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Cooked bean hardness is an important trait for the processing of soybean products such as nimame, natto, miso, and soy sauce. Previously, we showed that cooked bean hardness is primarily affected by the pectin methylesterase gene Glyma03g03360, and that calcium content has a secondary effect on this trait. To establish a simple and timely method for the evaluation of cooked bean hardness, primers of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were designed to detect a single-nucleotide polymorphism of Glyma03g03360 and subsequently used to evaluate three soybean progeny lines. The determined genotypes were compared to those identified using the cleaved amplified polymorphic sequence (CAPS) method. Seven out of 284 lines presented different genotypes, which were determined using the two methods: A genotypes were incorrectly assigned as heterozygous by CAPS, suggesting that ARMS-PCR is more reliable. Glyma03g03360 genotypes could be used to evaluate cooked bean hardness, except for intermediate values. Cooked bean hardness within the same genotype groups was significantly correlated with calcium contents. These findings indicate that ARMS-PCR is useful for a marker-assisted selection of soybean with soft-cooked beans and that calcium content may be used for additional selection.

Whole-genome re-sequencing is a powerful approach to detect gene variants, but it is expensive to analyse only the target genes. To circumvent this problem, we attempted to detect novel variants of flowering time-related genes and their homologues in soybean mini-core collection by target re-sequencing using AmpliSeq technology. The average depth of 382 amplicons targeting 29 genes was 1,237 with 99.85% of the sequence data mapped to the reference genome. Totally, 461 variants were detected, of which 150 sites were novel and not registered in dbSNP. Known and novel variants were detected in the classical maturity loci—E1, E2, E3, and E4. Additionally, large indel alleles, E1-nl and E3-tr, were successfully identified. Novel loss-of-function and missense variants were found in FT2a, MADS-box, WDR61, phytochromes, and two-component response regulators. The multiple regression analysis showed that four genes—E2, E3, Dt1, and two-component response regulator—can explain 51.1–52.3% of the variation in flowering time of the mini-core collection. Among them, the two-component response regulator with a premature stop codon is a novel gene that has not been reported as a soybean flowering time-related gene. These data suggest that the AmpliSeq technology is a powerful tool to identify novel alleles.

The common cutworm (CCW, Spodopteraab litura Fabricius) is one of the pests that most severely infect soybean (Glycine max L. Merr.). In a previous report, quantitative trait loci (QTL) analysis of CCW resistance using a recombinant inbred line derived from a cross between a susceptible cultivar ‘Fukuyutaka’ and a resistant cultivar ‘Himeshirazu’, identified two antixenosis resistance QTLs, CCW-1 and CCW-2. To reveal sequence variation between the aforementioned two cultivars, whole genome resequencing was performed using Illumina HiSeq2000 (75,632,747 and 91,540,849 reads). The generated datasets can be used for fine mapping and gene isolation of CCW-1 and CCW-2 as well as for revealing more detailed genetic differences between ‘Fukuyutaka’ and ’Himeshirazu’ .

Soybeans planted in early to mid-June (early) are less affected by rainfall during rainy season than those conventionally planted in early to mid-July in southwestern Japan. Also, narrow row cultivation is expected to increase soybean yield and save labor for inter-tillage and ridging. Field experiments were performed in 2014 and 2015 to test the effect of plant density (high, middle, and low) under early planting condition on growth, yield, and several agronomical traits of Sachiyutaka A1 and three new genotypes (Sakukei 155, Kanto 127 and Shikoku 15). Early planting was performed in mid- to late June, even though rainy season started in early June. Higher plant densities produced 13% greater yield than low plant density through an increase in biomass accumulation, especially at R5. Among yield components, only pods m−2 was significantly and positively correlated with yield, indicating that an increase in pods m−2 led to a greater yield with higher plant densities. The yields of Sachiyutaka A1 were relatively stable for two years, but the lodging resistance should be further improved. Shikoku 15 had greater yield potential and lodging resistance, but its resistance to damping-off disease should be improved. Sakukei 155 with medium plant density produced relatively high and stable yield with less lodging. Although the yield of Kanto 127 fluctuated between experimental years, this genotype showed higher yield potential in higher plant densities with less lodging in 2015. Thus, Sakukei 155 and Kanto 127 with high or medium density may be suitable for early planting in this region.

