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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

AFLP analysis of genetic relationships between barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) landraces from north Shewa in Ethiopia

Local cultivars adapted to specific environmental conditions are the chief source of seed for farmers in Ethiopia and deserve research priority. The aim of this study was, therefore, to determine the genetic relationships between different barley landraces, from north Shewa in Ethiopia so as to differentiate genotypes known by different local names and facilitate their conservation and use in breeding new varieties. Five AFLP primer combinations were analyzed for 19 barley landraces and five malting varieties. The number of scoreable fragments amplified by each AFLP primer combination varied from 49 to 118 with an average of 84.5 and polymorphic fragments for each primer combination varied from 27 to 77 with an average of 58.5. The average percent polymorphism was 69.9% with values ranging from 55.1% to 75.8%. Cluster analysis placed the accessions and malting varieties into one main group while all the farmers’ cultivars, with the exception of two, were in the other main group. It was shown that sampling of germplasm at a given locality might not represent the whole array of genetic variability of locally grown famers’ cultivars. A comprehensive study of all the farmers’ barley cultivars, grown in different parts of Ethiopia, is required to maximize the efforts of germplasm conservation and utilization in national and regional breeding programs.     

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.     

Semi-dwarf barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">brh2</Emphasis> and <Emphasis Type="Italic">ari-l</Emphasis> mutants are deficient in a U-box E3 ubiquitin ligase

Lodging is the process where crop plants fall over and lie on the ground due to strong winds and heavy precipitation. This problem reduces yield and increases the risk of fungal infections and pre-harvest germination. In order to avoid lodging, plant breeders utilize short-culm mutants, which often have a robust culm that can support the weight of a heavy spike. In barley (Hordeum vulgare L.), thousands of short-culm mutants have been isolated in breeding programs around the world. Our long-term goal is to reveal the genetic network underlying culm length, with the objective to provide an enlarged repertoire of genes and alleles suitable for future breeding of lodging resistant barley. In the present work we studied a group of allelic brh2 and ari-l mutants, which have a relatively strong semi-dwarf phenotype and are phenotypically similar to previously identified mutants deficient in brassinosteroid signalling or metabolism. The Brh2 gene is located in the centromeric region of chromosome 4H and we applied a candidate gene approach to identify the gene. Brh2 is orthologous to TUD1 in rice (Orysa sativa L.), which encodes a U-box E3 ubiquitin ligase. We identified one missense mutation, one nonsense mutation and four deletions of the complete Brh2 gene. The mutants could respond to exogenously applied brassinolide, which suggests that the apparent brassinosteroid deficient phenotype of barley brh2 and ari-l mutants is related to brassinosteroid metabolism rather than signalling.     

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.     

Polymorphism of hordein-coding loci in cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) in Afghanistan

Polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd F loci was analyzed in 84 accessions of local barley (Hordeum vulgare L.) varieties from major agricultural regions of Afghanistan using starch gel electrophoresis. Forty alleles of the Hrd A locus with the frequencies from 0.12 to 32.73%, 62 alleles of the Hrd B locus with the frequencies from 0.12 to 14.29%, and five alleles of the Hrd F locus with the frequencies from 0.59 to 32.15% have been identified. The conclusion about genetic similarity of barley populations from different regions of Afghanistan is made on the basis of cluster analysis of the matrix of allele frequencies in barley populations from 31 localities. The local barley populations form four unequal clusters. The largest cluster I includes populations from 14 localities of Afghanistan. The second largest cluster IV consists of populations from ten localities, and clusters II and III comprise populations from four and three localities, respectively. Each of the four clusters includes populations from different regions of northern and southern Afghanistan. Based on our results, we conclude that the diversity of hordein-coding loci and the distribution of their alleles among different regions of Afghanistan are the consequences of introduction of barley landraces and their distribution over trade routes.     

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.     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">Rph14</Emphasis> in <Emphasis Type="Italic">Hordeum vulgare</Emphasis>

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F₃ lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively.     

<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.     

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     

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.