Lathyrus sativus (grass pea) is a valuable crop for sustainable agriculture, offering dietary benefits and desirable agronomic traits. However, its yield stability is limited by diseases such as powdery mildew caused by Erysiphe pisi. Increasing fungal resistance to pesticides and environmental concerns demand the development of resistant crop varieties. To identify key defense mechanisms and effector genes involved in the Lathyrus sativus-Erysiphe pisi interaction we analyzed four L. sativus accessions exhibiting varying resistance to E. pisi (resistant, partially resistant, partially susceptible, and susceptible) using a dual RNA-Seq experiment across different time points. We observed a host biphasic response, characterized by an initial burst of gene expression, followed by a quiescent phase, and a subsequent wave of intense gene expression. Common L. sativus defense mechanisms included antifungal protein expression, cell wall reinforcement, and reactive oxygen species-mediated defense. These defenses involved respectively Bowman-Birk type proteinase inhibitors, peptidyl-prolyl cis-trans isomerases and mannitol dehydrogenases. The resistant accession specifically activated early reinforcement of structural barriers associated with lignin biosynthesis and the phenylpropanoid pathway, along with sustained chemical defenses (e.g. eugenol synthase 1), epigenetic regulation, and oxidative stress responses thorough peroxidases and heat shock proteins. The partial resistant accession exhibited a front-loaded defense response at early infection stages. Contrastingly, the partially susceptible accession exhibited a weaker baseline defense, with a slower and less robust response targeting pathogen infection. We identified potential E. pisi effectors, including genes involved in cell wall hydrolysis (e.g. mannosidase DCW1), nutrient acquisition (e.g. secreted alpha-glucosidase), and virulence (e.g. SnodProt1), with a higher diversity of effectors identified in the susceptible accession. In conclusion, this study identifies novel targets such as NLRs and effectors, antifungal proteins and genes related to cell wall reinforcement, within the complex Lathyrus sativus-Erysiphe pisi interaction to support future breeding programs aimed at enhancing resistance to E. pisi in L. sativus and related species.

AbstractLathyrus sativus(grass pea) is a valuable crop for sustainable agriculture, offering both dietary benefits and desirable agronomic traits. However, its yield stability is limited by different diseases such as powdery mildew caused byErysiphe pisi. Frequent fungal resistance to pesticides and growing environmental concerns highlight the need for research investment to develop resistant crop varieties. FourL. sativusaccessions, exhibiting varying levels of resistance toE. pisi(resistant, partially resistant, partially susceptible, and susceptible), were analysed using dual RNA-seq to identify key defence mechanisms and effector genes involved in this plant-pathogen interaction. The dual transcriptomic analysis highlighted a host biphasic response, characterised by an initial burst of gene expression, followed by a quiescent phase, and a second wave of intense gene expression at 72 hours after inoculation. CommonL. sativusdefence mechanisms, including antifungal protein expression, cell wall reinforcement, reactive oxygen species-mediated defence were activated by all accessions compared to susceptible accession. Unique responses in the resistant accession integrate early reinforcement of structural barriers with sustained chemical defences and stress responses. Overall, the partially resistant accession exhibited a front-loaded defence response, focused on biotic stimuli and interspecies interactions at early infection stages. In contrast, partial susceptible accessions exhibited a weaker baseline defence system, with a slower and less robust response specifically targeting pathogen infection. We identified potentialE. pisieffectors, including genes involved in cell wall hydrolysis, nutrient acquisition, and virulence, with a higher diversity of effectors identified in the susceptible accession. This study identifies novel targets within the complex defence mechanisms of theLathyrus sativus-Erysiphe pisiinteraction that will support to future breeding programs aimed at enhancing resistance toE. pisiinL. sativusand other related species.

