Differential Al resistance and citrate secretion in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

While barley ( Hordeum vulgare L.) is the most sensitive species to Al toxicity among small-grain crops, variation in Al resistance between cultivars does exist. We examined the mechanism responsible for differential Al resistance in 21 barley varieties. Citrate was secreted from the roots in response to Al stress. A positive correlation between citrate secretion and Al resistance [(root elongation with Al)/(root elongation without Al)] and a negative correlation between citrate secretion and Al content of root apices, were obtained, suggesting that citrate secretion from the root apices plays an important role in excluding Al and thereby detoxifying Al. The Al-induced secretion of citrate was characterized using an Al-resistant variety (Sigurdkorn) and an Al-sensitive variety (Kearney). In Sigurdkorn, Al-induced secretion of citrate occurred within 20 min, and the secretion did not increase with increasing external Al concentration. The Al-induced citrate secretion ceased at low temperature (6 degrees C) and was inhibited by anion-channel inhibitors. Internal citrate content of root apices was increased by Al exposure in Sigurdkorn, but was not affected in Kearney. The activity of citrate synthase was unaffected by Al in both Al-resistant and Al-sensitive varieties. The secretion rate of organic acid anions from barley was the lowest among wheat, rye and triticale.     

Inducing homozygosity in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.     

Quantification of the tissue-culture induced variation in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background  

When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.     

Hordein locus polymorphism of cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) in Turkey

Starch gel electrophoresis has been used to study the polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd F loci in 93 landrace specimens of barley assigned to 17 ancient provinces located in modern Turkey. Forty-five alleles of Hrd A with frequencies of 0.11–29.34%, 51 alleles of Hrd B with frequencies of 0.11–8.07%, and 5 alleles of Hrd F with frequencies of 0.75–41.29% have been detected. Cluster analysis of the matrix of allele frequencies has demonstrated that barley populations from different old provinces of Turkey are similar to one another. Cluster structure of local barley populations has been found, most populations (82%) falling into three clusters. The first cluster comprises barley populations from six provinces (Thracia, Bithynia, Pontus, Lydia, Cappadocia, and Armenia); the second cluster, populations from five provinces (Paphlagonia, Galatia, Lycaonia, Cilicia, and Mesopotamia); and the third one, populations from three provinces (Phrygia, Karia, and Lycia). Barley populations from Mysia, Pamphlya, and Syria do not fall in any cluster.     

Genetic variation of <Emphasis Type="Italic">Bmy1</Emphasis> alleles in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) investigated by CAPS analysis

The enzyme beta-amylase is one of the most important hydrolytic enzymes in the grain of malting barley and is encoded by the gene Bmy1. To learn more about its structure and function, a total of 657 barley accessions including 541 Hordeum vulgare ssp. vulgare (HV), and 116 H. vulgare ssp. spontaneum (HS) were selected for the cleaved amplified polymorphic sequence (CAPS) analysis. These materials, covering all the 16 kinds of beta-amylase phenotypes screened from more than 8,500 accessions of the world barley germplasm, were classified into 13 CAPS types in the present study. A combined assay of phenotypes and CAPS types revealed extensive genetic variation at the Bmy1 locus, and in total 23 Bmy1 allele types were identified. The newly identified alleles (A-I-11, A-II-6, A-II-7, A-II-10, B-I-3, B-I-12 and B-I-13) provided us with a novel resource for barley breeding and Bmy1 study. In HV barley, six out of seven major allele types (C-II-1, B-II-2, B-Ia-3, A-II-5, A-II-6, and A-II-7) were shared with HS barley; the B-I-8 allele, which was predominant in north European cultivated barley, was found to be unique. Remarkably, very low Bmy1 genetic variation was detected in Tibetan barleys, which puts the validity of the hypothesis that Tibet is one of the original centers of cultivated barley into question.     

Heterologous expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> haemoglobin in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.     

