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Evolution of the indole alkaloid biosynthesis in the genus Hordeum: distribution of gramine and DIBOA and isolation of the benzoxazinoid biosynthesis genes from Hordeum lechleri.

Two indole alkaloids with defense related functions are synthesized in the genus Hordeum of the Triticeae. Gramine (3(dimethyl-amino-methyl)-indole) is found in H. spontaneum and in some varieties of H. vulgare, the benzoxazinoid 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) is detected in H. roshevitzii, H. brachyantherum, H. flexuosum, H. lechleri. Biosynthesis of DIBOA and of gramine was found to be mutually exclusive in wild Hordeum species, indicating that there was selection against simultaneous expression of both pathways during evolution. The full set of genes required for DIBOA biosynthesis in H.lechleri was isolated and the respective enzyme functions were analyzed by heterologous expression. The cytochrome P450 genes Bx2-Bx5 demonstrate a monophyletic origin for H. lechleri, Triticum aestivum and Zea mays. HlBx2-HlBx5 share highest homology to the orthologous genes of T. aestivum. In contrast, the branch point enzyme of the DIBOA pathway, the indole-3-glycerol phosphate lyase BX1, might have evolved independently in H. lechleri. In all Hordeum species that synthesize DIBOA, DNA sequences homologous to Bx genes are found. In contrast, these sequences are not detectable in the genomes of H. vulgare and H. spontaneum that do not synthesize benzoxazinoids.

Alkaloids↗

Identification of intergenomic translocations involving wheat, Hordeum vulgare and Hordeum chilense chromosomes by FISH.

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were analyzed. Multicolor FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.

Chromosomes↗

Development and characterization of recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a Hordeum vulgare subsp. vulgare background.

The ancestor of barley (Hordeum vulgare subsp. spontaneum) may be a source of novel alleles for crop improvement. We developed a set of recombinant chromosome substitution lines (RCSLs) using an accession of H. vulgare subsp. spontaneum (Caesarea 26-24, from Israel) as the donor and Hordeum vulgare subsp. vulgare 'Harrington' (the North American malting quality standard) as the recurrent parent via two backcrosses to the recurrent parent, followed by six generations of selfing. Here we report (i) the genomic architecture of the RCSLs, as inferred by simple sequence repeat (SSR) markers, and (ii) the effects of H. vulgare subsp. spontaneum genome segment introgressions in terms of three classes of phenotypes: inflorescence yield components, malting quality traits, and domestication traits. Significant differences among the RCSLs were detected for all phenotypes measured. The phenotypic effects of the introgressions were assessed using association analysis, and these were referenced to quantitative trait loci (QTL) reported in the literature. Hordeum vulgare subsp. spontaneum, despite its overall inferior phenotype, contributed some favorable alleles for agronomic and malting quality traits. In most cases, the introgression of the ancestral genome resulted in a loss of desirable phenotypes in the cultivated parent. Although disappointing from a plant breeding perspective, this finding may prove to be a useful tool for gene discovery.

Alleles↗

Identification of all chromosome arms and their involvement in meiotic homoeologous associations at metaphase I in 2 Hordeum vulgare L. x Hordeum bulbosum L. hybrids.

We have identified all Hordeum bulbosum chromosomes in 2 diploid Hordeum vulgare x Hordeum bulbosum hybrids using suitable probes and fluorescence in situ hybridization. Using the parental idiograms allowed us to carry out a full analysis of chromosome associations among all chromosome arms in the hybrids. Association frequencies were generally lower for the short arms than for the long arms. There were also significant differences among the chromosome arms in association frequencies, partly correlated with the absolute length of the chromosome arm, as well as with the frequency of recombinant lines, which were recovered from partially fertile interspecific hybrids. The H. bulbosum idiogram will be useful for further chromosome association studies and will enable the identification of H. bulbosum chromosomes involved in chromosome addition or substitution lines.

Chimera↗

The 5S rRNA gene in sea barley (Hordeum marinum Hudson sensu lato): sequence variation among repeat units and relationship to the X haplome in barley (Hordeum).