Lathyrus sativus (grass pea) is a valuable crop for sustainable agriculture, offering both dietary benefits and desirable agronomic traits. However, its yield stability is limited by different diseases such as powdery mildew caused by Erysiphe pisi . Frequent fungal resistance to pesticides and growing environmental concerns highlight the need for research investment to develop resistant crop varieties. Four L. sativus accessions, exhibiting varying levels of resistance to E. pisi (resistant, partially resistant, partially susceptible, and susceptible), were analysed using dual RNA-seq to identify key defence mechanisms and effector genes involved in this plant-pathogen interaction. The dual transcriptomic analysis highlighted a host biphasic response, characterised by an initial burst of gene expression, followed by a quiescent phase, and a second wave of intense gene expression at 72 hours after inoculation. Common L. sativus defence mechanisms, including antifungal protein expression, cell wall reinforcement, reactive oxygen species-mediated defence were activated by all accessions compared to susceptible accession. Unique responses in the resistant accession integrate early reinforcement of structural barriers with sustained chemical defences and stress responses. Overall, the partially resistant accession exhibited a front-loaded defence response, focused on biotic stimuli and interspecies interactions at early infection stages. In contrast, partial susceptible accessions exhibited a weaker baseline defence system, with a slower and less robust response specifically targeting pathogen infection. We identified potential E. pisi effectors, including genes involved in cell wall hydrolysis, nutrient acquisition, and virulence, with a higher diversity of effectors identified in the susceptible accession. This study identifies novel targets within the complex defence mechanisms of the Lathyrus sativus-Erysiphe pisi interaction that will support to future breeding programs aimed at enhancing resistance to E. pisi in L. sativus and other related species. ### Competing Interest Statement The authors have declared no competing interest.

Common bean (Phaseolus vulgaris L.) is one of the most important food legumes worldwide, and its production is severely affected by fungal diseases such as powdery mildew. Portugal has a diverse germplasm, with accessions of Andean, Mesoamerican, and admixed origin, making it a valuable resource for common bean genetic studies. In this work, we evaluated the response of a Portuguese collection of 146 common bean accessions to Erysiphe diffusa infection, observing a wide range of disease severity and different levels of compatible and incompatible reactions, revealing the presence of different resistance mechanisms. We identified 11 incompletely hypersensitive resistant and 80 partially resistant accessions. We performed a genome-wide association study to clarify its genetic control, resulting in the identification of eight disease severity-associated single-nucleotide polymorphisms, spread across chromosomes Pv03, Pv09, and Pv10. Two of the associations were unique to partial resistance and one to incomplete hypersensitive resistance. The proportion of variance explained by each association varied between 15 and 86%. The absence of a major locus, together with the relatively small number of loci controlling disease severity, suggested an oligogenic inheritance of both types of resistance. Seven candidate genes were proposed, including a disease resistance protein (toll interleukin 1 receptor-nucleotide binding site-leucine-rich repeat class), an NF-Y transcription factor complex component, and an ABC-2 type transporter family protein. This work contributes with new resistance sources and genomic targets valuable to develop selection molecular tools and support powdery mildew resistance precision breeding in common bean.

Common bean (Phaseolus vulgaris) is one of the legume crops most consumed worldwide and bean rust is one of the most severe foliar biotrophic fungal diseases impacting its production. In this work, we searched for new sources of rust resistance (Uromyces appendiculatus) in a representative collection of the Portuguese germplasm, known to have accessions with an admixed genetic background between Mesoamerican and Andean gene pools. We identified six accessions with incomplete hypersensitive resistance and 20 partially resistant accessions of Andean, Mesoamerican, and admixed origin. We detected 11 disease severity-associated single-nucleotide polymorphisms (SNPs) using a genome-wide association approach. Six of the associations were related to partial (incomplete non-hypersensitive) resistance and five to incomplete hypersensitive resistance, and the proportion of variance explained by each association varied from 4.7 to 25.2%. Bean rust severity values ranged from 0.2 to 49.1% and all the infection types were identified, reflecting the diversity of resistance mechanisms deployed by the Portuguese germplasm.The associations with U. appendiculatus partial resistance were located in chromosome Pv08, and with incomplete hypersensitive resistance in chromosomes Pv06, Pv07, and Pv08, suggesting an oligogenic inheritance of both types of resistance. A resolution to the gene level was achieved for eight of the associations. The candidate genes proposed included several resistance-associated enzymes, namely β-amylase 7, acyl-CoA thioesterase, protein kinase, and aspartyl protease. Both SNPs and candidate genes here identified constitute promising genomics targets to develop functional molecular tools to support bean rust resistance precision breeding.