Temporal trends of genetic diversity in European barley cultivars (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The changes of genetic diversity over time were monitored in 504 European barley cultivars released during the 20th century by genotyping with 35 genomic microsatellites. For analysis, the following four temporal groups were distinguished: 1900–1929 (TG1 with 19 cultivars), 1930–1949 (TG2 with 40 cultivars), 1950–1979 (237 cultivars as TG3), and 1980–2000 (TG4 consisting of 208 cultivars). After rarefaction of allelic diversity data to the comparable sample size of 18 varieties, of the 159 alleles found in the first group (TG1) 134 were retained in the last group (TG4) resulting in a loss of only 15.7% of alleles. On the other hand 51 novel alleles were discovered in the group representing the last investigated time period (TG4) in comparison with the TG1. Novel alleles appeared evenly distributed over the genome, almost at all investigated genomic loci, with up to five such novel alleles per locus. Alleles specific for a temporal group were discovered for all investigated time periods, however analysis of molecular variance (AMOVA) did not reveal any significant population structure attributable to temporal decadal grouping. Only 2.77% of the total observed variance was due to differences between the four temporal groups and 1.42% between individual decades of the same temporal group, while 95.81% of the variance was due to variation within temporal groups. The distinction between two-rowed and six-rowed genetic types accounted for 19.5% of the total observed variance by AMOVA, whereas the comparison between ‘winter’ and ‘spring’ types accounted for 17% of the total observed variation. The analysis of linkage disequilibrium did not reveal statistically significant differences between the temporal groups. The results indicated that the impact of breeding effort and variety delivery systems did not result in any significant quantitative losses of genetic diversity in the representative set of barley cultivars over the four time periods.     

High-density AFLP map of nonbrittle rachis 1 (<Emphasis Type="Italic">btr1</Emphasis>) and 2 (<Emphasis Type="Italic">btr2</Emphasis>) genes in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Wild relatives of barley disperse their seeds at maturity by means of their brittle rachis. In cultivated barley, brittleness of the rachis was lost during domestication. Nonbrittle rachis of occidental barley lines is controlled by a single gene (btr1) on chromosome 3H. However, nonbrittle rachis of oriental barley lines is controlled by a major gene (btr2) on chromosome 3H and two quantitative trait loci on chromosomes 5HL and 7H. This result suggests multiple mutations of the genes involved in the formation of brittle rachis in oriental lines. The btr1 and btr2 loci did not recombine in the mapping population analyzed. This result agrees with the theory of tight linkage between the two loci. A high-density amplified fragment-length polymorphism (AFLP) map of the btr1/btr2 region was constructed, providing an average density of 0.08 cM/locus. A phylogenetic tree based on the AFLPs showed clear separation of occidental and oriental barley lines. Thus, barley consists of at least two lineages as far as revealed by molecular markers linked to nonbrittle rachis genes.Electronic Supplementary Material Supplementary material is available for this article at An erratum to this article can be found at     

High-resolution mapping of the <Emphasis Type="Italic">Alp</Emphasis> locus and identification of a candidate gene <Emphasis Type="Italic">HvMATE</Emphasis> controlling aluminium tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Aluminium (Al) tolerance in barley is conditioned by the Alp locus on the long arm of chromosome 4H, which is associated with Al-activated release of citrate from roots. We developed a high-resolution map of the Alp locus using 132 doubled haploid (DH) lines from a cross between Dayton (Al-tolerant) and Zhepi 2 (Al-sensitive) and 2,070 F₂ individuals from a cross between Dayton and Gairdner (Al-sensitive). The Al-activated efflux of citrate from the root apices of Al-tolerant Dayton was 10-fold greater than from the Al-sensitive parents Zhepi 2 and Gairdner. A suite of markers (ABG715, Bmag353, GBM1071, GWM165, HvMATE and HvGABP) exhibited complete linkage with the Alp locus in the DH population accounting 72% of the variation for Al tolerance evaluated as relative root elongation. These markers were used to map this genomic region in the Dayton/Gairdner population in more detail. Flanking markers HvGABP and ABG715 delineated the Alp locus to a 0.2 cM interval. Since the HvMATE marker was not polymorphic in the Dayton/Gairdner population we instead investigated the expression of the HvMATE gene. Relative expression of the HvMATE gene was 30-fold greater in Dayton than Gardiner. Furthermore, HvMATE expression in the F_2:3 families tested, including all the informative recombinant lines identified between HvGABP and ABG715 was significantly correlated with Al tolerance and Al-activated citrate efflux. These results identify HvMATE, a gene encoding a multidrug and toxic compound extrusion protein, as a candidate controlling Al tolerance in barley.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

Arabinogalactan proteins improve plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) anther culture

Androgenesis-based methods of doubled haploid (DH) production show considerable variation in efficiency in different barley genotypes. Arabinogalactan proteins (AGPs) have been shown to play a key role in several developmental processes, including embryogenesis, in different plant species. In this study we investigated the effect of exogenous AGPs from gum arabic on androgenesis and the regeneration efficiency in barley anther culture. Supplementation of the induction medium with 10 mg l^?1 gum arabic increased the total plant regeneration rate up to 2.8 times; when exposure to GA was extended to also include the pretreatment step, the regeneration rate was up to 6.6-times higher than in control. The effect of gum arabic was reversed by the Yariv reagent, an AGPs antagonist. This suggests a direct involvement of AGPs in androgenic development from barely microspores. Addition of gum arabic reduced cell mortality, increased the frequency of mitotic divisions of microspores and the number of multicellular structures (MCSs) when compared to control. The positive effect of gum arabic also included reduction in time required for the androgenic induction and substantially improved the quality of formed embryos. Observations made in this study imply a complex role of AGPs during androgenic development and confirmed the usefulness of gum arabic in production of barley androgenic plants.     