Using progeny of a cross between Japanese soybean Enrei and Chinese soybean Peking, we developed a high-density linkage map and chromosomal segment substitution lines (CSSLs). The map consists of 2,177 markers with polymorphism information for 32 accessions and provides a detailed genetic framework for these markers. The marker order on the linkage map revealed close agreement with that on the chromosome-scale assembly, Wm82.a2.v1. The differences, especially on Chr. 5 and Chr. 11, in the present map provides information to identify regions in the genome assembly where additional information is required to resolve marker order and assign remaining scaffolds. To cover the entire soybean genome, we used 999 BC3F2 backcross plants and selected 103 CSSLs carrying chromosomal segments from Peking in the genetic background of Enrei. Using these low-genetic-complexity resources, we dissected variation in traits related to flowering, maturity and yield into approximately 50 reproducible quantitative trait loci (QTLs) and evaluated QTLs with small genetic effects as single genetic factors in a uniform genetic background. CSSLs developed in this study may be good starting material for removing the unfavourable characteristics of Peking during pre-breeding and for isolation of genes conferring disease and stress resistance that have not yet been characterized.

In southwestern Japan, soil water fluctuations from flooding to drought cause unstable soybean yields. Water table management (WTM) with sub-irrigation/drainage systems will overcome the soybean yield instability by inhibiting these fluctuations. Narrow row cultivation is expected to increase soybean yields. The effects of WTM and row width on soybean growth and yield in this region are not clear. We evaluated the effects of WTM with sub-irrigation/drainage systems and row widths (35 or 70 cm) on the growth and yield of one conventional (tall main stem) and two newly developed (short main stem) soybean cultivars. The WTM consisted of (1) fluctuation of the water table between the natural water table depth and that at 30 cm depth according to the growth stage and weather conditions, especially rainfall events (newly developed); (2) maintaining the water table at a 30 cm constant depth throughout the growth period (recommended in Japan); and (3) the natural water table with an underdrain (control). No significant interaction was observed between the WTM and cultivar or row width treatment, indicating that cultivars and row width treatments responded similarly to WTM. WTM 1 and 2 decreased the soybean yield by approx. 5% when the natural water table depth in control existed at 50–60 cm depths throughout the growing period, indicating that the natural water table depth in control was near optimum for soybean growth and yield. Before performing WTM, therefore, the natural water table depth should be measured and considered. The combination of newly developed cultivars with narrow rows had similar or greater yields than conventional cultivation (cultivar and row width), due mainly to an increase in pods m−2 and a decrease in yield loss without severe lodging. Thus, yield potential in southwestern Japan could be increased by narrow row cultivation, but cultivars with short main stem lengths should be cultivated.

Seed coat cracking after soaking (SCAS) and after cooking (SCAC) are unfavorable traits associated with soybeans for food uses, such as cooked and seasoned beans (nimame) and fermented steamed beans (natto) because they cause an inferior appearance of the products and clogging of the production lines. The variation and causes of SCAS and SCAC among cultivars have not yet been clarified, but if they are determined genetically, genetic modification could be possible. Cultivars showed considerable variations in SCAS and SCAC. Significantly positive seasonal correlations of SCAS (Spearman’s rank correlation coefficient, ρ = 0.518) and of SCAC (ρ = 0.681) were observed among recombinant inbred lines (RILs) derived from the cross between cultivars Nattoshoryu and Hyokeikuro 3. Quantitative trait locus (QTL) analyses and statistical analyses using generalized linear models showed that QTLs for SCAS (qSCAS1, qSCAS2, and qSCAS3) and SCAC (qSCAC1 and qSCAC2) were located on chromosomes 4 (linkage group [LG]: C1), 6...

The common cutworm (CCW, Spodopteraab litura Fabricius) is one of the pests that most severely infect soybean (Glycine max L. Merr.). In a previous report, quantitative trait loci (QTL) analysis of CCW resistance using a recombinant inbred line derived from a cross between a susceptible cultivar ‘Fukuyutaka’ and a resistant cultivar ‘Himeshirazu’, identified two antixenosis resistance QTLs, CCW-1 and CCW-2. To reveal sequence variation between the aforementioned two cultivars, whole genome resequencing was performed using Illumina HiSeq2000 (75,632,747 and 91,540,849 reads). The generated datasets can be used for fine mapping and gene isolation of CCW-1 and CCW-2 as well as for revealing more detailed genetic differences between ‘Fukuyutaka’ and ’Himeshirazu’ .

Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

Radiocesium is an extremely harmful radionuclide because of its long half-life; it is important to reduce its transfer from contaminated soil into crops. Here we surveyed genetic variation for seed cesium (Cs) concentration in soybean mini-core collections representing large genetic diversity. The collections grown over 3 years in rotational paddy fields exhibited varying seed Cs concentrations with significant year-to-year correlations, although the phenotypic stability of Cs concentration was lower than that of the congeners potassium (K) and rubidium (Rb). Although Cs is supposedly accumulated in plants via the K transport system, there was no apparent relationship between Cs and K concentrations, whereas a clear positive correlation was observed between Cs and Rb concentrations. Cs and K concentrations in seed showed slightly positive and negative correlations, respectively, with days to flowering. We selected several high or low Cs accumulator candidates on the basis of the 3 years of seed concentration data. These two groups showed significantly different seed Cs concentrations in another field. The differences could not be explained by flowering time alone. These results suggest that genetic variation for seed Cs concentration is present in soybean germplasm and would be useful for breeding low Cs-accumulating varieties.

