The plant-specific NAC (NAM, ATAF, and CUC)-domain proteins play important roles in plant development and stress responses. Comparative time-course expression analyses were carried out to analyze the expression levels of 62 soybean NAC genes during drought stress in order to search for the stress-inducible NAC genes. Ten GmSNAC (Glycine max stress-inducible NAC) genes having the significant differential expression in response to the drought stress and abscisic acid (ABA) hormone application were further investigated for their expression profiles with various stresses such as drought, high salinity, cold and with ABA treatments by the quantitative real-time PCR analyses. In this research, the full-length cDNAs of eight GmSNAC were isolated for the further studies. Eight GmSNAC proteins were tested for their transcription activation in the yeast assay system. Two GmSNAC proteins showed the very high transcriptional activities and the other two GmSNAC proteins displayed moderate levels of transactivation while the remaining four GmSNAC proteins lacked transactivation in yeast. Subcellular localization of eight GmSNAC proteins was analyzed via the green fluorescent protein-GmSNAC fusion protein in tobacco plant cell. Three GmSNAC proteins with the C-terminal transmembrane domain were localized to the nucleus and cytoplasmic fractions. The other five GmSNAC proteins were targeted to the nucleus. The function of GmSNAC49 gene was further investigated using the overexpression transgenic Arabidopsis. Germination rate in transgenic plants over-expressing GmSNAC49 was delayed in the media supplemented with mannitol or ABA compared with that of wild-type (WT) plants. The 35S:GmSNAC49 transgenic Arabidopsis displayed improved tolerance to drought stress compared to the WT. The results of this systematic analysis of the GmSNAC family responsive to abiotic stress will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars

Comparative time-course expression analyses were carried out to analyze the expression levels of 60 soybean WRKY genes during abiotic stress in order to search for the stress-inducible WRKY genes. Five GmWRKY(Glycine max WKRY) genes having the significant differential expression in response to the drought stress and abscisic acid(ABA) hormone application were further investigated for their expression profiles with various stresses such as drought, high salinity, cold and with ABA treatments by the quantitative real-time PCR analyses. In this research, the full-length cDNAs of five GmWRKY were isolated for the further studies. Five GmWRKY proteins were tested for their transcription activation in the yeast assay system. GmWRKY3 proteins showed the very high transcriptional activities and the other two GmWRKY proteins displayed moderate levels of transactivation while the remaining two GmWRKY proteins lacked transactivation in yeast. Subcellular localization of five GmWRKY proteins was analyzed via the green fluorescent protein-GmWRKY fusion protein in tobacco plant cell and all of GmWRKY proteins were targeted to the nucleus. In order to analyze the function of GmWRKY genes in plant, 35S:GmWRKY overexpression(OE) transgenic Arabidopsis were generated. Root growth and germination rates in transgenic OE plants were investigated in the media supplemented with mannitol, NaCl or ABA compared with that of wild-type(WT) plants. The 35S:GmWRKY42 transgenic Arabidopsis displayed reduced tolerance to drought stress compared to the WT. The results of this systematic analysis of the GmWRKY family responsive to abiotic stress will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars

Soybean [Glycine max. (L.) Merr] is one of the most important legumes in the world. However, soybean varieties are sensitive to flooding stress and their seed yields are substantially reduced in response to the flooding stress. 192 soybean germplasm collection was screened to identify flooding tolerant germplasm at an early vegetative growth stage (V1). Soybean plants at V1 stage were waterlogged for 4 to 10 days. To evaluate flooding tolerance, survival rate were investigated as a time dependent manner. Jangbaegkong, Danbaegkong, Sowonkong, Socheong2 and Suwon269 showed flooding tolerance, while Shillog, T201, T181, NTS1116 and HP-963 showed flooding sensitivity. We also investigated effects of flooding stress on soybean morphology. The adventitious root development was greatly increased in flooding tolerant plants compared to it in flooding sensitive plants. In addition, root length and root number were analyzed. The significant reduction of root length and root number was observed in flooding sensitive plants. Thus, these results indicate that the morphological changes in roots are important for acclimation to flooding stress. Taken together, the relationship between the morphological changes in the roots and flooding tolerance may be useful in selecting a flooding tolerant soybean germplasm.

