In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. A cDNA coding for the C-terminus of spider silk protein (AvMaSp) was cloned from the spider Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvMaSp consists of 165 amino acids of are petitive region and 99 amino acids of a C-terminalnon-repetitive region. The peptide motifs found in spider silk proteins, GGX and An, were conserved in the repetitive region of AvDrag. The AvMaSp cDNA was expressed as a 28kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvMaSp was introduced into rice plant via Agrobacteriumtumefaciens-mediated gene transformation. Because of seeds pecific prolamin promoter, expression of AvMaSp protein has been achieved inriceseed. The introduction and copy number of the AvMaSp gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvMaSp expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immuno fluorescence staining with the AvMaSpantiserum revealed that the recombinant AvMaSp proteins were localized in transgenic rice seeds. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls. The present study is the first to show the expression of spider silk protein in rice seed.

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a significant disease in most rice cultivation areas. The present study was performed to identify new BB R-gene conferring resistance to Korea Xoo isolates, derived from IR65482-7-216-1-2 and to construct a physical map of the candidate gene. An F2 population derived from a cross between 11325 and Anda was used to determine the exact position of the nearest recombination event to the target region. The position of the R-gene was delimited by flanking markers, RM1233 and RM5766, on chromosome 11. Of the 56 markers designed in the flanking region, 20 were selected as anchor markers and the R-gene was mapped to a 295kb region on chromosome 11. To narrow down the interval spanning the R-gene, an additionally SSR marker, 20 STS markers, and CAPS marker between RM27320 and ID55.05-79 were developed using rice reference genome information. From the result the gene was defined by RM27320 and ID55.WA18-5 located in the BAC clone OSJNBa0036K13. The physical distance between these two markers is approximately 80kb. In a further study, gene expression analysis against listed candidate genes was investigated using semi-quantitative transcription PCR. These results will useful for future disease breeding as well as gene function studies regarding resistance genes.

Current agriculture encounters several challenges including increase in human population, increased meat consumption from income growth, climate change, and demand for healthier foods. Together this puts a tremendous strain on limited natural resources and on an increasingly fragile ecosystem. Today, 55% percent of habitable land is used for agriculture. Two-thirds (66%) of all annual fresh water withdrawals are used for irrigation. Energy is another vital input for agriculture productivity and experts are predicting increasing global competition for supply sources. Monsanto Company uses biotechnology, plant breeding, and agronomic solution to meet the increased food demand. In June 2008, we issued a three-fold commitment (produce more, conserve more and improve farmer’s lives for agriculture to be sustainable) that we call our Commitment to Sustainable Yield. Here, we present Monsanto pipeline to fulfill our commitment for sustainable agriculture.

FLOWERING LOCUS T (FT) is major determinant of the length of the vegetative phase in plants. To understand the role of FT homologs in flowering time control of soybean, we identified ten soybean FT genes and named GmFTs. Expression analysis of GmFT homologs showed that the transcripts of most FT clade genes are mainly expressed in leaves. The expression of GmFT2a, GmFT2b, GmFT5a, and GmFT6 strongly induced in response to floral inductive short-day condition, but GmFT4 and GmFT6 exhibited opposite expression pattern. To understand the biological function of each GmFT/TFL1 genes in flowering time control, we ectopically expressed GmFT cDNAs in Arabidopsis under the control of CaMV 35S promoter. Interestingly, while 35S:GmFT2a and 35S:GmFT5a transgenic plant showed extremely early flowering phenotype, overexpression of GmFT4 delayed flowering. Furthermore we analyzed expression patterns GmFT genes in the leaves of Korean soybean landraces showing various flowering time. The results showed that the transcript level of two FT homologs, GmFT2a and GmFT5a, was high in early flowering landraces, but low in late flowering landraces. In contrast, GmFT4 exhibited opposite expression pattern to those of GmFT2a and GmFT5a, suggesting that GmFT4 may function antagonistically to GmFT2a and GmFT5a in flowering time control of soybean. These results demonstrated that soybean FT homologs have both unique and conserved functions in the photoperiodic control of flowering compared with those in Arabidopsis.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

