Evapotranspiration and crop coefficients of rice (Oryza sativa L.) under sprinkler irrigation in a semiarid climate determined by the surface renewal method

The evapotranspiration (ETc) of sprinkler-irrigated rice was determined for the semiarid conditions of NE Spain during 2001, 2002 and 2003. The surface renewal method, after calibration against the eddy covariance method, was used to obtain values of sensible heat flux (H) from high-frequency temperature readings. Latent heat flux values were obtained by solving the energy balance equation. Finally, lysimeter measurements were used to validate the evapotranspiration values obtained with the surface renewal method. Seasonal rice evapotranspiration was about 750–800 mm. Average daily ETc for mid-season (from 90 to 130 days after sowing) was 5.1, 4.5 and 6.1 mm day−1 for 2001, 2002 and 2003, respectively. The experimental weekly crop coefficients fluctuated in the range of 0.83–1.20 for 2001, 0.81–1.03 for 2002 and 0.84–1.15 for 2003. The total growing season was about 150–160 days. In average, the crop coefficients for the initial (Kcini), mid-season (Kcmid) and late-season stages (Kcend) were 0.92, 1.06 and 1.03, respectively, the length of these stages being about 55, 45 and 25 days, respectively.

Ground cover and leaf area index relationship in a grass, legume and crucifer crop

Canopy characterization is essential for describing the interaction of a crop with its environment. The goal of this work was to determine the relationship between leaf area index (LAI) and ground cover (GC) in a grass, a legume and a crucifer crop, and to assess the feasibility of using these relationships as well as LAI-2000 readings to estimate LAI. Twelve plots were sown with either barley (Hordeum vulgare L.), vetch (Vicia sativa L.), or rape (Brassica napus L.). On 10 sampling dates the LAI (both direct and LAI-2000 estimations), fraction intercepted of photosynthetically active radiation (FIPAR) and GC were measured. Linear and quadratic models fitted to the relationship between the GC and LAI for all of the crops, but they reached a plateau in the grass when the LAI mayor que 4. Before reaching full cover, the slope of the linear relationship between both variables was within the range of 0.025 to 0.030. The LAI-2000 readings were linearly correlated with the LAI but they tended to overestimation. Corrections based on the clumping effect reduced the root mean square error of the estimated LAI from the LAI-2000 readings from 1.2 to less than 0.50 for the crucifer and the legume, but were not effective for barley.

Aplicación de imagen hiperespectral para observar el efecto de la salinidad en hojas de lechuga.

La salinización en suelos de cultivos es un fenómeno muy relevante en el Sureste español que se produce por el empleo de aguas de riego salinas o de mala calidad. La lechuga (Lactuca sativa L.) es uno de los cultivos hortícolas de mayor implantación en esta área, destinándose principalmente para consumo en fresco y en productos cuarta gama. La salinidad puede afectar a la productividad y calidad del cultivo, sin embargo, en determinadas concentraciones investigaciones previas muestran que la salinidad puede favorecer la conservación de las hojas tras el corte, disminuyendo los procesos de degradación enzimática y el desarrollo de microorganismos. El objetivo del presente trabajo es evaluar la viabilidad de la imagen hiperespectral (400 a 1000 nm) para identificar la influencia del estrés salino en lechuga ?baby? recién recolectada. Para ello, se han adquirido imágenes de 40 hojas de diferentes lechugas sometidas a tres soluciones salinas diferentes y a una solución control. Dichas imágenes fueron sometidas a preprocesado de espectros (suavizado con el algoritmo Savitsky-Golay + normalización SNV), combinado con Análisis de Componentes Principales. Las imágenes virtuales de scores generadas con el modelo muestran diferencias progresivas en los valores asignados a los píxeles de las imágenes a medida que aumenta la concentración salina de la solución aplicada al cultivo. Se observa cómo la solución salina afecta a la hoja cambiando la coloración de las zonas medias, posiblemente debido a la concentración de solutos. La interpretación de los loadings de estos modelos permite conocer cómo afecta la salinidad al comportamiento espectral de las hojas. La imagen hiperespectral puede tener un gran potencial para identificar los l��mites de salinidad tolerados y evitar concentraciones tóxicas que afecten negativamente la vida útil de las hortalizas de hoja. Este conocimiento permitirá desarrollar índices multiespectrales capaces de identificar hojas afectadas por salinidad durante su cultivo y procesado postcosecha.