Hardness of cooked soybeans [Glycine max (L). Merr.] is an important attribute in food processing. We found one candidate gene, Glyma03g03360, to be associated with the hardness of cotyledons of cooked soybeans, based on a quantitative trait locus and fine-scale mapping analyses using a recombinant inbred line population developed from a cross between two Japanese cultivars, "Natto-shoryu" and "Hyoukei-kuro 3". Analysis of the DNA sequence of Glyma03g03360, a pectin methylesterase gene homologue, revealed three patterns of mutations, two of which result in truncated proteins and one of which results in an amino acid substitution. The truncated proteins are presumed to lack the enzymatic activity of Glyma03g03360. We classified 24 cultivars into four groups based on the sequence of Glyma03g03360. The texture analysis using the 22 cultivars grown in different locations indicated that protein truncation of Glyma03g03360 resulted in softer cotyledons of cooked soybeans, which was further confirmed by texture analysis performed using F2 populations of a cross between "Enrei" and "LD00-3309", and between "Satonohohoemi" and "Sakukei 98". A positive correlation between hardness and calcium content implies the possible effect of calcium binding to pectins on the hardness of cooked soybean cotyledons.

In order to determine the time for terminating Ipomoea coccinea control in Japanese soybean-growing systems, the relationship between the relative photosynthetic photon flux density at the emergence time of I. coccinea seedlings and their fate in competition with a Japanese determinate soybean cultivar, “Hatayutaka”, was investigated. A 2 year field study was conducted, in which I. coccinea was seeded at 0, 5 and 7 weeks after seeding of the soybean in 2010 and once per week between 0 and 7 weeks after seeding in 2011, while soybean was seeded in six plots at three different seeding times and two different row-spacing arrangements with the same intrarow spacing. The results showed that the I. coccinea seedlings that emerged at a relative photosynthetic photon flux density of

The firmness of whole beans and of separated embryos and testas from 20 soybean cultivars grown in the same field conditions was estimated after 40 min cooking with AACC International Approved Method 56-36.01. There were considerable variations in the firmness of whole beans, embryos, and testas among cultivars. Testas were much firmer than embryos because of their leathery texture and resistance at slots in the shear cell during compression. For soft cultivars, the firmness after 10 min cooking was also measured, and an apparent first-order reaction kinetics model was applied to estimate the initial firmness at 0 min cooking and the softening rate constant from firmness data after 10 and 40 min cooking. The estimated initial firmness at 0 min cooking and softening rate constant of embryos and testas varied widely among seven cultivars, and these two parameters were considered to be controlled by different genetic mechanisms, because no correlation was found between the parameters. The softening ...

Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.

Boiled seed hardness is an important factor in the processing of soybean food products such as nimame and natto. Little information is available on the genetic basis for boiled seed hardness, despite the wide variation in this trait. DNA markers linked to the gene controlling this trait should be useful in soybean breeding programs because of the difficulty of its evaluation. In this report, quantitative trait locus (QTL) analysis was performed to reveal the genetic factors associated with boiled seed hardness using a recombinant inbred line population developed from a cross between two Japanese cultivars, ‘Natto-shoryu’ and ‘Hyoukei-kuro 3’, which differ largely in boiled seed hardness, which in ‘Natto-shoryu’ is about twice that of ‘Hyoukei-kuro 3’. Two significantly stable QTLs, qHbs3-1 and qHbs6-1, were identified on chromosomes 3 and 6, for which the ‘Hyoukei-kuro 3’ alleles contribute to decrease boiled seed hardness for both QTLs. qHbs3-1 also showed significant effects in progeny of a residual heterozygous line and in a different segregating population. Given its substantial effect on boiled seed hardness, SSR markers closely linked to qHbs3-1, such as BARCSOYSSR_03_0165 and BARCSOYSSR_03_0185, could be useful for marker-assisted selection in soybean breeding.

