Publications (19)41.44 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: In hexaploid crops, such as bread wheat, it should be possible to fine-tune phenotypic traits by identifying wild-type and null genes from each of the three genomes and combining them in a calculated manner. Here, we demonstrate this with gene combinations for two starch synthesis genes, SSIIa and GBSSI. Lines with inactive copies of both enzymes show a very dramatic change in phenotype, so to create intermediate phenotypes, we used marker-assisted selection to develop near-isogenic lines (NILs) carrying homozygous combinations of null alleles. For both genes, gene dosage effects follow the order B > D ≥ A; therefore, we completed detailed analysis of starch characteristics for NIL 3-3, which is null for the B-genome copy of the SSIIa and GBSSI genes, and NIL 5-5, which has null mutations in the B- and D-genome-encoded copies of both of these genes. The effects of the combinations on phenotypic traits followed the order expected on the basis of genotype, with NIL 5-5 showing the largest differences from the wild type, while NIL 3-3 characteristics were intermediate between NIL 5-5 and the wild type. Differences among genotypes were significant for many starch characteristics, including percent amylose, chain length distribution, gelatinization temperature, retrogradation, and pasting properties, and these differences appeared to translate into improvements in end-product quality, since bread made from type 5-5 flour showed a 3 day lag in staling.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Yellow mosaic disease, caused by wheat yellow mosaic virus (WYMV), is one of the most serious diseases of winter wheat (Triticum aestivum L.) in Japan. The three pathotypes of WYMV are distributed in different geographical areas: pathotype I is found mainly in western and central Japan (Kanto), pathotype II in northern Japan (Tohoku and Hokkaido) and pathotype III on the southern island of Japan (Kyushu). A total of 246 doubled-haploid (DH) lines, derived from a cross between ‘Yumechikara’ (resistant) and ‘Kitahonami’ (susceptible), were evaluated for 2 years for their resistance to WYMV pathotype I. A single major quantitative trait locus, Q.Ymym, mapping to chromosome 2D was associated with resistance to pathotype I in ‘Yumechikara’. This is the first time a QTL responsible for pathotype I resistance has been identified. Fine mapping of Q.Ymym indicated that it was on a tight linkage block originating from ‘Yumechikara’, and the markers associated with this block will accelerate the development of varieties resistant to WYMV pathotype I.
- [Show abstract] [Hide abstract] ABSTRACT: Durum wheat (Triticum turgidum L.) cultivars can benefit from having some level of seed dormancy to help reduce seed damage and lower grain quality caused by preharvest sprouting (PHS) occurring during wet harvesting conditions. Previously, a single chromosome substitution line carrying chromosome 2B of wild emmer [Triticum turgidum L. subsp. dicoccoides (Körn. ex Asch. & Graebn.) Thell.] in the durum cultivar Langdon background was found to have higher levels of seed dormancy and PHS tolerance. In this study, a population of recombinant substitution lines was developed and used to construct a single nucleotide polymorphism (SNP)-based high-density genetic linkage map. Seed germination tests were used to evaluate seed dormancy levels. Multiple interval mapping analysis revealed four quantitative trait loci (QTL) regions affecting seed dormancy. Two regions containing major-effect QTL contributed by wild emmer were consistently expressed in four environments and explained 5.89 to 11.14% of the phenotypic variation. One QTL region was located near the centromere and the other on the long arm of chromosome 2B in Bayes credible intervals of 4 and 7 cM, respectively. Recombinant inbred lines (RILs) carrying both QTL had an average of 35% reduced rate of germination measured by weighted germination index compared with RILs carrying neither QTL. The two QTL regions identified in this study should be useful for improving PHS tolerance in wheat. Efforts to transfer the two QTL into elite durum cultivars are in progress to examine the effects of genetic background and environment on QTL expression and to evaluate the performance of other agronomic traits in the presence of the QTL.