The purpose of this study was to establish a system for plant fluorescence image acquisition and to verify the possibility of plant fluorescence image analysis as a non-destructive method to screen the salt tolerance of soybean (Glycine max). Two-weeks-old seedlings of soybean at the V1 growth stage were treated with 0, 50, and 100 mM of NaCl for salt stress and plant fluorescence images were taken by CCD camera (EOS-600D, Canon, Japan) equipped with band pass filter (XNiteBPB, LPD LLC, USA) at 0, 15, 30, 60, 120 and 240 second after blue light exposure at 1 day after treatment. Red color intensity was extracted using MatLab 8.1 (The MathWorks Inc., USA) for estimation of plant fluorescence intensity. Red color intensity of soybean image decreased 0 (F0-10) to 240 (F240-250) second after blue light exposure irrespective of NaCl concentration, while F0-10/F240-250 decreased with NaCl concentration, resulting in significant relationship with plant fluorescence (Fv/Fm) and salt stress intensity. Therefore, our results suggest that our plant fluorescence image acquisition and analysis methods can be a part of high-throughput screening system for salt tolerance of soybean varieties

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

Undomesticated soybeans (Glycine soja) are an important source of genetic variation for introducing useful traits to domesticated soybeans (Glycine max). Although Korea is known as the origin of the soybean, a little is known about genetic diversity and structure analysis of G.soja. The objectives of this study were to investigate the genetic diversity and the structure analysis of wild soybeans, and to construct a core collection of G. soja accessions in Korea. To evaluate the genetic diversity and structure analysis of G.soja, we analyzed allelic profiles at 21 SSR loci of 1028 accessions using POWERMARKER V3.25. These markers generated a total of 581 alleles over all loci. The number of alleles per locus ranged from 21 to 40, with a mean of 28 alleles per locus and a mean gene diversity of 0.886 in this accessions tested. Polymorphic information content value ranged from 0.737 to 0.946, with an average of 0.877. Using STRUCTURE V2.34, wild soybean originated from Korea was divided into two distinct populations, largely corresponding to two geographic regions. Population 1 consisted of eight sub-groups corresponds to mountains; population 2 to entire regions in Korea. Based on theses 21 SSR markers, a core collection development was performed by POWERCORE V1.0. A G. soja core collection consisted of 148 accessions which were established from 1028 accessions in Korea. Most accessions of the core collection were belonged to population 2 and only four were belonged to population 1. These results of this study would provide valuable information for future breeding programs using the G. soja core collection.

Microsatellites are one of the most suitable markers for variety identification as it has great discrimination power for varieties with narrow genetic variation. The polymorphism level between forty microsatellite primer pairs and 148 soybean varieties was investigated through fluorescence based automatic detection system. A set of 16 primer pairs showed highly reproducible and polymorphic in these varieties. A total of 204 alleles were detected by using 16 microsatellite markers. The number of alleles per locus ranged from 6 to 28 with an average of 12.75 alleles per locus. The average polymorphism information content (PIC) was 0.86 ranging from 0.75 to 0.95. Two hundred four microsatellite loci were used to calculate Jaccard’s distance coefficients for unweighted pair group method using the arithmetic averages cluster analysis. These varieties were separated into several distinctive groups corresponding to varietal types. All of the varieties were perfectively discriminated by markers genotypes. This information may be useful to compare through genetic relationship analysis between existing and candidate varieties in distinctive tests and protection of plant breeders’ intellectual properties rights through variety identification.

Soybean [Glycine max (L.) Merrill] is one of the world’s most major crops as not only an important source of oil and protein, but also secondary metabolites. Intake of soybean is associated with decreased risk of cardiovascular disease and osteoporosis, as well as cancer, including breast and colon cancers. Seventy soybeans germplasms collected from 4 different countries, America (6 varieties), China (15 varieties), Japan (16 varieties), and Korea (33 varieties), were distributed by Chungbuk National University (Cheongju, Chungbuk, Korea) and cultivated in Konkuk University farm. This study investigated the isoflavones in seventy soybeans according to 4 different origins (America, China, Japan and Korea). Between 4 different origins, Korea showed highest concentrations of total isoflavones (1292.6 ± 438.6 ㎍ g-1) and China showed the lowest concentrations of total isoflavones (843.8 ± 365.7 ㎍ g-1). The total isoflavone contents in soybean of America and China ranged from 572.3 ㎍ g-1 to 2001.9 ㎍ g-1 and from 275.8 ㎍ g-1 to 1521.8 ㎍ g-1, respectively. And the isoflavone contents of Japan and Korea ranged from 473.3 ㎍ g-1 to 2314.6 ㎍ g-1 and from 419.0 ㎍ g-1 to 3010.7 ㎍ g-1, respectively. Malonylgenistin (356.9 ± 158.8 ㎍ g-1) was the major isoflavones among 12 isoflavones. Specially, glycoside and malonylglycosides constituted 49.2 % and 45.3 % of total isoflavones in soybeans, respectively.

