Trisomic analysis of ribosomal RNA cistron multiplicity in barley (Hordeum vulgare L.)

Ribosomal RNA cistron numbers in all the seven primary trisomics of diploid barley (Hordeum vulgare L.) were determined by DNA-rRNA filter hybridisation. Trisomies for the nucleolus organiser (NO) chromosomes 6 and 7 showed the highest levels of rDNA (DNA complementary to rRNA) indicating the localisation of rRNA cistrons on the NOs. Chromosomes 6 and 7 possessed 1,580 and 2,690 rRNA (18S + 5.8S + 26S) cistrons respectively. Trisomics for the other chromosomes (except for 3) also displayed levels of rDNA significantly higher (22–32%) than the diploid controls although the dosage of NOs was not altered. These non-specific increases were also present in trisomics for 6 and 7 (NOs) which showed further increases equivalent to their respective contributions. The nonspecific increases due to trisomy is indicative of rDNA compensation. Such increases did not persist in diploid sibs of the trisomics, demonstrating the nonheritable nature of the compensation.     

An embryo-specific protein of barley (Hordeum vulgare).

An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 microM) or mannitol (9% mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structure.     

Resistance genes in barley (Hordeum vulgare L.) and their identification with molecular markers

Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomalocation and the list of available DNA markers useful in characterising cultivars and breeding accessions.     

Cytogenetics of an unstable trisomie in barley (Hordeum vulgare).

The origin, identification, meiotic chromosome behavior, and breeding behavior of an unstable trisomic barley were studied. The extra chromosome originated by breakage and fusion of an acrocentric chromosome 3 in a plant from an F2 population of a cross between acrotrisomic 3L3S (2n = 14 + 1 acro3L3S) and a balanced lethal stock, xc. (xantha) ac (albino). The F2 population segregated only for the albino trait. The genotypic constitution of the trisomic plant was ac ac (for both normal chromosome 3) and Ac (for the unstable metacentric chromosome). The unstable extra metacentric chromosome was designated as metacentric 3B (abbreviated as meta3B). Meiotic chromosome behavior in plants with 2n = 14 + 1 meta3B differed from plant to plant and within spikes. Some plants showed only trisomic cells with a chromosome configuration of 1 III + 6 II and 7 II + 1 I at metaphase I, whereas other plants showed both trisomie and disomic cells (7 II) that resulted from the elimination of the extra meta3B. The frequency of ring trivalents was low (6.8%). An average transmission rate of unstable meta3B ranged from 4.3 to 12.9%. The elimination of meta3B, and hence loss of the dominant Ac allele, resulted in albino seedlings as well as white stripes on plants, leaves, and spikes. Chromosome numbers of albino seedlings in the progeny of 2n = 14 + 1 meta3B were all diploid (2n = 14), while green seedlings contained 2n = 14 + 1 meta3B. However, progenies of some spikes of one trisomic plant showed a low frequency of green diploids and metatrisomics (2n = 14 + 1 meta3B), which was attributed to crossing-over.     

Effects of chromosome reconstruction on deletion clustering in Hordeum vulgare

Summary The barley standard karyotype, two reconstructed karyotypes with all chromosomes interdistinguishable, and four translocation lines were treated with maleic hydrazide. A specific chromosomal site in satellite chromosome 7 (segment 44 adjacent to the nucleolus organizer region) of the standard karyotype was found to represent a deletion hot spot. A sample of specifically reconstructed karyotypes were used to check whether or not transposition of the hot spot region, or changes of its neighborhood, would affect its involvement in deletions. One of the seven karyotypes (translocation line T 505 with a pair of chromosomes having both nucleolus organizer regions and satellites in opposite arms) was without deletion clustering in segment 44. At the same time, a prominent Giemsa band close to the secondary constriction was absent from segment 44. These data show that the involvement in deletions of a certain chromosome segment is modifiable in certain cases by chromosome reconstruction. Similar observations have been made in Vicia faba.     

Identification of two forms of glutamine synthetase in barley (Hordeum vulgare).

