Genotyping, phenotyping and transcriptomic analysis of accessions of Vicia faba, Pisum sativum and Vigna unguiculata

Abstract

[SPA] Las legumbres son el segundo cultivo con mayor producción mundial después de los cereales, por lo que su importancia para consumo animal y humano es crucial. El haba (Vicia faba), el guisante (Pisum sativum) y el caupí (Vigna unguiculata) son especies de leguminosas con interés agronómico en la Unión Europea. El objetivo de esta tesis fue la selección de variedades de estas especies, mediante técnicas de fenotipado, genotipado y análisis transcriptómico, que permitirá la obtención de líneas de mejora con cualidades nutricionales y agronómicas óptimas para una producción sostenible y competitiva de proteínas en Europa. Dentro de las cualidades nutricionales nos centramos sobre todo en la cantidad de proteína en semilla. Y en cuanto a las características agronómicas nos centramos en la resistencia a estrés hídrico y en la productividad. Dentro de los estudios realizados con el caupí, en el primero de ellos se evaluaron 12 genotipos en tres localizaciones distintas de la Península Ibérica para determinar los componentes de la varianza y su estabilidad genética y ambiental. Los resultados mostraron variaciones interesantes entre las accesiones estudiadas las cuales podrían incorporarse en un programa de mejora. En cuanto al haba y el guisante, se hizo un análisis de genotipado de diferentes accesiones europeas. En este estudio se usó un protocolo simple codificando las muestras permitiendo el genotipado de material vegetal basado en un número mínimo de lecturas de secuencias mediante Next-Generation Sequencing (NGS). Los resultados demostraron la viabilidad de NGS para genotipar múltiples muestras usando la codificación de estas. Adicionalmente se estudió en caupí si la síntesis de proteínas de esta leguminosa está controlada por el reloj circadiano de la planta, donde se usó una línea de referencia. Para ello se analizaron la expresión de genes de referencia (como ELONGATION FACTOR 1-A), de ritmo circadiano (VuLHY, VuTOC1, VuGI y VuELF3,) y de almacenamiento de proteínas (VuLEG, VuLEGJ, y VuCVC) en distintos tejidos de la planta (hojas, vainas y semillas) con diferentes estadios de desarrollo. Con este estudio se confirmó que la síntesis de proteínas de almacenamiento puede estar regulada por el ritmo circadiano de la planta. Hay que tener en cuenta los cambios en la expresión génica afectados por el ritmo circadiano, para poder analizar los patrones de la expresión de estos genes, por ejemplo para analizar las accesiones que difieren en el contenido de proteínas. [ENG] Legumes are the second crop with the highest world production after cereals, so its importance for animal and human nutrition is crucial. Faba bean (Vicia faba), pea (Pisum sativum) and cowpea (Vigna unguiculata) are legume species with agronomic interest in the European Union. The main objective of this project was the selection of varieties of the above mentioned species, using techniques of phenotyping, genotyping and transcriptomic analysis. This techniques allow us the improvement of varieties with optimum nutritional and agronomic qualities, for a sustainable and competitive production of proteins in Europe. Within the nutritional qualities we focus mainly on the amount of seed protein. And as for the agronomic characteristics, we focus on resistance to drought. Among the studies carried out with cowpea, 12 genotypes were evaluated at three different locations in the Iberian Peninsula. Components of variance and their genetic and environmental stability were determined for these genotypes. The results demonstrate interesting variations among the studied accessions with which a breeding program could be performed. As for bean and pea, a genotyping analysis of different European accessions was perfomed with the aim to analyse the genetic relation among these accessions. Genotyping of accessions is a prerequisite for the exploitation of natural genetic variation. In this study we developed a simple protocol based on sample barcoding allowing the genotyping of plant material based on a minimum number of Next-Generation Sequencing (NGS) reads. The results demonstrated the feasibility of NGS-based genotyping using multiple barcoded samples. A further study was conducted on cowpea with the aim to investigate whether the protein synthesis is controlled by the circadian clock of the plant. A reference line of cowpea was used for this experiment. The expression of reference genes (such as ELONGATION FACTOR 1-A), circadian rhythm genes (VuLHY, VuTOC1, VuGI and VuELF3) and protein storage genes (VuLEG, VuLEGJ, and VuCVC) was measured in different tissues (leaves, pods and seeds) at different stages of development. This study confirmed that storage protein deposition may be circadian regulated by the circadian XIV clock. Diel changes in expression need to be taken into account when analysing expression patterns of these genes i.e. in accessions differing in protein content.

[ENG] Legumes are the second crop with the highest world production after cereals, so its importance for animal and human nutrition is crucial. Faba bean (Vicia faba), pea (Pisum sativum) and cowpea (Vigna unguiculata) are legume species with agronomic interest in the European Union. The main objective of this project was the selection of varieties of the above mentioned species, using techniques of phenotyping, genotyping and transcriptomic analysis. This techniques allow us the improvement of varieties with optimum nutritional and agronomic qualities, for a sustainable and competitive production of proteins in Europe. Within the nutritional qualities we focus mainly on the amount of seed protein. And as for the agronomic characteristics, we focus on resistance to drought. Among the studies carried out with cowpea, 12 genotypes were evaluated at three different locations in the Iberian Peninsula. Components of variance and their genetic and environmental stability were determined for these genotypes. The results demonstrate interesting variations among the studied accessions with which a breeding program could be performed. As for bean and pea, a genotyping analysis of different European accessions was perfomed with the aim to analyse the genetic relation among these accessions. Genotyping of accessions is a prerequisite for the exploitation of natural genetic variation. In this study we developed a simple protocol based on sample barcoding allowing the genotyping of plant material based on a minimum number of Next-Generation Sequencing (NGS) reads. The results demonstrated the feasibility of NGS-based genotyping using multiple barcoded samples. A further study was conducted on cowpea with the aim to investigate whether the protein synthesis is controlled by the circadian clock of the plant. A reference line of cowpea was used for this experiment. The expression of reference genes (such as ELONGATION FACTOR 1-A), circadian rhythm genes (VuLHY, VuTOC1, VuGI and VuELF3) and protein storage genes (VuLEG, VuLEGJ, and VuCVC) was measured in different tissues (leaves, pods and seeds) at different stages of development. This study confirmed that storage protein deposition may be circadian regulated by the circadian XIV clock. Diel changes in expression need to be taken into account when analysing expression patterns of these genes i.e. in accessions differing in protein content.