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Cadmium (Cd) is as an extremely toxic metal that can contaminate agricultural soils. To reduce the risk of Cd intake in food cereals, the development of cultivars with low grain Cd concentration (GCC) is an effective countermeasure. We analyzed quantitative trait loci (QTLs) for GCC in a doubled haploid (DH) common wheat (Triticum aestivum L.) population derived from ‘Chugoku 165’ (low GCC) × ‘Chukei 10-22’ (high GCC). We found novel loci for low GCC on the short arm of chromosome 4B and on the long arm of chromosome 6B. These QTLs accounted for 9.4%–25.4% (4B) and 9.0%–17.8% (6B) of the phenotypic variance in the DH population. An association analysis with 43 cultivars identified 3 loci at these QTLs: QCdc.4B-kita, QCdc.6B-kita1, and QCdc.6B-kita2. In contrast to durum wheat and barley, no QTL was detected on the chromosomes of homeologous group 5 for heavy metal P1B-type ATPase 3. These results will contribute to marker-assisted selection for low GCC in breeding of common wheat.

The number of wheat grains is one of the major determinant of yield. Many quantitative trait loci (QTLs) and some causal genes such as GNI-A1 and WAPO-A1 that are associated with grain number per spike (GNS) have been identified, but the underlying mechanisms remain largely unknown. We analyzed QTLs for grain number and other related traits using 188 doubled haploid lines derived from the Japanese high-yield variety, Kitahonami, as a parent to elucidate the genetic mechanism determining grain number. The major QTLs for grain number at the apical, central and basal parts of the spike were identified in different chromosomal regions. We considered GNI-A1 and WAPO-A1 as candidate genes controlling grain number at the central and basal parts of the spike, respectively. Kitahonami had the favorable 105Y allele of GNI-A1 and WAPO-A1b allele, and unfavorable alleles of QTLs for grain number at the apical part of spikes. Pyramiding the favorable alleles of these QTLs significantly increased GNS without significantly reducing thousand-grain weight (TGW). In contrast, the accumulation of favorable alleles of QTLs for TGW significantly decreased GNS, whereas days to heading positively correlated with GNS. Late heading increased the spikelet number per spike, resulting in a higher GNS. Pyramiding of the QTLs for TGW and days to heading also altered the GNS. In conclusion, GNS is a complex trait controlled by many QTLs, and it is essential for breeding to design.

Wheat flours are used to produce bread, pasta, breakfast cereals, and biscuits; the various properties of these end-products are attributed to the gluten content, produced as seed storage proteins in the wheat endosperm. Thus, genes encoding gluten protein are major targets of wheat breeders aiming to improve the various properties of wheat flour. Here, we describe a novel compensating wheat-Thinopyrum elongatum Robertsonian translocation (T1AS.1EL) line involving the short arm of wheat chromosome 1A (1AS) and the long arm of Th. elongatum chromosome 1E (1EL); we developed this line through centric breakage-fusion. Compared to the common wheat cultivars Chinese Spring and Norin 61, we detected two additional 1EL-derived high-molecular-weight glutenin subunits (HMW-GSs) in the T1AS.1EL plants. Based on the results of an SDS-sedimentation volume to estimate the gluten strength of T1AS.1EL-derived flour, we predict that T1AS.1EL-derived flour is better suited to bread-making than Chinese Spring- and Norin 61-derived flour and that this is because of its greater gluten diversity. Also, we were able to assign 33 of 121 wheat PCR-based Landmark Unique Gene markers to chromosome 1E of Th. elongatum. These markers can now be used for further chromosome engineering of the Th. elongatum segment of T1AS.1EL.