The Fabeae tribe comprises five legume genera, which include some of the most ancient and important crops, like peas, lentils, and faba beans. Biotic and environmental stresses are major threats to the stable and high productivity of Fabeae crops. The use of omics resources can provide breeders with the tools needed to develop new crop varieties in a more efficient and sustainable way. However, the genomic efforts on Fabeae crops have lagged behind compared to other legume species, mainly due to their large genome size and repeat content. The first annotated chromosome-level reference genome assembly in Fabeae was published for pea (Pisum sativum cv. Caméor) in 2019. Since then, many efforts have been made to sequence the genome of other species from this tribe. Currently, 17 genomes of Fabeae species are available for the scientific community; five of them are at the chromosome level. Fundamental knowledge and molecular tools for breeding have been boosted on the legume resistance/tolerance against biotic and abiotic stresses by the availability of some of these recent reference genomes, especially the pea cv. Caméor genome. This review provides a comparison of the Fabeae tribe genomes available and an overview of recent accomplishments in their application in abiotic and biotic stress research.

Metabolomics is one of the most powerful -omics to assist plant breeding. Despite the recognized genetic diversity in Portuguese common bean germplasm, details on its metabolomics profiles are still missing. Aiming to promote their use and to understand the environment's effect in bean metabolomics profiles, 107 Portuguese common bean accessions, cropped under contrasting environments, were analyzed using spectrophotometric, untargeted and targeted mass spectrometry approaches. Although genotype was the most relevant factor on bean metabolomics profile, a clear genotype x environment interaction was also detected. Multivariate analysis highlighted, on the heat-stress environment, the existence of higher levels of salicylic acid, and lower levels of triterpene saponins. Three clusters were defined within each environment. White accessions presented the lowest content and the colored ones the highest levels of prenol lipids and flavonoids. Sources of interesting metabolomics profiles are now identified for bean breeding, focusing either on local or on broad adaptation.

Mediterranean annual forage mixtures are facing the impact of climate change, especially higher frequencies of winter-time drought. Increased mixture plasticity to climate variability is needed to mitigate this impact. However, little information exists regarding the specificities and complementarities of each forage species component to potentiate mixture resilience under drought. In this study, we identified traits with breeding potential under water scarcity through a detailed characterization of leaf and root-related parameters of 10 legume and grass species components of Mediterranean annual forage mixtures, complemented by their photosynthetic response evaluation under well-watered and water deficit conditions. This integrated approach also allowed us to identify the most resilient species to water deficit. In particular, we found that the highest canopy height and root to shoot ratio of grass components complemented well the highest aerial and root biomass and superior photosynthetic performance of the legume components. Trifolium squarrosum and Triticosecale showed the most adequate combination of traits and the best photosynthetic performance under water deficit within each species family. Although some of these traits are not commonly used in annual forage selection, they may in part explain the potential higher resilience of the grass–legume mixture under water deficit and should be considered in forage breeding.

