Mass spectrometry based metabolic profiling of six-row barley (Hordeum vulgare L.) genotypes varying in resistance to Fusarium graminearum

Fusarium head blight (FHB) of barley (Hordeum vulgare L.) is a devastating disease, caused by Fusarium graminearum (teleomorph: Gibberella zea), resulting in reduced yield and quality of grain by producing mycotoxins. The resistance in barley to FHB is quantitative and controlled by several genes, thus making it difficult to breed for resistance. In wheat (Triticum aestivum L.) and barley more than 100 quantitative trait loci (QTL) for resistance have been reported against FHB, but the mechanisms of resistance controlled by these QTL are unknown. Metabolic profiling technology was applied to better understand the mechanisms of resistance and to phenotype resistance in barley genotypes against FHB. The current study aimed to: 1) identified the resistance related (RR) metabolites by comparing resistance in barley cultivars Chevron and Stander against FHB, and determined antimicrobial properties of selected RR metabolites under in vitro conditions; 2)determined the effects of selected RR metabolites on inhibition of trichothecene biosynthesis by F. graminearum under in vitro conditions; 3) identified biomarker metabolites, in six barley genotypes ('Chevron', H5277-44, H5277-164, M92-513,M122, and 'Stander') varying in resistance to FHB, for potential biomarker selection to screen barley genotypes for resistance. Barley genotypes were mock-inoculated or pathogen-inoculated under greenhouse conditions; metabolites were extracted using aqueous methanol and analyzed using LCESI-LTQ-Orbitrap. XCMS and CAMERA algorithms were used to process the LC/MS output. Significant metabolites were classified as RR constitutive, and RR induced based on their greater abundance in resistant genotypes. Deoxynivalenol (DON) and its detoxified metabolite DON-3-O-glucoside (D3G), designated here as resistance indicator metabolites, were detected in both resistant and susceptible genotypes. The resistant cultivar Chevron had the least DON accumulation and high level of DON conversion to D3G. The selected RR metabolites varied in their ability to inhibit mycelial biomass and trichothecene synthesis by F. graminearum in vitro. The major potential biomarkers selected were: p-coumaric acid, sinapic acid, naringenin, naringenin-glucoside, kaempferol-glucosides, jasmonic acid, methyl jasmonate, and linolenic acid. In conclusion, we have demonstrated here that the mass spectrometry tool can be used to better understand the mechanisms of quantitative resistance in barley against biotic stress and to select potential biomarkers to screen for FHB resistance.La fusariose de l'épi (FE) de l'orge est une maladie dévastatrice causée par Fusarium graminearum (Gibberella zea) et résultant en pertes de rendement et de qualité du grain dû à la production de mycotoxines. La résistance à la FE chez l'orge peut être quantifiée et est généralement contrôlée par plusieurs gènes, ce qui limite l'amélioration de ce trait par de simples croisements. Plus de 100 loci de caractères quantitatifs (LCQ) de résistance contre la FE ont été rapportés chez le blé et l'orge, mais les mécanismes de résistance contrôlés par ces LCQ sont inconnus. La technologie de profilage métabolique a été appliquée afin de mieux comprendre les mécanismes de résistance contre la FE et de 'phénotyper' la résistance de certains génotypes d'orge. Les objectifs de cette étude sont : 1) d'identifier les métabolites reliés à la résistance (RR) en comparant la résistance contre la FE descultivars Chevron et Stander et de déterminer les propriétés antimicrobiennes des métabolites RR sélectionnés in vitro; 2) de déterminer l'effet des métabolites RR sélectionnés sur l'inhibition de la biosynthèse du trichothécène par F. graminearum invitro; et 3) d'identifier des métabolites biomarqueurs chez six génotypes ('Chevron', H5277-44, H5277-164, M92-513, 'M122' et 'Stander') avec une résistance différente à la FE, afin de sélectionner des biomarqueurs permettant d'évaluer la résistance chez les génotypes d'orge. Les génotypes d'orge ont été inoculés avec de l'eau ou un pathogène enconditions de serre. Les métabolites ont été extraits avec du méthanol aqueux et analysés avec LC-ESI-LTQ-Orbitrap. Les algorithmes XCMS et CAMERA ont été utilisés pourtraiter le produit LC/MS. Des métabolites significatifs ont été classifiés en fonction de leur lien avec la résistance constitutive et exprimés en fonction de leur abondance qui est plus importante chez les génotypes résistants. Le déoxynivalénol (DON) et le glucoside DON-3-O, son métabolite détoxifié (D3G), désignés ici comme des métabolites indicateurs la résistance, ont été détectés dans les génotypes résistants et sensibles. Le cultivar résistant Chevron a produit le plus bas niveau de DON total et la plus grande proportion de DON converti en D3G. Les métabolites RR sélectionnés ont varié dans leur habileté à inhibiter la biomasse de mycélium et la synthèse du trichothécène par G. zeaein vitro. Les biomarqueurs potentiels qui ont été sélectionnés sont : l'acide p-coumarique, l'acide sinapique, la naringinine, le glucoside de naringinine, les glucosides de kaempférol, l'acide jasmonique, le jasmonate de méthyl et l'acide linolénique. En conclusion, nous avons démontré que la spectrométrie de masse peut être utilisée afin demieux comprendre les mécanismes de résistance quantitative chez l'orge contre le stress biotique et pour sélectionner des biomarqueurs potentiels permettant d'évaluer la résistance FE