Marker-assisted selection of crop plants requires DNA markers that can distinguish between the closely related strains often used in breeding. The availability of reference genome sequence facilitates the generation of markers, by elucidating the genomic positions of new markers as well as of their neighboring sequences. In 2017, a high quality genome sequence was released for the six-row barley (Hordeum vulgare) cultivar Morex. Here, we developed a de novo RNA-Seq-based genotyping procedure for barley strains used in Japanese breeding programs. Using RNA samples from the seedling shoot, seedling root, and immature flower spike, we mapped next-generation sequencing reads onto the transcribed regions, which correspond to ∼590 Mb of the whole ∼4.8-Gbp reference genome sequence. Using 150 samples from 108 strains, we detected 181,567 SNPs and 45,135 indels located in the 28,939 transcribed regions distributed throughout the Morex genome. We evaluated the quality of this polymorphism detection approach by analyzing 387 RNA-Seq-derived SNPs using amplicon sequencing. More than 85% of the RNA-Seq SNPs were validated using the highly redundant reads from the amplicon sequencing, although half of the indels and multiple-allele loci showed different polymorphisms between the platforms. These results demonstrated that our RNA-Seq-based de novo polymorphism detection system generates genome-wide markers, even in the closely related barley genotypes used in breeding programs.

Durum wheat (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 [ L. subsp. (Korn. 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.

Milling properties and flour color are essential selection criteria in soft wheat breeding. However, high phenotypic screening costs restrict selection to relatively few breeding lines in late generations. To achieve marker-based selection of these traits in early generations, we performed genetic dissection of quality traits using three doubled haploid populations that shared the high-quality soft wheat variety Kitahonami as the paternal parent. An amplicon sequencing approach allowed effective construction of well-saturated linkage maps of the populations. Marker-based heritability estimates revealed that target quality traits had relatively high values, indicating the possibility of selection in early generations. Taking advantage of Chinese Spring reference sequences, joint linkage maps of the three populations were generated. Based on the maps, multifamily quantitative trait locus (QTL) analysis revealed a total of 86 QTLs for ten traits investigated. In terms of target quality traits, 12 QTLs were detected for flour yield, and 12 were detected for flour redness (a* value). Among these QTLs, six for flour yield and nine for flour a* were segregating in more than two populations. Some relationships among traits were explained by QTL collocations on chromosomes, especially group 7 chromosomes. Ten different ideotypes with various combinations of favorable alleles for the flour yield and flour a* QTLs were generated. Phenotypes of derivatives from these ideotypes were predicted to design ideal genotypes for high-quality wheat. Simulations revealed the possibility of breeding varieties with better quality than Kitahonami.

In common wheat, the development of genotyping platforms has been hampered by the large size of the genome, its highly repetitive elements and its allohexaploid nature. However, recent advances in sequencing technology provide opportunities to resolve these difficulties. Using next-generation sequencing and gene-targeting sequence capture, 12,551 nucleotide polymorphisms were detected in the common wheat varieties ‘Hatsumochi’ and ‘Kitahonami’ and were assigned to chromosome arms using International Wheat Genome Sequencing Consortium survey sequences. Because the number of markers for D genome chromosomes in commercially available wheat single nucleotide polymorphism arrays is insufficient, we developed markers using a genome-specific amplicon sequencing strategy. Approximately 80% of the designed primers successfully amplified D genome-specific products, suggesting that by concentrating on a specific subgenome, we were able to design successful markers as efficiently as could be done in a diploid species. The newly developed markers were uniformly distributed across the D genome and greatly extended the total coverage. Polymorphisms were surveyed in six varieties, and 31,542 polymorphic sites and 5,986 potential marker sites were detected in the D genome. The marker development and genotyping strategies are cost effective, robust and flexible and may enhance multi-sample studies in the post-genomic era in wheat.

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.

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.

