Disease Resistance In Barley Research Articles

A comprehensive review on mutation breeding milestones in cereals: Conventional to advanced molecular approaches to achieve sustainable goals in trait improvement

The grass family includes the annual common grasses known as cereals. The 5 main types of cereals are rice, wheat, barley, rye and oats. As a result of their high vitamin (20-80%) and fiber content (90%), these grains can help treat conditions like type II diabetes, complex metabolic syndrome, obesity, gastrointestinal disorders, and high cholesterol. Cereals have inspired a long history of research into their genetics, development, and evolution due to their significance in both economic and scientific terms. They need to be improved because they play a big part in global food production and crop quality is declining. The crop improvement can be done by incorporating desired features from non-adapted landraces/wild resources. That induced mutagenesis has made a significant contribution to crop improvement initiatives by producing mutant varieties with better and desired genetic modifications in the crop plants mainly in agronomically significant features. The mutants produced by the induction of physical and chemical mutagens were highly useful. The notable traits obtained by the induced mutations are semidwarfness and early maturity in rice varieties, disease resistance in barley, salinity, and drought tolerance in wheat, and lodging and disease resistance in oat and rye. In this review, we focused on identifying and developing elite mutants, their genetic function, and their utilization in future breeding programs for various traits in cereal crops.

HvWRKY2 acts as an immunity suppressor and targets HvCEBiP to regulate powdery mildew resistance in barley

Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner. In barley, HvWRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). However, the molecular features of HvWRKY2 in its DNA-binding and repressor functions, as well as its target genes, are uncharacterized. We show that the W-box binding of HvWRKY2 requires an intact WRKY domain and an upstream sequence of ∼75 amino acids, and the HvWRKY2 W-box binding activity is linked to its repressor function in disease resistance. Chromatin immunoprecipitation (ChIP)-seq analysis identified HvCEBiP, a putative chitin receptor gene, as a target gene of HvWRKY2 in overexpressing transgenic barley plants. ChIP-qPCR and Electrophoretic Mobility Shift Assay (EMSA) verified the direct binding of HvWRKY2 to a W-box-containing sequence in the HvCEBiP promoter. HvCEBiP positively regulates resistance against Bgh in barley. Our findings suggest that HvWRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern (PAMP) recognition receptor genes, suggesting that HvCEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.

Identification microRNAs and target genes in Tibetan hulless barley to BLS infection

AbstractTibetan hulless barley (Hordeum vulgare L. variety nudum Hook. f.) is widely grown on the Qinghai‐Tibet Plateau, where it has served as a staple food for Tibetan people since the 5th century CE. Barley leaf stripe (BLS) is one of the most severe fungal diseases affecting the yield and quality of Tibetan hulless barley. Here, we compared the miRNA profiles before and after BLS in a BLS‐sensitive (‘Z1141’) and tolerant (‘Kunlun14’) Tibetan hulless barley for the first time. A total of 36 conserved and 56 novel miRNAs were identified, of which 24 differentially expressed miRNAs were found in Z1141 and Kunlun14, and an additional 546 putative target genes were predicted. Transcriptome sequencing analysis showed that among these genes, 131 were differentially expressed in normal and infected leaves of Kunlun14 and Z1141. Gene ontology (GO), pathway, and Blast analyses indicated 10 candidate target genes including seven functional genes and three transcription factors were involved in barley disease resistance. These functional genes were two members of the CYP450 genes, a RGA gene, a LIN gene, a SAM gene, a PSD gene, and a NDB gene; the three transcription factor genes were all from the WRKY family. These 10 candidate target genes were regulated by eight miRNAs (hvu‐miR168‐3p, hvu‐miR171‐5p, hvu‐miR159b, hvu‐miR156a, hvu‐novel‐91, hvu‐novel‐46, hvu‐novel‐52, and hvu‐novel‐11). According to quantitative real‐time polymerase chain reaction (PCR) results, the 10 targets were negatively correlated with their corresponding miRNAs after infection with BLS. Thus, these miRNAs and their target genes can be used to improve BLS resistance in Tibetan hulless barley though breeding programs or genetic engineering.

RNA-Spray-Mediated Silencing of Fusarium graminearum AGO and DCL Genes Improve Barley Disease Resistance.