A comparative proteomic study was performed to unravel the protein networks involved in cadmium stress response in soybean. Ten-day-old seedlings of contrasting cadmium accumulating soybean cultivars-Harosoy (high cadmium accumulator), Fukuyutaka (low cadmium accumulator), and their recombinant inbred line CDH-80 (high cadmium accumulator) were exposed to 100 μM CdCl(2) treatment for 3 days. Root growth was found to be affected under cadmium stress in all. Varietal differences at root protein level were evaluated. NADP-dependent alkenal double bond reductase P1 was found to be more abundant in low cadmium accumulating Fukuyutaka. Leaf proteome analysis revealed that differentially expressed proteins were primarily involved in metabolism and energy production. The results indicate that both high and low cadmium accumulating cultivars and CDH-80 share some common defense strategies to cope with the cadmium stress. High abundance of enzymes involved in glycolysis and TCA cycle might help cadmium challenged cells to produce more energy necessary to meet the high energy demand. Moreover, enhanced expressions of photosynthesis related proteins indicate quick utilization of photoassimilates in energy generation. Increased abundance of glutamine synthetase in all might be involved in phytochelatin mediated detoxification of cadmium ions. In addition, increased abundance of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase, catalase, ensures cellular protection from reactive oxygen species mediated damages under cadmium stress. Enhanced expression of molecular chaperones in high cadmium accumulating cultivar might be another additional defense mechanism for refolding of misfolded proteins and to stabilize protein structure and function, thus maintain cellular homeostasis.

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.

Phytophthora stem and root rot, caused by Phytophthora sojae, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.], and the incidence of this disease has been increasing in several soybean-producing areas around the world. This presents serious limitations for soybean production, with yield losses from 4 to 100%. The most effective method to reduce damage would be to grow Phytophthora-resistant soybean cultivars, and two types of host resistance have been described. Race-specific resistance conditioned by single dominant Rps (“resistance to Phytophthora sojae”) genes and quantitatively inherited partial resistance conferred by multiple genes could both provide protection from the pathogen. Molecular markers linked to Rps genes or quantitative trait loci (QTLs) underlying partial resistance have been identified on several molecular linkage groups corresponding to chromosomes. These markers can be used to screen for Phytophthora-resistant plants rapidly and efficiently, and to combine multiple resistance genes in the same background. This paper reviews what is currently known about pathogenic races of P. sojae in the USA and Japan, selection of sources of Rps genes or minor genes providing partial resistance, and the current state and future scope of breeding Phytophthora-resistant soybean cultivars.

β-Conglycinin, a major seed protein in soybean, is composed of α, α′, and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α′, and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, ‘QT2’, lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar ‘Fukuyutaka’, ‘QY7-25’, (its near-isogenic line carrying the Scg-1 gene), and the F2 population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

Significant inter-cultivar differences of soybean seed cadmium (Cd) concentrations arise from the inter-cultivar differences in root Cd accumulation ability. The Cd concentration in the shoots of plants at the vegetative stage is already controlled by the roots Cd concentration in the same way that it determines seed Cd concentration. Based on these results we conjectured that there is no need to wait until the full maturity stage because the inter-cultivar difference in seed Cd concentration can be predicted from the Cd concentration in the shoots of seedlings. To test this theory, we cultivated 150 cultivars/lines to the harvest stage in a field not contaminated with Cd and measured seed Cd concentration. We also planted seeds in pots filled with contaminated soil, cultivated them for 3 weeks, and measured the Cd concentration of the seedling obtained at the 5th-node (V5) stage when the 4th trifoliolate leaf had expanded. The 150 cultivars/lines were roughly divided into 2 groups based on the relationship between these 2 Cd concentrations. One group was cultivars in which seedlings and seeds both had low Cd concentrations (low Cd accumulation group, n = 129), and the other group was the opposite (high Cd accumulation group, n = 21). Further, when we predicted seed Cd concentration using the ratio of Cd and Zn concentrations in seedlings, we were able to clearly divide the 2 groups with no overlap. Measuring Cd/Zn in seedlings therefore makes it possible to select cultivars with low Cd accumulation tendency readily, without waiting to harvest the seeds. Additionally, by investigating genealogies we found that varieties in the high-Cd accumulation group were descended from certain cultivars such as Harosoy.

Cadmium (Cd) is a pollutant heavy metal that is naturally present in soils and highly toxic to biological organism even at low concentrations. Significant differences were observed among soybean [Glycine max (L.) Merr.] cultivars in the uptake of Cd by root and its posterior translocation to the shoot and grain. The aim of this research was to identify quantitative trait loci (QTLs) for seed Cd concentration in soybean. A single population derived from a cross between the cultivars Harosoy and Fukuyutaka, which differ in seed Cd levels, were used for QTL analysis. The population consisted of 93 F 5:6 recombinant inbred lines (RILs), 93 F 6:7 RILs, 93 F 7:8 RILs, and 100 F 6:7 near-isogenic lines (NILs). A total of 220 simple sequence repeat (SSR) markers were genetically mapped on the 93 F 5:6 RILs population and clustered into 22 molecular linkage groups (MLGs) which covered a genetic distance of approximately 2400 centimorgans (cM). Analysis revealed one major QTL (cd1) on Chromosome 9 (MLG K) associated with seed Cd concentration. The cd1 was stable across years-generations and accounted for 82, 57, and 75% of the genetic variation in the RILs populations tested in the field. Effect of cd1 was verified by means of a NIL population developed from the F 5:6 RILs. The followup survey results indicated that the novel SSR markers Gm09: 4770663 and Gm09: 4790483 assigned to MLG K seemed to be suitable markers for selecting genotypes with low seed Cd concentration in soybean.

