermodynamic properties of the oligonucleotide pairs, and computes the phylogenetic information content of the predicted amplicon sets from Shimodaira–Hasegawa-like branch support values of maximum likelihood phylogenies. A non-redundant set of primer formulations is returned, ranked according to their thermodynamic properties. An amplicon distribution map provides a convenient overview of the coverage of the target locus. Altogether these features greatly help the user in making an informed choice between alternative primer pair formulations. Primers4clades is available at two mirror sites: http://maya.ccg.unam.mx/primers4clades/and http://floresta.eead.csic.es/primers4clades/. Three demo data sets and a comprehensive documentation/tutorial page are provided for easy testing of the server's capabilities and interface., DGAPA/PAPIIT-UNAM (IN201806-2); CONACyT-Mexico (P1-60071); Consejo Superior de Investigaciones Científicas (200720I038). Funding for open access charge: Universidad Nacional Autónoma de México.

19 Pag., 4 Tabl., 14 Fig., Despite almost 40 years of molecular genetics research in Escherichia coli a major fraction of its Transcription Start Sites (TSSs) are still unknown, limiting therefore our understanding of the regulatory circuits that control gene expression in this model organism. RegulonDB (http://regulondb.ccg.unam.mx/) is aimed at integrating the genetic regulatory network of E. coli K12 as an entirely bioinformatic project up till now. In this work, we extended its aims by generating experimental data at a genome scale on TSSs, promoters and regulatory regions. We implemented a modified 5′ RACE protocol and an unbiased High Throughput Pyrosequencing Strategy (HTPS) that allowed us to map more than 1700 TSSs with high precision. From this collection, about 230 corresponded to previously reported TSSs, which helped us to benchmark both our methodologies and the accuracy of the previous mapping experiments. The other ca 1500 TSSs mapped belong to about 1000 different genes, many of them with no assigned function. We identified promoter sequences and type of σ factors that control the expression of about 80% of these genes. As expected, the housekeeping σ70 was the most common type of promoter, followed by σ38. The majority of the putative TSSs were located between 20 to 40 nucleotides from the translational start site. Putative regulatory binding sites for transcription factors were detected upstream of many TSSs. For a few transcripts, riboswitches and small RNAs were found. Several genes also had additional TSSs within the coding region. Unexpectedly, the HTPS experiments revealed extensive antisense transcription, probably for regulatory functions. The new information in RegulonDB, now with more than 2400 experimentally determined TSSs, strengthens the accuracy of promoter prediction, operon structure, and regulatory networks and provides valuable new information that will facilitate the understanding from a global perspective the complex and intricate regulatory network that operates in E. coli., National Institutes of Health, grant GM071962-03. Department of Energy, grant GTLDE-FC02-02ER63446. Consejo Nacional de Ciencia y Tecnologia (CONACYT, Mexico) grant 83686 G.I.

Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition., NIH grant RO1-GM071962 Fundación Agencia Aragonesa I+D

30 Pag., 6 Tabl., 2 Fig., A highly supported maximum-likelihood species phylogeny for the genus Bradyrhizobium was inferred from a supermatrix obtained from the concatenation of partial atpD, recA, glnII, and rpoB sequences corresponding to 33 reference strains and 76 bradyrhizobia isolated from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam. The power of the multigene approach using multiple strains per species was evaluated in terms of overall tree resolution and phylogenetic congruence, representing a practical and portable option for bacterial molecular systematics. Potential pitfalls of the approach are highlighted. Seventy-five of the isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense, B. yuanmingense, or B. elkanii, whereas one represented a novel Bradyrhizobium lineage. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence against the monophyly of the of B. japonicum I and Ia lineages was found. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage are misclassified as B. japonicum. The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones and had broad geographic and environmental distribution ranges, as revealed by mapping climate types and geographic origins of the isolates on the species tree., We thank the European Union for long-term support in INCO DEV project ICA4-CT-2001-10057 and the DEGAPA-PAPIIT from the National Autonomous University of Mexico for grant DGAPA IN201806-2 to P.V.

RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database offering curated knowledge of the transcriptional regulatory network of Escherichia coli K12, currently the best-known electronically encoded database of the genetic regulatory network of any free-living organism. This paper summarizes the improvements, new biology and new features available in version 6.0. Curation of original literature is, from now on, up to date for every new release. All the objects are supported by their corresponding evidences, now classified as strong or weak. Transcription factors are classified by origin of their effectors and by gene ontology class. We have now computational predictions for sigma(54) and five different promoter types of the sigma(70) family, as well as their corresponding -10 and -35 boxes. In addition to those curated from the literature, we added about 300 experimentally mapped promoters coming from our own high-throughput mapping efforts. RegulonDB v.6.0 now expands beyond transcription initiation, including RNA regulatory elements, specifically riboswitches, attenuators and small RNAs, with their known associated targets. The data can be accessed through overviews of correlations about gene regulation. RegulonDB associated original literature, together with more than 4000 curation notes, can now be searched with the Textpresso text mining engine.

7 Pág., 4 Fig., 2 Tabl. The original version is available at: http://bioinformatics.oxfordjournals.org/content/22/14/e74, Motivation: Comparative modelling is a computational method used to tackle a variety of problems in molecular biology and bio-technology. Traditionally it has been applied to model the structure of proteins on their own or bound to small ligands, although more recently it has also been used to model protein-protein interfaces. This work is the first to systematically analyze whether comparative models of protein-DNA complexes could be built and be useful for predicting DNA binding sites. Results: First, we describe the structural and evolutionary conservation of protein-DNA interfaces, and the limits they impose on modelling accuracy. Second, we find that side-chains from contacting residues can be reasonably modeled and therefore used to identify contacting nucleotides. Third, the DNASITE protocol is implemented and different parameters are benchmarked on a set of 85 regulators from Escherichia coli. Results show that comparative foot-printing can make useful predictions based solely on structural data, depending primarily on the interface identity with respect to the template used., This work has been supported by a postdoctoral fellowship from Universidad Nacional Autónoma de México awarded to B.C.M. and by NIH grant RO1-GM071962.