Over the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an in planta expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals in planta, plants can be protected from pathogens, and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed vs. manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

Genome Sequence Resource for Pathogen Bipolaris sorokiniana Shoemaker GN1 Causing Spot Blotch of Barley (Hordeum vulgare L.).

Spot blotch, caused by fungal pathogen Bipolaris sorokiniana Shoemaker, is one of the most frequent diseases affecting barley-growing regions worldwide. In this study, we reported the genome sequence of the highly virulent B. sorokiniana strain GN1 using the Illumina HiSeq 4000 platform. In total, 57 million 150-nucleotide paired-end clean reads were obtained and assembled into 96 scaffolds with an estimated genome size of 34.33 Mb. Furthermore, we identified genes that may be associated with strain-specific virulence and performed phylogenetic analysis of GN1 with five other Bipolaris spp. These results for GN1 will provide important information in understanding its molecular underpinning of pathogenicity and help identify novel sources of genetic resistance for improving disease resistance in barley.

Subfamily-Specific Specialization of RGH1/MLA Immune Receptors in Wild Barley.

The barley disease resistance (R) gene locus mildew locus A (Mla) provides isolate-specific resistance against the powdery mildew fungus Blumeria graminis hordei and has been introgressed into modern cultivars from diverse germplasms, including the wild relative Hordeum spontaneum. Known Mla disease resistance specificities to B. graminis hordei appear to encode allelic variants of the R gene homolog 1 (RGH1) family of nucleotide-binding domain and leucine-rich repeat (NLR) proteins. Here, we sequenced and assembled the transcriptomes of 50 H. spontaneum accessions representing nine populations distributed throughout the Fertile Crescent. The assembled Mla transcripts exhibited rich sequence diversity, linked neither to geographic origin nor population structure, and could be grouped into two similar-sized subfamilies based on two major N-terminal coiled-coil (CC) signaling domains that are both capable of eliciting cell death. The presence of positively selected sites located mainly in the C-terminal leucine-rich repeats of both MLA subfamilies, together with the fact that both CC signaling domains mediate cell death, implies that the two subfamilies are actively maintained in the population. Unexpectedly, known MLA receptor variants that confer B. graminis hordei resistance belong exclusively to one subfamily. Thus, signaling domain divergence, potentially as adaptation to distinct pathogen populations, is an evolutionary signature of functional diversification of an immune receptor. Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Speed breeding for multiple disease resistance in barley

To respond faster to the changing climate, evolving pathogens and to feed a global population of 9–10 billion by 2050, plant breeders are exploring more efficient crop improvement strategies. In this study, we applied novel methodology for rapid trait introgression to the European two-rowed barley cultivar Scarlett. Scarlett is widely-grown in Argentina and is preferred for malting and brewing, yet lacks adequate disease resistance. We used four donor lines combining multiple disease resistance (i.e. leaf rust, net and spot forms of net blotch and spot blotch) in a modified backcross strategy, which incorporated both multi-trait phenotypic screens and the rapid generation advance technology ‘speed breeding’, to develop 87 BC1F3:4 Scarlett introgression lines (ILs) within two years. Phenotyping this set of lines in disease nurseries located in Australia and Uruguay revealed the ILs had high levels of multiple disease resistance. Preliminary yield testing of the 12 most promising ILs in Argentina identified three ILs that were significantly higher yielding than Scarlett at Balcarce, whereas all 12 ILs displayed yield equivalent to Scarlett at Tres Arroyos. We propose that this approach is useful to rapidly transfer genes for multiple target traits into adapted cereal cultivars or pyramiding desirable traits in elite breeding material.

An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew.

Following the detection of pathogen cognate effectors, plant Nod-like receptors (NLRs) trigger isolate-specific immunity that is generally associated with cell death. The regulation of NLR stability is important to ensure effective immunity. In barley (Hordeum vulgare), the allelic Mildew locus A (MLA) receptors mediate isolate-specific disease resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Currently, how MLA stability is controlled remains unknown. Here, we identified an MLA-interacting RING-type E3 ligase, MIR1, that interacts with several MLAs. We showed that the carboxyl-terminal TPR domain of MIR1 mediates the interaction with the coiled-coil domain-containing region of functional MLAs, such as MLA1, MLA6, and MLA10, but not with that of the nonfunctional MLA18-1. MIR1 can ubiquitinate the amino-terminal region of MLAs in vitro and promotes the proteasomal degradation of MLAs in vitro and in planta. Both proteasome inhibitor treatment and virus-induced gene silencing-mediated MIR1 silencing significantly increased MLA abundance in barley transgenic lines. Furthermore, overexpression of MIR1 specifically compromised MLA-mediated disease resistance in barley, while coexpression of MIR1 and MLA10 attenuated MLA10-induced cell death signaling in Nicotiana benthamiana Together, our data reveal a mechanism for the control of the stability of MLA immune receptors and for the attenuation of MLA-triggered defense signaling by a RING-type E3 ligase via the ubiquitin proteasome system.

