Large-scale gene gains and losses molded the NLR defense arsenal during the Cucurbita evolution

[EN] Main conclusion Genome-wide annotation reveals that the gene birth-death process of the Cucurbita R family is associated with a species-specific diversification of TNL and CNL protein classes. The Cucurbitaceae family includes nearly 1000 plant species known universally as cucurbits. Cucurbita genus includes many economically important worldwide crops vulnerable to more than 200 pathogens. Therefore, the identification of pathogen-recognition genes is of utmost importance for this genus. The major class of plant-resistance (R) genes encodes nucleotide-binding site and leucine-rich repeat (NLR) proteins, and is divided into three sub-classes namely, TIR-NB-LRR (TNL), CC-NB-LRR (CNL) and RPW8-NB-LRR (RNL). Although the characterization of the NLR gene family has been carried out in important Cucurbita species, this information is still linked to the availability of sequenced genomes. In this study, we analyzed 40 de novo transcriptomes and 5 genome assemblies, which were explored to investigate the Cucurbita expressed-NLR (eNLR) and NLR repertoires using an ad hoc gene annotation approach. Over 1850 NLR-encoding genes were identified, finely characterized and compared to 96 well-characterized plant R-genes. The maximum likelihood analyses revealed an unusual diversification of CNL/TNL genes and a strong RNL conservation. Indeed, several gene gain and loss events have shaped the Cucurbita NLR family. Finally, to provide a first validation step Cucurbita, eNLRs were explored by real-time PCR analysis. The NLR repertories of the 12 Cucurbita species presented in this paper will be useful to discover novel R-genes.Open access funding provided by Universita degli Studi di Napoli Federico II within the CRUI-CARE Agreement.Andolfo, G.; Sáez-Sánchez, C.; Cañizares Sales, J.; Picó Sirvent, MB.; Ercolano, MR. (2021). Large-scale gene gains and losses molded the NLR defense arsenal during the Cucurbita evolution. Planta. 254(4):1-14. https://doi.org/10.1007/s00425-021-03717-xS114254

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

[EN] Background: K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design. Results: As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%. Conclusion: K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.This work was supported by the University Polytechnic of Valencia, Grant Number 20180051 "Desarrollo de herramientas para la identificacion de genes y loci de interes en la mejora genetica del tomate y otras horticolas".Ziarsolo, P.; Hasing, T.; Hilario, R.; García-Carpintero, V.; Blanca Postigo, JM.; Bombarely, A.; Cañizares Sales, J. (2021). K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology. Plant Methods. 17(1):1-11. https://doi.org/10.1186/s13007-021-00733-6S11117

Haplotype analyses reveal novel insights into tomato history and domestication driven by long-distance migrations and latitudinal adaptations

A novel haplotype-based approach that uses Procrustes analysis and automatic classification was used to provide further insights into tomato history and domestication. Agrarian societies domesticated species of interest by introducing complex genetic modifications. For tomatoes, two species, one of which had two botanical varieties, are thought to be involved in its domestication: the fully wild Solanum pimpinellifolium (SP), the wild and semi-domesticated Solanum lycopersicum var. cerasiforme (SLC) and the cultivated S. l. var. lycopersicum (SLL). The Procrustes approach showed that SP evolved into SLC during a gradual migration from the Peruvian deserts to the Mexican rainforests and that Peruvian and Ecuadorian SLC populations were the result of more recent hybridizations. Our model was supported by independent evidence, including ecological data from the accession collection site and morphological data. Furthermore, we showed that photosynthesis-, and f lowering time-related genes were selected during the latitudinal migrations

Long-term and transgenerational phenotypic, transcriptional and metabolic effects in rabbit males born following vitrified embryo transfer

