This study aimed to determine the optimal harvesting time for wheat to make grain silage, in Honam region of Korea. We harvested wheat grain every third day from 30 to 42 days after heading (DAH). The moisture content decreased from 61.6% at 30 DAH to 42.8% at 42 DAH. Yield of wheat grain significantly increased from 30 to 42 DAH (p< 0.05). Yield at 39 DAH (3.46T/ha) was not significantly different from that at 42 DAH (p< 0.05). With respect to the feed value of wheat grain silage, the amount of crude protein, crude fiber, and crude ash was different by harvesting time (p< 0.05). However, the amount of total digestible nutrients (TDN) from 30 and 42 DAH was not significantly different. The pH of wheat grain silage from 30 to 42 DAH was between 3.8 and 4.5 and it was stable until 39 DAH (p< 0.05). The lactic acid content of wheat grain silage from 30 to 42 DAH decreased from 3.08% to 1.10%. With respect to moisture content, yield, feed value, and fermentation, the optimal harvesting time for wheat grain silage was 39 DAH.

Vigna is a genus of flowering plants in the legume family, and about 100 species belong to this genus, including azuki bean, blackgram and mungbean which are well-known in Asia. Among the species in Vigna genus, Vigna reflexo-pilosa has its own unique characteristic form as allotetraploid (2n=4x=44), whereas other Vigna species exist as a diploid form (2n=2x=22). In this study, we de novo assembled V. reflexo-pilosa genome and 22 accessions of 18 Vigna species’ transcriptome using NGS platform and calculated the Ks values of synteny blocks within V. reflexo-pilosa to determine the whole-genome duplication event existence. Two WGD events had occurred on V. reflexo-pilosa unlike V. radiata which only occurred once. Also, we tried to find out which progenitor had contributed to formation of V. reflexo-pilosa using transcriptome assemble results of 22 Vigna accessions. Ks value calculation between the transcriptome assemble results and predicted gene sets of V. reflexo-pilosa has been excuted. V. trinervia showed 2 peaks (0.0075, 0.0495) on its Ks value bin distribution and confirmed as A-type genome donor. Based on the peak value, we considered the predicted genes of V. reflexo-pilosa in Ks value range from 0.033 to 0.072 as B-type genes, inherited from other progenitor. Re-calculating the Ks values of 4,796 predicted genes in B-type with the transcriptome assemble results of 22 Vigna accessions, we were able to find 6 Vigna species, V. umbelleta(CIAT34386), V. umbelleta(2004T2), V. nakashimae, V. nepalensis, V. riukiuensis, V. minima as candidate B-type genome donor.

UVB radiation which causes dermatosis, cancroid, and necrosis in living organisms is mostly absorbed by ozone layer, resulting in transmission of only small UVB proportion to earth surface. Recently, however, rapid increases of pollutants like CFCs have accelerated depletion of stratospheric ozone layer. Increased UVB irradiation alters affects biomolecule interinity such as DNA, RNA and protein. To understand DNA mutation spectra in response to UVB, in the present study, we used two soybean cultivars, Buseok and Cheongja 3, which were screened as the most UVB tolerant and sensitive genotypes among 140 soybean germplasms, respectively. Whole genomes of Buseok and Cheongja were sequenced at low-coverage depth by illumina Hiseq2000, and we also sequenced 6 hr UVB irradiated genomes of two cultivars. Raw sequence reads were aligned to the soybean reference sequences (cv. Williams 82) by BWA aligner software. To identify DNA mutations induced by UV-B irradiation, multiple comparisons between non-irradiated and irradiated genomes in these two soybean genotypes were conducted using samtools and GenomeAnalyzerTK packages and homebrew python codes. A total, 13,992 and 17,078 single nucleotide polymorphisms (SNPs) were indentified between non-irradiated and irradiated genomes of Buseok and Cheongja 3, respectively. In addition, Buseok and Cheongja 3 have 423 and 465 insertions/deletions induced by UVB, respectively. Approximately 58% of the identified SNPs were C to T or CC to TT transversions, consistent with the previous studies. Chromosomal distributions of the SNPs likely showed differences in UV-B mutation positions depending on the soybean genotype

Toxic (high phorbol esters) and nontoxic (low phorbol esters) jatropha accessions cannot be distinguished morphologically. Their seeds must be chemically analyzed through a complex and costly process using HPLC method. EST-SSR markers can be used to classify jatropha accessions with high and low phorbol esters. In this study, ninety-seven EST-SSR markers amplified the genomic DNA and showed polymorphism among 5 high phorbol esters accessions and 10 low phorbol esters accessions. These markers can be further exploited for jatropha improvement through marker-assisted breeding.