<Emphasis Type="Italic">TaMSH7</Emphasis>: A cereal mismatch repair gene that affects fertility in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background  

Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family.     

Exogenous 2-aminoethanol can diminish paraquat induced oxidative stress in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Effects of unfavourable environmental conditions (stresses) induce stressor specific and unspecific short- and long-term responses in plants. Long-term responses depend on intensity and duration of the stress. Short-term effects comprise the accumulation of reactive oxygen species (ROS), membrane damages by the oxidation of fatty acids, and the release of amino alcohols. They can incite higher stress tolerance in plants. In the present study, shoots of barley (Hordeum vulgare) were pre-treated with 2-aminoethanol, and, 2 days later, with the oxidative stress inducing herbicide, paraquat. Pre-treatments with 2-aminoethanol increased the stress tolerance in barley by the stabilization of the cell membranes, the enhanced production of superoxide dismutase and catalase, and the stimulation of glutathione metabolism (GSH, GST). These mechanisms of stress tolerance activation by 2-aminoethanol are discussed.     

Impact of the 7-bp deletion in <Emphasis Type="Italic">HvGA20ox2</Emphasis> gene on agronomic important traits in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Alike to Reduced height-1 (Rht-1) genes in wheat and the semi dwarfing (sd-1) gene in rice, the sdw1/denso locus involved in the metabolism of the GA, was designated as the ‘Green Revolution’ gene in barley. The recent molecular characterization of the candidate gene HvGA20ox2 for sdw1/denso locus allows to estimate the impact of the functional polymorphism of this gene on the variation of agronomically important traits in barley.

Results

We investigated the effect of the 7-bp deletion in exon 1 of HvGA20ox2 gene (sdw1.d mutation) on the variation of yield-related and malting quality traits in the population of DHLs derived from cross of medium tall barley Morex and semi-dwarf barley Barke. Segregation of plant height, flowering time, thousand grain weight, grain protein content and grain starch was evaluated in two diverse environments separated from one another by 15° of latitude. The 7-bp deletion in HvGA20ox2 gene reduced plant height by approximately 13 cm and delayed flowering time by 3–5 days in the barley segregating DHLs population independently on environmental cue. On other hand, the sdw1.d mutation did not affect significantly either grain quality traits (protein and starch content) or thousand grain weight.

Conclusions

The beneficial effect of the sdw1.d allele could be associated in barley with lodging resistance and extended period of vegetative growth allowing to accumulate additional biomass that supports higher yield in certain environments. However, no direct effect of the sdw1.d mutation on thousand grain weight or grain quality traits in barley was detected.

     

Acid phosphatases and growth of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars under diverse phosphorus nutrition

The morphological and physiological responses of barley to moderate Pi deficiency and the ability of barley to grow on phytate were investigated. Barley cultivars (Hordeum vulgare L., Promyk, Skald and Stratus) were grown for 1–3 weeks on different nutrient media with contrasting phosphorus source: KH₂PO₄ (control), phytic acid (PA) and without phosphate (−P). The growth on −P medium strongly decreased Pi concentration in the tissues; culture on PA medium generally had no effect on Pi level. Decreased content of Pi reduced shoot and root mass but root elongation was not affected; Pi deficit had slightly greater impact on growth of barley cv. Promyk than other varieties. Barley varieties cultured on PA medium showed similar growth to control. Extracellular acid phosphatase activities (APases) in −P roots were similar to control, but in PA plants were lower. Histochemical visualization indicated for high APases activity mainly in the vascular tissues of roots and in rhizodermis. Pi deficiency increased internal APase activities mainly in shoot of barley cv. Stratus and roots of cv Promyk; growth on PA medium had no effect or decreased APase activity. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoforms may be affected by Pi deficiency or growth on PA medium; two of four isoforms in roots were strongly induced by conditions of Pi deficit, especially in barley cv. Promyk. In conclusion, barley cultivars grew equally well both on medium with Pi and where the Pi was replaced with phytate and only slightly differed in terms of acclimation to moderate deficiency of phosphate; they generally used similar pools of acid phosphatases to acquire Pi from external or internal sources.     