Several species of the genus Aegilops, wild relatives of wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) carry gametocidal (Gc) genes. Gc genes kill the gametes without themselves by causing chromosomal breakage during post-meiotic cell divisions, and therefore are strong segregation distorters. The Gc gene Gc3-C1 derived from chromosome 3C of Ae. triuncialis (2n = 4x = 28, CCUU) induces chromosomal breakage in wheat cultivar 'Chinese Spring' (CS) but not in cultivar 'Norin 26' (N26). This cultivar-specific inhibition of Gc function is caused by a suppressor gene Igc1 located on chromosome 3B of N26. Igc1 is presumed to be a modified Gc gene without breakage function because of its homoeology to Gc3-C1. Here we report the results of linkage and physical mapping of Igc1 to help elucidate the molecular mechanisms underlying Gc action. Segregation analysis of the phenotypic data in BC(1)F(1) mapping population of the cross between (CSxN26)F(1) and CS + 3C" showed a 1:1 segregation ratio indicating that Igc1 is a dominant gene. In the linkage analysis, three molecular marker loci Xgwm285, Xgwm376, and Xcfp1886 cosegregated with the Igc1 locus. Bin mapping assigned the loci Xgwm285 and Xcfp1886 to bin C-3BS1-0.33 and Xgwm376 to bin C-3BL2-0.22. Physical mapping using Gc-induced chromosomal deletion lines of chromosome 3B of N26 revealed that the Igc1 locus resides in 52.0% or 2.1% of bins C-3BS1-0.33 and C-3BL2-0.22, respectively. Pericentromeric localization of Igc1 in chromosome 3B of N26 may have a positive effect to keep the two-component system of the Gc action. Map-based cloning approach to isolate the Igc1 may be difficult because recombination is depleted in the pericentromeric region. As is shown in this study, the combination of genetic and physical mapping offers high efficiency to identify the regions where genes are located especially in regions with low levels of recombination.

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.

Rye chromosome 1R contains many agronomically useful genes. Physical dissection of chromosome 1R into segments would be useful in mapping 1R-specific DNA markers and in assembling DNA clones into contig maps. We applied the gametocidal system to produce rearranged 1R chromosomes of Imperial rye (1R(i)) added to common wheat. We identified rearranged 1R(i) chromosomes and established 55 1R(i) dissection lines of common wheat carrying a single rearranged 1R(i) chromosome. Fifty-two of the rearranged 1R(i) chromosomes had single breakpoints and three had double breakpoints. The 58 breakpoints were distributed in the short arm excluding the satellite (12 breakpoints), in the satellite (4), in the long arm (28), and in the centromere (14). Out of the 55 lines, nine were homozygous for the rearranged 1R(i) chromosomes, and the remaining lines were hemizygous. We developed 26 PCR-based EST markers that were specific to the 1R(i) chromosome, and nine of them amplified 1R(i) arm-specific PCR products without restriction-enzyme digestion. Using the nine EST markers and two previously reported 1R-specific markers, we characterized the 55 1R(i) dissection lines, and also proved that we can select critical progeny plants carrying specific rearranged 1R(i) chromosomes by PCR, without cytological screening, in 48 out of the 55 hemizygous dissection lines.

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1803-y) contains supplementary material, which is available to authorized users.

The soft winter wheat variety, Kitahonami, shows a superior flour yield in comparison to other Japanese soft varieties. In order to map quantitative trait loci (QTL) associated with the flour-yield trait, association mapping was performed using panel lines in Kitahonami’s pedigree, along with leading varieties and advanced breeding lines. Using a mixed linear model corrected for kernel types and familial relatedness, 62 marker-trait associations were identified and classified into 21 QTLs. Five out of eight QTLs tested were validated by linkage analyses using three sets of doubled haploid populations from crosses in which Kitahonami was used as a parent. Among them, QTLs on 3B and 7A chromosome showed highly significant effects and consistency across the three populations. A joint linkage map of 3B showed that the QTL on this chromosome was located at the same interval across the populations. By applying a meta-analysis approach, we have succeeded in identifying QTLs with consistent contributions to high flour yield across various genetic backgrounds.

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.

