New allele of <Emphasis Type="Italic">HvBRI1</Emphasis> gene encoding brassinosteroid receptor in barley

The aim of these studies was to characterize nucleotide substitutions leading to the phenotype of brassinosteroid-insensitive, semi-dwarf barley mutant 093AR. Two substitutions in the sequence of barley HvBRI1 gene, encoding leucine-rich repeats receptor kinase (LRR-RK), which participates in brassinosteroid (BR) signalling, were identified in this chemically-induced barley mutant of the cv. Aramir. The LRR-RK is a transmembrane protein phosphorylating downstream components. The identified substitutions CC>AA at positions 1760 and 1761 in the HvBRI1 gene of this mutant led to a missense mutation, causing the Thr-573 to Lys-573 replacement in the protein sequence. The threonine residue is situated in the distal part of a 70-amino acids island responsible for binding of BR molecules. As this residue is conserved among BRI1 protein homologs in Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Hordeum vulgare, it was postulated that this residue is crucial for the protein function. The genetic analyses indicated that the mutant 093AR was allelic to the spontaneous, semi-dwarf mutant uzu which carries A>G substitution at position 2612 of the HvBRI1 gene (GenBank acc. no. AB088206). A comparison of the genomic sequence of HvBRI1 in the mutants uzu, 093AR and in the cv. ‘Aramir’ confirmed the presence of the single-nucleotide A>G substitution at position 2612 in the sequence encoding kinase domain of HvBRI1 polypeptide in uzu, but not in 093AR mutant, indicating that a new allele of the HvBRI1 gene was identified.     

Molecular Characterization of Barley 3H Semi-Dwarf Genes

The barley chromosome 3H accommodates many semi-dwarfing genes. To characterize these genes, the two-rowed semi-dwarf Chinese barley landrace ‘TX9425’ was crossed with the Australian barley variety ‘Franklin’ to generate a doubled haploid (DH) population, and major QTLs controlling plant height have been identified in our previous study. The major QTL derived from ‘TX9425’ was targeted to investigate the allelism of the semi-dwarf gene uzu in barley. Twelve sets of near-isogenic lines and a large NILF₂ fine mapping population segregating only for the dwarfing gene from ‘TX9425’ were developed. The semi-dwarfing gene in ‘TX9425’ was located within a 2.8 cM region close to the centromere on chromosome 3H by fine mapping. Molecular cloning and sequence analyses showed that the ‘TX9425’-derived allele contained a single nucleotide substitution from A to G at position 2612 of the HvBRI1 gene. This was apparently the same mutation as that reported in six-rowed uzu barley. Markers co-segregating with the QTL were developed from the sequence of the HvBRI1 gene and were validated in the ‘TX9425’/‘Franklin’ DH population. The other major dwarfing QTL derived from the Franklin variety was distally located on chromosome 3HL and co-segregated with the sdw1 diagnostic marker hv20ox2. A third dwarfing gene, expressed only in winter-sown trials, was identified and located on chromosome 3HS. The effects and interactions of these dwarfing genes under different growing conditions are discussed. These results improve our understanding of the genetic mechanisms controlling semi-dwarf stature in barley and provide diagnostic markers for the selection of semi-dwarfness in barley breeding programs.     

Uzu mutation in barley (Hordeum vulgare L.) reduces the leaf unrolling response to brassinolide

A sensitive method to examine the brassinolide (BL) response of barley (Hordeum vulgare L.) using dark-grown leaf segments was established based on the known method for wheat. BL responses of 53 dwarf isogenic lines of barley were examined, and two lines were found having a uzu gene that doesn't respond significantly. These results indicate that uzu dwarfism may be caused by the non-responding character to BL.     

Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions

 A collection of 200 wheat (Triticum aestivum L. cv ‘Chinese Spring’) cytogenetic stocks (nullisomic, tetrasomic, nulli-tetrasomic, ditelosomic and deletion lines, addition and substitution stocks from intra- and inter-specific crosses) was utilized to determine the proteins encoded by some of the wheat and barley dehydrin genes, using a western blot procedure. Proteins extracted from seeds were reacted with antibodies that recognize dehydrins in a wide range of plants, including wheat and barley. Proteins encoded by dehydrin loci in chromosome arms 4DS, 5BL and 6AL of ‘Chinese Spring’ wheat were assigned by this method. There was also evidence of a regulatory factor on 5B in the vicinity of the dhn genes, and on 5H in wheat-barley addition lines, that is required for a normal level of expression of seed dehydrins in hexaploid wheat. Further understanding of this putative regulatory factor would be helpful for the interpretation of linkage studies that may relate dehydrin gene expression to phenotypes such as dehydration, salinity or low-temperature tolerance. Received: 27 August 1997 / Accepted: 4 February 1998     

Identification of QTLs controlling tissue-culture traits in barley ( Hordeum vulgare L.)

Quantitative trait loci (QTLs) controlling callus growth (CG), subsequent shoot differentiation ratio (SD) and green shoot ratio (GS) in immature embryo culture were identified in barley. A base map was developed from 99 recombinant inbred lines (RILs) of 'Azumamugi' 2 'Kanto Nakate Gold'. The tissue-culture traits were evaluated at the F₇ and F₁₀ generations of the RILs. The RILs showed wide and continuous variations in each of the three tissue-culture traits. Three QTLs for CG, three QTLs for SD and two QTLs for GS were detected by using composite interval mapping. A QTL for SD on chromosome 3H had a large effect, and 'Kanto Nakate Gold', which has a high differentiation ability, contributed to this QTL. The location of this QTL is identical to, or very close to, the uzu locus. We discuss the relationships between tissue-culture loci in 'Azumamugi' 2 'Kanto Nakate Gold' and those in other mapping populations.     

Long-term effects of blue or red light on ATP and ADP contents in primary barley leaves

Levels of ATP and ADP were studied in primary leaves of barley (Hordeum vulgare L. cv. Viner) seedlings grown under blue (BL) or red light (RL) of various irradiances. In mature leaf segments, BL stimulated a greater accumulation of adenylates than RL. Transfer of barley seedlings from RL to BL for 48 h caused about a twofold increase in the content of adenylates, probably due to de-novo synthesis of adenine nucleotides. Weak BL was found to stimulate an increase in the adenylate content and a higher irradiance enhanced the stimulatory effect. The adenylate content increased markedly from the base towards the tip of barley leaves grown under BL but not in those grown under RL. However, the adenylate content was higher in the basalmost segment of barley leaves grown under RL, indicating that the action of RL on adenylate content proceeded more rapidly than that of BL. The same conclusion could be drawn from the results of experiments with de-etiolated leaves. A linear relationship was established between the maximum rate of CO₂ fixation and the ATP or ADP content in mature segments of primary barley leaves. The possible involvement of two photoreceptors, phytochrome and cryptochrome, in the long-term light regulation of the total content of adenylates in primary barley leaves is discussed.Abbreviations BL blue light - Chl chlorophyll - RL red light     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Genotypic variation in response of barley to boron deficiency

Responses of a range of barley (Hordeum vulgare L.) genotypes to boron (B) deficiency were studied in two experiments carried out in sand culture and in the field at Chiang Mai, Thailand. In experiment 1, two barley genotypes, Stirling (two-row) and BRB 2 (six-row) and one wheat (Triticum aestivum L.) genotype, SW 41, were evaluated in sand culture with three levels of applied B (0, 0.1 and 1.0 μM B) to the nutrient solution. It was found that B deficiency depressed flag leaf B concentration at booting, grain number and grain yield of all genotypes. In barley Stirling, B deficiency also depressed number of spikes plant^-1, spikelets spike^-1 and straw yield. However, no significant difference between genotypes in flag leaf B concentration was found under low B treatments. Flag leaf B concentration below 4 mg kg^-1 was associated with grain set reduction and could, therefore, be used as a general indicator for B status in barley. In experiment 2, nine barley and two wheat genotypes were evaluated in the field on a low B soil with three levels of B. Boron levels were varied by applying either 2 t of lime ha^-1 (BL), no B (B0) or 10 kg Borax ha^-1 (B+) to the soil prior to sowing. Genotypes differed in their B response for grain spike^-1, grain spikelet^-1 and grain set index (GSI). The GSI of the B efficient wheat, Fang 60, exceeded 90% in all B treatments. The B inefficient wheat SW 41 and most of the barley genotypes set grain normally (GSI >80%) only at the B+. In B0 GSI of the barley genotypes ranged from 23% to 84%, and in BL from 19% to 65%. Three of the barley with severely depressed GSI in B0 and BL also had a decreased number of spikelets spike^-1. In experiment 3, 21 advanced barley lines from the Barley Thailand Yield Nursery 1997/98 (BTYN 1997/98) were screened for B response in sand culture with no added B. Grain Set Index of the Fang 60 and SW 41 checks were 98 and 65%, respectively, and GSI of barley lines ranged between 5 and 90%. One advanced line was identified as B efficient and two as moderately B efficient. The remaining lines ranked between moderately inefficient to inefficient. These experiments have established that there is a range of responses to B in barley genotypes. This variation in the B response was observed in vegetative as well as reproductive growth. Boron efficiency should be considered in breeding and selection of barley in low B soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

Pyramiding of <Emphasis Type="Italic">Ryd2</Emphasis> and <Emphasis Type="Italic">Ryd3</Emphasis> conferring tolerance to a German isolate of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate

Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

A barley Hordoindoline mutation resulted in an increase in grain hardness

Barley seed proteins, Hordoindolines, are homologues of wheat Puroindolines, which are associated with grain hardness. Barley Hordoindoline genes are known to comprise Hina and Hinb, and Hinb consists of two Hinb genes, Hinb-1 and Hinb-2. Two types of allele were found for Hina, Hinb-1 and Hinb-2 genes, respectively, among Japanese two- and six-rowed barley lines. One of the alleles of Hinb-2 (Hinb-2b) had a frame-shift mutation resulting in an in-frame stop codon. For two-rowed barley lines, grain hardness was significantly higher among lines with the Hinb-2b than those with the wild type Hinb-2 gene (Hinb-2a). Protein spots corresponding to HINa, HINb-1, and HINb-2 were identified by 2D-gel electrophoresis among barley lines with Hinb-2a. Among the lines with Hinb-2b, HINa and HINb-1 were expressed at similar levels as those in the wild type, but HINb-2 was not detected. A DNA (cleaved amplified polymorphic sequence) marker was developed to distinguish between the Hinb-2a and Hinb-2b gene sequences. Analysis of grain hardness among F₂ lines derived from a cross between a line with Hinb-2a (Shikoku hadaka 115) and a line with the Hinb-2b (Shikoku hadaka 84) showed significantly higher grain hardness in the mutant lines. From these results, the Hinb-2b frame-shift (null) mutation might play a critical role in barley grain hardness. The DNA marker will be useful in barley breeding to select lines having harder grain texture.     

Characterising seedling and adult plant resistance to Puccinia hordei in Hordeum vulgare

A set of 113 genotypes of barley (Hordeum vulgare subsp. vulgare), along with the susceptible control genotype Gus, was tested for response to the barley leaf rust pathogen Puccinia hordei in the greenhouse (as seedlings) and field (as adult plants). The tests revealed that 68 lines carried adult plant resistance (APR), 23 lines carried uncharacterised seedling resistance (USR) and that three lines carried the seedling resistance gene Rph3. Nineteen lines lacked detectable seedling resistance and were also susceptible in the field at adult plant growth stages. The presence of marker bPb‐0837, linked to the APR gene Rph20, in 35 of the 68 lines carrying APR, suggested they carry this gene. The remaining 33 lines, which lacked the Rph20 linked marker, are likely sources of new uncharacterised APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cv. Gull and to Hordeum laevigatum; two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog, suggesting that these genotypes may be the ancestral sources of the APR carried by each.     

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression of peroxisomal Ascorbate Peroxidase gene from wheat

A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase (pAPX) was cloned by Suppression Subtractive Hybridization (SSH) and in silico approach. The cDNA was 1027 bp in length and contained a complete ORF of 876 bp, which encodes a protein of 292 amino acid residues. Its deduced amino acids sequence had 84% identity with that of pAPX from barley. The gene was designated as Ta-pAPX. The Ta-pAPX homologous genes were mapped on wheat chromosome 7A and 7D using Chinese Spring nulli-tetrasomic lines analysis. Northern analysis indicated that, after inoculation by Erysiphe graminis Dc.f.sp. tritici, the expression of Ta-pAPX gene in Yangmai5 was enhanced, but its expression in wheat-Haynaldia villosa 6VS/6AL translocation lines changed a little. The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew. The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32 (a+) and a protein with the same deduced molecular weight (MW) was expressed in E. coli BL21 (DE3), which showed ascorbate peroxidase activity.     

Production of alloplasmic and euplasmic wheat-barley ditelosomic substitution lines 7H<Superscript>1</Superscript>L<Superscript>mar</Superscript>(7D) and analysis of the 18S/5S mitochondrial repeat in these lines

Alloplasmic lines of common wheat with disomic substitution of chromosome 7D for telocentric chromosome 7H¹L^mar of barley H. marinum subsp. gussoneanum Hudson were isolated from the plants of generation BC₃, produced as a result of backcrossing of barley-wheat hybrids H. marinum subsp. gussoneanum (2n = 28) × T. aestivum (2n = 42), Pyrotrix, cultivar, with 28 common wheat cultivars Pyrotrix 28 and Novosibirskaya 67. Chromosome substitution pattern was determined using SSR analysis and C-banding. In preliminary genomic in situ hybridization experiments, telocentric chromosomes were assigned to wild barley was established. In the BC₃F₈ generations of three alloplasmic lines with the 7H¹L^mar(7D) substitution type the differences in fertility manifestation were observed: most of the L-32(1) plants were sterile, in line L-32(2) only sporadic plants were sterile, and line L-32(3) was fertile. Simultaneously with these experiments, using selfpollinated progeny of the hybrids obtained in crosses of common wheat cultivar Saratovskaya 29 (2n = 41), monosomic for chromosome 7D, with common wheat cultivar Pyrotrix 28 with addition of pair of telocentric chromosomes 7H¹L^mar (7D) of barley H. marinum subsp. gussoneanum, euplasmic wheat-barley ditelosomic substitution 7H¹L^mar (7D) lines were isolated. The lines obtained had normal fertility. PCR analysis of the 18S/5S mitochondrial repeat (hereafter, mtDNA sequence) in alloplasmic and euplasmic ditelosomic substitution lines 7H¹L^mar(7D) was performed. In the plants from alloplasmic sterile line L-32(1), the sequences only of the barley (maternal) type were revealed, while the plants from alloplasmic fertile lines L-32(2) and L-32(3) demonstrated heteroplasmy (the presence of barley- and wheat-like sequences within one individual). In euplasmic ditelosomic substitution lines the presence of only wheat-like 18S/5S mitochondrial repeat sequences was observed. The results indicate that the presence of barley-like mtDNA sequences in alloplasmic substitution lines was not associated with the presence of barley chromosomes in their nuclear genomes.