One century of Nordic barley breeding: nitrogen use efficiency, agronomic traits and genetic diversity

SUMMARYThe current study aimed to evaluate breeding effect on nitrogen use efficiency (NUE), its components and some agronomic traits and disease resistance in barley by using extensive germplasm covering 72 landraces and 123 cultivars released since 1910. Trials were established in southern Finland with a modified strip-plot experimental design. Prior to sowing, blocks were placement fertilized with compound nitrogen : phosphorus : potassium (NPK) fertilizer (N-P-K: 20–3–8) at the rate of 35 and 70 kg N/ha and unfertilized plots were placed at the other end of the fertilization block. The germplasm collection was genotyped with 1536 single nucleotide polymorphism (SNP) markers and phenotyped during a 2-year field experiment in 2011/12. Independent of row type, a positive breeding effect was evident in NUE and for other plant N traits, except that grain N slightly decreased. Breeding has improved NUE by 0·08 kg/year (26% over the century). Nitrogen utilization and N uptake efficiencies were also improved by breeding as were straw length, lodging tolerance, grain yield and yield components, without any sign of levelling-off. Bred cultivars were more resistant to leaf-damaging diseases, especially to net blotch. The SNP data indicated no reduction in overall genetic diversity. However, genetic diversity differed along the barley chromosomes showing either reduced or increased diversity in certain regions when landraces were compared with modern varieties.

Magical mystery tour: MLO proteins in plant immunity and beyond.

Stable heritable restriction of the ubiquitous powdery mildew disease is a desirable trait for agri and horticulture. In barley (Hordeum vulgare), loss-of-function mutant alleles of the Mildew resistance locus o (Mlo) gene confer broad-spectrum resistance to almost all known isolates of the fungal barley powdery mildew pathogen, Blumeria graminis f.sp. hordei. Despite extensive cultivation of barley mlo genotypes, mlo resistance has been durable in the field. Mlo genes are present as small families in the genomes of all higher plant species. The presumed negative regulatory role of particular members in plant immunity is evolutionarily conserved, as powdery mildew resistant mlo mutants have also been described in Arabidopsis thaliana, tomato(Solanum lycopersicum) and pea (Pisum sativum). Barley Mlo encodes a plasma membrane-localized seven-transmembrane domain protein of unknown biochemical activity. Here, we review the known requirements for mlo-mediated disease resistance in barley and Arabidopsis and reflect current views regarding Mlo function. We discuss additional mlo mutant phenotypes recently discovered in Arabidopsis and present a meta-analysis of the phylogenetic relationships within the Mlo family. Finally, we consider the novel versatile tools for functional analysis and targeted genome modification that can be used to induce mlo-based powdery mildew resistance in virtually any plant species.

The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other's function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.

Structural Basis for the Interaction between the Potato Virus X Resistance Protein (Rx) and Its Cofactor Ran GTPase-activating Protein 2 (RanGAP2)

The potato (Solanum tuberosum) disease resistance protein Rx has a modular arrangement that contains coiled-coil (CC), nucleotide-binding (NB), and leucine-rich repeat (LRR) domains and mediates resistance to potato virus X. The Rx N-terminal CC domain undergoes an intramolecular interaction with the Rx NB-LRR region and an intermolecular interaction with the Rx cofactor RanGAP2 (Ran GTPase-activating protein 2). Here, we report the crystal structure of the Rx CC domain in complex with the Trp-Pro-Pro (WPP) domain of RanGAP2. The structure reveals that the Rx CC domain forms a heterodimer with RanGAP2, in striking contrast to the homodimeric structure of the CC domain of the barley disease resistance protein MLA10. Structure-based mutagenesis identified residues from both the Rx CC domain and the RanGAP2 WPP domain that are crucial for their interaction and function in vitro and in vivo. Our results reveal the molecular mechanism underlying the interaction of Rx with RanGAP2 and identify the distinct surfaces of the Rx CC domain that are involved in intramolecular and intermolecular interactions.