Evaluation of cover crops based on characteristics of nitrogen use, and aerial and root growth

La caracterización de los cultivos cubierta (cover crops) puede permitir comparar la idoneidad de diferentes especies para proporcionar servicios ecol��gicos como el control de la erosión, el reciclado de nutrientes o la producción de forrajes. En este trabajo se estudiaron bajo condiciones de campo diferentes técnicas para caracterizar el dosel vegetal con objeto de establecer una metodología para medir y comparar las arquitecturas de los cultivos cubierta más comunes. Se estableció un ensayo de campo en Madrid (España central) para determinar la relación entre el índice de área foliar (LAI) y la cobertura del suelo (GC) para un cultivo de gramínea, uno de leguminosa y uno de crucífera. Para ello se sembraron doce parcelas con cebada (Hordeum vulgare L.), veza (Vicia sativa L.), y colza (Brassica napus L.). En 10 fechas de muestreo se midieron el LAI (con estimaciones directas y del LAI-2000), la fracción interceptada de la radiación fotosintéticamente activa (FIPAR) y la GC. Un experimento de campo de dos años (Octubre-Abril) se estableció en la misma localización para evaluar diferentes especies (Hordeum vulgare L., Secale cereale L., x Triticosecale Whim, Sinapis alba L., Vicia sativa L.) y cultivares (20) en relación con su idoneidad para ser usadas como cultivos cubierta. La GC se monitorizó mediante análisis de imágenes digitales con 21 y 22 muestreos, y la biomasa se midió 8 y 10 veces, respectivamente para cada año. Un modelo de Gompertz caracterizó la cobertura del suelo hasta el decaimiento observado tras las heladas, mientras que la biomasa se ajustó a ecuaciones de Gompertz, logísticas y lineales-exponenciales. Al final del experimento se determinaron el C, el N y el contenido en fibra (neutrodetergente, ácidodetergente y lignina), así como el N fijado por las leguminosas. Se aplicó el análisis de decisión multicriterio (MCDA) con objeto de obtener un ranking de especies y cultivares de acuerdo con su idoneidad para actuar como cultivos cubierta en cuatro modalidades diferentes: cultivo de cobertura, cultivo captura, abono verde y forraje. Las asociaciones de cultivos leguminosas con no leguminosas pueden afectar al crecimiento radicular y a la absorción de N de ambos componentes de la mezcla. El conocimiento de cómo los sistemas radiculares específicos afectan al crecimiento individual de las especies es útil para entender las interacciones en las asociaciones, así como para planificar estrategias de cultivos cubierta. En un tercer ensayo se combinaron estudios en rhizotrones con extracción de raíces e identificación de especies por microscopía, así como con estudios de crecimiento, absorción de N y 15N en capas profundas del suelo. Las interacciones entre raíces en su crecimiento y en el aprovisionamiento de N se estudiaron para dos de los cultivares mejor valorados en el estudio previo: uno de cebada (Hordeum vulgare L. cv. Hispanic) y otro de veza (Vicia sativa L. cv. Aitana). Se añadió N en dosis de 0 (N0), 50 (N1) y 150 (N2) kg N ha-1. Como resultados del primer estudio, se ajustaron correctamente modelos lineales y cuadráticos a la relación entre la GC y el LAI para todos los cultivos, pero en la gramínea alcanzaron una meseta para un LAI>4. Antes de alcanzar la cobertura total, la pendiente de la relación lineal entre ambas variables se situó en un rango entre 0.025 y 0.030. Las lecturas del LAI-2000 estuvieron correlacionadas linealmente con el LAI, aunque con tendencia a la sobreestimación. Las correcciones basadas en el efecto de aglutinación redujeron el error cuadrático medio del LAI estimado por el LAI-2000 desde 1.2 hasta 0.5 para la crucífera y la leguminosa, no siendo efectivas para la cebada. Esto determinó que para los siguientes estudios se midieran únicamente la GC y la biomasa. En el segundo experimento, las gramíneas alcanzaron la mayor cobertura del suelo (83-99%) y la mayor biomasa (1226-1928 g m-2) al final del mismo. Con la mayor relación C/N (27-39) y contenido en fibra digestible (53-60%) y la menor calidad de residuo (~68%). La mostaza presentó elevadas GC, biomasa y absorción de N en el año más templado en similitud con las gramíneas, aunque escasa calidad como forraje en ambos años. La veza presentó la menor absorción de N (2.4-0.7 g N m-2) debido a la fijación de N (9.8-1.6 g N m-2) y escasa acumulación de N. El tiempo térmico hasta alcanzar el 30% de GC constituyó un buen indicador de especies de rápida cubrición. La cuantificación de las variables permitió hallar variabilidad entre las especies y proporcionó información para posteriores decisiones sobre la selección y manejo de los cultivos cubierta. La agregación de dichas variables a través de funciones de utilidad permitió confeccionar rankings de especies y cultivares para cada uso. Las gramíneas fueron las más indicadas para los usos de cultivo de cobertura, cultivo captura y forraje, mientras que las vezas fueron las mejor como abono verde. La mostaza alcanzó altos valores como cultivo de cobertura y captura en el primer año, pero el segundo decayó debido a su pobre actuación en los inviernos fríos. Hispanic fue el mejor cultivar de cebada como cultivo de cobertura y captura, mientras que Albacete como forraje. El triticale Titania alcanzó la posición más alta como cultiva de cobertura, captura y forraje. Las vezas Aitana y BGE014897 mostraron buenas aptitudes como abono verde y cultivo captura. El MCDA permitió la comparación entre especies y cultivares proporcionando información relevante para la selección y manejo de cultivos cubierta. En el estudio en rhizotrones tanto la mezcla de especies como la cebada alcanzaron mayor intensidad de raíces (RI) y profundidad (RD) que la veza, con valores alrededor de 150 cruces m-1 y 1.4 m respectivamente, comparados con 50 cruces m-1 y 0.9 m para la veza. En las capas más profundas del suelo, la asociación de cultivos mostró valores de RI ligeramente mayores que la cebada en monocultivo. La cebada y la asociación obtuvieron mayores valores de densidad de raíces (RLD) (200-600 m m-3) que la veza (25-130) entre 0.8 y 1.2 m de profundidad. Los niveles de N no mostraron efectos claros en RI, RD ó RLD, sin embargo, el