- [Show abstract] [Hide abstract] ABSTRACT: The winter wheat variety Kitahonami shows a superior flour yield in comparison to other Japanese soft wheat varieties. To map the quantitative trait loci (QTL) associated with this trait, association mapping was performed using a panel of lines from Kitahonami's pedigree, along with leading Japanese varieties and advanced breeding lines. Using a mixed linear model corrected for kernel types and familial relatedness, 62 marker-trait associations for flour yield were identified and classified into 21 QTLs. In eighteen of these, Kitahonami alleles showed positive effects. Pedigree analysis demonstrated that a continuous pyramiding of QTLs had occurred throughout the breeding history of Kitahonami. Linkage analyses using three sets of doubled haploid populations from crosses in which Kitahonami was used as a parent were performed, leading to the validation of five of the eight QTLs tested. Among these, QTLs on chromosomes 3B and 7A showed highly significant and consistent effects across the three populations. This study shows that pedigree-based association mapping using breeding materials can be a useful method for QTL identification at the early stages of breeding programs.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Near isogenic lines (NILs) of the eight haplotypes of starch synthase IIa (SSIIa) were used to analyze the effects of SSIIa gene dosage on branch chain length, gelatinization, pasting, retrogradation, and enzymatic hydrolysis of starches. Compared to wild-type, the amylopectin of lines missing one or more active SSIIa enzymes had increases in the proportion of short branch chains (DP6-10) and decreases in mid-length chains (DP11-24), and the size of these differences depended on the dosage of active SSIIa enzymes. Of the three loci, SSIIa-A1 had the smallest contribution to amylopectin structure and SSIIa-B1 the largest. The different effects of the three SSIIa enzymes on starch properties were also seen in gelatinization, retrogradation, pasting, and enzymatic hydrolysis properties. Such differences in starch properties might be useful in influencing the texture and shelf life of food products.
- [Show abstract] [Hide abstract] ABSTRACT: The double mutant “sweet wheat” (SW), which produces substantial amounts of sugars in immature seeds, is missing two starch synthases, namely granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa). The lack of these two enzymes causes major changes in the attributes of SW seed, starch, and starch granules. SW seeds appear normal during early stages of development, but become shrunken when seeds begin to mature and dry. However, even in immature seed, starch granules are small and misshapen, and high levels of maltose are present throughout seed development. The crystallinity of SW starch is altered in that a major peak typical of the cereal A-type diffraction pattern is absent, and the gelatinization temperature of SW starch is considerably lower than that of wild-type starch. Amylopectin from SW seed has a substantially lower molecular weight than that from wild-type seed, and a low molecular weight peak with a bimodal distribution is found only in SW starch. This peak contains linear malto-oligosaccharides as well as short, branched glucans. SW starch has an increased proportion of branches with DP < 10, and chains with DP 2 and 3 are particularly increased. These changes suggest that sweet wheat starch is being modified in an atypical manner by isoamylases and/or β-amylases.
- [Show abstract] [Hide abstract] ABSTRACT: Japanese barnyard millet is an important food source in East Asian countries. However its crumbly texture limits desirability and consumption. Controlling amylose level in the endosperm is important to improve the eating quality of the millet. Because it is well known that the waxy gene determines the amylose level in the endosperm, we conducted a molecular analysis of the gene. Segregation analysis revealed that wild-type cultivars had three functional genes while low-amylose cultivars had one. We determined complete sequences of the three homoeologous waxy structural genes, EeWx1, EeWx2 and EeWx3, in a wild-type cultivar. These sequences showed high homology in the exon regions (97 %), and lower homology in the introns (82 %). Two spontaneous mutations were characterized in the low-amylose cultivars. In addition, one induced mutation was found in the fully waxy cultivar, Chojuromochi. Spontaneous mutations are deletions of whole and terminal regions in the EeWx2 and EeWx3 alleles, respectively. The induced mutation is a single-base deletion that led to a premature termination codon in EeWx1. These findings led us to develop useful markers for selecting low-amylose and waxy lines in millet.
- [Show abstract] [Hide abstract] ABSTRACT: Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition. Whole grain flour of SW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient.
- [Show abstract] [Hide abstract] ABSTRACT: Approximately 70% of wheat endosperm consists of starch, and variations in the quality and quantity of starch affect the processing characteristics of wheat flour. Amylose content in particular has a major effect on Asian noodle quality, and selection of wheat lines with slightly lower amylose levels is an important goal in Japanese wheat breeding programs. Accurately measuring amylose content by direct methods such as colorimetric assays was found to be problematic, suggesting there was a need for a more efficient and accurate method of screening for reduced amylose content. Therefore, we characterized mutations in the wheat waxy genes, which control amylose synthesis, and developed DNA markers for the identification of null waxy alleles. In this review, we describe the development of these markers and outline their utility for wheat breeding programs.
- [Show abstract] [Hide abstract] ABSTRACT: Waxy protein (granule-bound starch synthase I) is a key enzyme in the synthesis of amylose in endosperm tissue. The amylose content of wheat flour plays a significant role in determining Japanese udon noodle quality. Most wheat cultivars suitable for producing udon noodles have a low amylose level due to a lack of Wx-B1 protein conditioned by null Wx-B1 alleles. It was previously determined that the entire coding region of the wheat Wx-B1 gene is deleted in the most common null allele. However, the extent and breakpoints of the deletion have not been established. In this study, the position of the 3' deletion breakpoint was refined by mapping with PCR-based markers. Using information from this analysis, a chromosome walk was initiated and the DNA sequence flanking the deletion breakpoints was obtained. The deletion included a 3,872 bp region downstream from the termination codon of Wx-B1 gene. Based on similarity with T. monococcum sequences, it was estimated that approximately 60 kb upstream of the Wx-B1 gene was also deleted. Using this sequence information, a codominant marker for the identification of the Wx-B1 null allele was developed. This marker can unambiguously identify heterozygous plants, which will accelerate the selection of partial waxy mutants carrying the Wx-B1 null allele.