QTL mapping of chromosomal regions conferring reproductive frost tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Spring radiation frost is a major abiotic stress in southern Australia, reducing yield potential and grain quality of barley by damaging sensitive reproductive organs in the latter stages of development. Field-based screening methods were developed, and genetic variation for reproductive frost tolerance was identified. Mapping populations that were segregating for reproductive frost tolerance were screened and significant QTL identified. QTL on chromosome 2HL were identified for frost-induced floret sterility in two different populations at the same genomic location. This QTL was not associated with previously reported developmental or stress-response loci. QTL on chromosome 5HL were identified for frost-induced floret sterility and frost-induced grain damage in all three of the populations studied. The locations of QTL were coincident with previously reported vegetative frost tolerance loci close to the vrn-H1 locus. This locus on chromosome 5HL has now been associated with response to cold stress at both vegetative and reproductive developmental stages in barley. This study will allow reproductive frost tolerance to be seriously pursued as a breeding objective by facilitating a change from difficult phenotypic selection to high-throughput genotypic selection.     

Physiological and biochemical parameters: new tools to screen barley root exudate allelopathic potential (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>)

Morphological markers/traits are often used in the detection of allelopathic stress, but optical signals including chlorophyll a fluorescence emission could be useful in developing new screening techniques. In this context, the allelopathic effect of barley (Hordeum vulgare subsp. vulgare) root exudates (three modern varieties and three landraces) were assessed on the morphological (root and shoot length, biomass accumulation), physiological (F_v/F_m and F₀), and biochemical (chlorophyll and protein contents) variables of great brome (Bromus diandrus Roth., syn. Bromus rigidus Roth. subsp. gussonii Parl.). All the measured traits were affected when great brome was grown in a soil substrate in which barley plants had previously developed for 30 days before being removed. The response of receiver plants was affected by treatment with activated charcoal, dependent on barley genotype and on the nature of the growing substrate. The inhibitory effect was lower with the addition of the activated charcoal suggesting the release of putative allelochemicals from barley roots into the soil. The barley landraces were more toxic than modern varieties and their effect was more pronounced in sandy substrate than in silty clay sand substrate. In our investigation, the chlorophyll content and F_v/F_m were the most correlated variables with barley allelopathic potential. These two parameters might be considered as effective tools to quantify susceptibility to allelochemical inhibitors in higher plants.     

A major QTL controlling the tolerance to manganese toxicity in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Waterlogging stress disturbs plant metabolism through increased ion (manganese and iron) toxicity resulting from the changes in the soil redox potential under hypoxic conditions. Our previous study found a significant correlation between the tolerance to Mn²⁺ toxicity and waterlogging stress tolerance in barley, suggesting that waterlogging tolerance could be increased by improving the tolerance to Mn²⁺ toxicity. In this study, a doubled-haploid (DH) population from the cross between barley varieties Yerong and Franklin (waterlogging-tolerant and -sensitive, respectively) was used to identify QTL controlling tolerance to Mn²⁺ toxicity based on chlorophyll content and plant survival as selection criteria. Four significant QTL for plant survival under Mn²⁺ stress (QSur.yf.1H, QSur.yf.3H, QSur.yf.4H, and QSur.yf.6H) were identified in this population at the seedling stage. Two significant QTL (QLC.yf.3H and QLC.yf.6H) controlling leaf chlorosis under Mn²⁺ stress were identified on chromosomes 3H and 6H close to QSur.yf.3H and QSur.yf.6H. The major QTL QSur.yf.3H, located near the marker Bmag0013, explained 21% of the phenotypic variation. The major QTL for plant survival on 3H was validated in a different DH population (TX9425/Naso Nijo). This major QTL could potentially be used in breeding programmes to enhance tolerance to both manganese toxicity and waterlogging.     

Polymorphism of the cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) of South Arabia at hordein-coding loci

Electrophoresis in starch gel was used to study the polymorphism of hordeins controlled by loci Hrd A, Hrd B, and Hrd F in 89 accessions of the local barleys from South Arabia (Yemen). Overall, 36 alleles were detected for locus Hrd A; 48 alleles, for Hrd B; and 5 alleles, for Hrd F. The existence of the blocks of hordein components controlled by loci Hrd A and Hrd B was demonstrated. Calculation of genetic distances allows us to conclude that the barley populations from Yemen and Ethiopia are more similar compared with the populations from Egypt. This confirms the hypothesis of Bakhteev on the origin of Ethiopian barleys.