Shuhei Nasuda

We investigated the genetic diversity of the core collection of hexaploid wheat accessions in the Japanese wheat gene bank, NBRP-Wheat, with a focus on grain morphology. We scanned images of grains in the core collection, which consists of 189 accessions of Triticum aestivum, T. spelta, T. compactum, T. sphaerococcum, T. macha and T. vavilovii. From the scanned images, we recorded six metric characters (area size, perimeter length, grain length, grain width, length to width ratio and circularity) using the software package SmartGrain ver. 1.2. Statistical analyses of the collected data along with hundred-grain weight revealed that T aestivum has the largest diversity in grain morphology. Principal component analysis of these seven characters demonstrated that two principal components (PC(core)1 and PC(core)2) explain more than 96% of the variation in the core collection accessions. The correlation coefficients between the principal components and characters indicate that PC(core)1 is related to grain size and PC(core)2 to grain shape. From a genome-wide association study, we found a total of 15 significant marker-trait associations (MTAs) for grain morphological characters. More interestingly, we found mutually exclusive MTAs for PC(core)1 and PC(core)2 on 18 and 13 chromosomes, respectively. The results suggest that grain morphology in hexaploid wheat is determined by two factors, grain size and grain shape, which are under the control of multiple genetic loci.

In this study, we investigated the chromosome breakage caused by gametocidal (Gc) chromosome 3C^t and its interaction with the suppressor gene Igc1 (inhibitor of gametocidal gene 1) on wheat chromosome 3B. We demonstrated cytologically that patterns of 3C^t-induced chromosomal fragmentation in microspores differed from patterns observed for other Gc genes. Uninuclear microspores of the monosomic 3C^t addition line had high frequencies of micronuclei, possibly explaining its low fertility. Chromosome fragmentation was observed in prometaphase and metaphase of the first pollen mitosis in the monosomic 3C^t addition line. Patterns of chromosome fragmentation were different from those previously reported for Gc chromosomes 2S of Aegilops speltoides, 4S^sh of Ae. sharonensis and 2C^cy of Ae. cylindrica; many chromosome fragments were observed in prometaphase of the first pollen mitosis in the monosomic 3C^t addition plants. In anthers at the binuclear stage, many microspores at the uninuclear stage coexisted with normally developed microspores.

The current status of genome sequencing projects for hexaploid wheat and its relatives is reviewed. Wheat genome sequencing has become more feasible following the introduction of various techniques that reduce the complexity of its genome. Aneuploid stocks, namely ditelosomics, allowed us to sort individual chromosome arms by flow-sorting. Chromosome arm-specific DNA samples were prepared from the sorted chromosome arms for sequencing. The assemblies of the shotgun sequencing of the arm-specific DNA samples were published in 2004, where hypothetical wheat gene sequences can be found. This review is a milestone publication for wheat science that marks the dawn of the post-genomic era. I summarized the available molecular marker techniques for wheat.

A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91 % of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87 % of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F₁ hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T. turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae. tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae. tauschii’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.

Based on the similarity in gene structure between rice and wheat, the polymerase chain reaction (PCR)-based landmark unique gene (PLUG) system enabled us to design primer sets that amplify wheat genic sequences including introns. From the previously reported wheat PLUG markers, we chose 144 markers that are distributed on different chromosomes and in known chromosomal regions (bins) to obtain rye-specific PCR-based markers. We conducted PCR with the 144 primer sets and the template of the Imperial rye genomic DNA and found that 131 (91.0 %) primer sets successfully amplified PCR products. Of the 131 PLUG markers, 110 (76.4 %) markers showed rye-specific PCR amplification with or without restriction enzyme digestion. We assigned 79 of the 110 markers to seven rye chromosomes (1R to 7R) using seven wheat–rye (cv. Imperial) chromosome addition and substitution lines: 12 to 1R, 8 to 2R, 11 to 3R, 8 to 4R, 16 to 5R, 12 to 6R, and 12 to 7R. Furthermore, we located their positions on the short or long (L) chromosome arm, using 13 Imperial rye telosomic lines of common wheat (except for 3RL). Referring to the chromosome bin locations of the 79 PLUG markers in wheat, we deduced the syntenic relationships between rye and wheat chromosomes. We also discussed chromosomal rearrangements in the rye genome with reference to the cytologically visible chromosomal gaps.

Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.

An X-ray mutant showing an early flowering phenotype has been identified in einkorn wheat (Triticum monococcum L.), for which a major QTL for heading time was previously mapped in the telomeric region on the long arm of chromosome 3A. Recent advances in Triticeae genomics revealed that the gene order in this region is highly conserved between wheat and barley. Thus, we adopted a hypothetical gene order in barley, the so-called GenomeZipper, to develop DNA markers for fine mapping the target gene in wheat. We identified three genes tightly linked to the early heading phenotype. PCR analysis revealed that early-flowering is associated with the deletion of two genes in the mutant. Of the two deleted genes, one is an ortholog of the LUX ARRHYTHMO (LUX)/PHYTOCLOCK 1 (PCL1) gene found in Arabidopsis, which regulates the circadian clock and flowering time. We found distorted expression patterns of two clock genes (TOC1 and LHY) in the einkorn pcl1 deletion mutant as was reported for the Arabidopsis lux mutant. Transcript accumulation levels of photoperiod-response related genes, a photoperiod sensitivity gene (Ppd-1) and two wheat CONSTANS-like genes (WCO1 and TaHd1), were significantly higher in the einkorn wheat mutant. In addition, transcripts of the wheat florigen gene (WFT) accumulated temporally under short-day conditions in the einkorn wheat mutant. These results suggest that deletion of WPCL1 leads to abnormally higher expression of Ppd-1, resulting in the accumulation of WFT transcripts that triggers flowering even under short-day conditions. Our observations from gene mapping, gene deletions, and expression levels of flowering related genes strongly suggest that WPCL1 is the most likely candidate gene for controlling the early flowering phenotype in the einkorn wheat mutant.

MAD2 (mitotic arrest deficient 2) is a highly conserved protein involving in spindle checkpoint control. MAD2 locates at spindle-unattached kinetochores during prophase and dissolves from spindle-attached kinetochores towards metaphase. In this study, we isolated homologous genes encoding for MAD2 from hexaploid wheat. The three homoeologous genes on the long arms of the group-2 chromosomes shared approximately 99% similarity of the nucleotide sequence in coding regions. The intron-exon structures of the three homoeologues were also conserved, showing high similarities to that of the Arabidopsis MAD2 gene. All three homoeologues were transcribed in roots and spikes but not in leaves. We generated antibodies against the polypeptides with amino acid sequences derived from the cDNA sequences of the wheat MAD2 homologues. Using these antibodies, we found MAD2 in wheat root-tip cells to change in location and amount through the cell cycle, similar to those reported for human MAD2. Intense immunostaining signals were observed at the centromeres of all metaphase chromosomes when root-tips were treated with colchicine, a microtubule-destabilizing drug, but no signals were observed in untreated chromosomes. Thus, the wheat MAD2 protein could be a good marker for the functional kinetochores of metaphase chromosomes in wheat.

We conducted AFLP (Amplified Fragment Length Polymorphism) analysis with the six wheat-barley chromosome addition lines of common wheat cultivar Chinese Spring. We analyzed the AFLP fingerprints generated by 36 combinations of selective-amplification primers to find 103 markers specific to the barley chromosomes (2.9 markers per combination on average). The numbers of AFLP markers mapped to the barley chromosomes varied (one to 16) depending of the primer combinations. Each barley chromosome had 10 to 27 AFLP markers (17.2 markers on average). We identified the chromosome arms in which these markers are located using the barley telocentric addition lines (one to 20 markers per chromosome arm). The AFLP markers were not distributed evenly among chromosomes and chromosome arms. We could not determine the chromosome-arm locations for some of the barley-specific markers, either because such markers were found in both the short- and long-arm telocentric lines, or in neither line.