- [Show abstract] [Hide abstract] ABSTRACT: PCR-based Landmark Unique Gene (PLUG) markers are EST-PCR markers developed based on the orthologous gene conservation between rice and wheat, and on the intron polymorphisms among the three orthologous genes derived from the A, B and D genomes of wheat. We designed a total of 960 primer sets from wheat ESTs that showed high similarity with 951 single-copy rice genes. When genomic DNA of Chinese Spring wheat was used as a template, 872 primer sets amplified one to five distinct products. Out of these 872 PLUG markers, 531 were assigned to one or more chromosomes by nullisomic-tetrasomic analysis. For each wheat chromosome, the number of loci detected ranged from 32 for chromosome 6A to 73 for chromosome 7D, with an average of 48 loci per chromosome. Several novel synteny perturbations were identified using deletion bin-mapping of markers. Furthermore, we demonstrated that PLUG markers can be used as probes to simultaneously identify BAC clones that contain homoeologous regions from all three genomes.
Dataset: Additional file 3[Show abstract] [Hide abstract] ABSTRACT: Detailed data for 24 PLUG markers. The following data for the 24 PLUG markers are shown: TIGR rice locus ID, annotation of the rice locus, ID of wheat UniGene, accession number of the longest EST, sequences of forward and reverse primers, estimated sizes of rice and wheat PCR products, wheat PCR product sizes (assigned chromosome) and their HaeIII/TaqI-digested fragment sizes (assigned chromosome), results of BLASTN searches of the GrainGenes  databases, and results of sequence analysis.
Dataset: Additional file 1[Show abstract] [Hide abstract] ABSTRACT: List of TaEST-LUGs. TIGR rice locus IDs for the TaEST-LUGs extracted by the PLUG system are listed.
Dataset: Additional file 2[Show abstract] [Hide abstract] ABSTRACT: Numbers of LUGs and TaEST-LUGs. The worksheet shows the number of loci, LUGs, and TaEST-LUGs per 500 kb on each rice chromosome of the TIGR Pseudomolecules.
- [Show abstract] [Hide abstract] ABSTRACT: EST-PCR markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. However, because wheat is an allohexaploid plant, PCR products derived from homoeologous genes are often simultaneously amplified. Such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. However, genomic sequence data for wheat are limited; therefore it is difficult to predict the location of introns. By using the similarities in gene structures between rice and wheat, we developed a system called PLUG (PCR-based Landmark Unique Gene) to design primers so that PCR products include intron sequences. We then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes. The PLUG system consists of the following steps: (1) Single-copy rice genes (Landmark Unique Gene loci; LUGs) exhibiting high degrees of homology to wheat UniGene sequences are extracted; (2) Alignment analysis is carried out using the LUGs and wheat UniGene sequences to predict exon-exon junctions, and LUGs which can be used to design wheat primers flanking introns (TaEST-LUGs) are extracted; and (3) Primers are designed in an interactive manner. From a total of 4,312 TaEST-LUGs, 24 loci were randomly selected and used to design primers. With all of these primer sets, we obtained specific, intron-containing products from the target genes. These markers were assigned to chromosomes using wheat nullisomic-tetrasomic lines. By PCR-RFLP analysis using agarose gel electrophoresis, 19 of the 24 markers were located on at least one chromosome. In the development of wheat EST-PCR markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. Using the PLUG system, wheat EST sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. Hence, the PLUG system is an effective tool for large-scale marker development.
Article: Sweet Wheat[Show abstract] [Hide abstract] ABSTRACT: The major components of storage starch are amylose and amylopectin, and in wheat, both an amylose-free mutant lacking granule-bound starch synthase I and a high-amylose mutant lacking starch synthase IIa have been produced recently. Here, we report the production of an amylose-free/ high-amylose double mutant. This double mutant has kernel and carbohydrate characteristics that are remarkably different than those of either single mutant, including a dramatically shrunken seed shape. Surprisingly, the double mutant has maltose and sucrose levels that are high enough to make it worthy of being called "sweet wheat".
National Agriculture and Food Research OrganizationTsukuba, Ibaraki, Japan