A mutant induced in the malting barley cv Triumph with reduced dormancy and ABA response

 Induced mutants in the barley cultivar Triumph have been screened for reduced dormancy. One line, which germinated readily 2 weeks after harvest, was classified as ABA-insensitive, since it could tolerate a ten-fold increase in ABA, compared to its parent, before germination was inhibited. This mutant, designated TL43, was genotypically similar to Triumph and phenotypically similar under Scottish growing conditions, except for a slightly reduced grain size. In Spain, it showed considerable reductions in both grain yield and plant height, suggesting that it was less widely adapted than its parent. Levels of α-amylase activity were increased at both sites. The mutant appeared to be different from those with ABA insensitivity or altered dormancy previously documented in either barley or Arabidopsis. Received: 23 March 1998 / Accepted: 13 August 1998     

Identification of proteins associated with malting quality in a subset of wild barley introgression lines

Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters α-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.     

The gas environment of germinating barley in various microbial states during malting

Steeped barley was germinated in laboratory scale solid-state fermentors at various oxygen partial pressures. Experiments were performed with sterile living barley and with barley colonized by the natural microflora. On line monitoring of CO₂ concentration in the off-gas of the reactors reflected the metabolic activity more accurately than heat production measurement. When colonized by micro-organisms, the respiratory activity of germinating barley increased with the oxygen level in the air. However, CO₂ profiles of sterile barley did not alter at different oxygen concentrations in the inlet air of the reactor. It is concluded that the germination phase of malting can be optimized by controlling the oxygen supply to the barley based on CO₂ production measurements and taking into account microbial aspects.     

The assessment of two barley starch mutants as potential parents in a malting quality breeding programme

Grain and malt quality parameters were assessed in the barley starch mutants Waxy Hector, characterised by high levels of amylopectin, and Chalky Glenn, which has lines of cleavage across the large starch granules. Results were compared with those of their parental cultivars and suggested differences in the contribution of endosperm cell walls to milling energy. Although hot water extracts were not improved, other components, especially those associated with the fractured starch mutant, could be used in breeding programmes to improve aspects of quality, if they could be disassociated from deleterious effects of the starch mutation, e.g. poor grain filling.     

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Summary Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 31 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.     

Pyramiding QTLs to improve malting quality in barley: gains in phenotype and genetic diversity

The ability of barley (Hordeum vulgare L.) breeders to deliver germplasm that combine elite malt quality characteristics, disease resistances, and important agronomic traits has been greatly enhanced by the use of molecular marker technologies. These technologies facilitate the rapid transfer of desirable traits from diverse, elite, germplasm into locally adapted varieties. This present study sought to obtain an additive genetic effect by combining favourable alleles associated with the malting quality of two elite donor parents (Harrington and Morex) such that the resultant progeny would possess quality superior to either parent. Analysis of genetic diversity, based on whole-genome profiling with 700 DArT markers, showed clear separation of the BC₆F₁-derived doubled haploid lines from existing malting barley germplasm, indicating they represent a distinctly different source population for genetic improvement. Micro-malting quality results of the BC-derived lines showed substantial quality improvements, compared with the recurrent parent. Malt extract levels were increased by 1.5–2.0%, while diastase levels increased from approximately 320 WKE to 400–460 WKE. Similarly, α-amylase levels were increased from 160 units to between 218 and 251 units, and wort viscosities lowered from 1.90 cps to 1.72–1.82 cps. Other quality improvements include increases in β-glucanase levels from 375 to between 447 and 512 units, and reductions in wort β-glucan levels by 30–60%. Whilst the genetic gains compared to the recurrent parent were impressive, quality of the derived lines were largely equivalent to the levels now available in the recently released varieties, Buloke and Flagship. In a few cases, the backcross-derived lines also showed similarities to the original donors, Harrington and Morex, but in none of the cases did quality of these lines exceed those of either Harrington or Morex.     

Yeasts in an industrial malting ecosystem

The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37°C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of α-amylase, β-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.     