[EN] The advent of assisted reproductive technologies (ART) in mammals involved an extraordinary change in the environment where the beginning of a new organism takes place. Under in vitro conditions, in which ART is currently being performed, it likely fails to mimic optimal in vivo conditions. This suboptimal environment could mediate in the natural developmental trajectory of the embryo, inducing lasting effects until later life stages that may be inherited by subsequent generations (transgenerational effects). Therefore, we evaluated the potential transgenerational effects of embryo exposure to the cryopreservation-transfer procedure in a rabbit model on the offspring phenotype, molecular physiology of the liver (transcriptome and metabolome) and reproductive performance during three generations (F1, F2 and F3). The results showed that, compared to naturally-conceived animals (NC group), progeny generated after embryo exposure to the cryopreservation-transfer procedure (VT group) exhibited lower body growth, which incurred lower adult body weight in the F1 (direct effects), F2 (intergenerational effects) and F3 (transgenerational effects) generations. Furthermore, VT animals showed intergenerational effects on heart weight and transgenerational effects on liver weight. The RNA-seq data of liver tissue revealed 642 differentially expressed transcripts (DETs) in VT animals from the F1 generation. Of those, 133 were inherited from the F2 and 120 from the F3 generation. Accordingly, 151, 190 and 159 differentially accumulated metabolites (DAMs) were detected from the F1, F2 and F3, respectively. Moreover, targeted metabolomics analysis demonstrated that transgenerational effects were mostly presented in the non-polar fraction. Functional analysis of molecular data suggests weakened zinc and fatty acid metabolism across the generations, associated with alterations in a complex molecular network affecting global hepatic metabolism that could be associated with the phenotype of VT animals. However, these VT animals showed proper reproductive performance, which verified a functional health status. In conclusion, our results establish the long-term transgenerational effects following a vitrified embryo transfer procedure. We showed that the VT phenotype could be the result of the manifestation of embryonic developmental plasticity in response to the stressful conditions during ART procedures.Funding from the Ministry of Economy, Industry and Competitiveness (Research project: AGL2014-53405-C2-1-P) and Generalitat Valenciana (Research project: Prometeo II 2014/036) is acknowledged. X.G.D. was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429). 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Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit

[EN] Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO), Spain, grant number AGL2014-53405-C2-1-P and by Conselleria d'Educacio, Investigacio, Cultura i Esport, Spain, grant number Prometeo II 2014/036. Ximo Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness of Spain (BES-2015-072429).García-Domínguez, X.; Diretto, G.; Peñaranda, D.; Frusciante, S.; García-Carpintero, V.; Cañizares Sales, J.; Vicente Antón, JS.... (2021). Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit. International Journal of Molecular Sciences. 22(18):1-17. https://doi.org/10.3390/ijms22189716S117221

Flower induction and development in saffron: Timing and hormone signalling pathways

[EN] The demand for saffron is expected to rise in the coming years due to its nutraceutical and medicinal properties. To cope with this, it will be necessary to develop a mechanised production of saffron. Upgrading the production methods requires accurate control of the flowering time in this species. Nevertheless, little is known about the control of flowering time in Crocus sativus L. The aim of this study is to gain insight into the floral induction regulatory networks operating in this species. A transcriptomic analysis was performed from saffron main buds in different stages of development. The identification of putative integrators of flowering time signals, like FT, as well as meristem identity genes, such as LFY and TFL1, permitted the definition of the time of flowering induction of the buds, being able to use them as molecular markers. The identification of the transcripts encoded by a DROOPING LEAF-like (DL) gene is of particular relevance because this gene might be a novel factor for carpel specification in saffron. To elucidate the hormonal signalling networks working during flower induction, transcriptomic data were used, and the content of IAA, ABA and gibberellins was determined in competent and non-competent buds to flower, during the saffron life cycle. Our results suggested that ABA might be negatively regulating corm dormancy release, but its involvement in flower induction cannot be ruled out. ABI5 and the mediator of ABA regulated dormancy gene MARD1, could be key players of this pathway. In addition, a drop in GA4 levels may also be a necessary, but insufficient, condition for floral induction and development. DELLA, TFL1 and PIF3 genes might be involved in the gibberellin pathway. Notably, IAA seems to be a positive regulator of the process, involving MP/ARF5 and ANT genes in the pathway. Taken together, these results pave the way to the unveiling of the regulatory networks controlling the vegetative-to-reproductive phase change in saffron.The activities of this study have been supported by a project funded by the "Ministerio de Ciencia, Innovacion y Universidades de Espana" [AGL2016-77078-R].Renau-Morata, B.; Nebauer, SG.; García-Carpintero, V.; Cañizares Sales, J.; Minguet, E.; De Los Mozos, M.; Molina Romero, RV. (2021). Flower induction and development in saffron: Timing and hormone signalling pathways. Industrial Crops and Products. 164:1-19. https://doi.org/10.1016/j.indcrop.2021.113370S11916

Ceremonia de entrega del Premio Estudios Financieros 2017, Clausura del curso académico 2016-2017 del CEF.- y Acto de graduación de la VI promoción de la UDIMA