This study was conducted to investigate plant body temperature response of soybean (Glycine max) to saline stress. Two-weeks-old seedlings of soybean in V1 growth stage were treated with 0, 10, 20, 40, 80 and 160 mM of NaCl for salt stress. Thermal images acquired using Flir T-420 (US) were obtained at 4 days after treatment. Soybean leaf temperature increased with increasing NaCl concentration, resulting in significant positive correlation between soybean leaf temperature and stress intensity (P < 0.01). Leaf temperature of soybean was significantly different at 160 mM of NaCl, where no visual symptom was observed. Therefore, soybean leaf temperature can be used for evaluating the response of soybean to salt stress as a non-destructive and phenomic parameter. Non-destructive diagnosis of soybean leaf temperature may be a key parameter in a high throughput screening (HTS) system in breeding program for salt stress tolerance soybean cultivars.

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

In this study we established the high throughput screening system of high functional soybean cultivars using PLS modeling from FT-IR spectral data of soybean(Glycine max L) seeds. Crude extract of 20% methanol from soybean seed powders (153 lines) were used for FT-IR spectroscopy. Total fatty acid, carotenoids, flavonoids and phenolic compounds contents from soybean seed powders were analyzed using UV-spectrum and GC analysis respectively. PCA analysis showed that 153 soybean lines formed a single clusters with a few outlier. PC score 1 and 2 represented 39.5, 16.4% of total variation, respectively. And than showed change patten from the middle to outside for PCA plot. We conducted PLS regression analysis between FT-IR spectral data and fatty acids data. Palmitic acid showed the highest regression coefficient (R=0.78). This result implied that the content of palmitic acid could be predicted from FT-IR spectral data from soybean seed powders with relatively high fidelity. PLS modeling of total carotenoids also showed regression coefficient of 0.69. Regression coefficient of total flavnoids and phenolic compounds were 0.44, 0.39, respectively. At present, we are trying to confirm the accuracy of PLS prediction modeling using targeted metabolite analysis (GC-MS, LC-MS) from predicted soybean lines. To increase the accuracy of PLS modeling, we also trying to standardization of spectroscopy and spectral data processing. Furthermore we are going to develop PLS modeling from GC-MS, LC-MS data. The PLS prediction modeling established in this study could be applied for high throughput screening of other leguminous plant.

Recent release of whole genome draft sequences in legume species have led comparative genome studies among legume plants including Glycine max, G. soja, Cajanus cajan and Medicago truncatula. The majority of comparative genomic researches have been conducted based on synteny of coding sequences and coding sequence variations may be one of major forces for speciation and evolution. However, non-coding sequences have been also reported to be important phenotypic regulators. Especially, since short sequence motifs in the promoter regions are highly conserved, they are suggested to be another resources of interests in comparative studies. In this study, we predicted the conserved short sequence motifs by BLASTN algorithm using dicot promoter database from Softberry (http://www.softberry.com). A total of 37,396 conserved short sequence motifs were identified onto 2 kb upstreams of 46,367 high confident gene model of G. max (cv. Williams 82). Meanwhile, whole genome of 7 soybean landraces (G. max) and 7 wild soybean genotypes (G. soja) were sequenced at low depth of less than ten using Illumina Hiseq 2000. Among these genotypes, nucleotide variations were identified in predicted conserved regulatory motifs by mapping of short reads to the reference genome sequence using the Samtools program (http://samtools.sourceforge.net/). Fifteen and two genes, which have SNPs in regulatory motifs and no SNP in coding sequence, were identified by comparisons of inter-species and intra-species, respectively. qRT-PCR experiments are in progress for investigating differences of these 17 genes expressions at transcriptional level.