Most protein structure prediction methods use templates to assist in the construction of protein models. In this paper, we analyse the current state of template-based modelling approaches and reach an estimate of the empirical limits of these methods. Our analysis show that current prediction methods are already reaching these empirical accuracy limits in the easier cases, where finding a close homologue to the native target structure is not a problem. However, we find that even in the absence of alignment errors and using optimal templates, template-based methods have intrinsic limitations, suggesting that other methodologies, such as ab initio procedures, must be used if accuracy is ultimately to be improved.

Epithelial ovarian cancer (EOC), the leading cause of death from gynecological malignancy, is a poorly understood disease. The typically advanced presentation of EOC with loco-regional dissemination in the peritoneal cavity and the rare incidence of visceral metastases are hallmarks of the disease. These features relate to the biology of the disease, which is a principal determinant of outcome1,2. EOC arises as a result of genetic alterations sustained by the ovarian surface epithelium (OSE; ref. 3). The causes of these changes are unknown but are manifest by activation of oncogenes and inactivation of tumor-suppressor genes (TSGs). Our analysis of loss of heterozygosity at 11q25 identified OPCML (also called OBCAM), a member of the IgLON family of immunoglobulin (Ig) domain–containing glycosylphosphatidylinositol (GPI)-anchored cell adhesion molecules, as a candidate TSG in EOC. OPCML is frequently somatically inactivated in EOC by allele loss and by CpG island methylation. OPCML has functional characteristics consistent with TSG properties both in vitro and in vivo. A somatic missense mutation from an individual with EOC shows clear evidence of loss of function. These findings suggest that OPCML is an excellent candidate for the 11q25 ovarian cancer TSG. This is the first description to our knowledge of the involvement of the IgLON family in cancer.

13 Pag., 2 Tabl., 4 Fig., Background Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity., This work was supported by CONSI+D, DGA (Grant PM062/2007 to M.M. and I.Y.), the Spanish Ministry of Science and Innovation (BIO2007-65890-C02-01 and BIO2010-14983 to M.M.) and Gobierno de Aragón (DGA-GE B18 to M. M. and I.Y. and DGA-GC A06 to B.C-M).

24 Pags.- 2 Tabls.- 3 Figs. The definitive version is available at: https://link.springer.com/journal/11032, Six-rowed spike 1 (Vrs1) is a gene of major importance for barley breeding and germplasm management as it is the main gene determining spike row-type (2-rowed vs. 6-rowed). This is a widely used DUS trait, and has been often associated to phenotypic traits beyond spike type. Comprehensive re-sequencing Vrs1 revealed three two-rowed alleles (Vrs1.b2; Vrs1.b3; Vrs1.t1) and four six-rowed (vrs1.a1; vrs1.a2; vrs1.a3; vrs1.a4) in the natural population. However, the current knowledge about Vrs1 alleles and its distribution among Spanish barley subpopulations is still underexploited. We analyzed the gene in a panel of 215 genotypes, made of Spanish landraces and European cultivars. Among 143 six-rowed accessions, 57 had the vrs1.a1 allele, 83 were vrs1.a2, and three showed the vrs1.a3 allele. Vrs1.b3 was found in most two-rowed accessions, and a new allele was observed in 7 out of 50 two-rowed Spanish landraces. This allele, named Vrs1.b5, contains a ‘T’ insertion in exon 2, originally proposed as the causal mutation giving rise to the six-row vrs1.a2 allele, but has an additional upstream deletion that results in the change of 15 amino acids and a potentially functional protein. We conclude that eight Vrs1 alleles (Vrs1.b2, Vrs1.b3, Vrs1.b5, Vrs1.t1, vrs1.a1, vrs1.a2, vrs1.a3, vrs1.a4) discriminate two and six-rowed barleys. The markers described will be useful for DUS identification, plant breeders, and other crop scientists., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness grants AGL2010-21929, AGL2013-48756-R, RFP2012-00015-00-00, RTA2012-00033-C03-02, and EUI2009-04075 (national code for Plant-KBBE project ExpResBar). CPC was funded by the Spanish Ministry of Economy, Industry and Competitiveness grant no. BES-2011-045905 (linked to project AGL2010-21929). TK and SS were supported by a research fund by the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation grants no. TRS1002). SS was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for Research Abroad and a Grant-in-Aid for Young Scientists (B) (no. 16 K18635).

Ensembl Genomes (https://www.ensemblgenomes.org) provides access to non-vertebrate genomes and analysis complementing vertebrate resources developed by the Ensembl project (https://www.ensembl.org). The two resources collectively present genome annotation through a consistent set of interfaces spanning the tree of life presenting genome sequence, annotation, variation, transcriptomic data and comparative analysis. Here, we present our largest increase in plant, metazoan and fungal genomes since the project's inception creating one of the world's most comprehensive genomic resources and describe our efforts to reduce genome redundancy in our Bacteria portal. We detail our new efforts in gene annotation, our emerging support for pangenome analysis, our efforts to accelerate data dissemination through the Ensembl Rapid Release resource and our new AlphaFold visualization. Finally, we present details of our future plans including updates on our integration with Ensembl, and how we plan to improve our support for the microbial research community. Software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license). Data updates are synchronised with Ensembl's release cycle.