Hepatocytes of 14-day-old rats have no detectable glucokinase activity $\text{in}$$\text{vivo}$, but it was induced by insulin (10^?8M) in primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also inhibited its induction by insulin. Dexamethasone and testosterone, which alone had no inductive effect, strongly enhanced the induction by insulin. When hepatocytes of 14-day-old rats were cultured with 10^?7M insulin, 10^?6M dexamethasone and 10^?7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats.     

Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS

We have applied a refined microdissection procedure to create a plasmid library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The technical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection of the collection drop to avoid evaporation, and a motorized and programmable microscope stage. Thirteen readily-discernible telocentric chromosomes have been excised from metaphases of synchronized root-tip mitoses. After lysis in a collection drop (2 nl), the DNA was purified, restricted withRsaI, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant plasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analysis after colony hybridization with labelled total barley DNA. Analysis of 552 recombinant plasmids established that: (1) the insert sizes ranged between 70 and 1150 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecificH. vulgare xH. spontaneum F₂ population. One of these probes was found to be closely linked to theMla locus conferring mildew resistance.     

Production and identification of new structural chromosome mutations in barley (Hordeum vulgare L.)

A total of 52 reciprocal translocations and 9 pericentric inversions were induced and identified in both standard and cytologically marked barley karyotypes using gamma-rays as the clastogenic agent. An analysis based upon Giemsa N-banding patterns and arm length measurements of the reconstructed chromosomes enabled a rather precise cytological localization of intra- and interchange breakpoints. This analysis was significantly facilitated and improved, especially for the identification of pericentric inversions, when the reconstructed karyotype T-1586 was used as starting material. The majority, if not all, of the aberration breakpoints proved to be localized in interband regions or in medial and terminal parts of the chromosomes, i.e., in regions which are deficient in constitutive heterochromatin. A great number of the structural mutations produced in this study contain specific cytological markers covering nearly all of the chromosomes of barley karyotype. This material might be of considerable interest in solving various problems of barley cytogenetics and chromosome engineering and especially in constructing a physical map of barley genome.     

Molecular mapping of genes determining height, time to heading, and growth habit in barley (Hordeum vulgare).

A combination of random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers has been used to locate genes controlling important developmental characters in barley. The denso dwarfing gene has been mapped to the long arm of chromosome 3H. Stepwise multiple regression was also used to identify another region of the barley genome (on chromosome 7H), which contributed to variation in height. The denso locus was shown to be associated with delaying time to heading. A protein (WSP2) and an RAPD marker on barley chromosomes 5H and 6H, respectively, were also associated with time to heading. These results are discussed in relation to the genetic analysis of developmentally important traits and the development of dwarfing genes in barley breeding programs.     

Hordoindolines are associated with a major endosperm-texture QTL in barley (Hordeum vulgare).

Endosperm texture has a tremendous impact on the end-use quality of wheat (Triticum aestivum L.). Cultivars of barley (Hordeum vulgare L.), a close relative of wheat, also vary measurably in grain hardness. However, in contrast to wheat, little is known about the genetic control of barley grain hardness. Puroindolines are endosperm-specific proteins found in wheat and its relatives. In wheat, puroindoline sequence variation controls the majority of wheat grain texture variation. Hordoindolines, the puroindoline homologs of barley, have been identified and mapped. Recently, substantial allelic variation was found for hordoindolines among commercial barley cultivars. Our objective was to determine the influence of hordoindoline allelic variation upon grain hardness and dry matter digestibility in the 'Steptoe' x 'Morex' mapping population. This population is segregating for hordoindoline allele type, which was measured by a HinA/HinB/Gsp composite marker. One-hundred and fifty lines of the 'Steptoe' x 'Morex' population were grown in a replicated field trial. Grain hardness was estimated by near-infrared reflectance (NIR) and measured using the single kernel characterization system (SKCS). Variation attributable to the HinA/HinB/Gsp locus averaged 5.7 SKCS hardness units (SKCS U). QTL analysis revealed the presence of several areas of the genome associated with grain hardness. The largest QTL mapped to the HinA/HinB/Gsp region on the short arm of chomosome 7 (5H). This QTL explains 22% of the SKCS hardness difference observed in this study. The results indicate that the Hardness locus is present in barley and implicates the hordoindolines in endosperm texture control.     

Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.)

 Boron toxicity has been recognised as an important problem limiting production in the low-rainfall regions of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in barley has been characterised but the mode of inheritance and the location of genes controlling tolerance were not previously known. A population of 150 doubled-haploid lines from a cross between a boron toxicity tolerant Algerian landrace, Sahara 3771, and the intolerant Australian cultivar Clipper was screened in four tolerance assays. An RFLP linkage map of the Clipper×Sahara population was used to identify chromosomal regions associated with boron tolerance in barley. Interval regression-mapping allowed the detection of four chromosomal regions involved in the boron tolerance traits measured. A region on chromosome 2H was associated with leaf-symptom expression, a region on chromosome 3H was associated with a reduction of the affect of boron toxicity on root growth suppression, a region on chromosome 6H was associated with reduced boron uptake, and a region on chromosome 4H was also associated with the control of boron uptake as well as being associated with root-length response, dry matter production and symptom expression. The benefits and potential of marker-assisted selection for boron toxicity tolerance are discussed. Received: 18 December 1997 / Accepted: 28 November 1998     

High resolution analysis of meiotic chromosome structure and behaviour in barley (Hordeum vulgare L.)

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM).     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm² filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley. Received: 20 September 1999 / Accepted: 25 March 2000     

Regulation of genes encoding β-d-glucan glucohydrolases in barley (Hordeum vulgare)

Expression patterns of barley β - d -glucan glucohydrolase genes were monitored using cDNAs encoding isoenzymes ExoI and ExoII. The cDNAs were isolated from 5-day-old seedling libraries. The enzymes are encoded by a small gene family, in which marked differences in codon usage are evident. The cDNAs can be used as specific probes for two subfamilies of β - d -glucan glucohydrolase genes. Genes of both subfamilies are transcribed in the scutellum of germinated grain, in elongating coleoptiles, and in young roots and leaves. Low levels of mRNA for the isoenzyme ExoI gene subfamily could be detected in aleurone layers of germinated grain. Most of the β - d -glucan glucohydrolase activity can be extracted from tissues with dilute aqueous buffers. Enzyme activity is highest in young leaves and elongating coleoptiles, but is not well-correlated with mRNA levels. The expression patterns are consistent with proposed roles for β -glucan glucohydrolases in the turnover or modification of cell-wall (1→3,1→4)- β - d -glucans in elongating coleoptiles and in young vegetative tissues.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

A molecular,isozyme and morphological map of the barley (Hordeum vulgare) genome

A map of the barley genome consisting of 295 loci was constructed. These loci include 152 cDNA restriction fragment length polymorphism (RFLP), 114 genomic DNA RFLP, 14 random amplified polymorphic DNA (RAPD), five isozyme, two morphological, one disease resistance and seven specific amplicon polymorphism (SAP) markers. The RFLP-identified loci include 63 that were detected using cloned known function genes as probes. The map covers 1,250 centiMorgans (cM) with a 4.2 cM average distance between markers. The genetic lengths of the chromosomes range from 124 to 223 cM and are in approximate agreement with their physical lengths. The centromeres were localized to within a few markers on all of the barley chromosomes except chromosome 5. Telomeric regions were mapped for the short (plus) arms of chromosomes 1, 2 and 3 and the long (minus) arm of chromosomes 7.This research was also supported by other members of the NABGMP: K. Kasha, Department of Crop Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1; W. Kim, Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, Canada R3T 2M9; A. Laroche, Agriculture Canada Research Station, P.O. Box 3000 Main, Lethbridge, Alberta, Canada,TU 4B1; S. Molnar, Plant Research Centre Agriculture Canada, Central Experimental farm, Ottawa, Ontario, Canada K1A 0C6; G. Scoles, Department of Crop Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWOThis research is part of the North American Barley Genome Mapping Project, R. A. Nilan and K. Kasha, Coordinator and Associate Coordinator, respectively Permanent address: Department of Plant Genetics, NI Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow