Series of resistance genes in barley (Hordeum vulgare L.) that control Barley yellow mosaic virus multiplication and the root-to-leaf systemic movement.

The infection of young winter barley (Hordeum vulgare L.) root system in winter by Barley yellow mosaic virus (BaYMV) can lead to high yield losses. Resistance breeding is critical for managing this virus, but there are only a few reports on resistance genes that describe how the genes control BaYMV propagation and the systemic movement from the roots to the leaves. Here we report a real-time quantitative PCR analysis of the virus in barley roots and leaves carrying BaYMV resistance genes (rym1-rym15 and an unknown gene) to elucidate the molecular mechanisms underlying the barley response to BaYMV. The resistance mechanism directly targets the virus. Moreover, the resistance genes/cultivars were classified into the following three groups according to their BaYMV titer: (1) immune (BaYMV was undetectable in the roots or leaves); (2) partially immune (BaYMV was detected in the roots, but not in the leaves); (3) susceptible (BaYMV was detected in the roots and leaves). Our results clarified the functions of the resistance genes in barley roots and leaves following a BaYMV infection. We anticipate our analysis to be a starting point for more understanding the correspondence between resistance genes of Triticeae and the soil-borne viruses.

Organ-enriched gene expression during floral morphogenesis in wild barley.

Floral morphology varies considerably between dicots and monocots. The ABCDE model explaining how floral organ development is controlled was formulated using core eudicots and applied to grass crops. Barley (Hordeum. vulgare) has unique floral morphogenesis. Wild barley (H. vulgare ssp. spontaneum), which is the immediate ancestor of cultivated barley (H. vulgare ssp. vulgare), contains a rich reservoir of genetic diversity. However, the wild barley genes involved in floral organ development are still relatively uncharacterized. In this study, we generated an organ-specific transcriptome atlas for wild barley floral organs. Genome-wide transcription profiles indicated that 22 838 protein-coding genes were expressed in at least one organ. These genes were grouped into seven clusters according to the similarities in their expression patterns. Moreover, 5619 genes exhibited organ-enriched expression, 677 of which were members of 47 transcription factor families. Gene ontology analyses suggested that the functions of the genes with organ-enriched expression influence the biological processes in floral organs. The co-expression regulatory network showed that the expression of 690 genes targeted by MADS-box proteins was highly positively correlated with the expression of ABCDE model genes during floral morphogenesis. Furthermore, the expression of 138 genes was specific to the wild barley OUH602 genome and not the Morex genome; most of these genes were highly expressed in the glume, awn, lemma, and palea. This study revealed the global gene expression patterns underlying floral morphogenesis in wild barley. On the basis of the study findings, a molecular mechanism controlling floral morphology in barley was proposed.

Genetic resistance in barley against Japanese soil-borne wheat mosaic virus functions in the roots.

Infection by the Japanese soil-borne wheat mosaic virus (JSBWMV) can lead to substantial losses in the grain yield of barley and wheat crops. While genetically based resistance to this virus has been documented, its mechanistic basis remains obscure. In this study, the deployment of a quantitative PCR assay showed that the resistance acts directly against the virus rather than by inhibiting the colonization of the roots by the virus' fungal vector Polymyxa graminis. In the susceptible barley cultivar (cv.) Tochinoibuki, the JSBWMV titre was maintained at a high level in the roots during the period December-April, and the virus was translocated from the root to the leaf from January onwards. In contrast, in the roots of both cv. Sukai Golden and cv. Haruna Nijo, the titre was retained at a low level, and translocation of the virus to the shoot was strongly suppressed throughout the host's entire life cycle. The roots of wild barley (Hordeum vulgare ssp. spontaneum) accession H602 responded in the early stages of infection similarly to those of the resistant cultivated forms, but the host was unable to suppress the translocation of the virus to the shoot from March onwards. The virus titre in the root was presumed to have been restricted by the action of the gene product of Jmv1 (on chromosome 2H), while the stochastic nature of the infection was suppressed by the action of that of Jmv2 (on chromosome 3H), a gene harbored by cv. Sukai Golden but not by either cv. Haruna Nijo or accession H602.

Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Genome-Wide Analysis of Snf2 Gene Family Reveals Potential Role in Regulation of Spike Development in Barley.

