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.     

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.     

Polymorphism of hordein-coding loci in barley (Hordeum vulgare L.) populations of Iran and Central Asian countries

Starch gel electrophoresis was performed to study polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd Floci in 366 local old barley accessions from Iran and Central Asian countries, including Turkmenistan, Uzbekistan, Tajikistan (Mountain Badahsan), and Kirgizia. In total, 60 alleles with frequencies of 0.0003-0.2818 were observed for the Hrd A locus, 106 alleles with frequencies of 0.0003-0.1603 were observed for the Hrd B locus, and five alleles with frequencies of 0.0164-0.4131 were observed for the Hrd Flocus. The alleles and allele frequencies displayed irregular distributions in barley populations of the above countries. Cluster analysis of the matrix of allele frequencies in populations from known collection sites revealed a cluster structure of local barley populations within each country. Local populations formed five differently sized clusters in Iran, six in Turkmenistan, three in Uzbekistan, and three in Kirgizia. The variation and allele frequency distribution of the hordein-coding loci in Iran and Central Asian countries were assumed to result from the introduction and spreading of barley forms via migrations of husbandmen.     

Polymorphism of hordei-coding loci in Near Eastern local populations of cultivated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Electrophoresis in starch gel has been used to study the polymorphism of hordeins encoded by loci Hrd A, Hrd B, and Hrd F in 140 local barley populations from the Near East, including 60, 34, 33, 8, and 5 populations from Syria, Jordan, Iraq, Palestine, and Israel, respectively. Fifty-seven Hrd A, 87 Hrd B, and 5 Hrd F alleles have been found. The alleles of these loci considerably differ in frequencies and distribution in populations from different Near Eastern countries. Cluster analysis of the matrix of the frequencies of alleles of hordei-coding locus alleles in barley populations from the Near East, North Africa, Ethiopia, and South Arabia has yielded two clusters. The first cluster includes barley populations from Israel, Palestine, Morocco, Tunisia, Algeria, and Egypt; the second cluster, populations from Iraq, Syria, Jordan, Yemen, and Ethiopia.     

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.     

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.     

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.     

Current state of the genetic polymorphism in spring barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) from Russia assessed by the alleles of hordein-coding loci

Starch gel electrophoresis was performed to study the polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd F loci in 211 varieties of spring barley. For 41 of these varieties, the genetic formulas were established for the first time. In the two samples of varieties, the comparative analysis of allelic diversity and allele frequencies of hordein-coding loci was carried out. The first sample consisted of 101 spring barley varieties approved for the use on the territory of the Russian Federation in 1999, while the second sample included 160 spring barley varieties that were approved in 2014; 49 of these varieties were common for both samples. It is demonstrated that the current tendency to reduction of the proportion of heterogeneous spring barley varieties is mainly due to the introduction of foreign varieties homogeneous for the hordein-coding loci. At the same time, there is an increase in polymorphism of hordein-coding loci in modern spring barley varieties. The number of alleles for the Hrd A locus increased by five alleles, and for the Hrd B locus, by nine alleles. Along with the alleles recorded earlier in barley landrace populations and varieties bred in 20th century, three novel alleles of the Hrd A locus and four alleles of the Hrd B locus were identified. The number of alleles of the Hrd F locus remained unchanged (four), and the changes in their frequencies were small. At the same time, the changes in frequency observed for some alleles of the Hrd A and Hrd B loci were statistically significant. All newly identified alleles of hordein-coding loci were found with low frequencies (from 0.003 to 0.006), so despite the increased number of alleles, no statistically significant increase in genetic diversity in terms of μ and PIC indices was observed.     

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.     

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.     

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.     

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.     

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.     

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     

<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.     

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.