As soybean (Glycine max) is known for its high nutritional value of oil and protein, soybean has been domesticated and cultivated by one specific character trait based on human selection. Importantly, tracing back in time where G. max and G. soja, the undomesticated ancestor of G. max have diverged plays an important role in studying of genetic diversity and in investigating the common ancestor of soybean. In this study, we sequenced 6 G. max and 6 G. soja using Illumina’s Hiseq 2000 with a low coverage sequencing technology to estimate the divergence of times between genotypes and populations. A total of the 12 genotypes were sequenced at the average depth of 6.5 and resulted 892.5 Mb and 903.3 MB consensus sequences with the coverage of 91.54% and 92.65% for G. max and G. soja, respectively. The whole genome SNP analysis showed that G. max had lower frequency levels of polymorphism (~0.1%) than G. soja (~0.25%). And, a high number of SNPs located in introns were found among 6 G. soja genotypes as SNPs were approximately twice than those found in 6 G max. The number of SNPs in G. max intronic regions was 53,134, whereas a total of 133,329 SNPs were discovered in G. soja introns. Almost an equal number of SNPs were discovered in 5’ UTR and exon regions; however, different numbers of SNP in CDS and 3′ UTR were identified. By the rate of nonsynonymous change, divergence of time between G. soja and G. max would be investigated.

Soybean (Glycine max, 2n = 2x = 40) is broadly distributed throughout East and South East Asia, and important crop as a source of protein, oil, food and animal feed. In order to better understand the morphological differentiation of soybean germplasm collected from China, Japan, Korea, Philippines, America, we analyzed the morphological variabilities among 629 soybeans with 11 morphological traits, such as growth type, leaflet, flower color, trichome, seed coat color, color inside seed etc. and measured the fatty acid composition. The result of the principal coordinates analysis (PCoA) based on the 11 morphological traits revealed diversity among all accessions. The PCoA separated the accessions into two main groups, each group with distinctive features. Among tested germplasm, the contents of five fatty acids were as follows: linolenic acid (2.8%－16.23%), linoleic acid (27.4%－56.6%), oleic acid (9.2%－35.0%), stearic acid (2.9%－8.8%), and palmitic acid (8.7%－17.1%). The fatty acid composition has not shown significant variation among all accessions. IT 22268 was the highest linolenic acid composition (16.2%), while IT 154687 was the lowest (2.8%). Forty three of 629 accessions showed the arachidic acid (0.5%-3.6%), which is the saturated fatty acid with a 20 carbon chain and is as a minor constituent of peanut oil (1.1%-1.7%). This result of this characterization served as reliable resources for detailed description and new functional plant breeding of soybean varieties.

We improved the separation of the basic proteins from the soybean cotyledon, Glycine max L. Merr. by searching N-terminal sequences data in proteins isolated by two-dimensional electrophoresis (2-DE). After removed Hexane, proteins were extracted from cotyledon with a urea/Triton/2-mercaptoetanol solution. Using this method, the highly reproducible isoelectric focusing (IEF) can formed with polyacrylamide gels with pH 4.0-9.8. The IEF tube gels were used as the first dimension, and proteins were visualized by second-dimensional gel electrophoresis, and identify a number of soybean cotyledon proteins using mass spectrometry in the proteome analysis. These instruments of 2-DE and IEF tube gels were used 27 cm and investigate under various conditions. The total number of spots and features was obtained by PDQuest software (Bio-Rad). In this experiments performed, the IEF tube gels and instruments afforded good reproducibility in the number of PDQuest-detected spots from gel to gel while IPG offered better reproducibility in the total number of manually detected spots from gel to gel. In conclusion, we have separated of the basic 13 proteins in soybean. The glycinin subunit separations are also considered to play important roles in soybean breeding and biochemical characterization. The improved technique will be useful to dissect the genetic control of glycinin expression in soybean.