一般にダイズは，自殖率が極めて高い作物であるが，開花期前に低温に遭遇すると，花粉の形成不良による不稔が原因となり他殖率が増大する可能性が指摘されている．そこで本研究では，開花期前の低温処理が自然交雑率（他殖率）に及ぼす影響を調査した．ダイズ品種「青丸くん」の種子の子葉色は緑色であるが，子葉色が黄色の品種と交雑すると，交雑種子（F1）の子葉色は黄色になるので，これを交雑判定に利用した．開花期約1週間前の 「青丸くん」を人工気象室に入れ，8～15℃で7日間低温処理を施した．処理個体および対照個体を圃場の花粉源となるダイズのそばに設置し，自然交雑率を調査した．その結果，対照区の交雑率は0～0.18％に対して低温区では0.1～0.62％で，開花期前の低温処理は交雑率を著しく増大させた．また，花粉を媒介するミツバチの巣箱設置の効果も調査したところ，交雑率はミツバチ設置区では0.21％で非設置区の0.07％よりも高かった．本研究により，開花期前の低温がダイズの交雑率を増大させる一因であることが確かめられた．

A major quantitative trait locus (QTL) controlling pod dehiscence (shattering) in soybean, designated qPDH1, has previously been identified using progeny of shattering-resistant cultivars derived from a Thai cultivar, SJ2. The QTL was located near a simple sequence repeat marker, Sat_366, on linkage group J. To determine whether shattering-resistance genes originating from different resources are located at qPDH1 in general, we conducted genetic analysis using DNA markers for several populations. In an F2 population derived from a cross between a shattering-susceptible cultivar, Toyomusume, and a shattering-resistant cultivar, Harosoy, a major QTL for pod dehiscence was identified in the region near qPDH1, which was confirmed in the progeny of F4:5 populations. A major QTL was identified near qPDH1 also in F2 populations derived from crosses including Wasekogane and Kariyutaka as shattering-resistant parents. The heterozygous genotypes at the QTL showed high degrees of pod dehiscence, suggesting that shattering resistance behaves as a nearly recessive trait. In F2 populations derived from crosses between shattering-resistant cultivars, heterozygous genotypes at the Sat_366 locus were shattering-resistant. These results suggest that shattering-resistant cultivars harbor recessive shattering-resistance allele(s) at qPDH1 regardless of their origin and that molecular markers near qPDH1 could be used for marker-assisted selection for shattering resistance in soybean.

Pod dehiscence (shattering) is a major source of yield loss in mechanical harvest of soybean. To develop a marker-assisted selection system for a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1, we confirmed the usefulness of flanking markers and the effect of qPDH1 under different genetic backgrounds. The progeny of a residual heterozygous line for the genomic region around qPDH1 was screened for four flanking markers to obtain various recombinants in the vicinity of the QTL. The analysis of the relationship between the pod dehiscence degree and the graphical genotype of these lines confirmed the presence of qPDH1 in the region between the SSR markers, Sat_366 and Sat_093, on linkage group J. At these marker loci, the alleles from a Thai cultivar, SJ2, the donor of the shattering resistance, were inherited by most of the shattering-resistant, SJ2-derived cultivars and were distinct from those of the shattering-susceptible cultivars tested. The effect of the allele from SJ2 at qPDH1 was confirmed by association tests under four genetic backgrounds derived from crosses with three susceptible cultivars in the northern to southwestern regions of Japan and a susceptible accession of Indonesian origin at three locations. These results suggest that the allele from SJ2 at qPDH1 and the linked markers could be widely used for the improvement of the shattering resistance in soybean.