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. We constructed a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis and to enable a better understanding of the evolution of leguminous species. The draft genome sequence covers 80% of the estimated genome, of which 50.1% consists of repetitive sequences. In total, 22,427 high confidence protein-coding genes were predicted. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (Vigna reflexo-pilosa var. glabra) provided genomic evidence of a recent allopolyploid event. To further study speciation, we compared de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max and Cajanus cajan. The species tree was constructed by a Bayesian Markov chain Monte Carlo method using highly confident orthologs shared by all 24 accessions. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.

Although much effort has been made to find agronomically important loci in the soybean plant, extensive linkage disequilibrium and genome duplication have limited efficient genome-wide linkage analyses that can identify important regulatory genes. In this respect, recombination block-based analysis of cultivated plant genomes is a potential critical step for molecular breeding and target locus screening. We propose a new three-step method of detecting recombination blocks and comparative genomics of bred cultivars. It utilizes typical reshuffling features of their genomes, which have been generated by the recombination processes of breeding ancestral genomes. To begin with, mutations were detected by comparing genomes to a reference genome. Next, sequence blocks were examined for likenesses and difference with respect to the reference genome. The boundaries between the blocks were taken as recombination sites. All recombination sites found in the cultivar set were used to split the genomes, and the resulting sequence fragments were named as core recombination blocks (CRBs). Finally, the genomes were compared at the CRB level, instead of at the sequence level. In the genomes of the five Korean soybean cultivars used, the CRB-based comparative genomics method produced long and distinct CRBs that are as large as 22.9 Mb. We also demonstrated efficiency in detecting functionally useful target loci by using indel markers, each of which represents a CRB. We further showed that the CRB method is generally applicable to both monocot and dicot crops, by analyzing publicly available genomes of 31 soybeans and 23 rice accessions.

Resequencing data is actively used for searching QTL or analyzing genetic diversity in the crops. However, the complexity of genome, caused by genome duplication, limits the utility of genome-wide association studies and linkage analyses to identify genes that regulate agronomically valuable traits. Here, we propose a comparative genomics approach based on core or common variation-based recombination blocks (CRB) using single nucleotide variation (SNV) density information. We found that the soybean genomes are assembled with long and distinct CRBs as large as 10Mb. CRB-based comparative genomics enabled us to accurately identify recombination blocks at the whole-chromosome level. We identified the Ih locus that determines the yellow hilum color in soybeans using CRB-based mapping with representative indel markers. These results suggest that the CRB-based comparison method is a promising platform for molecular breeding and map-based cloning.

The purposes of this research project are to identify quantitative trait loci (QTLs) associated with yield-related traits using a recombinant inbred line (RIL) population derived from a cross between a high-yield soybean genotype SS0404-T5-76 and Daewonkong and to develop high-yield soybean and lodging-resistant sprout soybean cultivars. For development of DNA markers and identification of functional sequence variations, firstly, whole genome of five soybean genotypes, Sinpaldalkong 2, SS2-2, Pungsanamulkong, SS0404-T5-76 and Daewongkong, were sequenced using Illumina Hi-Seq technology. SS2-2 is a EMS-induced mutant of Sinpadalkong 2. SS0404-T5-76 showing high-yield is a F8 RIL derived from a cross of Pungsanamulkong x SS2-2. Daewonkong is a elite cultivar with high-protein. Furthermore, to construct a genetic linkage map, we are advancing F4 lines of SS0404-T5-76 x Daewonkong by single seed-descent. Secondly, we developed high-protein and high-yield soybean lines and lodging-resistant sprout lines. Area-adaptability tests of these promising lines are performing in three different locations including Jeju, Naju, and Suwon. Based on the results of area adaptability tests, we are planing to conduct cultivar registration of the promising soybean lines.

R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain. Some soybean NBS-LRR genes have also been reported to function in disease resistance. A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS-LRR genes have disease resistance functions in the soybean genome. The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.

