Inheritance of resistance to yellow mosaic virus in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of resistance to mungbean yellow mosaic virus (MYMV) was studied in blackgram (Vigna mungo (L.) Hepper). The highly resistant donors Pant U-84 and UPU-2 and a highly susceptible line, UL-2, their F₁'s, F₂'s and backcrosses were grown with spreader located every 5 to 6 rows. The resistance was found to be digenic and recessive in all the crosses and free from cytoplasmic effect.     

Rapid production of recombinant barley yellow mosaic virus resistant Hordeum vulgare lines by anther culture

Summary In a winter barley breeding program for barley yellow mosaic virus (BaYMV) resistance, the resistant six-rowed cv. Franka was crossed to 17 susceptible and two resistant cultivars, three of which were tworowed. A total of 233,445 anthers of the 19 hybrids and their parents were cultured and 831 green plants regenerated. Anther culture responsiveness varied greatly between genotypes, and the responsiveness of F₁hybrids was generally related to that of the more responsive (high) parent. On average, 3.6 green plants were recovered from 1,000 cultured anthers, almost twice as many as in comparable spring barley experiments. Androgenetic green plants were tested for their reaction to mechanical inoculation of BaYMV. In crosses of resistant parents, all the cross progeny proved to be resistant, which indicates that both parents carry identical gene(s). In the crosses of the resistant cv. Franka to susceptible parents, an average of 62% of the androgenetic progenies were resistant, which indicates that probably more than one gene is responsible for Franka's BaYMV-resistance. From the crosses of Franka to two-rowed cultivars, 282 androgenetic plants were produced. When 132 of these were tested for their reaction to BaYMV, 79 (59.8%) were resistant, and 30 of the latter were shown to be two-rowed recombinant lines. Doubled haploid lines are field-tested for other agronomic characters including grain yield and its components.     

Association of two barley yellow mosaic virus (RNA 2) encoded proteins with cytoplasmic inclusion bodies revealed by immunogold localisation

Summary Antisera were raised against the RNA 2-encoded proteins of 28 kDa and 70 kDa of barley yellow mosaic virus (BaYMV) by using the corresponding cDNA sequences of a German isolate for protein overexpression inEscherichia coli BL 21 and subsequent purification. The proposed processing of a 98 kDa precursor polyprotein encoded by the long open reading frame of RNA 2 to two proteins of 28 kDa and 70 kDa could be confirmed by immunoprecipitation of the in vitro transcribed and translated cDNA-clone of RNA 2 and Western blot analysis of fragmentated protein extracts of BaYMV-infected winter barley plants. In situ localisation studies of infected leaf tissue using immunogold labeling techniques for electron microscopy revealed that both viral proteins of BaYMV (RNA 2) were associated with the crystal-like cytoplasmic inclusion bodies. No other parts of the cells and no other inclusions (pinwheelstructures or aggregated virus particles) showed any gold labeling when the 28 kDa and 70 kDa antisera were used. We suppose that both RNA 2-encoded proteins take part in the formation of the crystal-like cytoplasmic inclusion bodies which are the most dominant structures in the cytoplasm of BaYMV-infected tissue. Possible functions of the 28 kDa and 70 kDa protein of BaYMV (RNA 2) are discussed.Abbreviations PBS phosphate-buffered saline - CEA chicken egg albumin - BaYMV barley yellow mosaic virus - BaMMV barley mild mosaic virus     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

Delivery of foreign genes to intact barley cells by high-velocity microprojectiles

Summary Foreign DNA was introduced through the cell walls of intact suspension culture cells of barley (Hordeum vulgare L.) by utilizing the particle acceleration approach. DNA-coated microscopic tungsten particles were accelerated to velocities that permitted their penetration of intact cells. Chimaeric constructs of -glucuronidase and neomycin phosphotransferase II under the control of the dual Agrobacterium T_R 12 promoter or the cauliflower mosaic virus 35S promoter served as reporter genes. Three days after particle delivery, high-level expression of both reporter genes was observed. That plasmid size could be critical for stabilizing DNA in the course of particle delivery will be discussed.     