We aimed to detect the allergen proteins in food materials using recently developed near-infrared fluorescent probes. Sensitivities of this method were comparable to chemiluminescence detection methods, which are known to be sensitive. In addition, the sensitivities of this near-infrared fluorescent method were at least 10-50-times higher than those of the conventional visible fluorescent methods using Cy3 and Cy5 dyes. This method was effectively applicable to immunoblotting, dot-blotting and plate-assay (direct FLISA : fluorescence-linked immunosorbent assay) with ELISA plate. Allergen levels of the food sample were quantified by standard curves using standard allergen protein using the dot-blotting technique. This highly sensitive detection system also provided multiple detections of different allergens for different antibodies and dyes with distinct properties of wavelength. This enables high-throughput screening of characteristic allergen contents of target food materials, or cultivars. Generally, allergen proteins are recognized by patient's serum IgE. Therefore, we tried to detect patient's IgE-binding proteins, the putative allergens in foodstuffs. In this detection system, it was possible to detect IgE-binding proteins with sensitivity almost equivalent to a chemiluminescent detection system. Taken together, it was shown that this novel detection system was an effective technique for the sensitive detection and screening of food allergens.

To examine the capacity for plant regeneration through somatic embryogenesis, Japanese soybean cultivars were investigated by 3-step-somatic embryogenesis: 1) induction of somatic embryos (SEs) from immature cotyledons, 2) proliferation of SEs in liquid medium and 3) differentiation of SEs into cotyledon-stage embryos. At each step, properties of the cultures varied among cultivars. Notably, significant differences were observed on 1) efficiencies of SE induction, 2) compactness of individual SEs during proliferation, and 3) yields of differentiated embryos (numbers of cotyledon-stage embryos differentiated from 100 mg globular SEs). We found that the compactness of SEs in liquid medium was very important factor for the recovery of well differentiated embryos. In fact, Yuuzuru and Yumeyutaka which indicated high ratios of compact SEs at proliferation step showed high yields of well differentiated embryos. The abilities of these Japanese cultivars to yield cotyledon-stage embryos were superior or comparable to North American cultivar 'Jack', which is used as one of the most preferable genotypes for somatic embryogenesis, indicating their high potentials for plant regeneration.

Seed storage protein β-conglycinin of soybean contains major allergens in its subunits, and displays a lower amino acid score and lower functional gelling properties than glycinin. Therefore, decrease in the content of β-conglycinin is one of the objectives of soybean breeding programs. A β-conglycinin-deficient mutant QT2 was identified from a wild soybean in Kumamoto prefecture, and the phenotype was found to be controlled by a single dominant gene Scg-1 (Suppressor of β-conglycinin). Fukuyutaka and a near-isogenic line of Fukuyutaka, QY7-25 harboring the Scg-1 gene were used for development of DNA markers associated with β-conglycinin deficiency. Ten single nucleotide polymorphisms (SNPs) in the β subunit genes were detected between Fukuyutaka and QY7-25. Two β subunit gene loci were found to cosegregate with β-conglycinin deficiency with the DNA marker based on SNPs in a F2 population derived from a cross between Fukuyutaka and QY7-25. The DNA marker also enabled to detect polymorphisms between QY7-25 and major soybean cultivars and could be used as a practical tool for the introduction of Scg-1 gene into soybean. The chromosome region associated with β-conglycinin deficiency was located on linkage group I of a soybean genetic linkage map with the developed marker using a F2 population from the parents, Misuzudaizu and Moshidou Gong 503.

大豆タンパク質のSS結合をジチオスレイトールで還元し,蛍光色素モノブロモバイメイン(mBBr)で標識し,電気泳動で解析した.グリシニンの酸性サブユニット,塩基性サブユニット,Bowman-Birkプロテアーゼインヒビターに由来する主要バンドが見出された.二次元電気泳動上のスポットのうち,17個のポリペプチドがmBBr蛍光標識で検出された.国産大豆33品種について,mBBr標識されたバンドの蛍光強度と,豆腐の物性の関係を調べた.グリシニン塩基性サブユニットおよび酸性サブユニットが,それぞれ相関係数0.69および0.39で豆腐の破断応力と正の相関があったが,Bowman-Birkプロテアーゼインヒビターには豆腐破断応力との相関関係が見られなかった.豆腐のゲル形成にはグリシニンの塩基性サブユニット成分が寄与するものと推定された.