Variation in the ratio of physical to genetic distance in intervals adjacent to theMla locus on barley chromosome 1H

Variants of the pulsed-field gel electrophoresis technique were used in conjunction with two-dimensional DNA gel electrophoresis (2-DDGE) to determine the ratio of physical to genetic distance in two genetically defined intervals on barley chromosome 1H.2-DDGE analysis demonstrated that two loci that define a 0.3 cM interval, as determined by hybridization with BCD249, reside on a single 450-kbMluI fragment. This result indicates a maximum ratio of physical to genetic distance in this interval of 1500 kb/cM as compared to 3.7–4.2 Mb/cM for the barley genome as a whole. High molecular weight (HMW) DNA restricted withNotI and probed sequentially with MWG068 and BCD249 yield diffuse bands at approximately 2.8 Mb and 3.0 Mb in the C.I. 16151 and C.I. 16155 parental lines, respectively. These results suggest the maximum ratio of physical to genetic distance in the interval defined by these probes is 7.8 Mb/cM. unique HMW DNA restriction fragment length polymorphisms (RFLP) were attributed to the presence of recombination breakpoints. Data from the recombination breakpoint analysis were used to estimate a ratio of physical to genetic distance of 2.5 Mb/cM in theXbcd249.2-Xmwg068 interval and 0.465 Mb/cM in theXbcd249.1-Xbcd249.2 interval. Both physical linkage and recombination breakpoint analysis indicate theXbcd249.1-Xbcd249.2 interval is approximately five-fold smaller, physically, than theXbcd249.2-Xmwg068 interval.Names are necessary to report factually on available data; however the USDA neither guarantees nor warrants the standard of the product and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

QTL analysis of a cross between European and North American malting barleys reveals a putative candidate gene for β-glucan content on chromosome 1H

To determine the genetic factors influencing grain β-glucan content, that were effective in a population of two-row barley grown in very contrasting environments, 102 doubled haploid lines from the cross Beka × Logan were sown at two sites, Lleida (N.E. Spain) and Dundee (E. Scotland) in 2002. Following harvest, grain samples were assessed for total β-glucan content. Beka had lower β-glucan content than Logan at both sites but, while there was transgressive segregation among the DH lines, this was primarily amongst lines with higher β-glucan than Logan. In addition to differences between DH lines, there were differences between the sites and there was also genotype × site interaction. Three QTLs for β-glucan content were detected at both sites, but their contribution to β-glucan content was, in all cases, higher at Lleida compared to Dundee. One QTL was located in the distal end of the long arm of chromosome 1H, in the same region as a gene for UDP-glucose-4-epimerase, an enzyme known to be involved in the synthesis of cell wall polysaccharides, while another was located in the same area of chromosome 5H as a genetic factor shown previously, in the same cross, to influence grain protein content. The third was in the centromeric region of chromosome 7H, close to the gene for naked (hulless) grain. These findings will be important in designing crosses and devising selection strategies in breeding of both low β-glucan, malting barley and high β-glucan, hulless barley for human food use.     

Dissection of a malting quality QTL region on chromosome 1 (7H) of barley

Malting and brewing are major uses of barley (Hordeum vulgare L.) worldwide, utilizing 30–40% of the crop each year. A set of complex traits determines the quality of malted barley and its subsequent use for beer. Molecular genetics technology has increased our understanding of genetic control of the many malting and brewing quality traits, most of which are quantitatively inherited. The objective of this study was to further dissect and evaluate a known major malting quality quantitative trait locus (QTL) region of about 28 cM on chromosome 1 (7H). Molecular marker-assisted backcrossing was used to develop 39 isolines originating from a Steptoe / Morex cross. Morex, a 6–row malting type, was the donor parent and Steptoe, a 6–row feed type, was the recurrent parent. The isolines and parents were grown in four environments, and the grain was micro-malted and analyzed for malting quality traits. The effect of each Morex chromosome segment in the QTL target region was determined by composite interval mapping (CIM) and confirmed and refined by multiple interval mapping (MIM). One QTL was resolved for malt extract content, and two QTLs each were resolved for -amylase activity, diastatic power, and malt -glucan content. One additional putative malt extract QTL was detected at the plus border of the target region by CIM, but not confirmed by MIM. All QTLs were resolved to intervals of 2.0 to 6.4 cM by CIM, and to intervals of 2.0 cM or less by MIM. These results should facilitate marker-assisted selection in breeding improved malting barley cultivars.     