Genetic analysis of preharvest sprouting in a six-row barley cross

Preharvest sprouting (PHS) can be a problem in barley (Hordeum vulgare L.) especially malting barley, since rapid, uniform, and complete germination are critical. Information has been gained by studying the genetics of dormancy (measured as germination percentage, GP). The objective of this study was to determine if the quantitative trait loci (QTLs) discovered in previous research on dormancy are related to PHS. PHS was measured as sprout score (SSc) based on visual sprouting in mist chamber-treated spikes and as alpha-amylase activity (AA) in kernels taken from mist chamber-treated spikes that showed little or no visible sprouting. GP was also measured. All traits were measured at 0 and 14 days after physiological maturity. Evaluation of the spring six-row cross, Steptoe (dormant)/Morex (non-dormant) doubled haploid mapping population grown in greenhouse and field environments revealed QTL regions for SSc, AA, and GP on five, four, and six of the seven barley chromosomes, respectively. In total, seven and eight regions on five and six chromosomes had effects ranging from 4 to 31% and 3 to 39% on PHS and dormancy, respectively. One chromosome 3H and three chromosome 5H QTLs had the greatest effects. All PHS QTLs coincide with known dormancy QTLs, but some QTLs appear to be more important for PHS than for dormancy. Key QTLs identified should benefit breeding of barley for a suitable balance between PHS and dormancy.     

Chromosomal localization of four isozyme loci by trisomic analysis in rice (Oryza sativa L.)

Summary Four genes coding for isozymes in rice (Oryza sativa L.), were located to respective chromosmes through trisomic analysis. Twelve primary trisomics in IR36 background were crossed with 2 lines having contrasting alleles at four loci. For each gene, all 12 disomic and trisomic F₁ hybrids were screened for allele dosage effects. Either F₂ or BC1 populations of all cross combinations were assessed for gene segregtion. Evidence from both sources indicated the following locations: Pgi-1 on chromosome 4, Sdh-1 on chromosome 6, Est-8 on chromosome 7 and Adh-1 on chromosome 11. The location of Sdh-1 was further confirmed through the production of triallelic heterozygotes with trisomic 6.     

The Yd2 gene for barley yellow dwarf virus resistance maps close to the centromere on the long arm of barley chromosome 3

Barley yellow dwarf luteovirus (BYDV) causes serious yield losses in all cereals worldwide. The Yd2 gene from a number of Ethiopian barleys (Hordeum vulgare L.) has been the most effective means of providing resistance against BYDV in cultivated barley. Isolation of the Yd2 gene will enable characterisation of the molecular basis of the Yd2-BYDV interaction. This paper describes the first stage in a project to isolate the gene: the construction of a detailed linkage map of the Yd2 region. The map encompasses 27.6 centiMorgans (cM) of chromosome 3 and contains 19 RFLPs, 2 morphological marker loci, the centromere and Yd2. In the mapping population of 106 F₂ individuals, Yd2 perfectly cosegregated with the RFLP loci Xwg889 and XYlp, which were located on the long arm, 0.5 cM from the centromere. The two morphological marker loci, uzu dwarfand white stripe j, both mapped distal to Yd2. The protein product of the gene at the XYlp locus will provide a convenient assay for the selection of Yd2 during the breeding of BYDV-resistant barley varieties.     

Chromosomal location of isozyme markers in wheat-barley addition lines

Summary The peroxidase (CPX, PER), -amylase (-AMY), acid and alkaline phosphatase (PHE, PHS) and esterase (EST) zymogram phenotypes of Chinese Spring wheat, Betzes barley and a number of presumptive Betzes chromosome additions to Chinese Spring were determined. It was found that five disomic chromosome addition lines could be distinguished from one another and from the other two possible lines on the basis of the zymogram phenotypes of these isozymes. The structural genes Cpxe-H1 and Cpxe-H2 were located in Betzes chromosome 1, the Perl-H5 and Perl-H6 in chromosome 2, the -Amy-H2 and -Amy-H3 in chromosome 7, the Phs-H5 and Phs-H4 in chromosomes 1 and 3 respectively, the Phe-H2, Phe-H3 and Phe-H4 in chromosome 1, the Phe-H1 in chromosome 3, the Ests-H4, Este-H2 and Ests-H6, Este-H8 in chromosomes 1 and 3 respectively and the Estl-H10 and Estl-H2 structural genes were related to chromosomes 3 and 6 respectively. These gene locations provide evidence of homoeology between Betzes chromosomes 1, 2, 3, 6 and 7 and the rye chromosomes 7, 2, 3, 6 and 5, respectively, and also between Betzes chromosomes 1, 2, 3, 6 and 7 and the Chinese Spring homoeologous groups 7, 2, 3, 6 and 5, respectively.     

