Identification of a candidate dwarfing gene in Pallas, the first commercial barley cultivar generated through mutational breeding.

Many induced mutants are available in barley (Hordeum vulgare L.). One of the largest groups of induced mutants is the Erectoides (ert) mutants, which is characterized by a compact and upright spike and a shortened culm. One isolated mutant, ert-k.32, generated by X-ray treatment and registered in 1958 under the named "Pallas", was the first ever induced barley mutant to be released on the market. Its value was improved culm strength and enhanced lodging resistance. In this study, we aimed to identify the casual gene of the ert-k.32 mutant by whole genome sequencing of allelic ert-k mutants. The suggested Ert-k candidate gene, HORVU.MOREX.r3.6HG0574880, is located in the centromeric region of chromosome 6H. The gene product is an alpha/beta hydrolase with a catalytic triad in the active site composed of Ser-167, His-261 and Asp-232. In comparison to proteins derived from the Arabidopsis genome, ErtK is most similar to a thioesterase with de-S-acylation activity. This suggests that ErtK catalyzes post-translational modifications by removing fatty acids that are covalently attached to cysteine residues of target proteins involved in regulation of plant architecture and important commercial traits such as culm stability and lodging resistance.

FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Analysis of barley mutants ert-c.1 and ert-d.7 reveals two loci with additive effect on plant architecture.

MAIN CONCLUSION: Both mutant ert-c.1 and ert-d.7 carry T2-T3 translocations in the Ert-c gene. Principal coordinate analyses revealed the translocation types and translocation breakpoints. Mutant ert-d.7 is an Ert-c Ert-d double mutant. Mutations in the Ert-c and Ert-d loci are among the most common barley mutations affecting plant architecture. The mutants have various degrees of erect and compact spikes, often accompanied with short and stiff culms. In the current study, complementation tests, linkage mapping, principal coordinate analyses and fine mapping were conducted. We conclude that the original ert-d.7 mutant does not only carry an ert-d mutation but also an ert-c mutation. Combined, mutations in Ert-c and Ert-d cause a pyramid-dense spike phenotype, whereas mutations in only Ert-c or Ert-d give a pyramid and dense phenotype, respectively. Associations between the Ert-c gene and T2-T3 translocations were detected in both mutant ert-c.1 and ert-d.7. Different genetic association patterns indicate different translocation breakpoints in these two mutants. Principal coordinate analysis based on genetic distance and screening of recombinants from all four ends of polymorphic regions was an efficient way to narrow down the region of interest in translocation-involved populations. The Ert-c gene was mapped to the marker interval of 2_0801to1_0224 on 3HL near the centromere. The results illuminate a complex connection between two single genes having additive effects on barley spike architecture and will facilitate the identification of the Ert-c and Ert-d genes.

Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain.

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain.

Molecular mapping of adult plant resistance to Parastagonospora nodorum leaf blotch in bread wheat lines 'Shanghai-3/Catbird' and 'Naxos'.

KEY MESSAGE: The field resistance to Parastagonospora nodorum leaf blotch in SHA3/CBRD is based on many genes with minor effects. Parastagonospora nodorum leaf blotch is a severe wheat disease in Norway and other regions with humid and rainy climate. It causes grain shriveling and reduced yield in years of epidemics. Shanghai-3/Catbird (SHA3/CBRD), a CIMMYT breeding line, was observed to be resistant to P. nodorum leaf blotch in the field. The objective of the current study was to map the genetic factors related to its resistance. A recombinant inbred line population from a cross between SHA3/CBRD and the susceptible German spring cv. Naxos was tested in field trials over 4 years (2010, 2011, 2012 and 2013) with natural infection supplied with mist irrigation. Leaf blotch severity was scored together with plant height, heading date and maturity date in these trials. A testing data set was also available from other field trials with the same population. Totally, two major and six minor QTL were detected for leaf blotch resistance. The major QTL on chromosome 3BL with resistance contributed by Naxos was consistent across all environments and explained up to 12 % of the phenotypic variation. Another major QTL on 3B with resistance from SHA3/CBRD was significant in 2010, 2013 and the testing data set and explained up to 12 % of the phenotypic variation. Minor QTL were detected on 1B, 3AS, 5BS, 5BL, 7A and 7B. The 5BS QTL was likely caused by Snn3-B1, with sensitivity contributed by Naxos. The 5BL QTL mapped to the Tsn1 region, but was likely caused by other mechanisms since both parents were insensitive to ToxA.

