Ryoko Ohno

Kinetics of transcript and protein accumulation of a low-molecular- weight wheat LEA D-11 dehydrin in response to low temperature

We studied the kinetics of induction of the wheat (Triticum aestivum L.) gene Wdhn 13, which encodes a predominantly cold-responsive protein belonging to the LEA D-11 dehydrin family. The deduced polypeptide WDHN 13 (MW = 12.8 kDa) represented the smallest dehydrin member in cereals with three lysine-rich K-segments. Purified WDHN 13 was boiling-stable due to its high hydrophilicity. Western blot analysis using the polyclonal anti-WDHN 13-antibody revealed a number of cross-reacting proteins in mature embryos and endosperms and in seedling leaves under normal temperature (25 degrees C), while a single major protein and a transcript were induced in response to low temperature (4 degrees C) in the leaves. An increase in the amount of mRNA was temporary with a peak occurring at day 3 to 5 of the low temperature treatment, while the protein accumulation proceeded with a significant time lag and continued until the end of the experiment (day 10). Steady-state levels of the transcript and protein were much higher in the leaves than in the roots of low temperature-treated seedlings and were apparently modulated by light/dark conditions. The light/dark modulation of the transcript and protein levels suggested stabilization of the mRNA under the low temperature condition. Genomic Southern blot analysis showed that Wdhn 13 is located on the homoeologous group 7 chromosomes, unlike all other wheat Dhn genes that are located on the group 6 chromosomes.

A High-Density Genetic Map with Array-Based Markers Facilitates Structural and Quantitative Trait Locus Analyses of the Common Wheat Genome

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map.

Global gene expression profiling related to temperature-sensitive growth abnormalities in interspecific crosses between tetraploid wheat and Aegilops tauschii

Triploid wheat hybrids between tetraploid wheat and Aegilops tauschii sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat APETALA1-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes SPLs, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/SPLs module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.

Three dominant awnless genes in common wheat: Fine mapping, interaction and contribution to diversity in awn shape and length

The awn is a long needle-like structure formed at the tip of the lemma in the florets of some grass species. It plays a role in seed dispersal and protection against animals, and can contribute to the photosynthetic activity of spikes. Three main dominant inhibitors of awn development (Hd, B1 and B2) are known in hexaploid wheat, but the causal genes have not been cloned yet and a genetic association with awn length diversity has been found only for the B1 allele. To analyze the prevalence of these three awning inhibitors, we attempted to predict the genotypes of 189 hexaploid wheat varieties collected worldwide using markers tightly linked to these loci. Using recombinant inbred lines derived from two common wheat cultivars, Chinese Spring and Mironovskaya 808, both with short awns, and a high-density linkage map, we performed quantitative trait locus analysis to identify tightly linked markers. Because this linkage map was constructed with abundant array-based markers, we converted the linked markers to PCR-based markers and determined the genotypes of 189 hexaploids. A significant genotype-phenotype correlation was observed at the Hd and B1 regions. We also found that interaction among these three awning inhibitors is involved in development of a membranous outgrowth at the base of awn resembling the Hooded mutants of barley. For the hooded awn phenotype, presence of the Hd dominant allele was essential but not sufficient, so B2 and other factors appear to act epistatically to produce the ectopic tissue. On the other hand, the dominant B1 allele acted as a suppressor of the hooded phenotype. These three awning inhibitors largely contribute to the genetic variation in awn length and shape of common wheat.

Genotypic effects on sugar and by-products of liquid hydrolysates and on saccharification of acid-insoluble residues of from wheat straw

Wheat straw is one of the major attractive resources for low-cost raw materials for renewable energy, biofuels and biochemicals. However, like other sources of lignocellulosic biomass, straw is a heterogeneous material due to its mixed origin from different tissue and cell types. Here, to examine the genotypic effects on biorefinery usage of wheat straw, straw obtained from different wheat cultivars and experimental lines was pretreated with dilute acid. Significant differences between cultivars were observed in the concentrations of glucose and toxic by-products of the liquid hydrolysates. A higher content of xylose than glucose was found in liquid hydrolysates from wheat straw, and the xylose content appeared to be affected by both environmental and genetic factors. Analysis using chromosome substitution lines of the common wheat cultivar Chinese Spring showed that chromosomes 2A and 3A from other wheat cultivars, Hope and Timstein, significantly increased the xylose content. However, no significant relationship was observed between the liquid hydrolysate xylose content and the glucose content obtained from enzymatic saccharification of the acid-insoluble residue. These results highlight the potential of wheat breeding to improve biomass-related traits in wheat straw.

Development of the BAC Physical Maps of Wheat Chromosome 6B for Its Genomic Sequencing

For a purpose of better understanding the genome structure of wheat and accelerating the development of DNA markers for gene isolations and breeding, the Japanese research group, as a member of The International Wheat Genome Sequencing Consortium, is now conducting the physical mapping and genomic sequencing of wheat chromosome 6B of ‘Chinese Spring’ (CS). BAC libraries were constructed respectively using the short and long arm-specific DNAs extracted from the flow-sorted chromosome 6BS and 6BL of double ditelosomic 6B lines of CS. With a sequence-based finger printing method and contig assembly, the BAC physical maps for the 6BS and 6BL have been successfully established. For validation and chromosomal landing of the BAC contigs, we have developed a large number of 6B-specific DNA markers using the public resources including available EST databases, and 6B survey sequence. In parallel, three reference maps, a highresolution radiation hybrid map, a gametocidal system-derived deletion map and a genetic linkage map, are also under construction for anchoring the BAC contigs to their specific genomic regions.

Extracellular trafficking of a wheat cold-responsive protein, WLT10

A cold-responsive wheat gene, WLT10, encodes a member of the cereal-specific low temperature-responsive/cold-responsive protein family, which contains a hydrophobic N-terminal 20 amino acid sequence that corresponds to signal peptides associated with extracellular trafficking. To verify the subcellular localization of WLT10 and the function of its putative signal peptide, we constructed three chimeric genes in which either the WLT10 signal peptide, a signal peptide with only 6 additional amino acids, or the full-length WLT10 polypeptide was fused to the N-terminus of green fluorescent protein (GFP). These fusion constructs were transiently introduced into onion epidermal cells by particle bombardment. GFP signals were observed not only in the extracellular space (ECS) but also in the endoplasmic reticulum (ER) and Golgi apparatus. The time course of GFP signal localization suggests the movement of WLT10 through the ER/Golgi pathway and into the ECS. Thus, WLT10 is a cold-responsive secreted protein, and its N-terminal 20 amino acid region is important for transport to the ECS.