Genome-wide and Mla locus-specific characterisation of Latvian barley varieties

Genetic diversity in locally adapted germplasm forms the basis for crop improvement through breeding. While single loci have been routinely used for studies of genetic diversity, the highthroughput genotyping platforms that have recently become available for large genome cro plants offer an unbiased view on genetic diversity on a genome-wide scale. We assessed genetic diversity in Latvian barley varieties and some progenitors using DArT markers and studied the extent of linkage disequilibrium in Latvian germplasm. Further, genetic diversity at three loci flanking the barley powdery mildew Mla locus conferring race-specific resistance was studied in Latvian barley germplasm. The Mla locus encompasses several closely related resistance gene homologues with a complex evolutionary history, which complicates the design of molecular markers for different Mla genes. We observed significant linkage disequilibrium between the single nucleotide polymorphisms (SNPs) at the three loci, 206i20_T7, ABC15612, and 538P8, flanking the Mla locus. SNP haplotypes were largely in agreement with known phenotypic data and, thus, may be potentially diagnostic for Mla resistance genes in hybrids.publishersversionPeer reviewe

Development and Validation of a Reversed-Phase Liquid Chromatography Method for the Simultaneous Determination of Indole-3-Acetic Acid, Indole-3-Pyruvic Acid, and Abscisic Acid in Barley (Hordeum vulgare L.)

A simple, sensitive, precise, and specific reverse HPLC method was developed and validated for the determination of plant hormones in barley (Hordeum vulgare L.). The method includes extraction in aqueous organic solvent followed by solid-phase extraction, sample evaporation, and reversed-phase HPLC analysis in a general purpose UV-visible (abscisic acid (ABA)) and fluorescence detection (indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA)), high-performance liquid chromatography system. The separation was carried out on Zorbax Eclipse XDB C8 column (150  ×  4.6  mm I.D) with a mobile phase composed of methanol and 1% acetic acid (60 : 40 v/v) in isocratic mode at a flow rate of 1 ml min−1. The detection was monitored at 270 nm (ABA) and at 282 nm (Ex) and 360 nm (Em) (IAA, IPA). The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The determined validation parameters are in the commonly acceptable ranges for that kind of analysis

Differential disease resistance response in the barley necrotic mutant nec1

<p>Abstract</p> <p>Background</p> <p>Although ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. <it>Arabidopsis thaliana </it>CNGC4 mutants <it>hlm1 </it>and <it>dnd2 </it>display an impaired hypersensitive response (HR), retarded growth, a constitutively active salicylic acid (SA)-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant <it>nec1 </it>comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses <it>PR-1</it>, yet it is not known what effect the <it>nec1 </it>mutation has on barley resistance against different types of pathogens.</p> <p>Results</p> <p><it>nec1 </it>mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating <it>nec1 </it>disease resistance demonstrated positive effect of <it>nec1 </it>mutation on non-host resistance against <it>Pseudomonas syringae </it>pv. <it>tomato </it>(<it>Pst</it>) at high inoculum density, whereas at normal <it>Pst </it>inoculum concentration <it>nec1 </it>resistance did not differ from wt. In contrast to augmented <it>P. syringae </it>resistance, penetration resistance against biotrophic fungus <it>Blumeria graminis </it>f. sp. <it>hordei </it>(<it>Bgh</it>), the causal agent of powdery mildew, was not altered in <it>nec1</it>. The <it>nec1 </it>mutant significantly over-expressed race non-specific <it>Bgh </it>resistance-related genes <it>BI-1 </it>and <it>MLO</it>. Induction of <it>BI-1 </it>and <it>MLO </it>suggested putative involvement of <it>nec1 </it>in race non-specific <it>Bgh </it>resistance, therefore the effect of <it>nec1</it>on <it>mlo-5</it>-mediated <it>Bgh </it>resistance was assessed. The <it>nec1</it>/<it>mlo-5 </it>double mutant was as resistant to <it>Bgh </it>as <it>Nec1</it>/<it>mlo-5 </it>plants, suggesting that <it>nec1 </it>did not impair <it>mlo-5 </it>race non-specific <it>Bgh </it>resistance.</p> <p>Conclusions</p> <p>Together, the results suggest that <it>nec1 </it>mutation alters activation of systemic acquired resistance-related physiological markers and non-host resistance in barley, while not changing rapid localized response during compatible interaction with host pathogen. Increased resistance of <it>nec1 </it>against non-host pathogen <it>Pst </it>suggests that <it>nec1 </it>mutation may affect certain aspects of barley disease resistance, while it remains to be determined, if the effect on disease resistance is a direct response to changes in SA signaling.</p