Large-Scale Data Integration Reveals Colocalization of Gene Functional Groups with Meta-QTL for Multiple Disease Resistance in Barley

Race-nonspecific and durable resistance of plant genotypes to major pathogens is highly relevant for yield stability and sustainable crop production but difficult to handle in practice due to its polygenic inheritance by quantitative trait loci (QTL). As far as the underlying genes are concerned, very little is currently known in the most important crop plants such as the cereals. Here, we integrated publicly available data for barley (Hordeum vulgare subsp. vulgare) in order to detect the most important genomic regions for QTL-mediated resistance to a number of fungal pathogens and localize specific functional groups of genes within these regions. This identified 20 meta-QTL, including eight hot spots for resistance to multiple diseases that were distributed over all chromosomes. At least one meta-QTL region for resistance to the powdery mildew fungus Blumeria graminis was found to be co-linear between barley and wheat, suggesting partial evolutionary conservation. Large-scale genetic mapping revealed that functional groups of barley genes involved in secretory processes and cell-wall reinforcement were significantly over-represented within QTL for resistance to powdery mildew. Overall, the results demonstrate added value resulting from large-scale genetic and genomic data integration and may inform genomic-selection procedures for race-nonspecific and durable disease resistance in barley.

Differential disease resistance response in the barley necrotic mutant nec1

BackgroundAlthough 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. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 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 nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens.Resultsnec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst) at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance.ConclusionsTogether, the results suggest that nec1 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 nec1 against non-host pathogen Pst suggests that nec1 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.

A synteny map and disease resistance gene comparison between barley and the model monocotBrachypodium distachyon

Grass species have coevolved with current economically important crop pathogens over millions of years. During this time, speciation of current domestic crops has occurred, resulting in related yet divergent genomes. Here, we present a synteny map between the crop species Hordeum vulgare and the recently sequenced Brachypodium distachyon genome, focusing on regions known to harbor important barley disease resistance genes. The resistance genes have orthologous genes in Brachypodium that show conservation of the form and likely the function of the genes. The level of colinearity between the genomes is highly dependent on the region of interest and, at the DNA level or protein level, the gene of interest. The stem rust resistance gene Rpg1 has an ortholog with a high level of identity at the amino acid level, while the stem rust resistance gene Rpg5 has two orthologs with a high level of identity, one corresponding to the NBS-LRR domain and the other to the serine/threonine protein kinase domain, on different contigs. Interestingly, the predicted product of the Brachypodium Rpg1 ortholog contained a WD40 domain at the C-terminal end. The stem rust resistance gene rpg4 (actin depolymerizing factor 2) also has an ortholog with a high level of identity, in which one of the three residues indicated by allele sequencing in barley cultivars to be important in disease resistance is conserved. The syntenous region of the seedling spot blotch resistance locus, Rcs5, has a high level of colinearity that may prove useful in efforts to identify and clone this gene. A synteny map and orthologous resistance gene comparisons are presented.

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

Validation of Quantitative Trait Loci for Multiple Disease Resistance in Barley Using Advanced Backcross Lines Developed with a Wild Barley

ABSTRACTQuantitative trait loci (QTLs) for resistance to spot blotch {Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur [anamorph: Bipolaris sorokiniana (Sacc. in Sorok.) Shoem.]}, net type net blotch (NTNB, Pyrenophora teres f. teres), Septoria speckled leaf blotch (SSLB, primarily Septoria passerinii Sacc.), and leaf scald [caused by Rhynchosporium secalis (Oudem.) Davis] were previously detected in a recombinant inbred line (RIL) population derived from a cross between the wild barley [Hordeum vulgare subsp. spontaneum (K. Koch) Thell.] accession OUH602 and the two‐rowed malting barley (H vulgare L.) cultivar Harrington. Our objectives in this study were to develop and genetically characterize an advanced backcross (AB) population with OUH602 as the donor parent and Harrington as the recurrent parent, to validate seedling disease resistance QTLs detected in the OUH602/Harrington RIL population, and to identify adult plant spot blotch resistance QTLs. We developed an AB population (BC2F6:8) and genotyped the population with a set of 111 simple sequence repeat (SSR) markers. The estimated donor‐parent genome introgression averaged 11.25%. A QTL analysis of the AB population validated all major QTLs previously identified in the OUH602/Harrington RIL population. No new QTLs for seedling resistance were identified in the AB population, indicating that AB populations do not provide an advantage compared with RIL populations for detecting seedling disease resistance QTLs. A previously unidentified QTL conferring adult plant spot blotch resistance was identified on chromosome 4(4H). The resistance alleles for each of the QTLs were contributed by OUH602. The AB population is a valuable resource for further genetic studies and breeding applications.

