Molecular and genetic approaches for oat breeding for biotic and abiotic stress resistance

La avena es un cultivo de origen mediterráneo presente de forma tradicional como cultivo de grano y forraje, siendo el cuarto cereal en importancia en España, solo superado por trigo, cebada y maíz. Sin embargo, la sequía y enfermedades causadas por hongos biotrófos, tales como la roya de la corona (Puccinia coronata f.sp. avenae) y el oídio (Blumeria graminis f. sp. avenae), mitigan fuertemente su producción. El hecho que tanto la tolerancia a la sequía como la resistencia a ambas enfermedades sean extremadamente complejas, incluyendo un amplio rango de procesos entre los que están incluidos importantes interacciones entre moléculas señalizadoras, hacen que el conocimiento de las bases moleculares de la resistencia a dichos estreses sea fundamental para la mejora genética de este cultivo. Además, conocer un poco más sobre los efectos sinérgicos y antagónicos de las moléculas implicadas en la resistencia a ambos estreses es fundamental para promover una mejora genética de la avena más eficiente y con ello poder desarrollar plantas con una resistencia más amplia pudiendo hacer frente a diversos estreses. Teniendo en cuenta esto, en el capítulo 1 de la presente tesis estudiamos el papel de las poliaminas en la resistencia a oidio. Así, se monitorizó el contenido de poliaminas en dos genotipos, resistente y susceptible, de avena a oidio durante la interacción huésped y nohuésped. Los resultados mostraron diferencias significativas en los niveles de algunas poliaminas solubles entre los genotipos resistente y susceptible en ambas interacciones, así como en los productos de degradación de las poliaminas relacionados con las especies reactivas de oxígeno (ROS). Esto sugiere un papel importante de las poliaminas en la resistencia a este hongo fitopatógeno. En el segundo capítulo se llevó a cabo el estudio del papel de las poliaminas durante la infección de roya en cultivares resistentes y susceptibles de avena y en particular en los diferentes mecanismos de resistencia. Se confirmaron aumentos de espermidina y espermina asociados a mecanismos de resistencia pre- y durante la penetración de la células del mesófilo, que se corroboró con el aumento de la resistencia en el cultivar susceptible tras la aplicación exógena de las mismas. En el cultivar resistente Saia observó además un aumento en la actividad DAO y PAO unida a pared y en la producción de DAP en etapas tempranas, lo que sugiere que la implicación de los mismos en la resistencia a la penetración o la contribución al endurecimiento de la pared celular o lignificación. En capitulo 3 se monitorizaron los niveles de óxido nítrico (NO) endógeno en cultivares de avena susceptible y resistente a la sequía confirmando una reducción de los niveles de NO asociados a la tolerancia. Para confirmar estos resultados se han utilizado líneas transgénicas de cebada...Oat is a Mediterranean crop used for grain and fodder and ranking fourth in importance in Sapin, following wheat, barley and maize. However drought stress and fungal diseases such as the crown rust (Puccinia coronata f.sp. avenae) and the powdery mildew (Blumeria graminis f. sp. avenae) strongly constrain its yield. The high complexity of the resistance responses to both, drought and fungi, including the crosstalk with other signaling molecules, made that a deep knowledge of the molecular bases of the resistance to these stresses is crucial for breeding the crop. In addition, knowledge of the synergic and antagonic effects that different molecules involved in both kind of stresses may have is important to promote a more efficient breeding of the crop. Taking this into account in the chapter 1 of the present thesis we studied the role of polyamines in the resistance to powdery mildew. Thus, polyamine content in two, powdery mildew resistant and susceptible oat genotypes was monitored during a host and non-host interactions. Results showed significant differences in the levels of particular soluble polyamines between resistant and susceptible genotypes in both interactions. In addition, resistant and susceptible genotypes differed in the content of important polyamine degradation products, suchas reactives oxygen species (ROS). This suggests an important role for polyamines in the oat resistance to these phytopathogenic fungi. In the second chapter, we carried out an study on the role of polyamines during the oat-rust interaction focusing in particular disease resistance mechanism. We confirmed an increase on spermidine and spermine associated with the pre- and penetration resistance. These results were supported with bioassays of exogenous polyamine application that increase the resistance in the susceptible cultivar. In addition in the resistant cultivar Saia, we observed an early increase in the cell-wll bound DAO and PAO activity and in the generation of DAP, suggesting its implication in the penetration resistance for instance in the cell-wall reinforcement or lignification. In chapter 3 we monitored the levels of nitric oxide (NO), in resistant and susceptible oat cultivars and confirmed an NO reduction associated with drought tolerance. In order to confirm the results and to determine the role of NO during drought in relation with polyamine metabolism, barley transgenic plants overexpressing the hemoglobin gen HvHb1 and hence with lower level of NO were used. Results showed a higher drought resistance in the barley lines overexpressing the hemoglobin gene compared with the wild type and modification in these plants in the level of specific polyamines. In addition we confirmed the influence of NO in the polyamine biosynthetic pathway at different levels: influencing nitrogen..

