Metabolomics profile responses to changing environments in a common bean (Phaseolus vulgaris L.) germplasm collection

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 × 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.To FCT, Portugal, in BEGEQA project (PTDC/AGR-TEC/3555/2012), E.M. PhD fellowship (SFRH/BD/89287/2012), as well as to R&D unit, UIDB/04551/2020 (GREEN-IT – Bioresources for sustainability) and COST Action FA1403 (STSM-FA1403-290815-063873) for funding. The authors also acknowledge PORTUGAL 2020 to the Portuguese Mass Spectrometry Network, grant number LISBOA-01-0145-FEDER-402-022125. The project NETDIAMOND (SAICTPAC/0047/2015), financially supported by FEEI (Lisboa 2020 and FCT/POCI-01-0145-FEDER-016385), to the iNOVA4Health (UID/Multi/04462/2013), financially supported by FCT and co-funded by FEDER under the PT2020 Partnership Agreement, as well as to POCI-01-0145-FEDER-029702, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES

Unravelling the genetic control of combined biotic/abiotic stress resistance in Phaseolus vulgaris L. The fusarium wilt - drought interaction

Tesis Doctoral.-- Dissertation presented to obtain the Ph.D degree in Plant Sciences - Plants for Life Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa.[EN] Common bean (Phaseolus vulgarisL.) is among the most important grain legumes for human consumption worldwide. Portugal has a promising commonbeangermplasmthat resultedfrom more than five centuries of natural adaptation and farmers’selection. Nevertheless, limited characterization of this resource hampers its exploitationby breeding programs. To support a more efficient conservation of the national beangermplasm and promote its use in crop improvement, we performed, for the first time, asimultaneous molecular marker and seed and plant morphological characterization of 175accessions collected from Portuguese mainland and islands traditional bean-growing regions.Most of the Portuguese accessions grouped with theracerepresentatives and wild relatives from the Andean region. One-third of the nationalgermplasmhad an admixed genetic origin andmight represent putative hybrids among genepools from the two original centers of domestication in the Andes and Mesoamerica. Themolecularmarker-based classification was largely congruent with the threemost frequentphaseolin haplotype patterns observed in the accessions analyzed.Additionally, we developed a core collection with 37 accessions that, by maximizing the genetic and morphological diversity of the original collection, represents the Portuguese common bean germplasm with minimum repetitiveness.Fungal diseases, such as fusarium wilt, and water deficit are among the major constraints to common bean production worldwide, limiting yield, farmer’s income, and commercialization. Toidentify new sources of fusarium wilt resistance and detect resistance-associated SNPs, we explored, for the first time, the Portuguese diverse common bean germplasmusing genome-wide association analyses. The collection was evaluated for fusarium wilt resistance under growth chamber conditions, using the highly virulent Fusarium oxysporumf. sp. phaseoli(Fop) strain, FOP-SP1 race 6. Fourteen accessions were considered highly resistant and71 intermediate. The same collection was genotyped with single nucleotide polymorphism (SNP)-based arrays and SNP-resistance associations were tested using a mixed linear model accounting for the genetic relatedness among accessions.The results from the genome-wide association study (GWAS) revealed nine SNPs associated with resistance oncommon beanchromosomes Pv04, Pv05, Pv07, and Pv08, indicating that fusarium wilt resistance is under oligogenic control. Putative candidate genes related to phytoalexinsbiosynthesis,hypersensitive response, and plant primary metabolism were identified. Moreover, photosynthesis-related traits, namely leaf gas exchange parameters and photosynthetic pigments,were evaluated in the same Portuguese common bean collection, under both well-watered(WW)and water-deficit(WD)conditions.Once again, the natural variation of the collection was explored and a GWAS performed to identify SNP alleles and candidate genes associated withthe studiedphotosynthesis-related traits. A total of 133 SNP-trait associations were identified for net CO2assimilation rate, transpiration rate, and stomatal conductance, and for chlorophylls aand b, carotenes and xanthophylls contents. Ninety of these associations were detected under WDand 43 under WWconditions. The identified candidate genes revealed that stomatal regulation, membrane translocation of proteins, mechanisms of redox, hormone and osmotic stress signaling were the most relevant processes related to the studied common bean response to water deficit.Finally, to understand the unique and shared responses of common bean to Fopand WDstresses, we characterizedthe differentially expressed genes in a double resistant and a double susceptible accessionwhen these stresses were appliedsinglyor combined. The susceptible accession revealed a clearly higher level of transcriptional changes in relation to the control condition, in all the three treatments –Fopand WD single stresses and in the combined FopWD stress -compared with the resistant accession (944 versus 228 differentially expressed genes (DEGs)).The most represented functional categories within the DEGs were “RNA”, “secondary metabolism”, “stress”, “signaling”, “hormone metabolism”, “transport”, “protein” and “cell wall”. The resistance-related genes found might be targets for functional characterization and development of molecular tools, to expedite the screening and improvement of common bean multiple resistance to biotic and abiotic stresses.In conclusion, the work developedunder this Ph.D. thesisunlocked the door of futurecommon beanbreeding in Portugal, improved the knowledge on the molecular and morphological characterization of this national resource,identified new sources of resistance to a highly virulent Fopisolate and accessions more tolerant to WD conditions,and unveilthe genetic control of disease resistance and photosynthesis-relatedtraits in common bean.