incremento de N favoreció la proliferación de raíces de veza en la asociación en capas profundas del suelo, con un ratio cebada/veza situado entre 25 a N0 y 5 a N2. La absorción de N de la cebada se incrementó en la asociación a expensas de la veza (de ~100 a 200 mg planta-1). Las raíces de cebada en la asociación absorbieron también más nitrógeno marcado de las capas profundas del suelo (0.6 mg 15N planta-1) que en el monocultivo (0.3 mg 15N planta-1). ABSTRACT Cover crop characterization may allow comparing the suitability of different species to provide ecological services such as erosion control, nutrient recycling or fodder production. Different techniques to characterize plant canopy were studied under field conditions in order to establish a methodology for measuring and comparing cover crops canopies. A field trial was established in Madrid (central Spain) to determine the relationship between leaf area index (LAI) and ground cover (GC) in a grass, a legume and a crucifer crop. Twelve plots were sown with either barley (Hordeum vulgare L.), vetch (Vicia sativa L.), or rape (Brassica napus L.). On 10 sampling dates the LAI (both direct and LAI-2000 estimations), fraction intercepted of photosynthetically active radiation (FIPAR) and GC were measured. A two-year field experiment (October-April) was established in the same location to evaluate different species (Hordeum vulgare L., Secale cereale L., x Triticosecale Whim, Sinapis alba L., Vicia sativa L.) and cultivars (20) according to their suitability to be used as cover crops. GC was monitored through digital image analysis with 21 and 22 samples, and biomass measured 8 and 10 times, respectively for each season. A Gompertz model characterized ground cover until the decay observed after frosts, while biomass was fitted to Gompertz, logistic and linear-exponential equations. At the end of the experiment C, N, and fiber (neutral detergent, acid and lignin) contents, and the N fixed by the legumes were determined. Multicriteria decision analysis (MCDA) was applied in order to rank the species and cultivars according to their suitability to perform as cover crops in four different modalities: cover crop, catch crop, green manure and fodder. Intercropping legumes and non-legumes may affect the root growth and N uptake of both components in the mixture. The knowledge of how specific root systems affect the growth of the individual species is useful for understanding the interactions in intercrops as well as for planning cover cropping strategies. In a third trial rhizotron studies were combined with root extraction and species identification by microscopy and with studies of growth, N uptake and 15N uptake from deeper soil layers. The root interactions of root growth and N foraging were studied for two of the best ranked cultivars in the previous study: a barley (Hordeum vulgare L. cv. Hispanic) and a vetch (Vicia sativa L. cv. Aitana). N was added at 0 (N0), 50 (N1) and 150 (N2) kg N ha-1. As a result, linear and quadratic models fitted to the relationship between the GC and LAI for all of the crops, but they reached a plateau in the grass when the LAI > 4. Before reaching full cover, the slope of the linear relationship between both variables was within the range of 0.025 to 0.030. The LAI-2000 readings were linearly correlated with the LAI but they tended to overestimation. Corrections based on the clumping effect reduced the root mean square error of the estimated LAI from the LAI-2000 readings from 1.2 to less than 0.50 for the crucifer and the legume, but were not effective for barley. This determined that in the following studies only the GC and biomass were measured. In the second experiment, the grasses reached the highest ground cover (83- 99%) and biomass (1226-1928 g/m2) at the end of the experiment. The grasses had the highest C/N ratio (27-39) and dietary fiber (53-60%) and the lowest residue quality (~68%). The mustard presented high GC, biomass and N uptake in the warmer year with similarity to grasses, but low fodder capability in both years. The vetch presented the lowest N uptake (2.4-0.7 g N/m2) due to N fixation (9.8-1.6 g N/m2) and low biomass accumulation. The thermal time until reaching 30% ground cover was a good indicator of early coverage species. Variable quantification allowed finding variability among the species and provided information for further decisions involving cover crops selection and management. Aggregation of these variables through utility functions allowed ranking species and cultivars for each usage. Grasses were the most suitable for the cover crop, catch crop and fodder uses, while the vetches were the best as green manures. The mustard attained high ranks as cover and catch crop the first season, but the second decayed due to low performance in cold winters. Hispanic was the most suitable barley cultivar as cover and catch crop, and Albacete as fodder. The triticale Titania attained the highest rank as cover and catch crop and fodder. Vetches Aitana and BGE014897 showed good aptitudes as green manures and catch crops. MCDA allowed comparison among species and cultivars and might provide relevant information for cover crops selection and management. In the rhizotron study the intercrop and the barley attained slightly higher root intensity (RI) and root depth (RD) than the vetch, with values around 150 crosses m-1 and 1.4 m respectively, compared to 50 crosses m-1 and 0.9 m for the vetch. At deep soil layers, intercropping showed slightly larger RI values compared to the sole cropped barley. The barley and the intercropping had larger root length density (RLD) values (200-600 m m-3) than the vetch (25-130) at 0.8-1.2 m depth. The topsoil N supply did not show a clear effect on the RI, RD or RLD; however increasing topsoil N favored the proliferation of vetch roots in the intercropping at deep soil layers, with the barley/vetch root ratio ranging from 25 at N0 to 5 at N2. The N uptake of the barley was enhanced in the intercropping at the expense of the vetch (from ~100 mg plant-1 to 200). The intercropped barley roots took up more labeled nitrogen (0.6 mg 15N plant-1) than the sole-cropped barley roots (0.3 mg 15N plant-1) from deep layers.