New eSSR and gSSR markers added to Australian barley maps

To enhance genetic maps of barley previously developed in Australia for identifying markers useable in molecular breeding, a new set of simple sequence repeat (SSR) and indel markers was added to the maps. These markers were developed through (i) database mining of barley expressed sequence tag (EST) sequences, (ii) comparative barley-rice genome analysis, and (iii) screening of a genomic library with SSR probes. The primer set selected for this study comprised 216 EST-SSR (eSSR) and 25 genomic SSR (gSSR) markers, which were screened for polymorphism on 4 doubled haploid (DH) or recombinant inbred line (RIL) populations. In total, 81 new markers were added to the maps, with good coverage on all 7 chromosomes, except 6H, which only had 2 new markers added. The marker order of previously published maps was re-evaluated by comparing recombination fractions calculated by 2 methods to discover the best position for each marker. The new SSR markers were then added to the updated maps. Several of these new markers are linked to important barley disease resistance genes such as those for cereal cyst nematode, spot form of net blotch, and leaf scald resistance, and are readily useable for marker-assisted barley breeding. The new maps are available on-line at www.genica.net.au.

The complex developmental expression of a novel stress-responsive barley Ltp gene is determined by a shortened promoter sequence

The search for a cereal promoter capable of driving preferential transgene expression in the pericarp epidermis (epicarp) of developing barley (Hordeum vulgare L.) resulted in the cloning of a novel gene. This encoded a polypeptide of 124 amino acids showing 87 identity with WBP1A, a wheat lipid transfer protein (LTP), but much lower homology to other barley LTPs. In addition to the epicarp, this Ltp-like gene, Ltp6, is highly expressed in coleoptiles and embryos under normal growth conditions. Messenger RNA levels increased in seedling tissues during salt and cold treatments and under applied abscisic acid (ABA) and salicylic acid (SA). Taken together, Ltp6 tissue-specific and response patterns are distinct from other known barley Ltp genes. Inverse PCR was used to derive 2345 bp of upstream Ltp6 sequence. The level of transcription conferred by different promoter deletion constructs was assessed by quantitative real time RT-PCR using gfp as a reporter in transient expression assays. All constructs containing at least 192 bp of upstream sequence and the 5'UTR conferred tissue-specific expression and retained most of the promoter strength. Deletion of 64 bp (-192/-128) from this upstream sequence reduced expression levels by 80. Moreover, a minimal 247 bp Ltp6 promoter continuously drove gfp expression during spike development, from early ovary differentiation through its final expression in the epicarp and during embryogenesis and germination in transgenic barley, reproducing the expression pattern of the native gene. The potential use of this promoter sequence for targeting transgene-mediated disease resistance in barley and wheat is discussed.

The molecular genetics of disease resistance in barley

The molecular genetics of disease resistance of barley and its wild relatives is reviewed, and the implications of recent findings for resistance breeding and the potential for disease control using gene technologies are discussed. As a resource for barley researchers and breeders, a chromosome map and list of mapped resistance genes, their source, and associated molecular markers are presented, updated to ultimo 2002. Genetic mapping of major genes and quantitative trait loci for many major diseases is revealing a heterogeneous distribution of resistance loci on chromosomes, with more than half of mapped loci occurring in clusters. Relatively few resistance loci have been identified in the cultivated barley germplasm. Studies have shown that wild Hordeum species contain resistance genes for the major diseases, although their allelic relationship to previously mapped genes is unknown. The structure of genes involved in race-specific and race-non-specific barley powdery mildew resistance has been determined. Isolation of resistance genes for other major diseases is essential and may be accelerated via genomics techniques such as EST sequencing, subtractive hybridisation, or expression profiling. Current strategies for molecular manipulation of barley disease resistance are based on the over-expression of defence-related or disease-signalling genes from other species.