Sucrose nonfermenting 2 (Snf2) family proteins, as the catalytic core of ATP-dependent chromatin remodeling complexes, play important roles in nuclear processes as diverse as DNA replication, transcriptional regulation, and DNA repair and recombination. The Snf2 gene family has been characterized in several plant species; some of its members regulate flower development in Arabidopsis. However, little is known about the members of the family in barley (Hordeum vulgare). Here, 38 Snf2 genes unevenly distributed among seven chromosomes were identified from the barley (cv. Morex) genome. Phylogenetic analysis categorized them into 18 subfamilies. They contained combinations of 21 domains and consisted of 3 to 34 exons. Evolution analysis revealed that segmental duplication contributed predominantly to the expansion of the family in barley, and the duplicated gene pairs have undergone purifying selection. About eight hundred Snf2 family genes were identified from 20 barley accessions, ranging from 38 to 41 genes in each. Most of these genes were subjected to purification selection during barley domestication. Most were expressed abundantly during spike development. This study provides a comprehensive characterization of barley Snf2 family members, which should help to improve our understanding of their potential regulatory roles in barley spike development.

Two dominant genes in barley (Hordeum vulgare L.) complementarily encode perfect resistance to Japanese soil-borne wheat mosaic virus.

Japanese soil-borne wheat mosaic virus (Furovirus) is a damaging pathogen of wheat and barley. This virus can survive in the soil for several decades, so the deployment of resistant cultivars represents the only practical control measure. Here, a genetic analysis has identified two regions of the barley genome-one on chromosome 2H and the other on chromosome 3H-as harboring gene(s) encoding resistance to this virus. The joint presence of both loci, termed Jmv1 and Jmv2, made the plants essentially immune, with resistance being dominant over susceptibility at each locus. Phylogenetic analysis showed that the virus is not closely related to the type Furovirus species Soil-borne wheat mosaic virus. There was a difference between the RNA1- and RNA2-based phylogenies of the virus species in Furovirus implying the independent segregation of the virus subgenomes.

The Brittle Rachis Trait in Species Belonging to the Triticeae and Its Controlling Genes Btr1 and Btr2.

In many non-cultivated angiosperm species, seed dispersal is facilitated by the shattering of the seed head at maturity; in the Triticeae tribe, to which several of the world's most important cereals belong, shattering takes the form of a disarticulation of the rachis. The products of the genes Btr1 and Btr2 are both required for disarticulation to occur above the rachis nodes within the genera Hordeum (barley) and Triticum/Aegilops (wheat). Here, it has been shown that both Btr1 and Btr2 are specific to the Triticeae tribe, although likely paralogs (Btr1-like and Btr2-like) are carried by the family Poaceae including Triticeae. Aegilops tauschii (the donor of the bread wheat D genome) lacks a copy of Btr1 and disarticulation in this species occurs below, rather than above the rachis node; thus, the product of Btr1 appears to be required for disarticulation to occur above the rachis node.

Comparative Analysis of Root Transcriptome Reveals Candidate Genes and Expression Divergence of Homoeologous Genes in Response to Water Stress in Wheat.

Crop cultivars with larger root systems have an increased ability to absorb water and nutrients under conditions of water deficit. To unravel the molecular mechanism of water-stress tolerance in wheat, we performed RNA-seq analysis on the two genotypes, Colotana 296-52 (Colotana) and Tincurrin, contrasting the root growth under polyethylene-glycol-induced water-stress treatment. Out of a total of 35,047 differentially expressed genes, 3692 were specifically upregulated in drought-tolerant Colotana under water stress. Transcription factors, pyrroline-5-carboxylate reductase and late-embryogenesis-abundant proteins were among upregulated genes in Colotana. Variant calling between Colotana and Tincurrin detected 15,207 SNPs and Indels, which may affect protein function and mediate the contrasting root length phenotype. Finally, the expression patterns of five triads in response to water, high-salinity, heat, and cold stresses were analyzed using qRT-PCR to see if there were differences in homoeologous gene expression in response to those conditions. The five examined triads showed variation in the contribution of homoeologous genes to water, high-salinity, heat, and cold stresses in the two genotypes. The variation of homoeologous gene expression in response to environmental stresses may enable plants to better cope with stresses in their natural environments.

Combined QTL mapping and RNA-Seq profiling reveals candidate genes associated with cadmium tolerance in barley.