[PT] O feijão comum (Phaseolus vulgarisL.), ou simplesmente feijão, está entre as leguminosasde grão mais importantes para consumo humano em todo o mundo. Portugal possui um germoplasma de feijão com grande potencial, resultante da adaptação natural edaseleção efetuada pelos agricultores ocorridasnos últimos cinco séculos. No entanto, a caracterização desse recurso é limitada, o que dificulta a sua utilização em programas de melhoramento. Para promover uma conservação mais eficiente do germoplasma nacional de feijão e fomentar o seu uso em programas de melhoramento, foi realizado, pela primeiravez neste trabalho de doutoramento, uma caracterização molecular e morfológica de sementes e plantas de 175 acessos de feijão colhidos em diferentes regiões de Portugal continental e ilhas. A classificação baseada em marcadores moleculares foi amplamente congruente com os três haplótipos de faseolina mais frequentemente observados nos acessos analisados. A maioria dos acessos portugueses agrupou-se com os acessos representantes da região andina. Um terço do germoplasma nacional tem uma origem genética mista, sendo híbridos putativos entre os grupos genéticos dos dois centros originais de domesticação desta espécie, Andes e Mesoamérica. Adicionalmente,foi desenvolvida uma coleção nuclearcom 37 acessos, representativa da diversidade genética e morfológica da coleção original de feijão português.As doenças fúngicas, como a fusariose, e a secaestão entre os principais problemas que o cultivo de feijão enfrenta em todo o mundo, limitando a sua produção, o lucro do agricultor e a comercialização. Para identificar novas fontes de resistência à fusariose e detectar alelos de marcadores molecular associados a genes de resistência, foi explorada, pela primeira vez, a diversidade natural presente no germoplasma de feijão português, utilizando o mapeamento por associação ao longo de todo o genoma de feijão (GWAS, genome-wide asssociation study, em inglês). A coleção de acessos de feijão foi avaliada quanto à resistência à fusariose, numa câmara de crescimento de plantas com condições controladas, utilizando um isolado muito virulento de Fusarium oxysporumf. sp. phaseoli(Fop), denominado FOP-SP1 raça 6. Catorze acessos foram considerados altamente resistentes e 71 intermédios.A mesma coleção foi genotipada com arraysbaseados em polimorfismo de nucleotídeo único (SNP, single-nucleotide polymorphism,em inglês) e as associações entre os níveis de resistência e os alelos dos SNPs foram testadas usando um modelo linear misto, tendo em conta a relação genética entre os acessos. Os resultados do estudo de associação (GWAS) revelaram alelos de nove SNPs associados à resistência à fusariose nos cromossomas de feijão Pv04, Pv05, Pv07 e Pv08, indicando que esta resistência está sob controlo oligogénico. Foram identificados genes candidatos envolvidos na biossíntese de fitoalexinas, resposta hipersensitivae metabolismo primário da planta.Adicionalmente foram avaliadas,na mesma coleção de feijoeiros,características relacionadas com a fotossíntese, nomeadamente parâmetros relacionados com as troca gasosas foliares e com os pigmentos fotossintéticos, em condições bem irrigadas (WW, well-wateredem inglês) e em déficit hídrico (WD, water deficitem inglês). Uma vez mais, a variação natural da coleção de acessos de feijão foi explorada e um GWAS realizado para identificar alelos de SNPs e genes candidatos associados a características relacionadas com a fotossíntese. Foram identificadas 133 associações entre os alelos dos SNPs e as taxas de assimilação líquida de CO2e de transpiração, a condutância estomática, e o conteúdo em clorofilas ae b, e em carotenos e xantofilas. Noventa destas associações foram detetadas em condições bem irrigadas e 43 em déficit hídrico. Os genes candidatos identificados revelaram que a regulação estomática, a translocação de proteínas pelas membranascelulares, os mecanismos de redox, a sinalização de stresses hormonal e osmótico foram os processos mais relevantes relacionados com a resposta do feijoeiro ao déficit hídrico estudado.Finalmente, com o objetivo de estudar as respostas comuns eúnicas do feijoeiro à fusariose e ao déficit hídrico, foram identificados os genes diferencialmente expressos em dois acessos –um resistente e um suscetível a ambos os stresses –quando os stresses foram aplicados isoladamente ou em combinação. O acesso suscetível revelou um nível notoriamente mais elevado de alterações transcricionais em comparação com o acesso resistente, nos três tratamentos aplicados –stresses individuais de Fope WD e no stress combinado FopWD.As categorias funcionais mais representadas nos genes identificados foram "ARN", "metabolismo secundário", "stress", "sinalização", "metabolismo hormonal", "transporte", "proteína" e "parede celular". Os genes identificados envolvidos na resistência das plantas serão alvos preferenciais para a caracterização funcional e para o desenvolvimento de ferramentas moleculares que auxiliem o desenvolvimento de acessos de feijão com resistência múltipla a stresses bióticos e abióticos.Em conclusão, o trabalho descrito nestatese de doutoramento (Ph.D.) abriu caminhopara que se desenvolva um programa de melhoramento de feijão em Portugal com vista ao desenvolvimento de variedades mais adaptadas a múltiplosstresses.Foi caracterizada a diversidade molecular e morfológica deste recurso genético nacional, identificadasnovas fontes de resistência a Fusariume de tolerânciaacondições de déficit hídrico,e ampliou-se oconhecimento existente docontrolo genético da resistência a doenças fúngicas e de características relacionadas com a fotossíntese no feijoeiro