The relationship between the protein content of soybean seeds and the consistency of tofu was examined for six Japanese soybean varieties, Enrei, Fukuyutaka, Sachiyutaka, Ayakogane, Hatayutaka and Tachinagaha. The seed protein content was estimated by determining the nitrogen content using the Dumas method. Tofu was prepared from a raw homogenate of water-soaked soybeans by heating and by the addition of MgCl_2 as a coagulant. The tofu consistency was evaluated by measuring the breaking stress of tofu curd using a Creep meter. The breaking stress of tofu increased when the concentrations of MgCl_2 in soymilk increased above 0.20%. The breaking stress reached a maximum value at concentrations of around 0.40%, with differences among soybean varieties and cultivation conditions of the soybeans. There was a significant positive correlation (r = 0.87) between the maximum breaking stress of tofu and the seed protein content for the six varieties. In contrast, the breaking stress of tofu prepared with 0.25% MgCl_2 did not show a significant correlation (r = 0.27) with the seed protein content for the six varieties but was significantly correlated (r = 0.52), when the data of Sachiyutaka were excluded. Fukuyutaka and Ayakogane required a lower MgCl_2 concentration for the maximum breaking stress of tofu than Sachiyutaka, Enrei, Tachinagaha and Hatayntaka, which required a MgCl_2 concentration above 0.40% for the maximum breaking stress of tofu. Especially, Sachiyutaka required the highest MgCl_2 concentration, 0.45% on the average, for the maximum breaking stress of tofu among the six varieties. Sachiyutaka-tofu showed the lowest breaking stress on the average at a concentration of 0.25% MgCl_2, which is the concentration generally used by manufacturers, in spite of its high content in seed protein. In contrast, Fukuyutaka required the lowest MgCl_2 concentration, 0.34% on the average, for the maximum breaking stress and the highest breaking stress of tofu prepared with 0.25% MgCl_2. That is one of reasons why manufacturers prefer to use Fukuyutaka for producing tofu. Concentration of a coagulant for the maximum breaking stress as well as seed protein content could become criteria for quality evaluation of soybeans for tofu processing.

Oxygenation of lipids during the processing soybeans affects the flavor properties of soy products. We prepared tofu under anaerobic conditions and then evaluated its sensory properties and the compositions of volatiles and oxidized lipids. Anaerobic processing resulted in tofu with less intense richness (kokumi) concomitant with reductions in the amounts of oxidized lipids and volatile compounds.

Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world’s leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.

The QT2 Iine, which lacks β-conglycinin in the seed storage protein, was detected from our wild soybean germplasm collection. Although induced mutant lines lacking β-conglycinin were reported to be non viable in previous studies, the QT2 Iine grew normally and produced successive generations. To distinguish it from the non viable β-conglycinin-lacking line, this pheno-type was referred to as the “QT2-type”. In these studies, we investigated the mode of inheritance of the QT2-type and the genetic relationships between the QT2-type and some other soybean storage proteins. In spite of the fact that β-conglycinin was composed of three major subunits, genetic analysis of the QT2 Iine indicated that the QT2-type was controlled by one single dominant gene. The new genetic symbol of Scg was assigned to the QT2 type in this report. Furthermore, the mode of inheritance of the QT2-type was independent of that of the α'-subunit of β-conglycinin and seed lipoxygenases. The QT2-type hybrids, even without the presence of seed lipoxygenases, did not show any physiological abnormalities. These results indicate that the QT2 Iine is a suitable gene source to breed soybean varieties lacking β-conglycinin.

Hydroperoxide and n-hexanal production by lipoxygenase action is an important problem in the effective use of soybean seeds for food ingredients. We examined here the additional effect of unsaturated fatty acids on the 1,3-diethyl-2-thiobarbituric acid (DETBA) value (index for hydroperoxide production) and n-hexanal production with two normal soybean cultivars; three mutants partially lacking lipoxygenase and containing only L-1, L-2, or L-3; and one mutant completely lacking lipoxygenase (L0). Among the soybeans tested, the L0 soybean had the lowest levels of both DETBA value and n-hexanal production. The low DETBA values were maintained over a wide range of pH (4−10) and temperature (4−70 °C) and were not influenced by the addition of unsaturated fatty acids. These results suggest that the L0 soybean could be an excellent soybean source for making soybean products with a combination of soybean and other food ingredients. Keywords: Soybean; lipoxygenase isozymes; hexanal; DETBA value; lipid peroxidation;...

Soybean is recognized as a beneficial food with various functional components, such as β-conglycinin, which improves lipid metabolism. We evaluated the effects of the β-conglycinin-rich soybean Nanahomare on triglyceride (TG) levels. In this randomized, double-blind, placebo-controlled study, we divided 134 adult subjects into test and placebo groups that consumed processed food containing enriched-β-conglycinin soybean or low-β-conglycinin soybean. Hematological tests and body composition measurements were performed at weeks 0 (baseline), 4, 8, and 12 of the study period. TG levels significantly decreased in the test group compared with the placebo group at weeks 4 (change from baseline to week 4, placebo: 0.27 ± 44.13 mg/dL, test: −20.31 ± 43.74 mg/dL, p = 0.035) and 12 (change from baseline to week 12, placebo: −0.14 ± 65.83 mg/dL, test: −21.30 ± 46.21 mg/dL, p = 0.041). In addition, among subjects whose baseline TG levels were ≥100 mg/dL, the levels significantly improved in the test group at weeks 4 (p = 0.010) and 12 (p = 0.030), whereas the levels were not different between the test and placebo groups among those whose baseline levels were

