Identification of novel sources of partial and incomplete hypersensitive resistance to rust and associated genomic regions 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 ß-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.

Office endometrial sampling: effectiveness and predictive factors of success in Novak versus Endosampler devices.

BACKGROUND: The study aimed to evaluate the rate of endometrial sampling (ES) failure, predictive factors of success, and reliability as diagnostic methods of Endosampler versus Novak. METHODS: A retrospective single-center study was carried out with all patients who underwent ES via Endosampler or Novak in 2020 and 2021. Demographic data, personal background, and histopathologic results were evaluated. RESULTS: Eighty-six patients underwent ES by Novak and 90 by Endosampler. The failure rate of ES was 43.2% with lower values for Endosampler (33.3% vs. 53.5%, P<0.05). Age, biopsy device, menopausal status, indication for biopsy, and amount of sample collected were predictive factors of failure. Analyzing each device, Endosampler was only affected by menopausal status. Only 50% in Novak and 62.5% in the Endosampler group of endometrial neoplasia cases were detected by these methods. Analyzing the performance for endometrial neoplasia (EN), we obtained higher values of sensitivity and accuracy for Endosampler (62.5% vs. 50.0% and 83.3% vs. 72.7%), respectively. CONCLUSIONS: In our study, the failure rate obtained was in line with other previous studies. Menopausal status, age, type of biopsy device, indication for biopsy, and amount of sample collected affected ES performance. Analyzing diagnostic performance for EN, we found that these methods have better reliability for positive results than for negative ones, which may indicate the need for further evaluation in cases of high clinical suspicion. In short, we obtain a higher rate of success rate in Endosampler devices and better performance in diagnosing EN, which is the major objective of an ES.

Oligogenic Control of Quantitative Resistance Against Powdery Mildew Revealed in Portuguese Common Bean Germplasm.

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.

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.

Ovarian vein thrombosis: a rare but not to be forgotten cause of abdominal pain.

A healthy 38-year-old woman, with no significant medical or surgical history, was referred to the gynaecology emergency department with acute low quadrant abdominal pain and nausea. On examination, painful palpation of right iliac fossa, abdominal guarding and positive Blumberg's sign were noted. An abdominal-pelvic CT scan showed a hypodense tubular structure with peripheral enhancement to the right of the inferior vena cava, suggestive of thrombosis of the right ovarian vein. The only risk factor identified was the use of the combined contraceptive pill.

Towards a Trait-Based Approach to Potentiate Yield under Drought in Legume-Rich Annual Forage Mixtures.

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.

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.

Common bean SNP alleles and candidate genes affecting photosynthesis under contrasting water regimes.

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 CO2 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.

Portuguese Common Bean Natural Variation Helps to Clarify the Genetic Architecture of the Legume's Nutritional Composition and Protein Quality.

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.

Natural Variation in Portuguese Common Bean Germplasm Reveals New Sources of Resistance Against Fusarium oxysporum f. sp. phaseoli and Resistance-Associated Candidate Genes.

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.

Characterization of Soaking Process' Impact in Common Beans Phenolic Composition: Contribute from the Unexplored Portuguese Germplasm.

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).

First genetic linkage map of Lathyrus cicera based on RNA sequencing-derived markers: Key tool for genetic mapping of disease resistance.

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.

Establishing the Bases for Introducing the Unexplored Portuguese Common Bean Germplasm into the Breeding World.

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.

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.

Transferability of molecular markers from major legumes to Lathyrus spp. for their application in mapping and diversity studies.

