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Additional file 2:Gene ontology distribution of R. balthica contigs. Pie charts showing the distribution of contigs > 200bp, giving identical BLAST X gene hits for the NGEN and OASES k mer-31 assemblies, into the three main gene ontology categories. (PDF 706 KB)

Additional file 5:Rearing conditions subjected to Radix balthica individuals. (DOC 34 KB)

Additional file of Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance

Additional file 6:RNA quality inspection. RNA quality, RNA normalization, and RNA size selection gel pictures. (DOC 2 MB)

Additional file 4:Screen shot contig assembly vs. mitochondrial genome. The effect of a subsequent meta-assembly on contig redundancy is depicted here: a) Coverage of the mitochondrial gene COIII after the first separate assemblies and b) after the meta-assembly. (PDF 89 KB)

Additional file 1:Net coverage bp of R. balthica mitochondrial genes. Coverage bp of R. balthica mitochondrial genes by contigs > 80% bp identity. Numbers in parentheses indicate the number of contigs with identities > 80% matching the respective gene, and thus are included in the net coverage calculation. Combined coverage: overall coverage, thus bp identity of all first assembly contigs taken together. (DOC 48 KB)

Supplementary methods, supplementary results, supplementary table S1, supplementary figures S1-S6. (DOCX 555 kb)

Table S5. Details of enriched GO terms among differentially expressed transcripts. GO terms (biological process and molecular function) are grouped according to REVIGO semantic categories and generic GO slim terms. (DOC 145Â kb)

Trabajo presentado en el IV International Ascochyta Workshop (Ascochyta 2016), celebrado en Tróia (Portugal) el 10 y 11 de octubre de 2016., Efforts to control ascochyta blight in chickpea have mostly been based on the identification of resistance genes in the host. The identification of A. rabiei pathogenicity determinants will open a wide range of new alternatives to control ascochyta blight disease and increase our knowledge about Ascochyta-legume interactions. However, very little is known about the pathogenicity factor of A. rabiei. Equally, little information about A. rabiei is available at the molecular level. We have used Illumina and PacBio RS II platforms for sequencing the genome and transcriptome of an isolate of A. rabiei belonging to pathotype IV. A. rabiei secretome was also predicted. In addition, an accurate transc riptomic analysis using Massive Analysis of cDNA Ends (MACE) was performed to compare the gene expression profile of the fungus infecting chickpea with that of the fungus growing in absence of its host (1). A detailed analysis of the genes up-regulated dur ing infection identified a collection of candidate pathogenicity factors and unravelled the strategies that allow the pathogen to cause disease: A. rabiei produces a battery of cell wall-degrading enzymes, used by the pathogen to penetrate its host, secret es toxins to kill the host cells, and is able to detoxify the fungitoxic compounds produced by the plant as defense.

Trabajo presentado en la 3rd Annual Conference of the COST Action Sustain (FA1208), celebrada en Banyuls s/ Mer (France) del 17 al 19 de febrero de 2016., The most important foliar diseases in legumes worldwide are ascochyta blights. Health or disease is the result of a battle between plants and their pathogens. However, in the Ascochyta-legume pathosystem most studies focused on the identification of resistance genes in the host, while very little is known about the pathogenicity factors of Ascochyta spp. This study aimed at the identification of pathogenicity factors of ascochyta blight pathogens using Ascochyta rabiei as a model. Towards this objective we used NGS for the de novo sequencing of the A. rabiei transcriptome, and to identify genes differentially expressed by the fungus during infection of chickpea leaves in comparison to the fungus growing under artificial conditions. Combining RNA-Seq and MACE data we generated a comprehensive transcriptome data base comprising 22,725 assembled A. rabiei contigs with an average length of 1178 bp. Since pathogenicity factors are usually secreted, we predicted the A. rabiei secretome, yielding 550 putatively secreted proteins. Accurate transcriptome quantification by MACE identified 597 transcripts that were up-regulated during infection. An analysis of these genes identified a collection of candidate pathogenicity factors and effectors such as cell wall-degrading enzymes, toxins and genes involved in the detoxification of fungitoxic compounds produced by the plant as a defense.

Trabajo presentado en el XL Congreso de la Sociedad Española de Genética, celebrado en Córdoba del 16 al 18 de septiembre de 2015., Las leguminosas son un cultivo importante e interes ante. Además de ser una fuente barata y versátil de proteínas, tanto para consumo humano como animal, actúan como fertilizantes naturales, enriqueciendo el suelo con nitrógeno gracias a su simbiosis con bacterias fijadoras de nitrógeno. El rendimiento de las leguminosas se ve seriamente afectado por el ataque de las enfermedades. Entre ellas, la enfermedad foliar más importante es la ascoquitosis o rabia. Esta enfermedad está causada por distintas especies de Ascochyta spp. según el huésped y produce graves pérdidas de rendimiento en cultivos como el garbanzo, guisante, lenteja o habas. La salud o la enfermedad dependen de los componentes genéticos de las dos partes de la interacción planta-patógeno. Sin embargo, los estudios realizados hasta ahora en el patosistema Ascochyta-leguminosas se han centrado sólo en una de las partes de la interacción, la planta. Así, los esfuerzos realizados para controlar la ascoquitosis han tenido como objetivo principal la identificación de genes y fuentes de resistencia en la planta, mientras que no se sabe prácticamente nada sobre los factores de patogenicidad de las Ascochytas. Este estudio ha tenido como objetivo final identificar los factores de patogenicidad de las Ascochytas, en concreto de Ascochyta rabiei, el agente causal de la rabia del garbanzo. Con este objetivo se han utilizado técnicas de secuenciación masiva para secuenciar el genoma y transcriptoma de A. rabiei y para comparar el perfil de expresión de los genes del patógeno creciendo en medio artificial en ausencia de su huésped con el del hongo infectando hojas de garbanzo. Como resultado, en este trabajo se presenta la secuenciación de novo y caracterización del genoma y transcriptoma y de A. rabiei y la predicción de su secretoma. Además, se han identificado más de 500 genes que están más expresa dos, o sólo expresados, durante la infección. El análisis de estos genes revela las es trategias que utiliza A. rabiei para infectar a su huésped. Los genes candidatos de patogenicidad identificados incluyen enzimas degradativas, genes implicados en la defensa frente a los compuestos fungotóxicos producidos por la planta y en la producción de fitotoxinas., Este estudio ha sido financiado por los proyectos BMZ/GTZ project AscoSeq 331 501 0004 y FP7-P.

Overview of the heat stress conditions used in the study. (A) Pictograph showing the stress regime used for plant treatment as described in material and methods. (B) Representative photos of flower buds corresponding to different pollen stages. (C) Photos of pollen cells from different developmental stages, stained with the Alexander dye. Positive staining indicates viable pollen. (D) Bright field image of pollen at tetrad stage and fluorescent image of post-meiotic and mature pollen stained with DAPI, showing the single and double nuclei, respectively. (DOCX 2811 kb)

Primer sequences of selected sncRNAs for qPCR validation. (DOCX 13 kb)

Trabajo presentado en la International Conference Plant Diseases and Resistance Mechanics, celebrada en Viena del 20 al 22 de febrero de 2013., The MACE (Massive Analysis of cDNA Ends) technology is presented. MACE allows ultrdeep transcription profiling without PCR introduced bias by combining second generation sequencing and the TreQuant technology.

Background: The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database. Methods: Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions. Results: We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found. Conclusions: The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

Póster presentado en la First Legume Society Conference, celebrada en Novi Sad (Serbia) del 9 al 11 de mayo de 2013., Didymella pinodes, causing ascochyta blight, is one of the most important pea pathogens. Despite the devastating consequences of this disease, very little is known about the mechanisms of resistance in the host. We employed the open2architecture transcriptome profiling technique deepSuperSAGE, coupled with next2generation sequencing, to identify pea2specific genes involved in the resistance to this important disease in the resistant Pisum sativum ssp. syriacum accession P665.Two deepSuperSAGE libraries were constructed from leaf RNA of infected and control plants, yielding a total of 17,561 different UniTags. 70% of them could be assigned to known sequences from pea or other plants. 509 UniTags were significantly differentially expressed (p < 0.05; fold change ≥ 2, ≤ 2) in inoculated versus control plants. This study provides a detailed picture of all expressed genes and metabolic pathways differentially regulated during D. pinodes3Pisum sativum interaction and contribute to the identification of candidate resistance genes. Protease inhibitors, antifungal compounds, strengthening of host cell walls, detoxification of D. pinodes toxins and repair of cell walls could contribute to resistance. Ethylene, ABA and indole232acetic acid pathways were up2, while the GA pathway was down2regulated.