We have investigated the molecular diversity of the 5S rDNA units in sea barley, comprising Hordeum marinum and Hordeum geniculatum. Although we were unable to detect "short" units after screening of 639 clones, we found two unit classes, one 602-607 bp long and the other 507-512 bp long. We classify the shortest unit class of the two as belonging to the "long H1" unit class, identified in previous papers. The longest unit class is not similar to any unit class so far identified, and is therefore unique. It was coined by us as the "long X1," to reflect the X haplome. We present a summary of all the unit classes so far described in Hordeum. We carried out a cladistic analysis, based on the "long H1" (orthologous) sequences, that included H. vulgare, H. spontaneum, H. bulbosum, H. marinum, H. geniculatum, and H. bogdanii. As a result, the first three grouped in one clade, and the other three in the other clade, with the latter clade being more isolated. These results reflect current knowledge of relationships based on morphology, cytology, and genome analysis. Furthermore, the sequences from the 5S unit classes may be potentially useful as DNA probes for genomic identification and genetic transfer in the Triticeae.

Base Sequence↗

The 5S rRNA gene in wall barley (Hordeum murinum L. sensu lato): sequence variation among repeat units and relationship to the Y haplome in the genus Hordeum (Poaceae: Triticeae).

The molecular diversity of the 5S rDNA units in 13 accessions of wall barley, which include Hordeum murinum, H. leporinum, and H. glaucum, is reported. Our analyses, based on 54 sequenced clones, indicate the presence of two sequence classes not previously seen in other barley species; namely, the long Y1 unit class and the short Y1 unit class. In addition, the accumulation of new sequence information has allowed us to refine previous groups. Using these new results, along with previously published work, we present a summary of all the unit classes described to date and potential correspondences between 5S rDNA unit classes and haplomes identified previously. In H. murinum, we found the long H1 and long X2 unit classes, and in one of six accessions referable to H. glaucum we found the unique short Y1 unit class. Our cladistic analyses, using orthologous sequences, provide support for the current model for the relationships among several species within the Triticeae.

Base Sequence↗

Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl- into the shoots.

Eight wild Hordeum species: H. bogdanii, H. intercedens, H. jubatum, H. lechleri, H. marinum, H. murinum, H. patagonicum, and H. secalinum, and cultivated barley (H. vulgare) were grown in nutrient solution containing 0.2 (control), 150, 300, or 450 mol m(-3) NaCl. In saline conditions, the wild Hordeum species (except H. murinum) had better Na+ and Cl- 'exclusion', and maintained higher leaf K+, compared with H. vulgare. For example, at 150 mol m(-3) NaCl, the K+:Na+ in the youngest, fully expanded leaf blades of the wild Hordeum species was, on average, 5.2 compared with 0.8 in H. vulgare. In H. marinum grown in 300 mol m(-3) NaCl, K+ contributed 35% to leaf psi(pi), whereas Na+ and Cl- accounted for only 6% and 10%, respectively. By comparison, in H. vulgare grown at 300 mol m(-3) NaCl, K+ accounted for 19% and Na+ and Cl- made up 21% and 25% of leaf psi(pi), respectively. At 300 mol m(-3) NaCl, glycinebetaine and proline together contributed almost 15% to psi(pi) in the expanding leaf blades of H. marinum, compared with 8% in H. vulgare. Decreased tissue water content under saline conditions made a substantial contribution to declines in leaf psi(pi) in the wild Hordeum species, but not in H. vulgare. A number of the wild Hordeum species were markedly more salt tolerant than H. vulgare. H. marinum and H. intercedens, as examples, had relative growth rates 30% higher than H. vulgare in 450 mol m(-3) NaCl. Hordeum vulgare also suffered up to 6-fold more dead leaf material (as a proportion of shoot dry mass) than the wild Hordeum species. Thus, several salt-tolerant wild Hordeum species were identified, and these showed an exceptional capacity to 'exclude' Na+ and Cl- from their shoots.

Chlorides↗

Molecular phylogeny of the genus Hordeum using three chloroplast DNA sequences.

The genus Hordeum consists of three cytotypes (2x, 4x, and 6x). Its reproductive isolation has been incomplete between closely related species and hence the genetic relationship is reticulate and complex. We used 32 taxa of Hordeum and three chloroplast DNA sequences, matK, atpB-rbcL, and trnL-trnF in the current study. Molecular phylogenetic analysis based on sequence data of the three chloroplast DNA regions clearly demonstrated genetic relationships among taxa and origin of polypoids. The formation of H. secalinum likely involved hybridization between Hordeum marinum subsp. marinum and a Eurasian diploid possessing the H genome. The formation of hexaploid Hordeum brachyantherum involved hybridization between tetraploid H. brachyantherum and diploid H. marinum subsp. gussoneanum. The formation of three tetraploids, H. brachyantherum, Hordeum jubatum, and Hordeum guatemalense, probably involved hybridization between H. brachyantherum subsp. californicum and an altered H genome diploid. The formation of Hordeum arizonicum involved the two taxa Hordeum pusillum and H. jubatum.