Anther extrusion and plant height are associated with Type I resistance to Fusarium head blight in bread wheat line 'Shanghai-3/Catbird'.

Fusarium head blight (FHB) is a destructive wheat disease of global importance. Resistance breeding depends heavily on the Fhb1 gene. The CIMMYT line Shanghai-3/Catbird (SHA3/CBRD) is a promising source without this gene. A recombinant inbred line (RIL) population from the cross of SHA3/CBRD with the German spring wheat cv. Naxos was evaluated for FHB resistance and related traits in field trials using spray and spawn inoculation in Norway and point inoculation in China. After spray and spawn inoculation, FHB severities were negatively correlated with both anther extrusion (AE) and plant height (PH). The QTL analysis showed that the Rht-B1b dwarfing allele co-localized with a QTL for low AE and increased susceptibility after spawn and spray inoculation. In general, SHA3/CBRD contributed most of the favorable alleles for resistance to severity after spray and spawn inoculation, while Naxos contributed more favorable alleles for reduction in FDK and DON content and resistance to severity after point inoculation. SHA3/CBRD contributed a major resistance QTL close to the centromere on 2DLc affecting FHB severity and DON after all inoculation methods. This QTL was also associated with AE and PH, with high AE and tall alleles contributed by SHA3/CBRD. Several QTL for AE and PH were detected, and low AE or reduced PH was always associated with increased susceptibility after spawn and spray inoculation. Most of the other minor FHB resistance QTL from SHA3/CBRD were associated with AE or PH, while the QTL from Naxos were mostly not. After point inoculation, no other QTL for FHB traits was associated with AE or PH, except the 2DLc QTL which was common across all inoculation methods. Marker-assisted selection based on the 2DLc QTL from SHA3/CBRD combined with phenotypic selection for AE is recommended for resistance breeding based on this valuable source of resistance.

QTL mapping of adult-plant resistance to stripe rust in a population derived from common wheat cultivars Naxos and Shanghai 3/Catbird.

Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Erikss., is a severe foliar disease of common wheat (Triticum aestivum L.) worldwide. Use of adult-plant resistance (APR) is an efficient approach to provide long-term protection of crops from the disease. The German spring wheat cultivar Naxos showed a high level of APR to stripe rust in the field. To identify the APR genes in this cultivar, a mapping population of 166 recombinant inbred lines (RILs) was developed from a cross between Naxos and Shanghai 3/Catbird (SHA3/CBRD), a moderately susceptible line developed by CIMMYT. The RILs were evaluated for maximum disease severity (MDS) in Sichuan and Gansu in the 2009-2010 and 2010-2011 cropping seasons. Composite interval mapping (CIM) identified four QTL, QYr.caas-1BL.1RS, QYr.caas-1DS, QYr.caas-5BL.3 and QYr.caas-7BL.1, conferring stable resistance to stripe rust across all environments, each explaining 1.9-27.6, 2.1-5.8, 2.5-7.8 and 3.7-9.1 % of the phenotypic variance, respectively. QYr.caas-1DS flanked by molecular markers XUgwm353-Xgdm33b was likely a new QTL for APR to stripe rust. Because the interval between flanking markers for each QTL was less than 6.5 cM, these QTL and their closely linked markers are potentially useful for improving resistance to stripe rust in wheat breeding.

Partial resistance to powdery mildew in German spring wheat 'Naxos' is based on multiple genes with stable effects in diverse environments.