Phomopsis seed decay (PSD), primarily caused by Phomopsis longicolla, is a major contributor to poor soybean seed quality and significant yield loss, particularly in early maturing soybean genotypes. However, it is not yet known whether PSD resistance is associated with early maturity. This study was conducted to identify quantitative trait loci (QTLs) for resistance to PSD and maturity time using a recombinant inbred line (RIL) population derived from a cross between the PSD-resistant Taekwangkong and the PSD-susceptible SS2-2. Based on a genetic linkage map incorporating 117 simple sequence repeat markers, QTL analysis revealed two and three QTLs conferring PSD resistance and maturity time, respectively, in the RIL population. Two QTLs (PSD-6-1 and PSD-10-2) for PSD resistance were identified in the intervals of Satt100-Satt460 and Sat_038-Satt243 on chromosomes (Chrs) 6 and 10, respectively. These QTLs do not overlap with any previously reported loci for PSD resistance in other soybean genotypes. Two QTLs explained phenotypic variances in PSD resistance of 46.3% and 14.1%, respectively. Among three QTLs for maturity time, two (Mat-6-2 and Mat-10-3) were located at positions similar to the PSD resistance QTLs. The identification of the QTLs linked to both PSD resistance and maturity time indicates a biological correlation between these two traits. The newly identified QTLs for resistance to PSD associated with maturity time in Taekwangkong will help improve soybean resistance to P. longicolla.

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.

Mutagenesis approach in combination with whole genome sequencing has become an import role in genetic and molecular biological study and breeding of crop plants. In this study, we screened the fast neutron M4 10,000 soybean mutant plants based on morphological phenotypes of agronomically important traits and characterized the mutant of interest using resequencing. Fast neutron radiation has been known to be a very effective mutagen to cause large deletion in genome. The screened mutant showed abnormal phenotypes in plant heights, seed sizes, color of leaves, number of leaves, maturity and number of branches etc. Among them, the mutant displaying short plant height and bush type of growth habit was selected for identification of the altered genomic regions. Analysis of deletion sites of genome in interesting soybean mutant was performed using next generation sequencer Illumina Hi-seq. Mutant sequence reads generated by paired-end shotgun library were mapped on a draft soybean reference soybean (G. max cv. Williams 82). The paired-end DNA sequences of 21.6 Gb produced by Illumina Hi-seq produced 21 fold sequence depth. Among the predicted deletion sites, total 3 deletion regions confirmed by PCR. Glyma03g02390 gene and Glyma03g03560 gene were involved in the deletion regions. Glyma03g02390 gene was related to AMP binding, catalytic activity, cofactor binding and metabolic process of cell growth and Glyma03g03560 gene was concerned to oxygen binding, defense response to bacterium, and especially process of indole acetic acid (IAA) biosynthesis. These genes detected in this mutant will be studied about their molecular function in stunted phenotype.

Phytic acid, myo-inositol (1, 2, 3, 4, 5, 6)-hexakisphosphate, is a material that plants store phosphorus in seeds. Phytic acid is classified as an antinutrient because of indigestibility. Non-ruminant animals, such as human and swine, excrete unavailable phytic acid. The unavailable phytic acid run off to ground water, river, sea, causing eutrophication as a factor. Accordingly, low-phytic acid crops draw the attention due to both nutritional and environmental reasons. Using more than 900 Glycine accessions including G. max, G. soja and G. gracillis, colormetric method was applied for detecting low-phytic acid mutant. Two hundred fifty accessions were screened by the colormetric method so far, but no mutant was identified. Screening of mutants with the rest 710 accessions is in progress. MIPS1 (D-myo-inositol 3-phosphate synthase) is considered as gene related to phytic acid content in soybean. Also, lpa1 (Zea mays low phytic acid 1) known as controlling phytic acid content in maize was recently reported that homologs of lpa1 were responsible for phytic acid content in soybean and located on linkage groups L and N (Chromosomes 19 and 3). After primers were designed from these three candidate genes for phytic acid content, identification of genes responsible for low phytic acid and investigation of genetic variation among 960 accessions will be performed as further study.