Base Sequence↗

Genomic organization, evolution, and structural peculiarities of highly repetitive DNA of Hordeum vulgare.

A fraction of highly repeated DNA sequences of Hordeum vulgare has been investigated by cloning 19 separate highly repetitive sequences in the plasmid pBR327. Characteristics studied included genus specificity of isolated sequences, their prevalence, and genome organization. Sequences (pHv7161, pHv7191, pHv7179) have been identified that are the most widespread in the H. vulgare genome and have a complicated arrangement. A tandemly arranged sequence, pHv7141, was also identified. The primary structure of a 999 bp long, BamHI fragment of one of the most widespread sequences, pHv7161, as well as the adjacent pHv7302 and pHv7245 sequences was determined. The fragment abounds in inverted repeats, of which two are flanked by direct repeats, and contains short subrepeats, A, B, and C, and a great variety of potential protein-binding sites. A comparison is drawn between the content and genome organization of highly repeated DNA sequences of H. vulgare and those of the wild barley species Hordeum bulbosum, Hordeum jubatum, Hordeum geniculatum, Hordeum brevisubulatum, Hordeum turkestanicum, and Hordeum murinum. According to the above characters (close copy number and genome organization similarity of highly repetitive sequences) the species under discussion have been classified into four groups. This division is in good agreement with other data on interspecific crossing in Hordeum and on chromosome pairing in hybrid meiosis.

Base Sequence↗

A chloroplast genealogy of hordeum (poaceae): Long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference.

To analyze reasons for inconclusive results of earlier chloroplast phylogenies in the grass genus Hordeum, we established a genealogy of chloroplast haplotypes by sequencing the trnL-trnF region in 875 individuals, covering all 31 species of the genus. Although the outcomes of phenetic and parsimony analyses of 88 haplotypes were ambiguous, a network approach showed that in Hordeum ancient chloroplast types co-occur with their descendants. Moreover, we found up to 18 different chloroplast haplotypes within a single species and up to 6 species sharing single haplotypes. Persisting polymorphisms together with incomplete lineage sorting occurred preferentially in the rapidly speciating New World taxa of the genus, where ancient chloroplast types have survived for at least 4 Myr. Lineages-through-time plots and a high number of missing chloroplast haplotypes indicated far-reaching extinction of chloroplast lineages in Europe and particularly the Mediterranean. Survival of these lineages in East Asia and North America resulted in chloroplast relationships that markedly differed from nuclear estimations of species relationships. Thus, even for the deepest splits in the genus, reaching back more than 9 Myr, no safe phylogenetic inference from chloroplast data is possible in Hordeum. The chloroplast genealogy, however, revealed biogeographic patterns and indicated processes involved in speciation in Hordeum. We conclude that the described phenomena are not restricted to Hordeum and that the knowledge of the chloroplast relationships within a genus is indispensable to prevent misinterpretation of phylogeographic data within single species.

Chloroplasts↗

Multiple intercontinental dispersals shaped the distribution area of Hordeum (Poaceae).

The grass genus Hordeum (Poaceae, Triticeae), comprising 31 species distributed in temperate and dry regions of the world, was analysed to determine the relative contributions of vicariance and long-distance dispersal to the extant distribution pattern of the genus. Sequences from three nuclear regions (DMC1, EF-G and ITS) were combined and analysed phylogenetically for all diploid (20 species) and two tetraploid Hordeum species and the outgroup Psathyrostachys. Ages of clades within Hordeum were estimated using a penalized likelihood analysis of sequence divergence. The sequence data resulted in an almost fully resolved phylogenetic tree that allowed the reconstruction of intrageneric migration routes. Hordeum evolved c. 12 million years ago in South-west Asia and spread into Europe and Central Asia. The colonization of the New World and South Africa involved at least six intercontinental exchanges during the last 4 million years (twice Eurasia-North America, North America-South America, twice South America-North America and Europe-South Africa). Repeated long-distance dispersal between the northern and southern hemisphere were important colonization mechanisms in Hordeum.

Base Sequence↗

LINEs and gypsy-like retrotransposons in Hordeum species.