Multicriteria decision analysis applied to cover crop species andcultivars selection

Cover crop selection should be oriented to the achievement of specific agrosystem benefits. The covercrop, catch crop, green manure and fodder uses were identified as possible targets for selection. Theobjective was to apply multi-criteria decision analysis to evaluate different species (Hordeum vulgareL., Secale cereale L., ×Triticosecale Whim, Sinapis alba L., Vicia sativa L.) and cultivars according to theirsuitability to be used as cover crops in each of the uses. A field trial with 20 cultivars of the five specieswas conducted in Central Spain during two seasons (October?April). Measurements of ground cover, cropbiomass, N uptake, N derived from the atmosphere, C/N, dietary fiber content and residue quality werecollected. Aggregation of these variables through utility functions allowed ranking species and cultivarsfor each usage. Grasses were the most suitable for the cover crop, catch crop and fodder uses, while thevetches were the best as green manures. The mustard attained high ranks as cover and catch crop the firstseason, but the second decayed due to low performance in cold winters. Mustard and vetches obtainedworse rankings than grasses as fodder. Hispanic was the most suitable barley cultivar as cover and catchcrop, and Albacete as fodder. The triticale Titania attained the highest rank as cover and catch crop andfodder. Vetches Aitana and BGE014897 showed good aptitudes as green manures and catch crops. Thisanalysis allowed comparison among species and cultivars and might provide relevant information forcover crops selection and management.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.

