Empleo de herramientas genómicas para la búsqueda de genes candidatos relacionados con la resistencia a Ascochyta fabae y Orobanche crenata en habas (Vicia faba L.)

El objetivo principal de este trabajo ha sido desarrollar y explotar nuevas herramientas genómicas en habas para desvelar a nivel molecular posibles mecanismos de defensa frente al hongo patógeno Ascochyta fabae y la planta parásita Orobanche crenata, causantes de grandes pérdidas en su producción. Se ha pretendido ampliar la información genética sobre dichos mecanismos de resistencia e identificar posibles genes candidatos donde desarrollar marcadores diagnóstico que permitan extrapolar los resultados de QTL estables entre poblaciones y facilitar la selección precoz de materiales en sus programas de mejora. Para conseguir estos fines, se han empleado distintas herramientas genómicas (estructural, comparada y funcional). Así, en el capítulo II, se han utilizado la genómica estructural y la macrosintenia existente entre leguminosas, para saturar dos de los mapas genéticos disponibles por el grupo con marcadores relacionados con la resistencia a ambos patógenos, descritos en otras leguminosas. Dicha saturación, ha permitido refinar la posición de los QTLs existentes e identificar marcadores estrechamente ligados que podrían facilitar el proceso de selección. Además, el genotipado de estos nuevos marcadores ha ayudado al desarrollo de un mapa consenso para este cultivo. En el capítulo III, utilizando herramientas de genómica funcional, se ha tratado de profundizar en las rutas implicadas en la interacción planta-patógeno e identificar potenciales genes de resistencia que pudieran ser últiles en la mejora asistida por marcadores. Para ello se ha caracterizado el transcriptoma de dos líneas parentales (29H y Vf136) sometidas a la infección por A. fabae y obtenido sus perfiles de expresión génica. Aparte de proporcionar nueva información genómica, el estudio han revelado una amplia variedad de posibles mecanismos y rutas implicadas. El gran número SNPs e InDels identificados en los transcritos ofrece, además, una posibilidad rentable de saturar los mapas disponibles y de identificar genes responsables de la resistencia. Para finalizar, en el capítulo IV, se ha genotipado un conjunto de SNPs expresados diferencialmente entre ambos parentales, relacionados con mecanismos de resistencia. Ello ha permitido situar nuevos marcadores más robustos y transferibles (SNPs y ESTs) dentro de los intervalos de confianza de los QTLs de resistencia ya descritos, facilitando su comparación con otras poblaciones de habas y con otras leguminosas.The main objective of this work has been to develop and exploit new genomic tools in faba bean in order to reveal at molecular level possible defense mechanisms against the pathogenic fungus Ascochyta fabae and the parasitic plant Orobanche crenata, causing severe yield losses in this crop. The approach aimed to expand the genetic information on the resistance mechanisms involved and to identify possible candidate genes useful to develop diagnostic markers for validation of stable QTL among populations and for the efficient selection of superior materials in breeding programs. To achieve these ends, different genomic tools (structural, comparative and functional) have been applied. Thus, in Chapter II, both the structural genomics and the synteny among legumes have been exploited to saturate two of the genetic maps available by the group using markers related with the resistance to both pathogens, described in other legumes. The saturation allowed to refine the position of the underlying QTLs and to identify closely linked markers that could facilitate the selection process. In addition, the new markers were used as a bridge to develop a consensus map in this crop. In Chapter III, functional genomics tools have been applied to gain insight into the pathways involved in the plant-pathogen interactions and to identify potential resistance genes to be further used in marker assisted selection. The faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to A. fabae infection were characterized.in order to obtain its gene expression profiles. Apart from providing new genomic information, the study has revealed a wide variety of possible mechanisms and pathways involved. The large number of SNPs and InDels identified in the transcripts also offers a cost-effective way to saturate available maps and to identify genes responsible for resistance. Finally, in chapter IV, a set of differentially expressed SNPs between both parental lines, related with resistance mechanisms has been genotyped. The approach has allowed placing more robust and transferable markers (SNPs and ESTs) within the confidence intervals of the resistance QTLs already described, facilitating their comparison among faba bean and legume species