Powdery mildew is one of the most important wheat diseases in temperate regions of the world. Resistance breeding is considered to be an economical and environmentally benign way to control this disease. The German spring wheat cv. 'Naxos' exhibits high levels of partial and race non-specific resistance to powdery mildew in the field and is a valuable source in resistance breeding. The main objective of the present study was to map the genetic factors behind the resistance in Naxos, based on a population of recombinant inbred lines (RIL) from a cross with the susceptible CIMMYT breeding line SHA3/CBRD. Powdery mildew severity was evaluated in six field trials in Norway and four field trials in China. The major quantitative trait locus (QTL) with resistance from Naxos was detected close to the Pm3 locus on 1AS in all environments, and explained up to 35% of the phenotypic variation. Naxos was shown to carry another major QTL on 2DL and minor ones on 2BL and 7DS. QTL with resistance from SHA3/CBRD were detected on 1RS, 2DLc, 6BL and 7AL. The QTL on the 1B/1R translocation showed highly variable effects across environments corresponding to known virulence differences against Pm8. SHA3/CBRD was shown to possess the Pm3 haplotype on 1AS, but none of the known Pm3a-g alleles. The RIL population did not provide any evidence to suggest that the Pm3 allele of SHA3/CBRD acted as a suppressor of Pm8.

[Cloning and expression analysis of two salt and Fusarium graminearum stress associated UDP-glucosyltransferases genes in wheat].

Glycosyltransferases (GTs) play important roles in stress responses of plants by glycosylating hormones and secondary metabolites. UDP-glucosyltransferases (UGTs), which use UDP-glucuronic acid in animals, UDP-glucose, UDP-galactose, and UDP- rhamnose in plant as sugar donors, belong to family 1 of GTs. As a secondary metabolite produced by Fusarium graminearum during infection of grains, deoxynivalenol (DON) is not only harmful to human and animal's health by inhibiting protein synthesis, but also acts as a virulence factor during fungal pathogenesis. In order to study expression profile of UGT genes in wheat, two UGTs, designated TaUGT1 and TaUGT2, were isolated from bread wheat (Triticum aestivum L.) by reverse transcriptase-polymerase chain reaction. The genomic sequences of both genes had one intron. Their coding sequences shared 91% and 90% similarities at nucleic acid level and the deduced protein sequence level. The analysis of conserved domain revealed that these two cDNAs encoded UDP-glucoronosyl and UDP-glucosyl transferase with PSPG (Putative secondary plant glycosyltransferase) domain. Real-time PCR was carried out to detect the expression profiles of the two UGTs in wheat under various stress conditions. In young spikes infected by Fusarium graminearum, TaUGT2 was induced but TaUGT1 was repressed. These two genes were upregulated under higher NaCl concentration. In conclusion, TaUGT2 may participate in wheat resistance to Fusarium head blight in which mycotoxin DON plays a role, and these two genes might be involved in responses of wheat to salt stress.

[Development of the monoclonal antibody to deoxynivalenol].

OBJECTIVE: Deoxynivalenol (DON) is a trichothecene mycotoxin produced by Fusarium graminearum, a pathogen causing Fusarium Head Blight of wheat. It is necessary to establish a rapid and simple assay to detect DON. METHODS: High affinity monoclonal antibodies (Mab) against DON were produced by cell fusion with 500 mg/mL Polyethylene Glycol 400, and cell sub-cloning in HAT (H: hypoxanthine, A: aminopterin; T: thymidine) culture medium for screening and limiting dilution. Hybridoma lines were screened for specificity to DON by Enzyme-linked Immunosorbent Assay (ELISA). One hybridoma cell line (3B2) secreting monoclonal antibody (MAb) against DON was produced by fusing mouse myeloma cells (SP 2/0) with spleen cells from BAL B/C mice which were immunized by the artificial antigen conjugated with Ovalbumin (OVA). RESULTS: The MAb obtained in this experiment could specifically react with DON without cross-reactivity to DON related compounds except 3-acetyldeoxynivalenol (3-Ac-DON), with the titres of ascitic fluids up to 1 x 10(-7) by indirect ELISA. Isotype and subclass of the monoclonal cell line (3B2) showed that it belonged to IgG1. The light chain of the MAb was identified to be kappa. Ascites antibodies generated by hybridoma of 3B2 cells were purified. Inhibition rate studies showed that the detection limit of the ELISA was 8 microg/L, with the regression equation of indirect ELISA Y = 0.7331g(x)-0.572, R2=0.9652, IC50 value being 29 ug/L. CONCLUSION: The MAb can be used to prepare the reagents for analyzing DON residue.