パンコムギ品種「関東107号」におけるWx-B1遺伝子の変異は，麺用コムギに適する「やや低アミロース」形質の要因であり，育種における重要な選抜指標となっている．その変異の原因がコード領域全体の欠失であることは既に報告されているが，そのゲノム上での欠失点はまだ明らかにされていない．そこで，本研究では，コムギEST情報，オオムギおよびヒトツブコムギの waxy遺伝子を含むBACクローン塩基配列情報，さらにはイネゲノム情報を利用してマーカーを作製することにより，1） Wx-B1遺伝子下流の欠失点は，終止コドンから3.3 kbから6 kbの間に存在すること，2）同欠失により， Wx-B1遺伝子の下流に存在する機能未知の遺伝子も同時に欠失していることを明らかにした．

The Japanese rice cultivar Asominori shows high bacterial leaf blight resistance (BLBR). The resistance locus is closely linked with the Ph gene on chromosome 4. Japanese rice breeders noticed that lines resistant to both bacterial leaf blight (BLB) and rice leaf blast (LB) segregated from descendants of crosses with Asominori; however, the cause of this cosegregation was unclear. In 2004, we obtained five Asominori backcrossed inbred lines (BILs). Firstly, we evaluated the resistance to LB and BLB in these BILs. The partial resistance to LB was high, similar to criterial varieties such as Ouu320 and Chubu45. The BILs were also resistant to the BLB strain T-7174 (race1). Using 21 PCR-based DNA markers, we analyzed the genotypes of the BILs on chromosome 4, where BLBR and Ph genes are located. All five BILs carried a common Asominori-derived chromosomal segment estimated to be 1336 kbp long. Secondly, segregation of BLBR and LBR was monitored using 118 F3 lines obtained from a cross between Ukei854 and Hitomebore, a BLB- and LB-susceptible cultivar. Most resulting F3 lines showing BLBR also carried LBR; however, several lines showed only BLBR or BLR, indicating that BLBR and LBR were controlled by very closely linked loci. Using RM5473-2, we surveyed the genotypes of the 118 F3 lines located on the common segment. Most F3 lines with a high LBR showed genotypes identical to Asominori. Thus, the LBR locus in Asominori was tightly linked to the BLBR locus on chromosome 4, and the LBR gene was probably located near RM5473-2.

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.

BackgroundEST-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.ResultsThe 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.ConclusionIn 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.

Oncocytes are characterized under light microscope by swollen cytoplasm with acidophilic and granular appearence and frequently by pyknotic nuclei. These cells can be found not only in salivary glands and other exocrine and endocrine organs but also in some tumors. Generally, oncocytes appear to increase in frequency with advancing age, but their genesis and biologic significance remain still obscure. In this study, the appearence and the histological pattern of oncocytes in the human labial salivary glands were systematically examined, and the possible mode of development and the significance of these cells were also discussed. One hundred cases of the labial salivary glands were randomly collected from autopsies, and serial sections were made. The designation of oncocytes was in accordance with Hamperl's description. The results were as follows;1) Oncocytes were found in 85 cases (85.0%), ranging in age from two to 93. Although oncocytes occurred in greater number in elder cases, oncocytes were found in 50-to-70% of those who was under forty. The frequency was slightly higher in females than in males. 2) The frequent presence of oncocyte was noted in the duct systems, especially in the interlobular, intralobular and main excretory ducts. 3) In both acini and ducts, oncocytes were generally gathered in small groups but also occurred singly. Therefore, all of these epithelial elements ofthe salivary glands may be able to transform into oncocytes. 4) When oncocytes were found in moderately to heavy concentration, the oncocytes stratified in papillary projection, adenomatous pattern, conglomeration microtubular structures, etc., and it is thought that these findings suggest proliferative disposition of oncocytes. The highest changes of these were found in 8 cases of 50 to 70 years old. 5) There was no definite correlation between oncocytic changes and fibrosis, atrophy with fatty replacement, regenerative process due to radiation or lymphoid cell infiltration, etc. 6) From the above-mentioned light microscopic findings of oncocytes in the human labial salivary glands, it is assumed that the occurrence of oncocytic changes have some relationship with the aging. However, oncocytic changes may not be the result of degenerative one of the salivary glands associated with aging. 7) Under electron microscopy, the cytoplasm of oncocyte was completely filled with nonoriented mitochondria of various sizes and shapes. Moreover, the cristae of mitochondria were markedly increased in number and in length, and there were no intramitochondrial granules. Organellae, other than mitochondria, were sparse.As a results of this investigation and review of various reference literature, a hypothesis is put foward that the significance of oncocytes is a manifestation trying to compensate for functional defects by an increased number of mitochondria and the surface area of menbrane. The mitochondrial proliferation may occur in the abscence of obvious functional needs. Another possibility is that the oncocytic transformation represents a “mitochondrial disease” or “mitochondriopathia” of epithelial cells. The determination of the function of oncocytes willrequire more specific physiological and biochemical studies. And it must be decided whether oncocytes occurring in normal salivary glands and tumors are same cytological characteristics or not.