Large-Scale Transcriptome Analysis in Faba Bean (Vicia faba L.) under Ascochyta fabae Infection

Faba bean is an important food crop worldwide. However, progress in faba bean genomics lags far behind that of model systems due to limited availability of genetic and genomic information. Using the Illumina platform the faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to Ascochyta fabae infection have been characterized. De novo transcriptome assembly provided a total of 39,185 different transcripts that were functionally annotated, and among these, 13,266 were assigned to gene ontology against Arabidopsis. Quality of the assembly was validated by RT-qPCR amplification of selected transcripts differentially expressed. Comparison of faba bean transcripts with those of better-characterized plant genomes such as Arabidopsis thaliana, Medicago truncatula and Cicer arietinum revealed a sequence similarity of 68.3%, 72.8% and 81.27%, respectively. Moreover, 39,060 single nucleotide polymorphism (SNP) and 3,669 InDels were identified for genotyping applications. Mapping of the sequence reads generated onto the assembled transcripts showed that 393 and 457 transcripts were overexpressed in the resistant (29H) and susceptible genotype (Vf136), respectively. Transcripts involved in plant-pathogen interactions such as leucine rich proteins (LRR) or plant growth regulators involved in plant adaptation to abiotic and biotic stresses were found to be differently expressed in the resistant line. The results reported here represent the most comprehensive transcript database developed so far in faba bean, providing valuable information that could be used to gain insight into the pathways involved in the resistance mechanism against A. fabae and to identify potential resistance genes to be further used in marker assisted selection.This research was supported by the European Community's Seventh Framework Programme (FP7/ 2007-2013) under the grant agreement n°FP7-613551, LEGATO project; the 2014-2020 "Programa Operativo de Crecimiento Inteligente" (RTA2013-00025) and RTA2010-00059, the IPT-2011-1259-010000 project and the P10-CVI-6075, all co-financed by ERDF and the last by the Andalusian regional government.Peer reviewe

Análisis de la expresión diferencial de cDNAs y mapeo de genes candidatos para saturar QTLs de resistencia a Ascochyta en habas (Vicia faba L.)

Trabajo presentado en el XL Congreso de la Sociedad Española de Genética, celebrado en Córdoba del 16 al 18 de septiembre de 2015.Aunque las habas (Vicia faba L.) son un importante cultivo alimentario en todo el mundo y una fuente de proteínas en la dieta de países en vías de desarrollo, su información genética y genómica están aún muy por detrás de las especies modelo. Con el fin de contribuir en su progreso, en este estudio se ha realizado una caracterización exhaustiva del transcriptoma de dos genotipos de haba (29H y Vf136) con reacción diferencial frente a la infección por el hongo Ascochyta fabae utilizando la plataforma de secuenciación Illumina. A partir de la información obtenida tras el ensamblaje del nuevo transcriptoma se han identificado 21.243 unigenes que han sido anotados con diferentes bases de datos. De ellos se ha obtenido un subconjunto de 850 expresados diferencialmente entre el parental resistente (29H) y el susceptible (Vf136) a p < 0.05. Además se han identificado 39.060 SNPs y 3.669 INDELs. De los 850 unigenes se han seleccionado 128 por presentar algún SNP y se utilizaron para genotipar los individuos de esta población mediante los sistemas de genotipado KASPar e iPLEX-Sequenom. Estos unigenes expresados diferencialmente tras la infección del hongo Ascochyta fabae pueden identificar genes candidatos asociados a la resistencia a estreses bióticos en esta leguminosa. La integración de estos genes en el mapa de referencia disponible nos ayudará a actualizar el mapa de ligamiento de la población y a saturar QTLs relacionados con la resistencia a Ascochyta identificados en estudios previos (1, 2, 3). Los resultados serán un importante recurso para futuros estudios básicos y aplicados en leguminosas de importancia económica, proporcionand o información valiosa para comprender mejor las vías involucradas en el mecanismo de resistencia contra A. fabae y para identificar genes de resistencia potenciales para ser utilizado en la selección asistida por marcadores.N

Large-scale transcriptome analysis in faba bean (Vicia faba L.)