Soybean lipoxygenases are responsible for the generation of undesirable flavors which limit wide utilization of soybeans in food products. Lipoxygenase-lacking mutants are expected to be soybeans with improved flavors. In this study, the accumulation of hexanal and the 1,3-diethyl-2-thiobarbituric acid (DETBA) value in homogenates were determined from two normal soybean cultivars (L 123(S) and L 123(F) , three soybean cultivars with L-1 (L 1 ), L-2 (L 2 ), and L-3 (L 3 ), and one soybean cultivar lacking all three lipoxygenase isozymes (L 0 ). Hexanal accumulation in the homogenates was in the order L 2 >L 123(S) >L 123(F) >L 1 >L 3 >L 0 . The DETBA value in the homogenates was in order L 123(S) , L 123(F) >L 2 , L 3 >L 1 , L 0 . Such cultivar differences were induced after the soaked soybeans were ground or after the soybean flour was wetted. These results suggested that L 0 could become a superior soybean for food ingredients, concerning flavors originating from lipoxygenases

A simple and rapid spectrophotometric and visual judging method was developed for the selective detection of individual lipoxygenase isozymes (L-1, L-2, and L-3) in soybean [Glycine max (L.) Merrill] seeds, based on the bleaching activities of these isozymes in contact with methylene blue and β-carotene. The method consists of three procedures designated tests I-III. The tests are performed by mixing soybean extract samples or wetted soybean flour with each dye-substrate of test I for the L-1 isozyme, test II for the L-2 isozyme, and test III for the L-3 isozyme, whereupon the presence of individual isozymes contained in the soybean is determined spectrophotometically or visually within 10 or 5 min, respectively. In the visual judging method available as a routine screening test, the total amount of soybean flour required for the three tests is only about 10 mg

Cadmium (Cd) is a pollutant metal present in soils and toxic to biologic organisms. Previous studies using recombinant inbred lines derived from a cross between soybean (Glycine max [L.] Merr.) cultivars Harosoy and Fukuyutaka revealed a major quantitative trait loci for seed Cd concentration (cd1) in chromosome 9. The genome sequence of Williams 82 suggested that a P(1B)-ATPase gene involved in the transport of metals was located in the vicinity of cd1. cDNA sequencing suggested existence of two types of transcripts: one (GmHMA1a) consisting of 9 exons and 8 introns and the other (GmHMA1b) consisting of 8 exons and 7 introns. The putative polypeptide, GmHMA1a, consisted of 885 amino acids, whereas premature termination of translation of GmHMA1b generated a putative polypeptide with 559 amino acids. GmHMA1a had a 49.8% similarity with AtHMA3, a P(1B)-ATPase of Arabidopsis. GmHMA1a of Fukuyutaka differed from that of Harosoy by a single-base substitution that led to an amino acid substitution from E to G at amino acid position 608. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to detect the base substitution, and this dCAPS marker was successfully associated with seed Cd concentration. Transgenic experiments may be necessary to verify that GmHMA1 actually corresponds to cd1.

‘Enrei’ is the second leading variety of soybean (Glycine max (L.) Merr.) in Japan. Its cultivation area is mainly restricted to the Hokuriku region. In order to expand the adaptability of ‘Enrei’, we developed two near-isogenic lines (NILs) of ‘Enrei’ for the dominant alleles controlling late flowering at the maturity loci, E2 and E3, by backcrossing with marker-assisted selection. The resultant NILs and the original variety were evaluated for flowering, maturity, seed productivity and other agronomic traits in five different locations. Expectedly, NILs with E2 or E3 alleles flowered later than the original variety in most locations. These NILs produced comparatively larger plants in all locations. Seed yields were improved by E2 and E3 in the southern location or in late-sowing conditions, whereas the NIL for E2 exhibited almost the same or lower productivity in the northern locations due to higher degrees of lodging. Seed quality-related traits, such as 100-seed weight and protein content, were not significantly different between the original variety and its NILs. These results suggest that the modification of genotypes at maturity loci provides new varieties that are adaptive to environments of different latitudes while retaining almost the same seed quality as that of the original.

Flavor properties are important factors of soybean seeds in their utilization as food materials. In order to isolate novel varieties and mutants of soybean having preferable flavor properties, a simple and efficient screening system was established using an automated headspace sampler coupled to gas chromatography. With this system, five major volatile compounds were analyzed within 12 min. By applying this screening system to 626 soybean varieties collected worldwide, we isolated four soybean varieties that showed unique compositions of volatile compounds. Through biochemical analysis, it was found that the uniqueness of three of them was possibly independent of lipoxygenase enzyme, and thus perhaps this screening system can expand the subject of flavor properties beyond lipoxygenase and thus be useful in discovering new types of soybeans.