ヒトの耳下腺は一般に漿液性の腺として認められているが, 新生児で導管部に粘液細胞の存在することは知られている。また, 唾液腺に原発する粘表皮腫は大・小の唾液腺のうちでも耳下腺に最も多い。正常のヒト耳下腺導管上皮に時に粘液細胞が存在することから, 粘表皮腫は唾液腺の導管上皮に由来するのであろうと考えられている。しかし, ヒト耳下腺における粘液細胞の出現頻度や分布状態などを詳細に系統的に検索した報告はほとんどみられない。本研究では, 剖検例より得た耳下腺380例について, 耳下腺組織内にみられた粘液細胞の出現頻度や出現部位などについての検索を行った。粘液細胞は検索例全体の約30%にみられ, 新生児と高年者に比較的多かった。出現部位は導管系, ことに葉間と小葉間の導管部に比較的多くみられた。また, 導管系で粘液細胞の出現をみた例の約72%で, 粘液細胞出現部導管周囲にリンパ性細胞浸潤をみたが, 基礎疾患との間には特別の関係は見出し得なかった。

HeLa S3細胞を用いて細胞増殖と細胞電気泳動度の相関関係およびCon Aの細胞電気泳動度に及ぼす影響について検討した。培養開始後日数と細胞電気泳動度の関係では, 培養開始後細胞増殖の最も著明な3～4日に細胞電気泳動度も最高値を示し, 両者の推移はほぼ平行関係にあった。Con Aで処理すると細胞電気泳動度は5μg/ml, 10μg/mlの低濃度では増加するが, 20μg/ml以上の高濃度になると減少し.2相性の変化を示した。またこのCon Aによる細胞電気泳動度の反応性変動の程度は, 再生増殖期にある細胞に特に著明であることが判明した。

Multinodular hyperplastic foci of oncocytes found in mouse submandibular glands were examined ultrastructurally, and their morphogenesis discussed. The oncocytic foci showed lobular architecture surrounded by myoepithelial cells, and tubular formations were frequently present. The cytoplasm of the oncocytes was distended by tightly-packed and non-oriented mitochondria which varied in size and shape. Furthermore, varying numbers of electron-dense fine granules, which were remnants of secretory granules of granular duct cells, were scattered in the cytoplasm. Within the salivary gland parenchyma neighboring the hyperplastic foci of oncocytes, intermediate-type cells between oncocytes and normal salivary gland epithelia were found. The result of the present study shows that the oncocytic change has a tendency to be more frequent in the granular ducts and less in the striated ducts, and that some oncocyte-transformed cells proliferate to form nodular hyperplastic foci.