LINE and gypsy-like retroelements were studied in the genome of Hordeum vulgare, and compared with the representatives of the major sections of the genus Hordeum. We isolated reverse transcriptase (RT) genes from four gypsy-like and three LINE families using PCR primers specific for the corresponding conserved domains. A full-length barley LINE of 6295 bp, named BLIN, was isolated from a BAC genomic library. BLIN looks alien in the barley genome because its G+C content is 62% compared to an average of 45%. The BLIN nucleotide sequence showed it was structurally intact with the features typical of non-LTR retrotransposons, including 16 bp target site duplications, two short cysteine motifs, and two degenerate open reading frames (ORFs). The high degeneracy was also found in RT domain of both gypsy-like and, particularly, LINE families. The copy numbers of the gypsy-like families were relatively low compared to well-characterized copia-like element BARE-1. Each gypsy-like family gave unique RFLP patterns when hybridized to genomic DNA from each of the four basic Hordeum genomes. H. vulgare (I genome) had accumulated more copies than the wild Hordeum species (H, X, Y genomes), with the other I genome species, H. bulbosum, being intermediate. Analysis of the BAC library and in situ hybridization with LINE RT domains showed the low copy number of the LINE families, but there was little correlation between hybridization patterns and the division of the genus into four basic genomes. The distribution and content of gypsy retrotransposons in the BAC library indicated that a few copies are nested, although most are present as single, distinct, copies. Our results suggest that the major groups of retroelements make individual contributions to the shape of the plant genome; the factors involved in their amplification and distribution are independent, also varying among species.

Amino Acid Sequence↗

Effect of rye A and B chromosomes on meiotic association of Hordeum marinum ssp. gussoneanum (4x) chromosomes in intergeneric hybrids.

Chromosome association at metaphase I was studied in PMCs of eight H. marinum ssp. gussoneanum (4x) x rye hybrids. Differences in the levels of association separated six hybrids with 2n = 23 including 14 Hordeum, 7 rye A and 2 rye B chromosomes into two groups of three plants each, a "low association" group with means of 0.03III + 4.43II (1.55 rings + 2.88 rods) + 5.10I and 6.03 chiasmata/cell, and a "high association" group with means of 0.01IV + 0.03III + 6.40II (3.55 rings + 2.85 rods) + 1.13I and 10.04 chiasmata/cell. The low number of plants studied prevents a safe estimate of the number of genes involved, but the significant difference between groups suggests the presence in the rye genome of two major genes, or two genotypes, for control of meiotic chromosome association. In two additional hybrids with 2n = 25, one of each above-mentioned group, the presence of two extra rye B chromosomes raised chiasma frequencies by ca 1.5, indicating a promoting effect on chromosome association. The level of Hordeum chromosome association in the "high association" group and the observation of up to 7 Hordeum ring bivalents in some cells agree with an autoploid origin of H. marinum ssp. gussoneanum (4x). Hordeum and rye chromosomes formed a few heterogenomic bi- and trivalents. Most rye A chromosomes formed univalents, but 2.7% were included in associations. Rye B chromosomes generally formed rod bivalents. The use of genome analysis in its traditional sense is discussed.

Chromosomes↗

Species relationships between antifungal chitinase and nuclear rDNA (internal transcribed spacer) sequences in the genus Hordeum.

The sequences of the chitinase gene (Chi-26) and the internal transcribed spacer of 18S - 5.8S - 26S rDNA (ITS1) were determined to analyze the phylogenetic relationships among species representing the four basic genomes of the genus Hordeum. Grouping analysis based on data for Chi-26 gene sequences placed Hordeum secalinum (H genome) near the Hordeum murinum complex (Xu genome), and Hordeum bulbosum distant from the other species that carried the I genome. ITS sequence data showed the expected grouping based on the genome classification of the species studied. Different sequences of ITS were detected even in the genomes of the diploid species. The results are interpreted in terms of defective or unfinished concerted evolution processes in each taxon.

Antifungal Agents↗

Ancient differentiation of the H and I haplomes in diploid Hordeum species based on 5S rDNA.