Compromised Photosynthetic Electron Flow and H2O2 Generation Correlate with Genotype-Specific Stomatal Dysfunctions during Resistance against Powdery Mildew in Oats

Stomatal dysfunction known as "locking" has been linked to the elicitation of a hypersensitive response (HR) following attack of fungal pathogens in cereals. We here assess how spatial and temporal patterns of different resistance mechanisms, such as HR and penetration resistance influence stomatal and photosynthetic parameters in oat (Avena sativa) and the possible involvement of hydrogen peroxide (H2O2) in the dysfunctions observed. Four oat cultivars with differential resistance responses (i.e., penetration resistance, early and late HR) to powdery mildew (Blumeria graminis f. sp. avenae, Bga) were used. Results demonstrated that stomatal dysfunctions were genotype but not response-type dependent since genotypes with similar resistance responses when assessed histologically showed very different locking patterns. Maximum quantum yield (Fv/Fm) of photosystem II were compromised in most Bga-oat interactions and photoinhibition increased. However, the extent of the photosynthetic alterations was not directly related to the extent of HR. H2O2 generation is triggered during the execution of resistance responses and can influence stomatal function. Artificially increasing H2O2 by exposing plants to increased light intensity further reduced Fv/Fm ratios and augmented the patterns of stomatal dysfunctions previously observed. The latter results suggest that the observed dysfunctions and hence a cost of resistance may be linked with oxidative stress occurring during defense induced photosynthetic disruption

Deciphering Main Climate and Edaphic Components Driving Oat Adaptation to Mediterranean Environments

Oat, Avena sativa, is an important crop traditionally grown in cool-temperate regions. However, its cultivated area in the Mediterranean rim steadily increased during the last 20 years due to its good adaptation to a wide range of soils. Nevertheless, under Mediterranean cultivation conditions, oats have to face high temperatures and drought episodes that reduce its yield as compared with northern regions. Therefore, oat crop needs to be improved for adaptation to Mediterranean environments. In this work, we investigated the influence of climatic and edaphic variables on a collection of 709 Mediterranean landraces and cultivars growing under Mediterranean conditions. We performed genotype–environment interaction analysis using heritability-adjusted genotype plus genotype–environment biplot analyses to determine the best performing accessions. Further, their local adaptation to different environmental variables and the partial contribution of climate and edaphic factors to the different agronomic traits was determined through canonical correspondence, redundancy analysis, and variation partitioning. Here, we show that northern bred elite cultivars were not among the best performing accessions in Mediterranean environments, with several landraces outyielding these. While all the best performing cultivars had early flowering, this was not the case for all the best performing landraces, which showed different patterns of adaption to Mediterranean agroclimatic conditions. Thus, higher yielding landraces showed adaptation to moderate to low levels of rain during pre- and postflowering periods and moderate to high temperature and radiation during post-flowering period. This analysis also highlights landraces adapted to more extreme environmental conditions. The study allowed the selection of oat genotypes adapted to different climate and edaphic factors, reducing undesired effect of environmental variables on agronomic traits and highlights the usefulness of variation partitioning for selecting genotypes adapted to specific climate and edaphic conditionsThis work was supported by the grant PID2019-104518RBI00 funded by MCIN/AEI/10.13039/501100011033. FC and LMG-S are holder of a FPI fellowship (BES-2014-071044) and (BES-2017-080152), respectively, from the Spanish Ministry of Economy and Competitiveness. GM-B is holder of a Junta de Andalucia grant for Doctors [DOC_00394]. We thank CRF and USDA for supplying the seed

Multi-environmental trials reveal genetic plasticity of oat agronomic traits associated with climate variable changes