Identification of polypeptide markers of barley yellow dwarf virus resistance and susceptibility genes in non-infected barley (Hordeum vulgare) plants

Summary Barley yellow dwarf (BYDV) is a group a closely related viruses which cause economic losses in a wide range of graminaceous species throughout the world. Barley plants can be protected from the effects of BYDV by the Yd2 resistance gene. Plants which contain the Yd2 gene also contain a constitutively expressed polypeptide which was not found in any plants without Yd2. Conversely, BYDV susceptible plants contain another constitutively expressed polypeptide which was not found in any of the BYDV-resistant lines examined. These two polypeptides appear to have the same molecular weight (as assessed by SDS-PAGE) and only slightly different iso-electric points. They also appear to contain an extensive range of similar antigenic determinants. Both polypeptides were found in F₁ hybrids made from resistant and susceptible plants. We suggest that these two polypeptides are the products of two allelic genes. Analysis of near-isogenic lines showed that the locus which controls the Yd2 resistance gene and the locus controlling the synthesis of the two polypeptides may be within ± 9 cM of each other. We have developed a Western blot technique which allows assessment of barley lines, 4-days after seed imbibition, for the presence of the Yd2 gene.     

Identification of QTLs associated with Fusarium head blight resistance in Zhedar 2 barley

Fusarium head blight (FHB) in barley and wheat, caused by Fusarium graminearum, is a continual problem worldwide. Primarily, FHB reduces yield and quality, and results in the production of the toxin deoxynivalenol (DON), which can affect food safety. Identification of QTLs for FHB severity, DON level and related traits heading-date (HD) and plant-height (HT) with consistent effects across a set of environments, would provide the basis for marker-assisted selection (MAS) and potentially increase the efficiency of selection for resistance. A segregating population of 75 double-haploid lines, developed from the three-way cross Zhedar 2/ND9712//Foster, was used for genome mapping and FHB severity evaluation. A linkage map of 214 RFLP, SSR and AFLP markers was constructed. Phenotypic data were collected in replicated field trials from five environments in two growing seasons. The data were analyzed using MQTL software to detect quantitative trait locus (QTL) × environment (E) interactions. Because of the presence of QTL × E, the MQM procedure in MAPQTL was applied to identify QTLs in single environments. We identified nine QTLs for FHB severity and five for low DON. Many of the disease-related QTLs identified were coincident with FHB QTLs identified in previous studies. Only two of the QTLs identified in this study were consistent across all five environments, and both were Zhedar 2 specific. Five of the FHB QTLs were associated with HD, and two were associated with HT. Regions that appear to be promising candidates for MAS and further genetic analysis include the two FHB QTLs on chromosome 2H and one on 6H, which were also associated with low DON and later heading-date in multiple environments. This study provides a starting point for manipulating Zhedar 2-derived resistance by MAS in barley to develop cultivars that will show effective resistance under disease pressure.Communicated by H.F. Linskens     

Genetic analysis of disease resistance to all strains of BaYMV in a Chinese barley landrace, Mokusekko 3

 A Chinese landrace of barley, Mokusekko 3, is unique in being completely resistant against all strains of barley yellow mosaic virus (BaYMV). The present investigation revealed that the resistance of Mokusekko 3 is governed by two recessive genes. As one of the resistance genes was known to be tightly linked with alleles at the Est complex locus, consisting of the Est1, Est2 and Est4 loci for esterase isozymes, each of the resistance genes could be separated by means of marker-assisted selection using an isozyme allelic combination as a marker. One of the resistance genes, ym1, is linked to K (hooded lemma) and gl3 (glossy leaf 3) with recombination values of 25.3% and 9.7% respectively, and these three genes are located in the order K-gl3-ym1 on chromosome 4. Another newly designated resistance gene, ym5, is linked to alleles at the Est complex locus and cu2 (curly growth 2), with recombination values of 1.9% and 19.5% respectively, in the order cu2-Est-ym5 from proximal to distal on the long arm of chromosome 3. The complete resistance of Mokusekko 3 is caused by combining two resistance genes, ym1 and ym5. However, almost all the “resistant” cultivars derived from crosses with Mokusekko 3 are susceptible to the recently detected strain BaYMV-III in Japan, since they contain only one resistance gene, ym5. Marker-assisted selection to combine resistance genes into a cultivar is discussed for the breeding of stabilizing resistance to BaYMV. Received: 23 September 1996 / Accepted: 8 November 1996     

