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.

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.

A single recessive gene, rxp, controls bacterial leaf pustule (BLP) resistance in a soybean. The Rxp locus appears to be linked to the malate dehydrogenase (Mdh) locus and Satt372 on linkage group (LG) D2. Around the Rxp locus, four bacterial artificial chromosome (BAC) clones are anchored by Satt486, Satt498, BARC-022037-04263, and BARC-040963-07870. Using these BAC clone sequences, possible orthologous region of Rxp locus was identified: Medicago truncatula contig 962 at chromosome 3 and contig 283 and contig 1108 at chromosome 8. Sequence analysis of contig 962 had revealed microsynteny with three soybean BAC clones on LG A1, which are duplicated with other two soybean BAC clones anchored by Satt486 and Satt498. After BLAST search was performed with M. truncatula contig 962 against soybean ESTs, several soybean ESTs were identified. With developed single nucleotide polymorphism (SNP) markers and the RIL population from the cross of Pureunkong and Jinpumkong 2, SNP genotyping was able to locate twos oybean ESTs: CO979743 at 1 cM away from Satt195 on LG C1 and BE021935 at 5 cM away from Satt363 on LG C2. Thus, our results indicate that structure of soybean genome around Rxp locus is very complicated.

Improvement of crop yield can be achieved through understanding genetic variation in reproductive characters and its impact on yield components. The present study was performed to evaluate genetic diversity for reproductive growth characters in exotic germplasm resources and to determine the relationships between developmental and growth periods with yield and yield components in soybean cultivar groups. For phenotypic evaluation such as reproductive and agronomic traits, a total of 80 indigenous and exotic soybean cultivars collected from four different geographical regions (China, Japan, Korea, and Vietnam) were grown from May to November of 2003 at the Seoul National University Farm, Suwon, Korea (127~circ02 longitude, 37~circ26 latitude). Most of all the characters exhibited wide range of phenotypic variation, of which pod number, seed number, and plant yield showed greater range as compared to other characters. Korean cultivar groups showed greater diversity than the other cultivar groups in seven characters. Correlation analysis showed that days to flowering (DTF) and days to maturity (DTM) had close association with agronomic traits as well as yield and yield components. Both DTF and DTM had positive correlation with the other characters except one hundred seed weight. Stepwise multiple linear regression revealed that seed and pod number were identified as being significant for plant yield. The results in this study indicated wide variation in agronomic traits including DTF and DTM, suggesting the valuable genetic resources in a soybean breeding program.

Single nucleotide polymorphisms (SNPs) are valuable DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed. In this report, one of effective and high throughput SNP genotyping assays, which was named the template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) was described. Although the most of this assay succeed, the objective of this work was to deter­mine the reasons for the failures, find ways to improve the assay and reduce the running cost. Ninety F2 -derived soybean, Glycine max (L.) Merr., RILs from a cross between 'Pureunkong' and 'Jinpumkong 2' were genotyped at four SNPs. FP measurement was done on Victot3 microplate reader (perkinelmer Inc., Boston, MA, USA). Increasing the number of thermal cycles in the single-base extension step increased the separation of the FP values between the products corresponding to different genotypes. But in some assays, excess of heterozygous genotypes was observed with increase of PCR cycles. We discovered that the excess heterozygous was due to misincorporation of one of the dye­terminators during the primer extension reaction. After pyrophosphatase incubation and thermal cycle control, misincoporation can be effectively prevented. Using long amplicons instead of short amplicons for SNP genotyping and decreasing the amount of dye terminator and Acyclopol Taq polymerase to 1/2 or 1/3 decreased the cost of the assay. With these minor adjustments, the FP-TDI assay can be used more accurately and cost-effectively.

In the absence of exogeneous nitrogen supply, evaluation of a symbiosis effectiveness of Bradyrhizobium japonicum USDA 110 in a supernodulating soybean mutant, SS2-2, its wild type, Sinpaldalkong 2, and control genotype, Jangyeobkong, was conducted in this study. Nodules in SS2-2 were initially white and similar to its wild type, Sinpaldalkong 2. At the late stage, the wild type nodules became dark pinkish by maturation, by contrast, mature nodules in SS2-2 remained light green to pinkish, indicating a lack of leghemoglobin. Tap root length was short in nodulated symbiotic SS2-2 than that of its wild type and the control genotype. Nodulated root length and nodule density on root length were significantly increased by B. japonicum inoculation, but no significant increase was observed on root length and percentage of nodulation to total root length. Regardless of Bradyrhizobium inoculation, SS2-2 showed higher nodule dry weight and higher acetylene reduction activity (ARA) when compared with its wild type and the control genotype. Inoculation of B. japonicum leaded the increase of ARA in 47 days after planting (DAP), in part because of nodule development. Supernodulating mutant, SS2-2, less responded to B. japonicum induction in terms of nitrogen fixation and nodulation characteristics than its wild type. Thus, interaction of supernodulating soybean mutant with Bradyrhizobium had less symbiotically associated response than normal nodulating soybean.