Water deficit is a major worldwide constraint to common bean ( Phaseolus vulgaris L.) production, being photosynthesis one of the most affected physiological processes. To gain insights into the genetic basis of the photosynthetic response of common bean under water-limited conditions, a collection of 158 Portuguese accessions was grown under both well-watered and water-deficit regimes. Leaf gas-exchange parameters were measured and photosynthetic pigments quantified. The same collection was genotyped using SNP arrays, and SNP-trait associations tested considering a linear mixed model accounting for the genetic relatedness among accessions. A total of 133 SNP-trait associations were identified for net CO 2 assimilation rate, transpiration rate, stomatal conductance, and chlorophylls a and b , carotenes, and xanthophyll contents. Ninety of these associations were detected under water-deficit and 43 under well-watered conditions, with only two associations common to both treatments. Identified candidate genes revealed that stomatal regulation, protein translocation across membranes, redox mechanisms, hormone, and osmotic stress signaling were the most relevant processes involved in common bean response to water-limited conditions. These candidates are now preferential targets for common bean water-deficit-tolerance breeding. Additionally, new sources of water-deficit tolerance of Andean, Mesoamerican, and admixed origin were detected as accessions valuable for breeding, and not yet explored.

Common bean (Phaseolus vulgaris L.), one of the most consumed food legumes worldwide, is threatened by two main constraints that are found frequently together in nature, water deficit (WD) and fusarium wilt (Fop). To understand the shared and unique responses of common bean to Fop and WD, we analyzed the transcriptomic changes and phenotypic responses in two accessions, one resistant and one susceptible to both stresses, exposed to single and combined stresses. Physiological responses (photosynthetic performance and pigments quantification) and disease progression were also assessed. The combined FopWD imposition negatively affected the photosynthetic performance and increased the susceptible accession disease symptoms. The susceptible accession revealed a higher level of transcriptional changes than the resistant one, and WD single stress triggered the highest transcriptional changes. While 89 differentially expressed genes were identified exclusively in combined stresses for the susceptible accession, 35 were identified in the resistant one. These genes belong mainly to "stress", "signaling", "cell wall", "hormone metabolism", and "secondary metabolism" functional categories. Among the up-regulated genes with higher expression in the resistant accession, the cysteine-rich secretory, antigen 5 and Pr-1 (CAP) superfamily protein, a ribulose bisphosphate carboxylase family protein, and a chitinase A seem promising targets for multiple stress breeding.

Common bean is a nutritious food legume widely appreciated by consumers worldwide. It is a staple food in Latin America, and a component of the Mediterranean diet, being an affordable source of protein with high potential as a gourmet food. Breeding for nutritional quality, including both macro and micronutrients, and meeting organoleptic consumers’ preferences is a difficult task which is facilitated by uncovering the genetic basis of related traits. This study explored the diversity of 106 Portuguese common bean accessions, under two contrasting environments, to gain insight into the genetic basis of nutritional composition (ash, carbohydrates, fat, fiber, moisture, protein, and resistant starch contents) and protein quality (amino acid contents and trypsin inhibitor activity) traits through a genome-wide association study. Single-nucleotide polymorphism-trait associations were tested using linear mixed models accounting for the accessions’ genetic relatedness. Mapping resolution to the gene level was achieved in 56% of the cases, with 102 candidate genes proposed for 136 genomic regions associated with trait variation. Only one marker-trait association was stable across environments, highlighting the associations’ environment-specific nature and the importance of genotype × environment interaction for crops’ local adaptation and quality. This study provides novel information to better understand the molecular mechanisms regulating the nutritional quality in common bean and promising molecular tools to aid future breeding efforts to answer consumers’ concerns.

The common bean (Phaseolus vulgaris L.) represents a sustainable and affordable source of protein, namely, to populations with vegetarian dietary habits. Despite the national germplasm genetic diversity, little is known about the Portuguese accessions’ nutritional and protein quality, leading to their underuse in breeding programs. To fill this gap, a representative collection (106 accessions) was cropped under two contrasting environments (traditional versus heat stress) and evaluated in terms of nutritional quality by near-infrared spectroscopy. Protein quality was assessed, under the stressful environment, considering the individual amino acid contents and the activity of trypsin inhibitors through mass spectrometry (LC-MS/MS) and spectrophotometry, respectively. On top of strong genotypic control, the nutritional composition (protein, fat, fiber, moisture and ash) was also highly influenced by the environment and by genotype × environment interaction, with a clear nutritional quality ranking change for the accessions in heat stress conditions. Classified into three clusters, the accessions from the cluster with the highest individual amino acid and protein contents also showed higher trypsin inhibitor activity (TIA). Since different levels of TIA had no translation into contrasting protein digestibility, breeders focusing on common beans’ protein quality improvement, especially under challenging warming climate conditions, may take advantage of this group of accessions.