β-carotene hydroxylase (BCH) has been implicated as a key enzyme conferring a stress tolerant mechanism in plants by production of carotenoids, which serve as protectants against photoinhibition and precursor of ABA biosynthesis. We previously cloned a gene encoding a novel cytosolic form of BCH (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In the present work we extended our study to three GmBCHs as soybean is an allotetraploid, and examined their possible role(s) in nodule development. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in E. coli. Localization of GmBCHs by transfecting Arabidopsis protoplasts with GFP fusions and by EM immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids while GmBCH1 appeared to be cytosolic. RNAi of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids, in nodules. We therefore examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules, and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

Plant specific gene family, NAC (NAM, ATAF, and CUC) transcription factors have been characterized for their roles in plant growth, development, and stress tolerance. In this study, we isolated OsNAC58 gene from rice and analysed expression level by inoculation of bacterial leaf blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo). NAC transcription factor family can be divided into five groups (I–V). On the basis of phylogenetic analysis, OsNAC58 was fall into group III. 35S::OsNAC58-GFP fusion protein was localized on the nuclei. To investigate its biological function in the rice, we constructed vector for overexpression in rice, and then generated transgenic rices. Gene expression of OsNAC58-overexpressed transgenic rice lines were analyzed by northern blot. Analysis of disease resistance to pathogen Xoo, twelve OsNAC58-overexpressed transgenic rice lines showing high expression level of OsNAC58 were shown more resistant than wild type. These results suggest that OsNAC58 gene may play regulatory role during pathogen infection.

Understanding salt tolerance mechanisms is important for the increase of crop yields, and so, several screening approaches were developed to identify plant genes which are involved in salt tolerance of plants. Here, we transformed the Arabidopsis cDNA library into a salt-sensitive calcineurin (CaN)-deficient (cnbD) yeast mutant and isolated the colonies which can suppress salt-sensitive phenotype of cnbD mutant. Through this functional complementation screen, a total of 34 colonies functionally suppressed the salt-sensitive phenotype of cnbD yeast cells, and sequencing analysis revealed that these are 9 genes, including CaS, AtSUMO1 and AtHB-12. Among these genes, the ectopic expression of CaS gene increased salt tolerance in yeast, and CaS transcript was up-regulated under high salinity conditions. CaS-antisense transgenic plants showed reduced root elongation under 100 mM NaCl treatment compared to the wild type plant, which survived under 150 mM NaCl treatment, whereas CaS-antisense transgenic plant leaves turned yellow under 150 mM NaCl treatment. These results indicate that the expression of CaS gene is important for stress tolerance in yeast and plants.

Capsicum diversity is getting lower in modern crops because of the genetic erosion. In Capsicum, breeders have been mainly focused on agriculturally important traits such as disease resistances, high yield and pungency. However, this narrow breeding pool hampered to develop improved cultivars. It has become a hot issue to conservation of genetic diversity and exploitation of wild germplasm in Capsicum. Analysis of genetic diversity and construction of core collection is the first step to make efficient use of germplasm. Although there have been several attempts to construct core collections in Capsicum, most of these works were limited due to handling small number of samples, relying mainly on the characterization of morphological traits or focusing only C. annuum species. To expand understanding of the structure and genetic diversity of germplasm in Capsicum, we need to have a highly efficient genotyping tool to handle large number of samples. Toward this end, we are analyzing 3,599 germplasm accessions including other cultivated species and wild species in Capsicum with 48 single nucleotide polymorphism (SNP) markers.