Diversity and Genetic structure of the Spanish collection of durum wheat (Triticum turgidum L) landraces

The objectives of this study were to assess diversity and genetic structure of a collection of Spanish durum wheat (Triticum turgidum L) landraces, using SSRs, DArTs and gliadin-markers, and to correlate the distribution of diversity with geographic and climatic features, as well as agro-morphological traits. A high level of diversity was detected in the genotypes analyzed, which were separated into nine populations with a moderate to great genetic divergence among them. The three subspecies taxa, dicoccon, turgidum and durum, present in the collection, largely determined the clustering of the populations. Genotype variation was lower in dicoccon (one major population) and turgidum (two major populations) than in durum (five major populations). Genetic differentiation by the agro-ecological zone of origin was greater in dicoccon and turgidum than in durum. DArT markers revealed two geographic substructures, east-west for dicoccon and northeast-southwest for turgidum. The ssp. durum had a more complex structure, consisting of seven populations with high intra-population variation. DArT markers allowed the detection of subgroups within some populations, with agro-morphological and gliadin differences, and distinct agro-ecological zones of origin. Two different phylogenetic groups were detected; revealing that some durum populations were more related to ssp. turgidum from northern Spain, while others seem to be more related to durum wheats from North Africa

Obtención de clones de alcornoque (Quercus suber L.) mediante embriogénesis somática: aplicación de medios l��quidos y biorreactores en la sincronización de los procesos de desarrollo y maduración de los embriones