Summary About 160 cases of odontogenic tumor were studied histologically and a classification of the odontogenic tumor was presented. No clear-cut example of melano-ameloblastoma, odontogenic sarcoma, ameloblastic neurinoma, dentinoblastoma was observed in this study. About a half of the ameloblastoma belonged to Type III. Ameloblastoma of the maxilla was principally Type I. More differentiated types were found mostly in the younger patients. For the reason of a predirection of the ameloblastoma in the mandibular molar region, the developmental relationship between tooth band and jaw bone in this region should be considered. Cyst in the parenchyma might be produced in three ways; keratinous degeneration of the squamous epithelial portion, fine granular lipo-protein or glucolipid degeneration of the epithelium without keratinous changes, and hydropic degeneration of the stellate reticulum. Hyalinous halo surrounding nests of epithelium indicate a simple hyalinous change of the stromal tissue adjacent to the basement membrane or true thickening of the basement membrane like membrana preformativa. Cyst formation of the stroma may be influenced not merely by circulatory disturbances of the stroma, but by some action on the adjoining epithelium. So-called adeno-ameloblastoma should be considered as a variant of ameloblastoma. Its adenomatous structure may be formed from atypical growth of the epithelium of dental origin and not a true glandular structure. Two cases of malignant ameloblastoma were observed in this series. In one instance metastases to the cervical lymphnodes and the upper lip were histologically demonstrated. When we diagnose the odontogenic mesodermal tumors, by means of only histological examination, we should have careful consideration in various instance. Three types of the odontogenic mixed tumor represented various differentiating grade of thetum or tissues. Ameloblastic hemangioma was supposed to be a variant of ameloblastoma with a dilatation of the stromal blood vessels and not with a true hemangioma tissue.

The ultrastructural changes in the terminal secretory units of the labial salivary glands, in which was demonstrated light microscopically the characteristic appearance of Sjogren's syndrome, were investigated in 12 cases. A small number of lymphocytes appeared interspersed in the dilated interepithelial spaces. Acinar cells showed regressive changes of various degrees, i. e. in the mature acinar cells the secretory granules were irregularly shaped and often found fused to each other while the intracytoplasmic organellae were swollen and destroyed in the immature acinar cells. These findings suggested that both the productive and secretory processes of the secretory granules in the acinar cells were disturbed. And finally the acinar cells appeared completely destroyed and their whole intracytoplasmic contents spilled into the acinar lumen. In some of the terminal units which consisted of immature acinar cells, the acinar cells were arranged irregularly and showed a syncytial appearance. This finding might suggest the socalled tubular metaplasia or canalicular regression. There was no regenerative appearance of the acinar cells. The myoepithelial cells, located in the basal portion of the acinar cells, were more resistant to the inflammatory changes than the acinar cells, however, pyknosis of the cytoplasm, formation of many thin cytoplasmic processes and increase in the number of pinocytotic vesiclae were found in the advanced stage. There were no proliferative changes in the myoepithelial cells.

Light and electron microscopic studies of ameloblastoma were reviewed. The 21 cases of ameloblastoma examined were classified into 12 cases of the plexiform type and nine of the follicular type. The average age of the patients with the plexiform type was 25.3 years, while that of those with the follicular type was 54.4 years. Histologically, in the follicular type, the tumor cells consisted of two cell types, central polyhedral and star-shaped cells resembling the stellate reticulum and peripheral cuboidal and columnar cells similar to the inner enamel epithelium. The resemblance between the tumor follicle and enamel organ was confirmed electron microscopically. In the plexiform type, however the tumor cells did not show two cell types, but resembled squamous epithelium. Electron microscopically, all cells of the tumor strands had relatively numerous bundles of tonofilaments and were joined together by desmosomes. Differentiation of tumor cells to squamous epithelium less evident than in normal surface epithelium. We speculate that these histological differences between plexiform and follicular types represent the differentiation tendency of the remnant of the dental lamina at the time of neoplastic transformation. What decides the histological pattern is unknown but age may be a factor. Central epidermization with keratinization or microcytic changes was frequently seen in the follicular type. Keratinization and microcystic changes rarely occurred in the plexiform type. We do not believe that these changes are a form of involution or result from multipotentiality of the tumor cells.

An autopsy case of fatal strongyloidiasis in a patient with pemphigus vulgaris receiving long-term administration of corticosteroids was presented. There might exist a subclinical stage of 26 years between the acquisition of infection and the appearance of its severe symptoms. It is suggested that the long-term administration of corticosteroids and debilitating diseases increase the pathogenecity of Strongyloides stercoralis by autoinfection, and that fatal strongyloidiasis could be a possible complication of treatment with corticosteroids.