A single recessive gene, rxp, controls the bacterial leaf pustule (BLP) resistance in soybean and in our previous article, it has been mapped on linkage group (LG) D2 of molecular genetic map of soybean. A total of 130 recombinant inbred lines (RILs) from a cross between BLP-resistant SS2-2 and BLP-susceptible Jangyeobkong were used to identify molecular markers linked to rxp. Fifteen simple sequence repeat (SSR) markers on LG D2 were screened to construct a genetic map of rxp locus. Only four SSR markers, Satt135, Satt372, Satt448, and Satt486, showed parental polymorphisms. Using these markers, genetic scaffold map was constructed covering 26.2cM. Based on the single analysis of variance, Satt372 among these four SSR markers was the most significantly associated with the resistance to BLP. To develop new amplified fragment length polymorphism (AFLP) marker linked to the resistance gene, bulked segregant analysis (BSA) was employed. Resistance and susceptible bulks were made by pooling equal amount of genomic DNAs from ten of each in the segregating population. A total of 192 primer combinations were used to identify specific bands to the resistance, selecting three putative AFLP markers. These AFLP markers produced the fragment present in SS2-2 and the resistant bulk, and not in Jangyeobkong and the susceptible bulk. Linkage analysis revealed that McctEact97 (P=0.0004,~;R2=14.67~%) was more significant than Satt372, previously reported as the most closely linked marker.

Somaclonal variation, defined as phenotypic and genetic variations among regenerated plants from a parental plant, could be caused by changes in chromosome structure, single gene mutation, cytoplasm genetic mutation, insertion of transposable elements, and DNA methylation during plant regeneration. The objective of this study was to evaluate DNA variations among somaclonal variants from the cotyledonary node culture in soybean. A total of 61 soybean somaclones including seven ~textrmR1 lines and seven ~textrmR2 lines from Iksannamulkong as well as 27 ~textrmR1 lines and 20 ~textrmR2 lines from Jinju 1 were regenerated by organogenesis from the soybean cotyledonary node culture system. Field evaluation revealed no phenotypic difference in major agronomic traits between somaclonal variants and their wild types. AFLP and SSR analyses were performed to detect variations at the DNA level among somaclonal variants of two varieties. Based on AFLP analysis using 36 primer sets, 17 of 892 bands were polymorphic between Iksannamulkong and its somaclonal variants and 11 of 887 bands were polymorphic between Jinju 1 and its somaclonal variants, indicating the presence of DNA sequence change during plant regeneration. Using 36 SSR markers, two polymorphic SSR markers were detected between Iksannamulkong and its somaclonal variants. Sequence comparison amplified with the primers flanking Satt545 showed four additional stretches of ATT repeat in the variant. This suggests that variation at the DNA level between somaclonal variants and their wild types could provide basis for inducing mutation via plant regeneration and broadening crop genetic diversity.

Genetic diversity and soybean sprout-related traits were evaluated in a total of 72 soybean accessions (60 Glycine max, 7 Glycine soja, and 5 Glycine gracilis). 100-seed weight (SW) was greatly varied and ranged from 3.2g to 32.3g in 72 soybean accessions. Positive correlation was observed between GR and hypocotyl length (HL), whereas negative correlation was observed between SW and hypocotyl diameter (HD). Re-evaluation by discarding two soybean genotypes characterized with low GR indicated that much higher correlation of sprout yield (SY) with HD and SW. Based on the principal component analysis (PCA) for sprout-related traits, 57 accessions were classified. Soybean genotypes with better traits for sprout, such as small size of seeds and high SY, were characterized with high PCA 1 and PCA 2 values. The seed size in second is small but showed low GR and SY, whereas the third has large seed, high GR and more than 400% SY. In genetic similarity analysis using 60 SSR marker genotyping, 72 accessions were classified into three major and several minor groups. Nine of twelve accessions that were identified as the representatives of soybean for sprout based on PCA were in a group by the SSR marker analysis, indicating the SSR marker selection of parental genotypes for soybean sprout improvement program.