Molecular marker-assisted selection for enhanced yield in malting barley

Brewers are reluctant to change malting barley (Hordeum vulgare ssp. vulgare L.) cultivars due to concerns of altered flavor and brewing procedures. The U.S. Pacific Northwest is capable of producing high yielding, high quality malting barley but lacks adapted cultivars with desirable malting characteristics. Our goal was to develop high yielding near isogenic lines that maintain traditional malting quality characteristics by transferring quantitative trait loci (QTL) associated with yield, via molecular marker-assisted backcrossing, from the high yielding cv. Baronesse to the North American two-row malting barley industry standard cv. Harrington. For transfer, we targeted Baronesse chromosome 2HL and 3HL fragments presumed to contain QTL that affect yield. Analysis of genotype and yield data suggests that QTL reside at two regions, one on 2HL (ABG461C-MWG699) and one on 3HL (MWG571A-MWG961). Genotype and yield data indicate that additional Baronesse genome regions are probably involved, but need to be more precisely defined. Based on yield trials conducted over 22 environments and malting analyses from 6 environments, we selected one isogenic line (00-170) that has consistently produced yields equal to Baronesse while maintaining a Harrington-like malting quality profile. We conclude there is sufficient data to warrant experiments testing whether the 2HL and 3HL Baronesse QTL would be effective in increasing the yield of other low yielding barley cultivars.     

Effects of foliar application of BAP on source and sink strength in four six-rowed barley (Hordeum vulgare L.) cultivars

A field experiment was conducted to investigate the effects of foliar application of a synthetic cytokinin (BAP) on source and sink strength of four different six-rowed barley (Hordeum vulgare L.) cultivars. Different spraying treatments consisting of spraying on whole plant, spraying only on leaves and spraying only on ears started at anthesis and continued for 7 days. One additional spraying was carried out on late period of grain filling. Results showed that spraying only on leaves did not affect ear weight, grain yield and 1,000-grain weight, while the two other treatments increased all above mentioned traits. Neither of treatments affected stem weight, biological yield and contribution of stem reserves in grain filling. Exogenous cytokinin did not increase photosynthetic rate and chlorophyll content in treated leaves until late period of grain filling, although there was no significant increase in final grain weight due to late application of BAP. Our results suggested that effects of foliar application of BAP were mostly due to increased sink size soon after anthesis and increased sink demand probably met by current photosynthesis of organs other than leaves, like ear green tissues. An erratum to this article is available at .     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Genes involved in resistance to powdery mildew in barley differentially modulate root colonization by the mycorrhizal fungus Glomus mosseae

 Arbuscular mycorrhizal fungi (AMF) and Erysiphe graminis are obligate biotrophic fungi with different outcomes in their interaction with plants, different targeted host tissues, but similar patterns of development and infection processes. These similarities raise the question of whether the two types of biotrophic fungal infections have common features in their regulation. To investigate this question, we compared a number of Ror and Rar barley mutants susceptible to E.graminis f. sp. hordei, as well as their resistant progenitors, for susceptibility to infection by the AMF Glomus mosseae. The two powdery mildew-resistant lines BC Ingrid and Sultan presented a similar reduction in G. mosseae development within roots when compared to the wildtype cultivar Ingrid, indicating a systemic effect of the altered genes in the plant. Ror and Rar mutants, in which susceptibility to powdery mildew is restored, showed increased resistance to AM fungal development in their roots when compared to their progenitors, which suggests that corresponding mutations must have affected genes which differentially modulate symbiotic and pathogenic biotrophic plant-fungus interactions. Accepted: 16 September 1999     

The allene oxide cyclase of barley (Hordeum vulgare L.)--cloning and organ-specific expression

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199-213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.     

In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.     

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Summary Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.Abbreviations CMTs cortical microtubules - EMTs endoplasmic microtubules - MT microtubules - PI propidium iodide - SC sensitive combination - RC resistant combination     

Quantitative trait locus effects and environmental interaction in a sample of North American barley germ plasm

Quantitative trait locus (QTL) and QTL x environment (E) interaction effects for agronomic and malting quality traits were measured using a 123-point linkage map and multi-environment phenotype data from an F₁-derived doubled haploid population of barley (Hordeum vulgare). The QTL × E interactions were due to differences in magnitude of QTL effects. Highly significant QTL effects were found for all traits at multiple sites in the genome. Yield QTL peaks and support intervals often coincided with plant height and lodging QTL peaks and support intervals. QTL were detected in the vicinity of a previously mapped Mendelian maturity locus and known function probes for- and-amylase genes. The average map density (9.6 cM) should be adequate for molecular marker-assisted selection, particularly since there were few cases of alternative favorable alleles for different traits mapping to the same or adjacent intervals.Oreg Agric Exp Stn J No. 10150