Common bean (Phaseolus vulgaris) is one of the most consumed legume crops in the world, and Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. phaseoli, is one of the major diseases affecting its production. Portugal holds a very promising common bean germplasm with an admixed genetic background that may reveal novel genetic resistance combinations between the original Andean and Mesoamerican gene pools. To identify new sources of Fusarium wilt resistance and detect resistance-associated single-nucleotide polymorphisms (SNPs), we explored, for the first time, a diverse collection of the underused Portuguese common bean germplasm by using genome-wide association analyses. The collection was evaluated for Fusarium wilt resistance under growth chamber conditions, with the highly virulent F. oxysporum f. sp. phaseoli strain FOP-SP1 race 6. Fourteen of the 162 Portuguese accessions evaluated were highly resistant and 71 intermediate. The same collection was genotyped with DNA sequencing arrays, and SNP-resistance associations were tested via a mixed linear model accounting for the genetic relatedness between accessions. The results from the association mapping revealed nine SNPs associated with resistance on chromosomes Pv04, Pv05, Pv07, and Pv08, indicating that Fusarium wilt resistance is under oligogenic control. Putative candidate genes related to phytoalexin biosynthesis, hypersensitive response, and plant primary metabolism were identified. The results reported here highlight the importance of exploring underused germplasm for new sources of resistance and provide new genomic targets for the development of functional markers to support selection in future disease resistance breeding programs.

Participatory selection—exploiting specific adaptation traits to target environments—helps to guarantees yield stability in a changing climate, in particular under low-input or organic production. The purpose of the present study was to identify reliable, low-cost, fast and easy-to-use tools to complement traditional selection for an effective participatory improvement of maize populations for drought resistance/tolerance. The morphological and eco-physiological responses to progressive water deprivation of four maize open-pollinated populations were assessed in both controlled and field conditions. Thermography and Chl a fluorescence, validated by gas exchange indicated that the best performing populations under water-deficit conditions were ‘Fandango’ and to a less extent ‘Pigarro’ (both from participatory breeding). These populations showed high yield potential under optimal and reduced watering. Under moderate water stress, ‘Bilhó’, originating from an altitude of 800 m, is one of the most resilient populations. The experiments under chamber conditions confirmed the existence of genetic variability within ‘Pigarro’ and ‘Fandango’ for drought response relevant for future populations breeding. Based on the easiness to score and population discriminatory power, the performance index (PIABS) emerges as an integrative phenotyping tool to use as a refinement of the common participatory maize selection especially under moderate water deprivation.

Despite the common beans' nutritional and phytochemical value, in Portugal its consumption decreased more than 50% in the last decade. The present study aimed to characterize phenolic composition of the Portuguese traditional varieties and corresponding soaked seed fractions (including soaking water). With such purpose, the phenolic composition (total content of soluble phenolics, flavonoids, and proanthocyanidins) and in vitro antioxidant activity were evaluated in the raw whole flour of 31 Portuguese common bean varieties. The phenolic composition of the soaked fractions was respectively compared to the raw flour. Phenolic compounds' identification and relative quantification were achieved by UPLC-TripleTOF-MS for one representative variety and their fractions. The highest phenolic content was found in colored varieties and the brown market class highlighted as the richest one. The loss of phenolic compounds to the soaking water was highly dependent on variety. The predominant phenolic compounds' classes were flavan-3-ols (soaking water and coats), flavonols (coats), and phenolic acids (cotyledons). This characterization study showed the diversity on the phenolic composition of Portuguese varieties and the need to adjust the soaking and peeling processes to the variety (considering the possible loss of potential health promoter compounds, e.g., phenolic compounds).