El alcornoque tiene un gran valor ambiental, como integrante de los ecosistemas forestales mediterráneos, e interés comercial por el valor de la bellota (alimentación del cerdo ibérico), el carbón, la madera y sobre todo por las aplicaciones industriales del corcho. Las posibilidades de mejora genética del alcornoque, como las de otras especies forestales, están limitadas por sus largos ciclos reproductivos y porque su propagación vegetativa mediante estaquillado solo es posible en estados muy juveniles. Por ello este sistema de propagación tiene muy poca, o ninguna, utilidad práctica en la mejora genética. La embriogénesis somática es la vía más apropiada para la clonación de muchas especies forestales y ha hecho posible el desarrollo a gran escala de plantaciones multivarietales de coníferas. En alcornoque es posible la regeneración completa de árboles adultos mediante embriogénesis somática. Con los protocolos actuales (en medio semisólido), los embriones se generan formando acúmulos y en la fase de multiplicación conviven embriones en distintos estados de desarrollo. Es un sistema asincrónico, con baja eficacia para la propagación en masa, que no elimina completamente las dificultades para el desarrollo de programas de mejora genética del alcornoque. En otras especies la utilización de medios l��quidos ha mejorado: la sincronización, productividad de los cultivos, el manejo y reducido los costes de producción. Por ello el desarrollo de suspensiones embriogénicas de alcornoque se plantea como una vía para aumentar la eficacia de la propagación clonal a gran escala. En la presente tesis se desarrollan cultivos embriogénicos de alcornoque en medio l��quido. El capítulo 3 aborda el establecimiento y mantenimiento de suspensiones, el capítulo 4 el desarrollo de una fase de proliferación en medio l��quido y el capítulo 5 la utilización de sistemas de cultivo en medio l��quido, estacionarios y de inmersión temporal, como vía para favorecer la maduración de los embriones somáticos. Para iniciar los cultivos en medio l��quido se emplearon agregados de embriones tomados de la fase de proliferación en medio semisólido. Cuando estos agregados se inocularon directamente en medio l��quido no se logró el establecimiento de las suspensiones. El establecimiento se consiguió empleando como inóculo las células y Resumen pequeños agregados embriogénicos, de tamaño comprendido entre 41 y 800 μm, desprendidas por agitación breve de los agregados de embriones. El mantenimiento se logró inoculando en baja densidad masas embriogénicas compactas de tamaño comprendido entre 0,8 y 1,2 mm. Estas suspensiones, muy heterogéneas, mantuvieron su capacidad de proliferación y de regeneración de embriones al menos durante diez subcultivos consecutivos. El protocolo de iniciación y mantenimiento, desarrollado inicialmente con un solo genotipo, fue eficaz cuando se probó sobre otros 11 genotipos de alcornoque. En la fase de proliferación se ensayaron tres tipos de envase y tres velocidades de agitación. La combinación envase × velocidad determinó el intercambio gaseoso, la disponibilidad de oxígeno y el estrés hidrodinámico. Los agregados embriogénicos de alcornoque crecieron incluso en condiciones de hipoxia no siendo la disponibilidad de oxígeno un factor limitante del crecimiento para tasas de trasferencia de oxígeno comprendidas entre 0,11 h-1 y 1,47 h-1. Por otra parte la producción de biomasa creció con el estrés hidrodinámico para valores de índice de cizalladura inferiores a 5 x 10-3 cm min-1. La mayor producción de biomasa se obtuvo con matraces Erlenmeyer de 100 ml y alta velocidad de agitación (160 rpm) mientras que la diferenciación de embriones se vio favorecida por bajas velocidades de agitación (60 rpm) asociadas con bajas disponibilidades de oxígeno. La posibilidad de madurar embriones de alcornoque en medio l��quido se estudió utilizando sistemas de inmersión permanente y sistemas de inmersión temporal. En inmersión permanente no se diferenciaron embriones cotiledonares (posiblemente por hiperhidricidad). Los sistemas de inmersión temporal permitieron obtener embriones maduros en estado cotiledonar y capaces de regenerar plantas in vitro. Concentraciones de sacarosa superiores a 60 g l-1 y frecuencias de inmersión iguales o inferiores a una diaria, tuvieron efectos negativos para el desarrollo de los embriones somáticos. En los sistemas de inmersión temporal los parámetros físico-químicos del medio de cultivo se mantuvieron estables y no se observó ninguna limitación de nutrientes. No obstante, estos sistemas se vieron afectados por la evaporación que generó el flujo de aire necesario para desplazar el l��quido en cada periodo de inmersión. Abstract ABSTRACT Cork oak is one of the most important tree species of the Mediterranean ecosystem. Besides its high environmental value has a great economic interest due to the sustainable production of acorns (to feed the Iberian pig) charcoal, timber and cork, which is a renewable natural product with various technological applications. As happens with other forest species, cork oak genetic improvement programs are limited by their long life cycles and because vegetative propagation by cuttings it´s only possible in very juvenile plants. Hence this propagation system is useless or has little practical use for breeding cork oak. Plant regeneration by somatic embryogenesis is the most suitable way for cloning many forest species, and it is the enabling technology which has allowed the establishment of large-scale conifer multi-varietal plantations. Clonal plant regeneration of mature cork oak trees can be achieved through somatic embryogenesis. Somatic embryos at different stages of development and forming clusters are produced during the multiplication phase with current protocols (using semisolid medium). This is an asynchronous low-efficient process not suitable for mass propagation, and therefore it does not solve the difficulties presented by cork oak breeding programs. Culture in liquid medium has been used with other species to improve: synchronization, yield, handling, and to reduce production costs. Thus the development of cork oak embryogenic suspension cultures is envisaged as a way to increase the efficiency of large scale clonal propagation. The thesis herein develops cork oak embryogenic cultures in liquid medium. In chapter 3 establishment and maintenance of suspension cultures are developed, chapter 4 studies proliferation phase in liquid medium and chapter 5 considers the use of different systems of culture in liquid medium, both stationary and temporary immersion, as a way to promote somatic embryos maturation. Clusters of embryos taken from proliferating cultures on semisolid medium were used to initiate the cultures in liquid medium. When these clusters were inoculated directly in liquid medium establishment of suspension cultures was not executed. However using, as initial inoculum, cells and cell aggregates with a size between 41 and 800 μm detached from these clusters of embryos, subjected to a brief shaking, suspension cultures could be established. Suspension maintenance was achieved by inoculating compact embryogenic Abstract clumps with a size between 0.8 and 1.2 mm at low density. The suspension cultures, very heterogeneous, retained both their proliferation and embryo regeneration capacity for at least ten consecutive subcultures. The initiation and maintenance protocol, initially developed with a single genotype, was effective when tested on 11 additional genotypes of cork oak. In proliferation phase three types of vessels and three different levels of agitation were assayed. The combination vessel × orbiting speed determined gas exchange, oxygen availability and hydrodynamic stress. Cork oak embryogenic aggregates grew even under hypoxia conditions; oxygen availability at transfer rates between 0.11 and 1.47 h-1 was not a limiting factor for growth. Furthermore the biomass production was increased with hydrodynamic stress when shear rate values were of less than 5 x 10-3 cm min-1. The highest biomass production was obtained with 100 ml Erlenmeyer flask and high stirring speed (160 rpm) while the differentiation of embryos was favored by low agitation speeds (60 rpm) associated with low oxygen availability. The possibility to mature cork oak somatic embryos in liquid medium was studied using both permanent immersion systems and temporary immersion systems. Cotyledonary embryos did not differentiate in permanent immersion conditions (probably due to hyperhydricity). Temporary immersion systems allowed obtaining mature cotyledonary embryos, which were able to regenerate plants in vitro. Sucrose concentrations above 60 g l-1 and immersion frequencies equal to or lower than one each 24 h had negative effects on somatic embryo development. Physicochemical parameters of the culture medium in temporary immersion systems were stable and showed no limitation of nutrients. However, these systems were affected by the evaporation generated by the airflow necessary to relocate the medium at each immersion period.