La XXVII edición del Premio Estudios Financieros tuvo su brillante colofón el pasado 20 de julio en una ceremonia de entrega que en esta ocasión se desarrolló en el histórico Ateneo de Madrid. Como no podía ser de otra manera, premiados, familiares y amigos, representantes de los jurados y personalidades llenaron la sala en un día en el que la labor de mecenazgo en aras de la investigación, que el Centro de Estudios Financieros (CEF.-) lleva haciendo desde 1990, se pone en relieve

Integrative Transcriptomic and Metabolomic Analysis at Organ Scale Reveals Gene Modules Involved in the Responses to Suboptimal Nitrogen Supply in Tomato

[EN] The development of high nitrogen use efficiency (NUE) cultivars under low N inputs is required for sustainable agriculture. To this end, in this study, we analyzed the impact of long-term suboptimal N conditions on the metabolome and transcriptome of tomato to identify specific molecular processes and regulators at the organ scale. Physiological and metabolic analysis revealed specific responses to maintain glutamate, asparagine, and sucrose synthesis in leaves for partition to sustain growth, while assimilated C surplus is stored in the roots. The transcriptomic analyses allowed us to identify root and leaf sets of genes whose expression depends on N availability. GO analyses of the identified genes revealed conserved biological functions involved in C and N metabolism and remobilization as well as other specifics such as the mitochondrial alternative respiration and chloroplastic cyclic electron flux. In addition, integrative analyses uncovered N regulated genes in root and leaf clusters, which are positively correlated with changes in the levels of different metabolites such as organic acids, amino acids, and formate. Interestingly, we identified transcription factors with high identity to TGA4, ARF8, HAT22, NF-YA5, and NLP9, which play key roles in N responses in Arabidopsis. Together, this study provides a set of nitrogen-responsive genes in tomato and new putative targets for tomato NUE and fruit quality improvement under limited N supply.This study was supported by grants from The National Institute for Agriculture and Food Research and Technology (CSIC-INIA) (RTA2015-00014-c02-00 to JMA and RTA2015-00014-c02-01 to SGN) and the Community of Madrid (AGRISOST-CM S2018/BAA-4330 to JMA). We also want to acknowledge the "Severo Ochoa Program for Centers of Excellence in R&D" from the Agencia Estatal de Investigacion of Spain (Grant SEV-2016-0672) for supporting the scientific services used in this study. J. Canales was supported by the Agencia Nacional de Investigacion y Desarrollo de Chile (ANID, FONDECYT 1190812) and ANID-Millennium Science Initiative Program (ICN17-022).Renau-Morata, B.; Molina Romero, RV.; Minguet, E.; Cebolla Cornejo, J.; Carrillo, L.; Martí-Renau, R.; García-Carpintero, V.... (2021). Integrative Transcriptomic and Metabolomic Analysis at Organ Scale Reveals Gene Modules Involved in the Responses to Suboptimal Nitrogen Supply in Tomato. Agronomy. 11(7):1-26. https://doi.org/10.3390/agronomy11071320S12611

European traditional tomatoes galore: a result of farmers' selection of a few diversity-rich loci

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types

Discovery of a major QTL controlling trichome IV density in tomato using K-seq genotyping

Trichomes are a common morphological defense against pests, in particular, type IV glandular trichomes have been associated with resistance against different invertebrates. Cultivated tomatoes usually lack or have a very low density of type IV trichomes. Therefore, for sustainable management of this crop, breeding programs could incorporate some natural defense mechanisms, such as those afforded by trichomes, present in certain Solanum species. We have identified a S. pimpinellifolium accession with very high density of this type of trichomes. This accession was crossed with a S. lycopersicum var. cerasiforme and a S. lycopersicum var. lycopersicum accessions, and the two resulting F2 populations have been characterized and genotyped using a new genotyping methodology, K-seq. We have been able to build an ultra-dense genetic map with 147,326 SNP markers with an average distance between markers of 0.2 cm that has allowed us to perform a detailed mapping. We have used two different families and two different approaches, QTL mapping and QTL-seq, to identify several QTLs implicated in the control of trichome type IV developed in this accession on the chromosomes 5, 6, 9 and 11. The QTL located on chromosome 9 is a major QTL that has not been previously reported in S. pimpinellifolium. This QTL could be easily introgressed in cultivated tomato due to the close genetic relationship between both species