Lathyrus research in Portugal

Lathyrus spp. in Portugal are nowadays underutilized crops. The main species grown are L. sativus (grass pea) for food and L. cicera (chickling vetch) for feed.Peer reviewe

Portuguese common bean landraces vs. Fusarium oxysporum: a diverse array of resistances (first steps into an unexploited source for resistance breeding)

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 the most important food legume worldwide. Portugal holds a very diverse common bean germplasm consisting of landraces that resulted from more than five centuries of natural adaptation and farmer’s mass selection, not yet fully explored in breeding. Fusarium wilt is among the diseases that cause major yield losses in this crop. The agronomical, morphological and molecular diversity observed in this Portuguese common bean germplasm anticipates the presence of diverse sources of resistance not yet explored in breeding and foresees the identification of novel resistance / effector target genes. In order to identify alternative sources of resistance for future allelic diversity breeding exploitation of potential resistance genes, we characterized the resistance to fusarium wilt of 150 Portuguese common bean landraces. Evaluation took place under controlled conditions. Seven-day-old seedlings were inoculated with Fusarium oxysporum f. sp. phaseoli, and symptoms assessed every three days, from 7th to 30th dai, using a visual disease index scale from 1 (healthy leaf) to 5 (dead leaf). These data were used to calculate the AUDPC (area under the disease progress curve) values. Infection responses revealed great variability among landraces, with the identification of an array of diverse sources of resistance. Representatives of the different levels of resistance were selected for a future more detailed molecular characterization of the resistance mechanisms. Once these mechanisms are characterized, this germplasm can be better exploited on common bean resistance breeding.N

Elucidation of the rust resistance genetic control in Portuguese common bean through a genime-wide association study

Trabajo presentado en el 15th Congress of the Mediterranean Phytopathological Union (Plant health sistaining Mediterranean ecosystems), celebrado en Córdoba (España) del 20 al 23 de junio de 2017.Common bean (Phaseolus vulgaris) is the most important grain legumes for human consumption worldwide, with recognized nutritional and environmental benefits. However, its vulnerability to several diseases leads to significant yield losses and limited cultivation in Europe.This research was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through the grant SFRH/BD/92160/2013, IF/01337/2014 FCT Investigator contract, the project Exploiting bean genetics for food quality and attractiveness innovation (PTDC/AGRTEC/3555/2012), the Research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013), and by COST Actions FA1208 and FA1306.Peer reviewe

Shared and tailored common bean transcriptomic responses to combined fusarium wilt and water deficit