Targeted mutagenesis in wheat microspores using CRISPR/Cas9

Abstract CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants

Single-nucleotide polymorphism identification and genotyping in Camelina sativa

Camelina sativa, a largely relict crop, has recently returned to interest due to its potential as an industrial oilseed. Molecular markers are key tools that will allow C. sativa to benefit from modern breeding approaches. Two complementary methodologies, capture of 3\u2032 cDNA tags and genomic reduced-representation libraries, both of which exploited second generation sequencing platforms, were used to develop a low density (768) Illumina GoldenGate single nucleotide polymorphism (SNP) array. The array allowed 533 SNP loci to be genetically mapped in a recombinant inbred population of C. sativa. Alignment of the SNP loci to the C. sativa genome identified the underlying sequenced regions that would delimit potential candidate genes in any mapping project. In addition, the SNP array was used to assess genetic variation among a collection of 175 accessions of C. sativa, identifying two sub-populations, yet low overall gene diversity. The SNP loci will provide useful tools for future crop improvement of C. sativa.Peer reviewed: YesNRC publication: Ye

The emerging biofuel crop camelina sativa retains a highly undifferentiated hexaploid genome structure

Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop

Alpha(2)-adrenergic receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats during development

The nucleus tractus solitarii (NTS), located in the brainstem, is one of the main nuclei responsible for integrating different signals in order to originate a specific and orchestrated autonomic response. Antihypertensive drugs are well known to stimulate alpha(2)-adrenoceptor (alpha(2R)) in brainstem cardiovascular regions to induce reduction in blood pressure. Because alpha(2R) impairment is present in several models of hypertension, the aim of the present study was to investigate the distribution and density of alpha(2R) binding within the NTS of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats during development (1,15,30 and 90 day-old) by an in vitro autoradiographical study. The NTS shows heterogeneous distribution of alpha(2R) in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. Alpha(2R) increased from rostral to caudal dorsomedial/dorsolateral subnuclei in 30 and 90 day-old SHR but not in WKY. Alpha(2R) decreased from rostral to caudal subpostremal subnucleus in 15, 30 and 90 day-old SHR but not in WKY. Medial/intermediate subnuclei did not show any changes in alpha(2R) according to NTS levels. Furthermore, alpha(2R) are decreased in SHR as compared with WKY in all NTS subnuclei and in different ages. Surprisingly, alpha(2R) impairment was also found in pre-hypertensive stages, specifically in subpostremal subnucleus of 15 day-old rats. Finally, alpha(2R) decrease from 1 to 90 day-old rats in all subnuclei analyzed. This decrease is different between strains in rostral dorsomedial/dorsolateral and caudal subpostremal subnuclei within the NTS. In summary, our results highlight the importance of alpha(2R) distribution within the NTS regarding the neural control of blood pressure and the development of hypertension. (C) 2011 Elsevier B.V. All rights reserved.FAPESPFAPESPCNPqCNPqCAPESCAPE

The emerging biofuel crop camelina sativa retains a highly undifferentiated hexaploid genome structure

Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

Background Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. Results We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. Conclusion The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species