Genetic characterization of chestnut (Castanea sativa Mill.) orchards and traditional nut varieties in El Bierzo, a glacial refuge and major cultivation site in Northwestern Spain

This paper presents a detailed genetic study of Castanea sativa in El Bierzo, a major nut production region with interesting features. It is located within a glacial refuge at one extreme of the distribution area (northwest Spain); it has a centenary tradition of chestnut management; and more importantly, it shows an unusual degree of genetic isolation. Seven nuclear microsatellite markers were selected to analyze the genetic variability and structure of 169 local trees grafted for nut production. We analyzed in the same manner 62 local nuts. The selected loci were highly discriminant for the genotypes studied, giving a combined probability of identity of 6.1 × 10−6. An unprecedented density of trees was sampled for this project over the entire region, and nuts were collected representing 18 cultivars marketed by local producers. Several instances of misclassification by local growers were detected. Fixation index estimates and analysis of molecular variance (AMOVA) data are supportive of an unexpectedly high level of genetic differentiation in El Bierzo, larger than that estimated in a previous study with broader geographical scope but based on limited local sampling (Pereira-Lorenzo et al., Tree Genet Genomes 6: 701–715, 2010a). Likewise, we have determined that clonality due to grafting had been previously overestimated. In line with these observations, no significant spatial structure was found using both a model-based Bayesian procedure and Mantel���s tests. Taken together, our results evidence the need for more fine-scale genetic studies if conservation strategies are to be efficiently improved.

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiol��gicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en l��neas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de l��tice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiol��gica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biol��gicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiol��gica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biol��gica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biol��gica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biol��gica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas l��neas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.

Effect of the Legume Plant Host on Rhizobial Soil Population Dynamics

Rhizobium leguminosarum bv viciae (Rlv) is a soil bacterium able to establish specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and Lathyrus. Rlv isolates from nodules of any of these legumes can nodulate any of them; however, it has been shown that plants select specific rhizobial genotypes from those present in the soil (1,2). We have previously shown this at the genomic level by following a population genomics approach. Pool genomic sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba and V. sativa, show different, specific profiles at the single nucleotide polymorphism (SNP) level for relevant genes. In this work, the extent of Rlv selection from a well-characterized soil population by different legume plant hosts: P. sativum, L. culinaris, V. faba and V. sativa, after a medium-term mesocosm study is described. Direct soil isolates from each of these mesocosm studies have been tested for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). Different populations were characterized further by Sanger sequencing of both the rpoB phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and size of the rhizobial population for each legume host showed changes during the medium-term mesocosm study. Particularly, a non-symbiotic group of rhizobia was enriched by all four hosts, in contrast to the symbiotic rhizobia profile, which was specific for each legume plant host.