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.Financial support by Fundação para a Ciência e Tecnologia (FCT), Portugal, is acknowledged through grant SFRH/BD/92160/2013 (S.T.L.), CEECIND/00198/2017 (C.S.), DL57 PhD holder contract (S.S.A.), research project BeGeQA (PTDC/AGR-TEC/3555/2012), and R&D Unit GREEN-IT—Bioresources for Sustainability (UIDB/04551/2020). PDR2020-784-042734 project is also acknowledged. S.S.A. also acknowledges the financial support from the 3i Bioeconomy project POCI-01-0246-FEDER-026758, funded by the Fundo Europeu de Desenvolvimento Regional (FEDER), under the “Programa Operacional Temático Competitividade e Internacionalização”—COMPETE 2020 and the program NORTE 2020 through the project NORTE-06-3559-FSE-000103 funded by the Fundo Social Europeu (FSE)

Allelic diversity in the transcriptomes of contrasting rust-infected genotypes of Lathyrus sativus, a lasting resource for smart breeding

[Background] Grass pea (Lathyrus sativus L.) is a valuable resource for potentially durable partial resistance to rust. To gain insight into the resistance mechanism and identify potential resistance genes, we generated the first comprehensive transcriptome assemblies from control and Uromyces pisi inoculated leafs of a susceptible and a partially rust-resistant grass pea genotype by RNA-seq.[Results] 134,914 contigs, shared by both libraries, were used to analyse their differential expression in response to rust infection. Functional annotation grouped 60.4% of the contigs present in plant databases (37.8% of total) to 33 main functional categories, being “protein”, “RNA”, “signalling”, “transport” and “stress” the most represented. Transcription profiles revealed considerable differences in regulation of major phytohormone signalling pathways: whereas Salicylic and Abscisic Acid pathways were up-regulated in the resistant genotype, Jasmonate and Ethylene pathways were down-regulated in the susceptible one. As potential Resistance-genes we identified a mildew resistance locus O (MLO)-like gene, and MLO-related transcripts. Also, several pathogenesis-related genes were up-regulated in the resistant and exclusively down regulated in the susceptible genotype. Pathogen effectors identified in both inoculated libraries, as e.g. the rust Rtp1 transcript, may be responsible for the down-regulation of defence-related transcripts. The two genotypes contained 4,892 polymorphic contigs with SNPs unevenly distributed between different functional categories. Protein degradation (29.7%) and signalling receptor kinases (8.2%) were the most diverged, illustrating evolutionary adaptation of grass pea to the host/pathogens arms race.[Conclusions] The vast array of novel, resistance-related genomic information we present here provides a highly valuable resource for future smart breeding approaches in this hitherto under-researched, valuable legume crop.This work was supported by the project LEGRESIST (GEN2006-27798-C6-5-/VEG) and by Fundação para a Ciência e a Tecnologia through Grants #PEst-OE/EQB/LA0004/2011 and #PTDC/AGR-GPL/103285/2008. NFA and MCVP were supported by Fundação para a Ciência e a Tecnologia (SFRH/BD/44357/2008 and Research Contracts by the Ciência 2008 program respectively).Peer reviewe

Unveilling the potential of Portuguese common bean varieties to genetic improvement: rust and fusarium wilt resistance

Trabajo presentado en la V Meeting Asociación Española de Leguminosas, AEL (Eucarpia International Symposium on Protein Crops), celebrada en Pontevedra del 4 al 7 de mayo de 2015.Common bean (Phaseolus vulgaris L.) is the worldwide most important food legume, being an excellent source of proteins, fiber, minerals (iron and zinc) and vitamins. In addition to its health benefits it is an environmentally friendly resource because of the capability of fixing atmospheric nitrogen as a result of the endosymbiotic association with the soil bacteria Rhizobium. However, due yield instability caused by biotic and abiotic stresses, this crop is underused in Europe. Therefore, it is essential to genetically improve common bean varieties in order to attain more stable, reliable and sustainable local production. A collection of 130 Portuguese common bean landraces, resulting from more than five centuries of adaptation and mass selection by farmers, was screened for resistance to rust and fusarium wilt under controlled conditions. The diseases caused by these two very different fungi (an airborne and a soilborne, respectively) are among the most important constraints to the bean production and quality being intimately related to yield loss. Rust infection type and disease severity were analyzed 12 days after inoculation using a 0-4 scale (Stakman et al., 1962). The most frequent infection type observed was 4, indicative of a compatible plant-pathogen interaction, with no macroscopically visible hypersensitivity. Disease severity values varied greatly, from <1 to 40 %. Sixteen landraces showed low infection types (0, 1 or 2), indicative of incompatible interaction. Forty landraces presented chlorotic halos surrounding the rust pustules on infected leaves. For the fusarium evaluation, symptoms were assessed every 3 days, from 7th to 30th days after inoculation, with ratings value based on a disease index scale ranging from 1 (healthy plant) to 5 (dead plant) (Rispail & Rubiales, 2014). A week after the inoculation, some landraces presented disease symptoms in the primary leaves, with curled yellow margins that progressed fast to a stage where whole leaves became wilted and dry. Two weeks after the infection, all plants from 5 land -races were already dead. On the other hand, 10 landraces showed plants with resistance to fusarium infection that presented no symptoms or only low levels of yellowing or discoloration on the leaves margins. These interesting sources of resistance may now be incorporated in pre-breeding materials aiming the development of attractive new varieties and will be study in detail to understand the depicted resistance mechanisms.N