Although oat cultivation around the Mediterranean basin is steadily increasing, its yield in these regions lags far behind those of Northern Europe. This results mainly from the poor adaptation of current oat cultivars to Mediterranean environments. Local landraces may act as reservoirs of favorable traits that could contribute to increase oat resilience in this region. To aid selection of suitable agro-climate adapted genotypes we integrated genome-wide association approaches with analysis of field assessed phenotypes of genetic variants and of the weight of associated markers across different environmental variables. Association models accounting for oat population structure were applied on either arithmetic means or best linear unbiased prediction (BLUPs) to ensure robust identification of associations with the agronomic traits evaluated. The meta-analysis of the six joint environments (mega-environment) identified several markers associated with several agronomic traits and crown rust severity. Five of these associated markers were located within expressed genes. These associations were only mildly influenced by climatic variables indicating that these markers are good candidates to improve the genetic potential of oat under Mediterranean conditions. The models also highlighted several marker-trait associations, strongly affected by particular climatic variables including high rain pre- or post-heading dates and high temperatures, revealing strong potential for oat adaptation to specific agro-climatic conditions. These results will contribute to increase oat resilience for particular climatic conditions and facilitate breeding for plant adaptation to a wider range of climatic conditions in the current scenario of climate change

Population genomics of Mediterranean oat (A. sativa) reveals high genetic diversity and three loci for heading date

KEY MESSAGE: Genomic analysis of Mediterranean oats reveals high genetic diversity and three loci for adaptation to this environment. This information together with phenotyping and passport data, gathered in an interactive map, will be a vital resource for oat genetic improvement. ABSTRACT: During the twentieth century, oat landraces have increasingly been replaced by modern cultivars, resulting in loss of genetic diversity. However, landraces have considerable potential to improve disease and abiotic stress tolerance and may outperform cultivars under low input systems. In this work, we assembled a panel of 669 oat landraces from Mediterranean rim and 40 cultivated oat varieties and performed the first large-scale population genetic analysis of both red and white oat types of Mediterranean origin. We created a public database associated with an interactive map to visualize information for each accession. The oat collection was genotyped with 17,288 single-nucleotide polymorphism (SNP) loci to evaluate population structure and linkage disequilibrium (LD); to perform a genome-wide association study (GWAs) for heading date, a key character closely correlated with performance in this drought-prone area. Population genetic analysis using both structure and PCA distinguished two main groups composed of the red and white oats, respectively. The white oat group was further divided into two subgroups. LD decay was slower within white lines in linkage groups Mrg01, 02, 04, 12, 13, 15, 23, 33, whereas it was slower within red lines in Mrg03, 05, 06, 11, 21, 24, and 28. Association analysis showed several significant markers associated with heading date on linkage group Mrg13 in white oats and on Mrg01 and Mrg08 in red oats. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00122-021-03805-2

Genome-wide association study for crown rust (Puccinia coronata f. sp. avenae) and powdery mildew (Blumeria graminis f. sp. avenae) resistance in an oat (Avena sativa) collection of commercial varieties and landraces

Diseases caused by crown rust (Puccinia coronata f. sp. avenae and powdery mildew (Blumeria graminis f. sp. avenae) are among the most important constraints for the oat crop. Breeding for resistance is one of the most effective, economical, and environmentally friendly means to control these diseases. The purpose of this work was to identify elite alleles for rust and powdery mildew resistance in oat by association mapping to aid selection of resistant plants. To this aim, 177 oat accessions including white and red oat cultivars and landraces were evaluated for disease resistance and further genotyped with 31 simple sequence repeat (SSR) and 15,000 Diversity Arrays Technology (DArT) markers to reveal association with disease resistance traits. After data curation, 1712 polymorphic markers were considered for association analysis. Principal component analysis and a Bayesian clustering approach were applied to infer population structure. Five different general and mixed linear models accounting for population structure and/or kinship corrections and two different statistical tests were carried out to reduce false positive. Five markers, two of them highly significant in all models tested were associated with rust resistance. No strong association between any marker and powdery mildew resistance at the seedling stage was identified. However, one DArT sequence, oPt-5014, was strongly associated with powdery mildew resistance in adult plants. Overall, the markers showing the strongest association in this study provide ideal candidates for further studies and future inclusion in strategies of marker assisted selection

Reduced nitric oxide levels during drought stress promote drought tolerance in barley and is associated with elevated polyamine biosynthesis