Scientific studies have shown that essential fatty acidintake can have a dramatic impact on human health. Soybean [Glycine max(L.) Merr.] oil from current commercial cultivars typically containsaround 8%linolenic acid (18:3) known as omega-3 fatty acid. Omega-3 fatty acid plays an important role to prevent cardiovascular disease and cancer. Relatively high 18:3 content in seed oil is a trait of the wild soybean (Glycine soja Sieb. and Zucc.) ancestor of modern soybean cultivars. Wild soybean is native to Korean peninsula and recently thousands of wild soybeans collected by soybean researchers in Korea. The objective of this study were to determine the linolenic acid content for wild soybean collection and to determine the stability of linolenic acid content derived from wild soybean over environments. Fatty acid profile for 1,806 wild soybean accessions collected from South Korea was determined by GC. The range of linolenic acid was 7.3 to 23.7% with an average 15.6%. We developed a recombinant inbred population from a cross PI483463 (wild soybean with 15% 18:3) and Hutcheson (cultivar with 8% 18:3). Three RILs, RIL156, RIL159 and RIL166, with high linolenic acid content (over 14%), parents and Williams 82 as checks were grown in nine environments over 2008-2011. Results showed that the content of linolenic acid for the PI483463, Hutcheson, and Williams 82 ranged from 14.8 to 17.1, 8.5 to 9.7, and 6.9 to 8.4 % and averaged 15.4, 9.2 and 8.0%, respectively. However selected RILs 156, 159, and 166 ranged from 10.7 to 15.7, 14 to 15.8, and 14.8 to 15.8, and averaged 13.9, 14.9, and 15.2, respectively. Among the tested accessions, RIL166 was the most stable with the lowest range and CV, and had a relatively lower stability coefficient value than other genotypes. Genes related to high linolenic acid from wild soybean may be useful in developing higher linolenic acid soybean genotypes and would broaden the use of soybean in food applications to improve human nutrition and health.

Soybean is desirable as a forage crop because of it has high protein and oil concentration. Wild soybean, a progenitor of cultivated soybean, has a softer stem and higher protein content in seed than cultivated soybean. There is little information on yield and forage quality for wild soybean and its derivatives. The objective of this study was to determine the forage yield and quality of wild soybeans and selected soybeans derived from a cross G. max ×G. soja. Forage yield and quality were assessed for three grain soybean cultivars, three wild soybeans and three selected lines from G. max×G. soja. Forage quality attributes such as crude protein (CP), crude fat (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), digestible dry matter (DDM), dry matter intake (DMI) and relative feed value (RFV) were determined at the R2, R4 and R6 developmental stages. Forage yield and CF were highest at stage R6 in G. max, G. soja and selected G. max×G. soja lines. CP content was similar between R2 and R4 but increased sharply after R4 and peaked at R6 in G. max and selected lines from G. soja×G. max. On the other hand, CP content was similar between R4 and R6 stage in wild soybeans. Generally, NDF and ADF were highest at stage R4 but decreased at stage R6. DDM, DMI, and RFV increased between R4 and R6. These results suggest that R6 was the optimal harvest stage to provide forage of highest quality and yield. A study was conducted in 2011 to evaluate forage yield and quality at stage R6 in 25 lines from PI483463 (G. soja)×Hutcheson (G. max) and four cultivated grain soybeans. Hutcheson had the highest forage yield with 24.7t/ha infresh weight (FW) among grain soybeans. Line W11 had the highest forage yield(25.7t/ha,FW) among G. soja×G. max selections and four other lines had similar forage yield compared to Hutcheson. Generally the 25 lines from this G. max×G. soja cross had thinner main stems and branches than cultivated soybeans. When the 25 lines were evaluated for their feed quality as per forage grade by AFGC, nine lines rated prime grade and all 25 lines were classified as forage Grade 1. Results of this study indicate crosses between wild and cultivated soybean show promise for improving soybean as a forage crop.

In order to get information on tocopherol content and composition in fifty-six soybean germplasms were evaluated by HPLC. From the principle component analysis, the first three components accounted for 71.6% of the total variance of tocopherol content in the germplasms. Principal component 1 showed significant correlations with all the morphological markers except 100-seed weight. Soybean germplasms were divided into three groups by the first two principal components. The highest content of α -tocopherol was 38.6~mug per g in Tanner, while that of ~gamma -tocopherol was 195.6~mug in PI 91073. In case of δ -tocopherol, IT 105622 showed the highest value as 29.8~mug . The contents of tocopherol were gradually increased from the late August to late September, which was 10 days before maturity, in Alchankong and PI 96322. The tocopherol content was higher in seeds from the plants sown early than those sown late. Total tocopherol contents of PI 96188, Geomjeongkong 2, and Suwon 183 grown in Gimje were higher than those grown in Iksan, but the difference was not observed in PI 96322. These results suggest that the contents of tocopherol in soybean were affected not only by the genotypes but also by environment.