A mutator nuclear gene inducing a wide spectrum of cytoplasmically inherited chlorophyll deficiences in barley

Summary Some striped plants were observed in plots of a long-grain mutant barley grown at a field nursery. All of the plants of these plots, which were naturally self pollinated, were individually harvested, and most of their progenies (92.5%) segregated seedlings carrying chlorophyll deficiencies (CD) as determined by greenhouse analysis. The majority of the mutant seedlings (84.3%) showed a pattern of longitudinal chlorophyll sectors. The spectrum of CD was wide among the solid mutant seedlings and consisted of three main types (albina, viridis and discontinuous). In association with some CD types morphological changes were frequently observed. Non-CD-associated morphological changes and diminished seed-set were scarce and, so far, none of them has proved to be inherited. Analysis of CD in reciprocal crosses and backcrosses proved that while CD were transmitted cytoplasmically their induction was controlled by a single nuclear mutator gene, active when homozygous. In addition once the CD were induced, they were expressed independently of the nuclear constitution. The results suggest that the mutator gene induces diverse mutational events on chloroplast (cp) DNA. In barley, as in other monocots, nuclear genes which are inductors of cytoplasmic genetic changes have been reported. However, all of them produced a narrower spectrum of CD and had a more rapid sorting-out of the cytoplasmic mutants than what we observed. On this basis a distinction between chloroplast and mitochondrial (mt) mutator genes is proposed. Accordingly, the chloroplast mutator here described would be the first one reported for monocots. Increased knowledge on this subject can play a fundamental role in elucidating organelle heredity and its interactions with the nuclear genome. Moreover, this material could be a valuable source of variability of the otherwise conservative genetic information encoded in the chloroplast.Paper GEN 792, Institute of Genetics, CICA, INTA, Castelar     

A barley RFLP map: alignment of three barley maps and comparisons to Gramineae species

Several gene linkage maps have been produced for cultivated barley. We have produced a new linkage map for barley, based on a cross between Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare (Hvs x Hvv), having a higher level of polymorphism than most of the previous barley crosses used for RFLP mapping. Of 133 markers mapped in the Hvs x Hvv F₂ population, 69 were previously mapped on other barley maps, and 26 were mapped in rice, maize, or wheat. Two known gene clones were mapped as well as two morphological markers. The distributions of previously mapped markers were compared with their respective barley maps to align the different maps into one consensus map. The distributions of common markers among barley, wheat, rice and maize were also compared, indicating colinear linkage groups among these species.To be considered dual first authorsPublished with the approval of the Director of the Colorado State University/Agricultural Experiment Station.     

Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development

Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis.     

The allelic relationship of genes giving resistance to mungbean yellow mosaic virus in blackgram

Summary The allelic relationship of resistance genes for MYMV was studied in blackgram (V. mungo (L.) Hepper). The resistant donors to MYMV — Pant U84 and UPU 2, and their F₁, F₂ and F₃ generations — were inoculated artificially using an insect vector, whitefly (Bemisia tabaci Genn.). The two recessive genes previously reported for resistance were found to be the same in both donors.Part of Ph.D. Thesis submitted by the senior author. Research Paper No. 4271     

Cadmium translocation and accumulation in developing barley grains

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. In order to study the transport of Cd into the developing grains, detached ears of two-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing the markers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in each part of detached spikes increased with external Cd levels, and Cd concentration in various organs over the three Cd levels of 0.5, 2, 8 μM Cd on 15-day Cd exposure was in the order: awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grains with the proportion of 51.0% relative to the total Cd amount in the five parts of detached spikes. Cd accumulation in grains increased not only with external Cd levels but the time of exposure contrast to stem, awn, rachis and glume. Those four parts of detached spike showed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 and increase again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cd concentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relative humidity (RH) also induced dramatic reduction in Cd transport to developing grains. The results indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as an important physiological process influencing the quality of harvested grains. Our results suggested that increasing RH to 90% and Zn addition in the medium at grain filling stage would be beneficial to decrease Cd accumulation in grains.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.