Genetic mapping of the barley nitrate reductase-deficient nar1 and nar2 loci

Summary The nar2 locus that codes for a protein involved in molybdenum cofactor function in nitrate reductase and other molybdoenzymes was mapped to barley chromosome 7. F₂ genotypic data from F₃ head rows indicated nar2 is located 8.4±2.1 and 23.0± 4.6 cm from the narrow leaf dwarf (nld) and mottled seedling (mt2) loci, respectively. This locates the nar2 locus at 54.7±3.1 cm from the short-haired rachilla (s) locus near the centromere of chromosome 7. Close linkage of nar2 with DDT resistance (ddt) and high lysine (lys3) loci was detected but could not be quantified due to deviations from the individual expected 121 segregations for the ddt and lys3 genes. Southern blots of wheat-barley addition lines probed with a nitrate reductase cDNA located the NADH : nitrate reductase structural gene, nar1, to chromosome 6.Scientific Paper No. 7762. College of Agriculture and Home Economics Research Center, Washington State University, Project No. 0745. This investigation was supported in part by United States Department of Agriculture Grant No. 86-CRCR-1-2004     

Characterisation of progeny from backcrosses of triploid hybrids between Hordeum vulgare L. (2x) and H. bulbosum L (4x) to H. vulgare

Interspecific hybridisations between Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) have been carried out to transfer desirable traits, such as disease resistance, from the wild species into barley. In this paper we report the results of an extensive backcrossing programme of triploid hybrids (H. vulgare 2x x H. bulbosum 4x) to two cultivars of H. vulgare. Progenies were characterised cytologically and by restriction fragment length polymorphism analysis and comprised (1) haploid and diploid H. vulgare plants, (2) hybrids and aneuploids, (3) single and double monosomic substitutions of H. bulbosum chromosomes into H. vulgare and (4) chromosomal rearrangements and recombinants. Five out of the seven possible single monosomic chromosome substitutions have now been identified amongst backcross progeny and will be valuable for directed gene introgression and genome homoeology studies. The presence amongst progeny of 1 plant with an H. vulgare-H. bulbosum translocated chromosome and one recombinant indicates the value of fertile triploid hybrids for interspecific gene introgression.     

Isolation and culture of barley megasporocyte protoplasts

An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were dissected out of ovaries under aseptic conditions, subjected to a brief enzymatic digestion, and then transferred to a modified Kao medium with 90 g/l sucrose and 20 mM CaCl₂. A small incision was made with a scalpel through the softened epidermal cell layer of the nucellus and the megasporocyte could then be liberated into the medium by applying gentle pressure on the nucellus. The megasporocyte appeared to be completely devoid of a wall and changed its in situ pyriform shape to completely spherical when extruded into the medium. Four to nine protoplasts could typically be isolated per spike. Protoplasts cultured in medium degenerated after a few days. Viability was dramatically improved if protoplasts were co-cultivated with barley microspores undergoing microspore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts stained with 4,6-diamidino-2-phenylindole (DAPI) or aniline blue revealed that the protoplasts remained uninuclear and reformed their callose wall.     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

Comparative mapping of the barley genome with male and female recombination-derived, doubled haploid populations

Male (anther culture) and female (Hordeum bulbosum) derived, doubled haploid populations were used to map the barley genome and thus determine the different recombination rates occurring during meiosis in the F1 hybrid donor plants. The anther culture-derived (male recombination) population showed an 18% overall increase in recombination rate. This increased recombination rate was observed for every chromosome and most of the chromosome arms. Examination of linkage distances between individual markers revealed eight segments with significantly higher recombination in the anther culture-derived population, and one in the Hordeum bulbosum-derived population. Very strong distortions of single locus segregations were observed in the anther culture-derived population, but map distances were not affected significantly by these distortions. There were 1.047 and 0.912 recombinations per chromosome in the anther culture and Hordeum bulbosum-derived doubled haploid populations, respectively.     

Genetic analyses of rDNA spacer-length variation in barley

Summary The genetic mechanism controlling the inheritance of single and multiple spacer-length variant (slv) phenotypes in barley was investigated in six F₂ segregating populations. The results indicated that two independently assorting loci, each with co-dominant alleles, govern genetic variability for rDNA in barley regardless of the number of bands expressed by a given phenotype. The following chromosomal locations are proposed: sl variants 1, 4, 5, 6, and 7 on chromosome 7, and sl variants 7, 8, 9, 12, and 13 on chromosome 6; sl variant 7 is thus located on both of the chromosomes.     

Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture and their potential for transformation by microinjection

Summary In order to identify microspores, suitable for transformation via microinjection of DNA, single microspores of barley (Hordeum vulgare L.) were selected after initial preculture of anthers floating on liquid media and analysed for their development in individual culture in microdroplets of culture medium. Conditions for microculture and plant regeneration from single selected embryogenie microspores were established. The technical feasability of intranuclear microinjection was demonstrated by injecting the fluorescent dye Lucifer Yellow. All essential procedures for a transformation system of barley based on microinjection into microspores have thus been performed successfully. Further efforts to increase efficiencies of culture and microinjection procedures are necessary, however, in order to improve the suitability of this approach towards transformation of barley.Abbreviations MES 2 (N-morpholino) ethanesulfonic acid - PEG polyethylene glycol     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

Embryology of barley. IV. Ultrastructure of the antipodal cells of Hordeum vulgare L. cv. Bomi before and after fertilization of the egg cell

The antipodal cells have been the stepchildren in most investigations of the female gametophyte. In Hordeum vulgare cv. Bomi, three antipodal cells are originally developed chalazally but because of differential growth of the embryo sac they soon become laterally situated and their number increases to 35–50 cells and the shape, size and structure of the cells change in the time before as well as after fertilization. The cells persist until about 60–70 h after pollination. At that time, the embryo consists of about 12–15 cells and a cellularization of the nuclear endosperm has started peripherally. The size of nuclei, and especially nucleoli, in the antipodal cells increases tremendously in the investigated period and the amounts of organelles also change. The walls of antipodals are diversified depending on which cells they are separating, and wall invaginations are developed especially between antipodal cells and surrounding nucellar cells in the placental region. The mitochondria appear in various shapes in section view, very often as cups or dumbbells with a rim in the ends containing cristae and a thin cristae-free base. These bases are sometimes stretched out as thin parts and at last a simple parting occurs. Binary fissions of the plastids happen especially in the hours before and just after egg fertilization. ER is extraordinarily well developed in the whole period of investigation and many ribosomes are attached to the membranes. Dictyosomes form numerous vesicles, especially in the antipodals near the nucellar cells in the placental region. These ultrastructural details support the opinion that antipodal cells may play an important role in the embryo sac and are able to be responsible for the supply of nutrition for the whole gametophyte and take part in the supply of nutrition during embryo formation.     

Geographic distribution of alleles at the Ga2 locus for segregation distortion in barley

Summary A distorted segregation of esterase alleles at the complex loci, Est1, Est2 and Est4, was found in an F₂ population. This distortion is typical for cross combinations between the Ga2Ga2 and ga2ga2 genotypes responsible for segregation distortion, since the Ga2 locus is linked with the complex loci encoding the esterase isozymes. The segregation of esterase isozyme patterns in F₂ populations between 473 varieties of barley and a tester of ga2ga2 genotype was examined, and the genotypes inducing segregation distortion were detected. Varieties with a ga2ga2 genotype are widely distributed throughout the world, whereas Ga2Ga2 varieties are found only in eastern and southern regions of Asia, from Japan to North India, with a low frequency. In varieties collected from these regions, some associations were detected between alleles at the Ga2 locus and esterase isozyme patterns. Additionally, most of the Ga2 barley varieties are naked and possess a BtBtbt2bt2 genotype for a non-brittle rachis.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.     

Gradual Disassembly of Photosystem 2 In Vivo Induced by Excess Irradiance. A Hypothesis Based on Changes in 77 K Fluorescence Spectra of Chlorophyll a in Barley Leaves

Effects of short-term exposure to different irradiances on the function of photosystem 2 (PS2) were studied for barley grown at low (LI; 50 μmol m⁻² s⁻¹) and high (HI; 1 100 μmol m⁻² s⁻¹) irradiances. HI barley revealed higher ability to down-regulate the light-harvesting within PS2 after exposure to high irradiance as compared to LI plants. This ability was estimated from the light-induced decreases of F685/F742 and E476/E436 in emission and excitation spectra of 77 K chlorophyll (Chl) a fluorescence in vivo which was 65 and 10 % for HI plants as compared to 30 and 2 % for LI plants, respectively. For LI plants this protective down-regulation of the light-harvesting of PS2 was saturated at 430 μmol m⁻² s⁻¹, and progressive PS2 photodamage was induced at higher irradiances. After exposure of LI segments to 2 200 μmol m⁻² s⁻¹ a pronounced maximum at 700 nm appeared in emission spectrum of 77 K Chl a fluorescence. Based on complementary analysis of 77 K excitation spectra measured at the emission wavelength 685 nm we suggest that this emission maximum may be attributed to the formation of aggregates of light-harvesting complexes of PS2 (LHC2) with part of PS2 core during progressive PS2 photodamage. Our results can be explained assuming different contributions of LHC2 and PS2 core to the total nonradiative dissipation of absorbed excitation energy for the LI and HI barley. This revised version was published online in August 2006 with corrections to the Cover Date.