Trabajo presentado en la 6th International Food Legume Research Conference y la 7th International Conference of Legume Genetics and Genomics (IFLRC VI & ICLGG VII), celebrada en Saskatoon (Canadá) del 7 al 11 de julio de 2014.Although faba bean is one of the biggest traded pulse crops, genomic resources are still limited. A comprehensive characterization of the faba bean transcriptome was performed using the Illumina platform. Two faba bean genotypes (Vf29H and Vf136) with differential reaction to Ascochyta fabae and 106.397 ESTs found in different public databases were used for de novo transcriptome assembly using a work flow with OASES, and CAP3. The resulting unigenes were annotated with Full-LengtherNext. The preliminary analysis of the 120.922 unigenes collection revealed that 20,804 out of the 39,269 were non-redundant transcripts based on orthologue ID. 2,752putative ncRNAs were found. Futhermore, 7,165 unigenes lacking homology could be considered as putative legume-specific genes. Single nucleotide polymorphisms identified through data mining provide a database for genotyping and integration of translational genomics in breeding programmes. In addition, the faba bean and chickpea transcriptome alignment revealed that 81.27% of the faba bean transcripts where present in the chickpea transcriptome despite of using a single faba bean source of tissue. As the genotypes sequenced show differential reaction to A. fabae and the cDNA libraries were constructed using inoculated and non-inoculated leaves, genes with differential expression in both genotypes were characterized. Thus, 348 and 327 transcripts where found to be unique in resistant and susceptible genotypes, respectively. On the other hand, 212 and 282 transcripts were found to be overexpressed in both lines. These transcripts might represent candidate genes associated with the A. fabae resistance.N

Strategies to identify candidate genes controlling Ascochyta blight in different legume crops

Trabajo presentado en la First Legume Society Conference, celebrada en Novi Sad (Serbia) del 9 al 11 de mayo de 2013.Until now complete resistance to Ascochyta blight has not been reported neither in chickpea nor in faba bean. However, several QTL`s have benn located in chickpeaLGs 2, 3, 4, 6 and 8. The most significant ones are those in LGs 2 (QTLar3) and 4 (QTLar1).Peer Reviewe

AutoFlow, a Versatile Workflow Engine Illustrated by Assembling an Optimised de novo Transcriptome for a Non-Model Species, such as Faba Bean (Vicia faba)

The use of workflows to automate routine tasks is an absolute requirement in many bioinformatics fields. Current workflow manager systems usually compromise between providing a user-friendly interface and constructing complex, scalable pipelines. We present AutoFlow, a Ruby-based workflow engine devoid of graphic interface and tool repository, that is useful in most computer systems and most workflow requirements in any scientific field. It accepts any local or remote command-line software and converts one workflow into a series of independent tasks. It has been supplied with control patterns that allow for iterative task capability, supporting static and dynamic variables for decision-making or chaining workflows, as well as debugging utilities that include graphs, file searching, functional consistency and timing. Two proof-of-concept cases are presented to illustrate AutoFlow capabilities, and a case-of-use illustrates the automated construction of the best transcriptome for a non-model species (Vicia faba) after analysis of several combinations of Illumina reads and Sanger sequences with different assemblers and different parameters in a complex and repetitive workflow where branching and convergent tasks were used and internal, automated decisions were taken. The workflow finally produced an optimal transcriptome of 118,188 transcripts, of which 38,004 were annotated, 10,516 coded for a complete protein, 3,314 were putatively new faba-specific transcripts, and 23,727 were considered the representative transcriptome of V. faba.Peer reviewe