Exploiting the forgotten common bean landrace phenotypes: disease resistance and genomic data integration

Trabajo presentado en la 2nd General Meeting of COST FA1306 (The quest for tolerant varieties - Phenotyping at plant and cellular level. PhenomenALL), celebrada en Copenhagen del 18 al 20 de abril de 2016.Common bean (Phaseolus vulgaris L.) is the worldwide most important grain legume for human consumption. In addition to its nutritional and health benefits, it is an environmentally friendly resource because of its ability to fix atmospheric nitrogen as a result of the endosymbiotic association with the soil bacteria Rhizobium. However, due to yield instability caused by biotic and abiotic stresses, this crop is underused in Europe. It is therefore essential to genetically improve common bean varieties stress resistance in order to attain more stable, reliable and sustainable local production. Fusarium wilt (caused by the soil borne fungus Fusarium oxysporum f. sp. phaseoli) is among the most important constraints to bean production, being intimately related to yield loss. Portugal holds a very promising national common bean germplasm collection, resulting from more than five centuries of natural adaptation and farmer’s mass selection, not yet fully explored in breeding. In this work we have used an integrated phenotyping approach to search for the genomic regions controlling fusarium wilt resistance in a collection of Portuguese landraces. This will pave the way for the future improvement of resistance in common bean, based on this valuable, highly diverse and unexplored genetic resource. To do so, a collection of 150 Portuguese common bean landraces was phenotyped for resistance to fusarium wilt under growth chamber controlled conditions. Disease symptoms were assessed, using a 1-5 visual scale, every 3 days, from the 7th to 30th day after inoculation. Two weeks after infection, observed disease severity varied greatly among landraces, ranging from landraces completely dead till landraces with no symptoms or only low levels of yellowing or discoloration on the leaves margins. The same collection was screened with more than 12.000 SNPs (Single Nucleotide Polymorphism) uniformly distributed throughout the genome. A genome-wide association study, joining the disease resistance phenotypic information with the genomic information, is currently ongoing using mixed linear models to account for population structure and familiar relatedness. This integration of phenotyping at different levels will allow the identification of molecular tools to assist future common bean resistance precision breeding.N

Genome-wide association study of common bean disease resistance in Portuguese germplasm

Trabajo presentado en el Second International Legume Society Conference "Legumes for a sustainable world" (ILS2), celebrado en Tróia (Portugal) del 11 al 14 de octubre de 2016.Common bean (Phaseolus vulgarisL.) is the world’s most important grain legume for human consumption. Portugal holds a very promising common bean germplasm, which results from more than five centuries of natural adaptation and farmer’s mass selection in the country, not yet fully explored in breeding. Fusarium wilt (Fusarium oxysporum f. sp. phaseoli) is among the diseases that cause major yield losses in this crop. In order to identify new sources of resistance and to unravel the associated genetic control we characterized the resistance to fusarium wilt of a collection of 150 Portuguese common bean accessions. Evaluation took place under growth chamber conditions, using a replicated complete randomized block design. Infection responses revealed great variability among the accessions with the identification of interesting sources of resistance. This collection was additionally screened with ~12000 molecular markers (SSR and SNPs from Illumina BeadChip and DArTseq arrays) uniformly distributed throughout the genome. A genome-wide association study, joining the disease resistance levels with the genomic information, is currently ongoing, using a mixed linear model approach accounting for population structure and familial relatedness. The putative statistically significant associations found will contribute to a more effective selection of common bean genetic resources in future disease resistance breeding programs.N