EMS was commonly used to induce mutations for various organisms, causing nucleotides to mispair with their complementary bases. So, chemical mutagenesis has become the best method for inducing mutations in genetic studies. Simple PCR-based detection and high-throughput technologies helped to screen and identify mutations. Degenerate oligonucleotide primed PCR (DOP-PCR) became getting attention for mutation survey because the requirement of sequence information and high cost for designing primers could be diminished. Also, high-throughput sequencing instruments, such as GS-FLX, allowed characterization of nucleic acids and massive mutant analysis. A total of 6,696 aligned pairs for Sinpaldalkong 2 vs. SS2-2 and 6,935 for Sinpaldalkong 2 vs. 25-1-1 were formed for mutation detection. A mutation every 437 bp in SS2-2 and every 402 bp in 25-1-1 was observed. About 2/3 of a total of mutations were single base variation in both comparisons. Mutated and non-mutated fragments from SS2-2 and 25-1-1 were distributed on all LGs. The 25-1-1 had more mutations than SS2-2 compared with their wild type, Sinpaldalkong 2. Local compositional bias was also observed around the mutated G. Our modified DOP-PCR primers were successfully amplified and their amplicons were located on randomly but somewhat targeted regions of soybean genome.

Expressed sequence taqs (ESTs) have accepted to be a valuable tool for discovering single nucleotide polymorphism (SNP) in many species. For detection putative SNPs in soybean genome, approximately 90,000 EST sequences from genotype Williams 82 were downloaded from NCBI. The paralog sequences of these ESTs were distinguished by TGI clustering tools (TGICL) performing megablast and EST cluster analysis, and the EST clusters were used as reference sequences for detection putative SNPs by in silico. The EST clusters were aligned with EST sequence from other cultivars of soybean by Polybase (computer software). The results revealed that putative SNPs were distributed in 5,677 clusters with frequency of 1 SNP per 333 nucleotide sites. For SNP validation, 43 primer pairs were designed from EST clusters containing putative SNPs for sequencing genomic DNA of Williams 82, Harosoy, Peking, Pureunkong, Jinpumkong 2, Hwangkeumkong and IT182932. From results of sequencing PCR, we totally found 99 SNPs from 33 primer pairs. Twenty-three and 47 out of 99 SNPs showed polymorphisms between Pureunkong and Jinpumkong 2, and Hwangkeumkong and IT182932, respectively. The SNPs discovered from this study can be used for genetic mapping in the four genotypes of soybean.

The control of earliness has an agronomic importance since it reduces growing and harvesting time. Earliness is controlled by multiple genes in multiple pathways and influenced by the environment. In Arabidopsis thaliana, many earliness related genes were identified. Among them, Arabidopsis Frigida (FRI) gene confers late flowering phenotype, which is reversed to earliness by vernalization. Blast search using FRI against soybean EST database at TIGR identified Isoflavone reductase-like gene (TC217830). Fifty seven SNPs were identified in a total of 4,242 bp lengths in genomic region of Isoflavone reductase-like from 62 soybean genotypes (31 early maturity group and 31 late maturity group). From the obtained sequences, we identified 6 haplotypes of Isoflavone reductase-like gene. Among them, three haplotypes showed a significant association with maturity, suggesting that Isoflavone reductase-like gene is tightly linked to flowering time or actual gene it-self. Thus, to delimit a putative genomic region for maturity and flowering time, SSR markers near Isoflavone reductase-like gene were designed and analyzed for their genetic diversity, assuming that highly selected regions might posses lower genetic diversity. Through these experiments, the region related to maturity and flowering was delimited to nearby ac_satc_4 in scaffold 16.

MADS-box genes encode a family of transcription factors which involve in diverse developmental processes in flowering plants. Because flowering time determines the timing of transition from vegetative to reproductive stage and time to harvest, it would be a significant trait not only to plant it-self but also to breeders. The sequences and gene structures of Arabidopsis MADS-box genes are conserved in model legumes. However, complex genome structure, in soybean, makes it difficult to identify actual genes related to flowering and maturity, although QTL researches have been generally conducted. Therefore, we hypothesized that putative MADS-box genes around the flowering time and maturity QTLs would be candidate genes for those loci. In this study, after surveying 84 QTLs highly associated with maturity and flowering, the QTLs were selected if they were located near 473 putative MADS-box genes. Finally, we found the highly associated 16 SNPs at non-coding region of the putative MADS-box gene around the QTL in 28 late maturity cultivars and 28 early maturity cultivars. Furthermore, by comparing genetic diversity in the cultivated soybeans of late and early maturity groups as well as 20 wild soybeans, selection pattern during domestication was predicted.