CARACTERIZAÇÃO DA PAISAGEM CÁRSTICA DO PARQUE ESTADUAL DE CAMPINHOS

Abstract Background Faba bean (Vicia faba L.) is among the earliest domesticated crops from the Near East. Today this legume is a key protein feed and food worldwide and continues to serve an important role in culinary traditions throughout Middle East, Mediterranean region, China and Ethiopia. Adapted to a wide range of soil types, the main faba bean breeding objectives are to improve yield, resistance to biotic and abiotic stresses, seed quality and other agronomic traits. Genomic approaches aimed at enhancing faba bean breeding programs require high-quality genetic linkage maps to facilitate quantitative trait locus analysis and gene tagging for use in a marker-assisted selection. The objective of this study was to construct a reference consensus map in faba bean by joining the information from the most relevant maps reported so far in this crop. Results A combination of two approaches, increasing the number of anchor loci in diverse mapping populations and joining the corresponding genetic maps, was used to develop a reference consensus map in faba bean. The map was constructed from three main recombinant inbreed populations derived from four parental lines, incorporates 729 markers and is based on 69 common loci. It spans 4,602 cM with a range from 323 to 1041 loci in six main linkage groups or chromosomes, and an average marker density of one locus every 6 cM. Locus order is generally well maintained between the consensus map and the individual maps. Conclusion We have constructed a reliable and fairly dense consensus genetic linkage map that will serve as a basis for genomic approaches in faba bean research and breeding. The core map contains a larger number of markers than any previous individual map, covers existing gaps and achieves a wider coverage of the large faba bean genome as a whole. This tool can be used as a reference resource for studies in different genetic backgrounds, and provides a framework for transferring genetic information when using different marker technologies. Combined with syntenic approaches, the consensus map will increase marker density in selected genomic regions and will be useful for future faba bean molecular breeding applications.This research has been funded by the Spanish projects AGL2008-02305/AGR, RTA2010-00059 and IPT-2011-1259-010000, co-financed by FEDER, and by the EU project GLIP (FP6- FOOD-CT-2004-506223). The authors thank Juan Prieto, Gloria de Lara and Ana Pozo for excellent technical assistance in the laboratory, greenhouse and field. We are grateful to A. Di Pietro for carefully reading the manuscriptPeer Reviewe

High-dose corticosteroid pulse therapy increases the survival rate in COVID-19 patients at risk of hyper-inflammatory response.

ObjectiveTest whether high dose corticosteroid pulse therapy (HDCPT) with either methylprednisolone or dexamethasone is associated with increased survival in COVID-19 patients at risk of hyper-inflammatory response. Provide some initial diagnostic criteria using laboratory markers to stratify these patients.MethodsThis is a prospective observational study, 318 met the inclusion criteria. 64 patients (20.1%) were treated with HDCPT by using at least 1.5mg/kg/24h of methylprednisolone or dexamethasone equivalent. A multivariate Cox regression (controlling for co-morbidities and other therapies) was carried out to determine whether HDCPT (among other interventions) was associated with decreased mortality. We also carried out a 30-day time course analysis of laboratory markers between survivors and non-survivors, to identify potential markers for patient stratification.ResultsHDCPT showed a statistically significant decrease in mortality (HR = 0.087 [95% CI 0.021-0.36]; P = 40 pg/ml, and/or two of the following: C-reactive protein > = 100 mg/L, D-dimer > = 1000 ng/ml, ferritin > = 500 ng/ml and lactate dehydrogenase > = 300 U/L).ConclusionsHDCPT can be an effective intervention to increase COVID-19 survival rates in patients at risk of developing a COVID-19 hyper-inflammatory response, laboratory marker tests can be used to stratify these patients who should be given HDCPT. This study is not a randomized clinical trial (RCT). Future RCTs should be carried out to confirm the efficacy of HDCPT to increase the survival rates of COVID-19