Single-feature polymorphism discovery in the barley transcriptome

A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes

Barley stem rust resistance genes: structure and function

Rusts are biotrophic pathogens that attack many plant species but are particularly destructive on cereal crops. The stem rusts (caused by Puccinia graminis) have historically caused severe crop losses and continue to threaten production today. Barley (Hordeum vulgare L.) breeders have controlled major stem rust epidemics since the 1940s with a single durable resistance gene Rpg1. As new epidemics have threatened, additional resistance genes were identified to counter new rust races, such as the rpg4/Rpg5 complex locus against races QCCJ and TTKSK. To understand how these genes work, we initiated research to clone and characterize them. The Rpg1 gene encodes a unique protein kinase with dual kinase domains, an active kinase, and a pseudokinase. Function of both domains is essential to confer resistance. The rpg4 and Rpg5 genes are closely linked and function coordinately to confer resistance to several wheat (Triticum aestivum L.) stem rust races, including the race TTKSK (also called Ug99) that threatens the world's barley and wheat crops. The Rpg5 gene encodes typical resistance gene domains NBS, LRR, and protein kinase but is unique in that all three domains reside in a single gene, a previously unknown structure among plant disease resistance genes. The rpg4 gene encodes an actin depolymerizing factor that functions in cytoskeleton rearrangement

Risk assessment of new sequencing data on GM maize event MIR604

In 2009 and 2010, the EFSA GMO Panel concluded the assessment of genetically modified (GM) maizes MIR604, MIR604 × GA21, MIR604 × Bt11 and MIR604 × GA21 × Bt11. These maizes were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 July 2015, the European Commission (EC) received from Syngenta new nucleic acid sequencing data on maize event MIR604 and updated bioinformatic analyses using the new sequencing data. EC tasked EFSA to analyse these data and to indicate whether the previous conclusions of the EFSA GMO Panel on the above-listed GM maizes remain valid. The EFSA GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. The new sequencing data indicated a single base pair difference compared to the sequencing data originally provided, located in a non-coding region of the insert. which had already been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event MIR604 as a single and as a part of stacked events remains valid

Updating risk management recommendations to limit exposure of non-target Lepidoptera of conservation concern in protected habitats to Bt-maize pollen

Using mathematical model ling , the EFSA GMO Panel ha s previously quantified the risk to non - target (NT) Lepidoptera of conservation concern, potentially occurring within protected habitats, associated with the ingestion of Bt - maize pollen deposited on their host plants. To reduce the estimated larval mortality to a negligible level, an isolation distance of 20 and 30 m was recommended between protected habitats and the nearest fields of maize MON 810/Bt11 and 1507, respectively. Here , the EFSA GMO Panel refines its model predictions , accounting for new ly reported information on maize pollen deposition over long distances . For its calculations , the EFSA GMO Panel considered three exposure scenarios at a range of isolation distances, at two protection levels and for a range of lepidopteran species, including hypothetical ones, with a wide spectrum of sensitivities to Bt toxins . An analysis of various sources of uncertainties affecting the exposure of NT Lepidoptera to Bt - maize pollen was conducted, in order to provide quantitative estimates of realistic exposure levels. The EFSA GMO Panel therefore provides risk managers with a tool to estimate and mitigate the risk for NT Lepidoptera of conservation concern. In contrast to its previous o utcomes obtained for unrealistically large levels of exposure that would not be expected in practice, the EFSA GMO Panel reports here mortality estimates for a more realistic level of exposure. The EFSA GMO Panel concludes that its previous recommendation for a 20 m isolation distance around protected habitats, within which maize MON810/Bt11 should not be cultivated, remains valid. New calculations show that the previously recommended isolation distance of 30 m from the nearest maize 1507 field would still protect NT Lepidoptera with known levels of sensitivity, including the ‘highly - sensitive’ Plutella xylostella . Should hypothetical species with greater sensitivities exist, larger isolation distances would be needed to ensure the desired level of protection

Scientific Opinion on a request from the European Commission related to the prolongation of prohibition of the placing on the market of genetically modified oilseed rape event GT73 for import, processing and feed uses in Austria

Following a request from the European Commission, the Panel on Genetically Modified Organisms of the European Food Safety Authority (EFSA GMO Panel) evaluated the documentation provided by Austria to support the prolongation of the safeguard clause measure prohibiting the placing on the market of the genetically modified oilseed rape event GT73 for import, processing and feed uses in Austria. The EFSA GMO Panel assessed whether the submitted documentation comprised new scientific information that would change or invalidate the conclusions of its previous risk assessments on oilseed rape GT73. The EFSA GMO Panel also considered the relevance of the concerns raised by Austria in the light of the most recent data published in the scientific literature. The authorised uses of oilseed rape GT73 exclude cultivation, but data on gene flow, persistence and invasiveness derived from cultivation were considered as a worst case, representing conditions where exposure and potential impact are expected to be the highest, to assess possible environmental impacts resulting from seed import spills. In the documentation provided by Austria and in the scientific literature, the EFSA GMO Panel could not identify new scientific evidence that indicates that the import, processing and feed uses of oilseed rape GT73 in the EU pose a significant and imminent risk to the environment. The EFSA GMO Panel does not consider the occurrence of occasional feral oilseed rape GT73 plants, pollen dispersal and consequent cross-pollination as environmental harm in itself. In conclusion, the EFSA GMO Panel considers that, based on the documentation supplied by Austria and a review of recent scientific literature, there is no specific scientific evidence in terms of risk to the environment that would support the notification of a safeguard clause measure under Article 23 of Directive 2001/18/EC nor its prolongation, and that would invalidate its previous risk assessments of oilseed rape GT73