5S rDNA clones from 12 South American diploid Hordeum species containing the HH genome and 3 Eurasian diploid Hordeum species containing the II genome, including the cultivated barley Hordeum vulgare, were sequenced and their sequence diversity was analyzed. The 374 sequenced clones were assigned to "unit classes", which were further assigned to haplomes. Each haplome contained 2 unit classes. The naming of the unit classes reflected the haplomes, viz. both the long H1 and short I1 unit classes were identified with II genome diploids, and both the long H2 and long Y2 unit classes were recognized in South American HH genome diploids. Based upon an alignment of all sequences or alignments of representative sequences, we tested several evolutionary models, and then subjected the parameters of the models to a series of maximum likelihood (ML) analyses and various tests, including the molecular clock, and to a Bayesian evolutionary inference analysis using Markov chain Monte Carlo (MCMC). The best fitting model of nucleotide substitution was the HKY+G (Hasegawa, Kishino, Yano 1985 model with the Gamma distribution rates of nucleotide substitutions). Results from both ML and MCMC imply that the long H1 and short I unit classes found in the II genome diploids diverged from each other at the same rate as the long H2 and long Y2 unit classes found in the HH genome diploids. The divergence among the unit classes, estimated to be circa 7 million years, suggests that the genus Hordeum may be a paleopolyploid.

DNA, Plant↗

The 5S rRNA gene units in ancestral two-rowed barley (Hordeum spontaneum C. Koch) and bulbous barley (H. bulbosum L.): sequence analysis and phylogenetic relationships with the 5S rDNA units of cultivated barley (H. vulgare L.).

5S rRNA genes from several accessions of Hordeum spontaneum and Hordeum bulbosum, wild relatives of cultivated barley, Hordeum vulgare, have been amplified by the polymerase chain reaction, cloned, and sequenced. Evaluation of aligned sequences along with principal coordinate analysis demonstrates that the two classes of 5S rDNA sequences found in cultivated barley, and subclasses (groups) of these sequences, can also be found in its closest wild relatives. The two classes of units, formerly categorized as containing short or long 5S rDNA repeats, are distinguishable by the presence or absence of a TAG repeating unit. Sequence comparisons of individual clones (units) isolated from different species have allowed us to confirm that orthology exists for several groups. This demonstration of orthologous groups suggests that the 5S rDNA sequence may be useful for further phylogenetic analysis in the genus Hordeum and possibly in the Triticeae.

Base Sequence↗

The Catabolism of (+/-)-Abscisic Acid by Excised Leaves of Hordeum vulgare L. cv Dyan and Its Modification by Chemical and Environmental Factors.

Excised light-grown leaves and etiolated leaves of Hordeum vulgare L. cv Dyan catabolized applied (+/-)-[2-(14)C]abscisic acid ([+/-]-[2-(14)C]ABA) to phaseic acid (PA), dihydrophaseic acid (DPA), and 2'-hydroxymethyl ABA (2'-HMABA). Identification of these catabolites was made by microchemical methods and by combined capillary gas chromatographymass spectrometry (GC-MS) following high dose feeds of nonlabeled substrate to leaves. Circular dichroism analysis revealed that 2'-HMABA was derived from the (-) enantiomer of ABA. By selecting tissue samples in which endogenous catabolites were undetectable by gas chromatography, it was possible to identify unequivocally ABA catabolites by GC-MS without the need to employ deuteriated substrate to distinguish the (+/-)-ABA catabolites from the same endogenous compounds. Refeeding studies were used to confirm the catabolic route. The methyl ester of (+/-)-[2(14)C]-ABA was hydrolyzed efficiently by light-grown leaves of H. vulgare. Leaf age played a significant role in (+/-)-ABA catabolism, with younger leaves being less able than their older counterparts to catabolize this compound. The catabolism of (+/-)-ABA was inhibited markedly in water-stressed Hordeum leaves which was characterized by a decreased incorporation of label into 2'-HMABA, DPA, and conjugates. The specific, mixed function oxidase inhibitor, ancymidol, did not inhibit, dramatically, (+/-)-ABA catabolism in light-grown leaves of Hordeum whereas the 80s ribosome, translational inhibitor, cycloheximide, inhibited this process markedly. The 70s ribosome translational inhibitors, lincomycin and chloramphenicol, were less effective than cycloheximide in inhibiting (+/-)-ABA catabolism, implying that cytoplasmic protein synthesis is necessary for the catabolism of (+/-)-ABA in Hordeum leaves whereas chloroplast protein synthesis plays only a minor role. This further suggests that the enzymes involved in (+/-)-ABA catabolism in this plant are cytoplasmically synthesized and are ;turned-over' rapidly, although the enzyme responsible for glycosylating (+/-)-ABA itself appeared to be stable.

Journal Article↗