Nitric oxide (NO) is a key messenger in plant stress responses but its exact role in drought response remains unclear. To investigate the role of NO in drought response we employed transgenic barley plants (UHb) overexpressing the barley non-symbiotic hemoglobin gene HvHb1 that oxidizes NO to NO3-. Reduced NO production under drought conditions in UHb plants was associated with increased drought tolerance. Since NO biosynthesis has been related to polyamine metabolism, we investigated whether the observed drought-related NO changes could involve polyamine pathway. UHb plants showed increases in total polyamines and in particular polyamines such as spermidine. These increases correlated with the accumulation of the amino acid precursors of polyamines and with the expression of specific polyamine biosynthesis genes. This suggests a potential interplay between NO and polyamine biosynthesis during drought response. Since ethylene has been linked to NO signaling and it is also related to polyamine metabolism, we explored this connection. In vivo ethylene measurement showed that UHb plants significantly decrease ethylene production and expression of aminocyclopropane-1-carboxylic acid synthase gene, the first committed step in ethylene biosynthesis compared with wild type. These data suggest a NO-ethylene influenced regulatory node in polyamine biosynthesis linked to drought tolerance/susceptibility in barley.publishersversionPeer reviewe

Changes in polyamine profile in host and non-host oat-powdery mildew interactions

Oat (Avena sativa L.) crop constitutes a rich source of biologically active secondary metabolites. Most of these compounds act as chemical signals and defense metabolites and constitute a potential source for the development of control methods for specific diseases. Polyamines are low molecular organic cations involved in various physiological events, particularly those related to abiotic stress responses, albeit recently their potential in disease resistance has been investigated. In this work we monitored the polyamine content in leaves of both resistant and susceptible oat cultivars in response to Blumeria graminis f.sp. avenae (Bga, host interaction) and with Blumeria graminis f.sp. hordei (non-host interaction). Our results show significant differences between the resistant and susceptible cultivars for specific free polyamine levels, and also with respect to the non-host interaction at crucial stages of the infection process. In addition, polyamine degradation products, such as 1.3-diamino propane increased following pathogen challenge, suggesting a role for reactive oxygen species derived from this pathway in resistance. Exogenous application of polyamines to leaf surface increased penetration resistance of oat against Bga. Overall, data support both, a direct and indirect role for polyamines in resistance in host and non-host interactions, in responses of oat against appropriate and inappropriate powdery mildew formae speciales. © 2014 Phytochemical Society of Europe.This work was supported by the Spanish Ministry of Science and Innovation [AGL2010-15936/AGR], and regional government through the AGR-253 group, the European Regional and Social Development Funds and a JAE fellowship from the CSIC to [GMB].Peer Reviewe

Digital image analysis for high throughput phenotyping of powdery mildew resistance in oats

Trabajo presentado en el II Spanish Symposium on Physiology and Breeding of Cereals (II SEFiMeC), celebrado en Córdoba (España) el 6 y 7 de marzo de 2019.-- Organized by excellence network AGL2016-81855-REDT.In the last years the decline of the cost of sequencing and increase in throughput have resulted in tremendous amounts of genomic data, creating many new opportunities, but at the same time making phenotyping the major bottleneck in plant breeding and fundamental plant science. This is particularly true for the assessment of plant diseases where in addition to the time consuming evaluations we have to manage sometimes with the subjectivity of macroscopic visual assessments. Furthermore, macroscopic assessments to not allow to discern the particular resistance mechanisms underlying the resistance response. This is crucial for breeding since some of these mechanisms have been proved to be durable in the field whereas others such as the hypersensitive response are usually easily overcome by new emerging pathogen isolates. In this work we set up a semiautomated method for evaluating the powdery mildew disease in oats obtaining great performance by using a simple smartphone camera and the free image software Fiji (developed from Image J). To this aim, we designed a detailed script for assessing rapidly high number of pictures through two steps. The first script can open an array of image files and apply a plugin to extract the leaf from the background. The second script, convert the picture in a black (leaf) and white (micellium) image, which can be confirmed or modified if necessary by the user through a threshold, and then, calculates the proportion of black (non-diseased) respect to white (diseased) pixels showing the disease severity in a table. We have also correlated this disease severity index with the percentage of haustoria within the cells. This will ease microscopic assessments by screening fast susceptible genotypes and focussing in the resistance mechanisms of resistant plants and allowing association studies that link markers not only with resistance but with durable resistance mechanisms.This research was financially supported by the Project AGL2016-78965-R (Spanish Ministry of Economy and Competitiveness) and the European Regional Development Funds (ERDF).Peer reviewe