Trabajo presentado en el 9th International Conference on Legume Genetics and Genomics (IGLCC 2019), delebrado en Dijon del 13 al 17 de mayo de 2019.

The Lathyrus cicera transcriptome was analysed in response to rust ( Uromyces pisi ) infection to develop novel molecular breeding tools with potential for genetic mapping of resistance in this robust orphan legume species. One RNA-seq library each was generated from control and rust-inoculated leaves from two L. cicera genotypes with contrasting quantitative resistance, de novo assembled into contigs and sequence polymorphisms were identified. In toto , 19,224 SNPs differentiate the susceptible from the partially resistant genotype’s transcriptome. In addition, we developed and tested 341 expressed E-SSR markers from the contigs, of which 60.7% varied between the two L. cicera genotypes. A first L. cicera linkage map was created using part of the developed markers in a RIL population from the cross of the two genotypes. This map contains 307 markers, covered 724.2 cM and is organised in 7 major and 2 minor linkage groups, with an average mapping interval of 2.4 cM. The genic markers also enabled us to compare their position in L. cicera map with the physical position of the same markers mapped on Medicago truncatula genome, highlighting a high macrosyntenic conservation between both species. This study provides a large new set of genic polymorphic molecular markers with potential for mapping rust resistances. It represents the first step towards genomics-assisted precision breeding in L. cicera .

Trabajo presentado en la 4th Annual Conference of the COST action SUSTAIN, celebrada en Bled (Slovenia) del 1 al 3 de marzo de 2017.

Common bean (Phaseolus vulgaris L.) is among the most important grain legumes for human consumption worldwide. Portugal has a potentially promising common bean germplasm, resulting from more than five centuries of natural adaptation and farmers' selection. Nevertheless, limited characterization of this resource hampers its exploitation by breeding programs. To support a more efficient conservation of the national bean germplasm and promote its use in crop improvement, we performed, for the first time, a simultaneous molecular marker (21 microsatellites and a DNA marker for phaseolin-type diversity analysis) and seed and plant morphological characterization (14 traits) of 175 accessions from Portuguese mainland and islands traditional bean-growing regions. A total of 188 different alleles were identified and an average pairwise Cavalli-Sforza and Edwards' chord genetic distance of 0.193 was estimated among accessions. To relate the Portuguese germplasm with the global common bean diversity, 17 wild relatives and representative accessions from the Andean and Mesoamerican gene pools were evaluated at the molecular level. No correlation was detected between the variability found and the geographic origin of accessions. Structure analysis divided the collection into three main clusters. Most of the Portuguese accessions grouped with the race representatives and wild relatives from the Andean region. One third of the national germplasm had admixed genetic origin and might represent putative hybrids among gene pools from the two original centers of domestication in the Andes and Mesoamerica. The molecular marker-based classification was largely congruent with the three most frequent phaseolin haplotype patterns observed in the accessions analyzed. Seed and plant morphological characterization of 150 Portuguese common bean accessions revealed a clear separation among genetic structure and phaseolin haplotype groups of accessions, with seed size and shape and the number of locules per pod the most discriminant traits. Additionally, we used molecular and morphological data to develop a series of smaller core collections that, by maximizing the genetic and morphological diversity of the original collection, represents the Portuguese common bean germplasm with minimum repetitiveness. A core collection with 37 accessions contained 100% of the genetic variation found in the entire collection. This core collection is appropriate for a more detailed characterization and should be explored, as a priority, in national and international common bean breeding efforts. Furthermore, the identified intermediate accessions (with admixed genetic origin) may have novel genetic combinations useful in future bean breeding.