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Heading/flowering at the appropriate time in each cultivation region can greatly contribute to stabilizing and maximizing yield. Genotype combinations of Vrn-1 and Ppd-1, the major genes determining heading time, can explain the variation in heading time. However, the genes that can explain the remaining variations in heading time are still unknown. In this study, we aimed to identify the genes conferring early heading using doubled haploid lines derived from Japanese wheat varieties. Quantitative trait loci (QTL) analysis revealed a significant QTL on the long arm of chromosome 1B in multiple growing seasons. Genome sequencing using Illumina short reads and Pacbio HiFi reads revealed a large deletion of a ~500 kb region containing TaELF-B3, an orthologue of Arabidopsis clock gene EARLY FLOWERING 3 (ELF3). Plants with the deleted allele of TaELF-B3 (ΔTaELF-B3allele) headed earlier only under short-day vernalization conditions. Higher expression levels of clock- and clock-output genes, such as Ppd-1 and TaGI, were observed in plants with the ΔTaELF-B3 allele. These results suggest that the deletion of TaELF-B3 causes early heading. Of the TaELF-3 homoeoalleles conferring early heading, the ΔTaELF-B3 allele showed the greatest effect on the early heading phenotype in Japan. The higher allele frequency of the ΔTaELF-B3 allele in western Japan suggests that the ΔTaELF-B3 allele was preferred during recent breeding to adapt to the environment. TaELF-3 homoeolog will help to expand the cultivated area by fine-tuning the optimal timing of heading in each environment.

Fine mapping by recombinant backcross populations revealed that a preharvest sprouting QTL on 2B contained two QTLs linked in coupling with different effects on the phenotype. Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait locus (QTL) revealed a major PHS QTL, QPhs.cnl-2B.1, located on chromosome 2B significant in 16 environments that explained from 5 to 31 % of the phenotypic variation. The objective of this project was to fine map the QPhs.cnl-2B.1 interval. Fine mapping was carried out in recombinant backcross populations (BC1F4 and BC1F5) that were developed by backcrossing selected doubled haploids to a recurrent parent and self-pollinating the BC1F4 and BC1F5 generations. In each generation, three markers in the QPhs.cnl-2B.1 interval were used to screen for recombinants. Fine mapping revealed that the QPhs.cnl-2B.1 interval contained two PHS QTLs linked in coupling. The distal PHS QTL, located between Wmc453c and Barc55, contributed 8 % of the phenotypic variation and also co-located with a major seed dormancy QTL determined by germination index. The proximal PHS QTL, between Wmc474 and CNL415-rCDPK, contributed 16 % of the variation. Several candidate genes including Mg-chelatase H subunit family protein, GTP-binding protein and calmodulin/Ca2+-dependent protein kinase were linked to the PHS QTL. Although many recombinant lines were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.

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.

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.

Association mapping identified quantitative trait loci (QTLs) and the markers linked to pre-harvest sprouting (PHS) resistance in an elite association mapping panel of white winter wheat comprising 198 genotypes. A total of 1,166 marker loci including DArT and SSR markers representing all 21 chromosomes of wheat were used in the analysis. General and mixed linear models were used to analyze PHS data collected over 4 years. Association analysis identified eight QTLs linked with 13 markers mapped on seven chromosomes. A QTL was detected on each arm of chromosome 2B and one each on chromosome arms 1BS, 2DS, 4AL, 6DL, 7BS and 7DS. All except the QTL on 7BS are located in a location similar to previous reports and, if verified, the QTL on 7BS is likely to be novel. Principal components and the kinship matrix were used to account for the presence of population structure but had only a minor effect on the results. Although, none of the QTLs was highly significant across all environments, a QTL on the long arm of chromosome 4A was detected in three different environments and also using the best linear unbiased predictions over years. Although previous reports have identified this as a major QTL, its effects were minor in our biparental mapping populations. The results of this study highlight the benefits of association mapping and the value of using elite material in association mapping for plant breeding programs.

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.

The soybean cultivar Yumeminori, which lacks the α′- and α-subunits of β-conglycinin, carries both naturally occurring and induced mutations. While the cause of the natural mutation resulting in the α′-subunit deficiency has been determined, the induced mutation in the CG-2 gene encoding the α-subunit has not been characterized at the molecular level. In this study, we identified a four base pair insertion in the first exon of CG-2, which introduced a premature stop codon. The insertion co-segregated with the lack of α-subunit, indicating that this mutation is the cause of the α-subunit deficiency. A multiplex PCR method of testing for the presence or absence of α′- and α-subunits was developed based on the sequences of mutated and wild-type alleles. This PCR-based test was also capable of detecting the presence of wild-type genes when Yumeminori DNA samples were contaminated with wild-type DNA at levels of 0.2% or greater. Thus, this method will be useful both for marker-assisted selection in soybean breeding programs, and for seed purity tests in food industries.

Crystalloids found in salivary duct cysts of the human parotid gland were examined by scanning electron microscopical observations with electron probe X-ray microanalysis. The cystic spaces were filled with numerous crystalloids which had a variety of forms with slight eosinophilic and glassy appearance. Scanning electron microscopically, crystalloids were hexagonal and rhombohedral in shape, and cutting the surface showed a polycyclic structure or regular parallel lamination. By electron probe X-ray microanalysis, sulphur was the only detected element. The present study suggests that crystalloids resulted from deposition from supersaturated saliva containing sulphur containing compounds into the cystic lumen or into epithelial cytoplasm.

Experimental autoallergic sialadenitis was induced in SL/Ni mice by one or two injections of syngeneic submandibular gland homogenate emulsified with adjuvant. Light microscopically, there were marked lymphoid cell infiltration in the submandibular glands with high incidence and proliferation of duct epithelia. Furthermore complete alteration of whole glandular lobules in some cases was observed. Ultrastructurally, small and medium sized lymphocytes and plasma cells constituted a major portion of the infiltrating cells, and lymphocytes were frequently observed inside the basal lamina of ductal and acinar regions, especially observed in the small ductal region. In the aggregates of infiltrating cells, the cell remnants of salivary gland epithelia were scattered. Furthermore some of the epithelial cell remnants in aggregates of infiltrating cells could be recognized as epithelial masses which were composed of proliferated duct epithelial cells, though no typical structure of epimyoepithelial islands seen in Sjogren's syndrome was found. Anti-salivary duct antibody was detected in only one case.