Lathyrus cicera L. (chickling pea) and L. sativus L. (grass pea) have great potential among grain legumes due to their adaptability to inauspicious environments, high protein content and resistance to serious diseases. Nevertheless, due to its past underused, further activities are required to exploit this potential and to capitalise on the advances in molecular biology that enable improved Lathyrus spp. breeding programmes. In this study we evaluated the transferability of molecular markers developed for closely related legume species to Lathyrus spp. (Medicago truncatula, pea, lentil, faba bean and lupin) and tested the application of those new molecular tools on Lathyrus mapping and diversity studies. Genomic and expressed sequence tag microsatellite, intron-targeted amplified polymorphic, resistance gene analogue and defence-related gene markers were tested. In total 128 (27.7 %) and 132 (28.6 %) molecular markers were successfully cross-amplified, respectively in L. cicera and L. sativus. In total, the efficiency of transferability from genomic microsatellites was 5 %, and from gene-based markers, 55 %. For L. cicera, three cleaved amplified polymorphic sequence markers and one derived cleaved amplified polymorphic sequence marker based on the cross-amplified markers were also developed. Nine of those molecular markers were suitable for mapping in a L. cicera recombinant inbred line population. From the 17 molecular markers tested for diversity analysis, six (35 %) in L. cicera and seven (41 %) in L. sativus were polymorphic and discriminate well all the L. sativus accessions. Additionally, L. cicera accessions were clearly distinguished from L. sativus accessions. This work revealed a high number of transferable molecular markers to be used in current genomic studies in Lathyrus spp. Although their usefulness was higher on diversity studies, they represent the first steps for future comparative mapping involving these species.

Conservative laparoscopic approach to a perforated pyomyoma after uterine artery embolization.

We describe a conservative laparoscopic approach to treatment of a perforated pyomyoma after uterine artery embolization. A 36-year-old woman came to our emergency department 8 weeks after undergoing uterine artery embolization. She reported painful abdominal cramps. Physical examination revealed fever, tachycardia, hypotension, abdominal pain with rebound tenderness and pain during bimanual examination. Ultrasonography showed a fundal/subserosal leiomyoma and a moderate amount of fluid in the abdominal cavity. Because the patient desired preservation of the uterus, a laparoscopic approach was used for drainage and lavage of a perforated pyomyoma. The patient had an uneventful recovery and remained well at follow-up visits. Second-look diagnostic laparoscopy enabled treatment of the adhesions formed and revealed patent fallopian tubes. Surgery has been the primary approach to pyomyoma. However, hysterectomy leads to irreversible sterility and myomectomy can be difficult to perform. Therefore, there is need for a less invasive intervention in women who seek conservative treatment. Recently, computed tomography-guided drainage was successfully performed in two patients with pyomyoma. To our knowledge, this is the first report of a successful conservative laparoscopic approach to pyomyoma.

A quantitative structure-property relationship study of the release of some esters and alcohols from barley and oat beta-glucan matrices.

This study investigates the release of selected strawberry flavor compounds from aqueous solutions of two barley and oat beta-glucan products at concentrations of 5, 10, and 15% (w/w). The flavor release of 12 esters and 3 alcohols was measured by dynamic headspace GC-MS. For each compound the ratio of the flavor release from the beta-glucan solution to the release from aqueous solution, A(rel), was recorded. In general, esters were retained in the beta-glucan matrices in a mass-dependent manner where heavier molecules were retained more. A(rel) for alcohols was found to be significantly larger than for the esters. Whereas A(rel) values for esters were always below unity, this parameter was above unity for alcohols in some preparations of beta-glucan. This implies that relative to esters, alcohols were rejected from some matrices. An increase in the concentration of the beta-glucan products was associated with an increased retention of alcohols and esters. For solutions of oat and barley beta-glucan products at the same concentration, the oat product retained the flavor compounds more strongly. This difference was more pronounced at low concentrations of the beta-glucan products. To investigate the potential of a multivariate approach for the analysis of the flavor release from beta-glucan products, partial least-squares regression was employed on a large selection of calculated molecular descriptors, yielding simple QSPR models capable of explaining the variation in A(rel). The robustness of the QSPR models was verified by cross-validation and permutation tests. The results indicate that the multivariate modeling approach might provide a useful tool for the investigation of flavor release systems similar to those studied here.