The high toxicity of cadmium (Cd) and its ready uptake by plants has become a major agricultural problem. To investigate the genetic architecture and genetic regulation of Cd tolerance in barley, we conducted quantitative trait loci (QTL) analysis in the phenotypically polymorphic Oregon Wolfe Barley (OWB) mapping population, derived from a cross between Rec and Dom parental genotypes. Through evaluating the Cd tolerance of 87 available doubled haploid lines of the OWB mapping population at the seedling stage, one minor and one major QTL were detected on chromosomes 2H and 6H, respectively. For chlorosis and necrosis traits, the major QTL explained 47.24% and 38.59% of the phenotypic variance, respectively. RNA-Seq analysis of the parental seedlings under Cd treatment revealed 542 differentially expressed genes between Cd-tolerant Rec and Cd-susceptible Dom genotypes. By analyzing sequence variations in transcribed sequences of the parental genotypes, 155,654 SNPs and 1,525 InDels were identified between the two contrasting genotypes and may contribute to Cd tolerance. Finally, by integrating the data from the identified QTLs and RNA-Seq analysis, 16 Cd tolerance-related candidate genes were detected, nine of which were metal ion transporters. These results provide promising candidate genes for further gene cloning and improving Cd tolerance in barley.

The Photoperiod-Insensitive Allele Ppd-D1a Promotes Earlier Flowering in Rht12 Dwarf Plants of Bread Wheat.

The gibberellin-responsive dwarfing gene Rht12 can significantly reduce plant height without changing seedling vigor and substantially increase ear fertility in bread wheat (Triticum aestivum. L). However, Rht12 delays heading date and anthesis date, hindering the use of Rht12 in wheat improvement. To promote early flowering of the Rht12 dwarf plants, the photoperiod-insensitive allele Ppd-D1a was introduced through a cross between Jinmai47 (Ppd-D1a) and Karcagi (Rht12). The results showed that Ppd-D1a can rescue the delaying effect of Rht12 on flowering time and promote earlier flowering by 9.0 days (163.2°Cd) in the Rht12 dwarf plants by shortening the late reproduction phase. Plant height was reduced by Rht12 (43.2%) and Ppd-D1a (10.9%), achieving dwarf plants with higher lodging resistance. Ear fertility, like the grain number per spike, was significantly increased by Rht12 (21.3%), while it was reduced by Ppd-D1a (6.5%). However, thousand kernel weight was significantly reduced by Rht12 (12.9%) but significantly increased by Ppd-D1a (16.9%). Finally, plant yield was increased by 16.4 and 8.2%, and harvest index was increased by 24.9 and 15.4% in the Rht12 dwarf lines and tall lines with Ppd-D1a, respectively. Clearly, there was an additive interaction between Rht12 and Ppd-D1 and the introduction of Ppd-D1a advanced the flowering time and improved the yield traits of Rht12 dwarf plants, suggesting that the combination of Rht12 and Ppd-D1a would be conducive to the successful use of Rht12 in wheat breeding programs.

The ß-Ketoacyl-CoA Synthase HvKCS1, Encoded by Cer-zh, Plays a Key Role in Synthesis of Barley Leaf Wax and Germination of Barley Powdery Mildew.

The cuticle coats the primary aerial surfaces of land plants. It consists of cutin and waxes, which provide protection against desiccation, pathogens and herbivores. Acyl cuticular waxes are synthesized via elongase complexes that extend fatty acyl precursors up to 38 carbons for downstream modification pathways. The leaves of 21 barley eceriferum (cer) mutants appear to have less or no epicuticular wax crystals, making these mutants excellent tools for identifying elongase and modification pathway biosynthetic genes. Positional cloning of the gene mutated in cer-zh identified an elongase component, ß-ketoacyl-CoA synthase (CER-ZH/HvKCS1) that is one of 34 homologous KCSs encoded by the barley genome. The biochemical function of CER-ZH was deduced from wax and cutin analyses and by heterologous expression in yeast. Combined, these experiments revealed that CER-ZH/HvKCS1 has a substrate specificity for C16-C20, especially unsaturated, acyl chains, thus playing a major role in total acyl chain elongation for wax biosynthesis. The contribution of CER-ZH to water barrier properties of the cuticle and its influence on the germination of barley powdery mildew fungus were also assessed.

Characterization and genetic mapping of the ß-diketone deficient eceriferum-b barley mutant.

KEY MESSAGE: The barley eceriferum-b.2 (cer-b.2) mutant produces glossy leaf sheaths and is deficient in the cuticular wax component 14,16-hentriacontanedione. The mutated gene maps to a 1.3-cM interval on chromosome 3HL flanked by the genes MLOC_10972 and MLOC_69561. The cuticular wax coating of leaves and stems in many grass species is responsible for the plants' glaucous appearance. A major component of the wax is a group of ß-diketone compounds. The barley eceriferum-b.2 (cer-b.2) mutant produces glossy leaf sheaths and is deficient for the compound 14,16-hentriacontanedione. A linkage analysis based on 708 gametes allowed the gene responsible for the mutant phenotype to be mapped to a 1.3-cM interval on chromosome 3HL flanked by the two genes MLOC_10972 and _69561. The product of the wild type allele may represent a step in the ß-diketone synthesis pathway.

A GDSL-motif esterase/acyltransferase/lipase is responsible for leaf water retention in barley.

The hydrophobic cuticle covers the surface of the most aerial organs of land plants. The barley mutant eceriferum-zv (cer-zv), which is hypersensitive to drought, is unable to accumulate a sufficient quantity of cutin in its leaf cuticle. The mutated locus has been mapped to a 0.02 cM segment in the pericentromeric region of chromosome 4H. As a map-based cloning approach to isolate the gene was therefore considered unlikely to be feasible, a comparison was instead made between the transcriptomes of the mutant and the wild type. In conjunction with extant genomic information, on the basis of predicted functionality, only two genes were considered likely to encode a product associated with cutin formation. When eight independent cer-zv mutant alleles were resequenced with respect to the two candidate genes, it was confirmed that the gene underlying the mutation in each allele encodes a Gly-Asp-Ser-Leu (GDSL)-motif esterase/acyltransferase/lipase. The gene was transcribed in the epidermis, and its product was exclusively deposited in cell wall at the boundary of the cuticle in the leaf elongation zone, coinciding with the major site of cutin deposition. CER-ZV is speculated to function in the deposition of cutin polymer. Its homologs were found in green algae, moss, and euphyllophytes, indicating that it is highly conserved in plant kingdom.

Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.

The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H(+) was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH.

Changes in genome-wide gene expression during allopolyploidization and genome stabilization in hexaploid wheat.

Allopolyploidization is an important evolutionary event in plants, but its genome-wide effects are not fully understood. Common wheat, Triticum aestivum (AABBDD), evolved through amphidiploidization between T. turgidum (AABB) and Aegilops tauschii (DD). Here, global gene expression patterns in the seedlings of a synthetic triploid wheat line (ABD), its chromosome-doubled hexaploid (AABBDD) and stable synthetic hexaploid (AABBDD), and the parental lines T. turgidum (AABB) and Ae. tauschii (DD) were compared using an oligo-DNA microarray to identify metabolic pathways affected by the genome conflict that occurs during allopolyploidization and genome stabilization. Characteristic gene expression patterns of non-additively expressed genes were detected in the newly synthesized triploid and hexaploid, and in the stable synthetic hexaploid. Hierarchical clustering of all differentially expressed and non-additively expressed genes revealed that the gene expression patterns of the triploid (ABD) were similar to those of the maternal parent (AABB), and that expression patterns in successive generations arising from self-pollination became closer to that of the pollen parent (DD). The non-additive gene expression profiles markedly differed between the triploid (ABD) and chromosome-doubled hexaploid (AABBDD), as supported by Gene Ontology (GOSlim) analysis. Four hundred and nineteen non-additively expressed genes were commonly detected in all three generations. GOSlim analysis indicated that these non-additively expressed genes were predominantly involved in "biological pathways". Notably, four of 11 genes related to sugar metabolism displayed elevated expression throughout allopolyploidization. These may be useful candidates for promoting heterosis and adaptation in plants.

Identification of differential gene expression for Kr1 gene in bread wheat using annealing control primer system.

Intergeneric hybridization is an important strategy to introgress alien genes into common wheat for its improvement. But presence of cross ability barrier mechanism regulated by Kr1 gene played a major destructive role for hybridization than other reported genes. In order to know the underlying molecular mechanism and to dissect out this barrier, a new annealing system, ACP (anneling control primer) system was used in chromosome 5B (containing Kr1 gene) specific Recombinant Inbred Line (RIL) population. Two differentially expressed fragments for Kr1 gene was identified, cloned and sequenced. Further the expression was confirmed by northern blotting analysis. Sequence analysis of the resulted clones revealed classes of putative genes, including stress responsive and signal transduction.

An efficient method for purifying high quality RNA from wheat pistils.

Many methods are available for total RNA extraction from plants, except the floral organs like wheat pistils containing high levels of polysaccharides that bind/or co-precipitate with RNA. In this protocol, a simple and effective method for extracting total RNA from small and feathery wheat pistils has been developed. Lithium chloride (LiCl) and phenol:chloroform:isoamylalcohol (PCI) were employed and the samples were ground in microcentrifuge tube using plastic pestle. A jacket of liquid nitrogen and simplified procedures were applied to ensure thorough grinding of the pistils and to minimize the samples loss. These measures substantially increased the recovery of total RNA (approximately 50%) in the extraction process. Reliable differential display by cDNA-AFLP was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method is also practicable for gene expression and gene regulation studies in floral parts of other plants.