Amaranths (Amaranthus sp.) are cosmopolitan and include grain, vegetable, ornamental and weed types. Forteen simple sequence repeat (SSR) markers were used to analyze the genetic diversity of 59 accessions of cultivated amaranth from Asian countries. A total of 63 alleles were detected with an average of 4.5 per locus. The averaged values of gene diversity and polymorphism information content (PIC) were 0.35 and 0.33, respectively. Alleles per locus in accessions from South Asia was 4.35, whereas 2.93 and 3.79 alleles per locus were found in Nepal and India, respectively. The mean gene diversity in Central Asia and East Asia was 0.36 and 0.28, respectively, whereas the mean PIC values were 0.27 and 0.22, respectively. The genetic diversity and PIC of the India amaranths were higher than that of other Asian countries. The model-based structure analysis revealed the presence of three subpopulations, which was basically consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 56.16% in contrast to 43.84% for the within-population component. The overall FST value was 0.56, reflecting genetic differentiation within Asian amaranths. These findings could be used for designing effective breeding programs aimed at broadening the genetic bases of commercially grown varieties.

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.

Our study is performed to confirm the level of genetic diversity and population structure with 80 maize inbred lines (40 waxy inbred lines and 40 flint inbred lines) and to explain the genetic basis of agronomic traits using an association mapping. The 200 SSR loci are confirmed a total of 1,610 alleles in total 80 maize inbred lines. The average number of alleles per locus was 8.05. The average GD was 0.72. The average PIC value was 0.68. The average MAF was 0.40. Population structure was revealed for K=2. Total 80 maize inbred lines were divided by groups I, II and admixed group. The 14 waxy inbred lines were assigned to group I. The 45 inbred lines include 5 waxy inbred lines and 40 flint inbred lines were contained to group II. The 21 waxy inbred lines were contained in the admixed group with lower than membership threshold 0.8. Association mapping between 200 SSR markers and 10 phenotypic traits of waxy/flint maize inbred lines were performed by Q GLM and Q+K MLM. In significant level at 0.01, 72 SSR markers were associated with 10 phenotypic traits using Q GLM. The 4 marker-trait association were detected in Q+K MLM. The results derived from this study will be used for designing efficient new maize breeding programs.

Abscission is an important developmental process used to shed organs such as leaves, flowers and fruits. Despite the detailed characterization of growth dynamics and hormonal balance during the early steps of fruit development, the molecular aspects remain unclear. Abscission of young fruit occurs by separation of cells in anatomically distinct regions between the pedicel and junction. Differences of gene expression between central pedicel and lateral pedicel were investigated by NGS. Partial cDNAs from 15 clones from both the central pedicel and lateral pedicel were selected for nucleotide sequence determination and homology searches, and 12 clones were subsequently selected for further analysis. In preliminary series of Real Time PCR analysis, 9 genes were confirmed as showing a higher expression level in lateral pedicel than in central pedicel. Many of these genes are expressed in a central or lateral pedicel in specific manner, and the expression profiles of the representative genes were confirmed. To clarify the mechanism of MdIAA14 transcription factor gene underlying abscission zone development, we are investigating the expression patterns between central and lateral pedicels in different apple cultivar using real-time PCR and constructing the vector for transformation into apple.

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.

AGenome-wide association studies (GWAS) have proven a useful technique for identifying genetic loci responsible for natural variation in rice. With the fast developed next-generation sequencing technology, it is possible for people to carry out GWAS by phenotyping different traits. However, how to make full use of huge data, abandon unnecessary data, and solve the problem of data application effectively seems still an obstacle for many researchers. Taking the case of whole-genome resequencing of Korean authentic rice core set, here we present a general technological path of GWAS including: 1) a schematic view of sequencing-based GWAS in rice; 2) a user-friendly and interactive web application for GWAS in rice by the aid of experience from Arabidopsis; 3) Haplotype and association analysis of candidate genes in a certain mechanism pathway, giving 10 starch synthesis genes as example; and 4) functional validation by Trans- and Mata-Omics analysis.

Drought and salinity are two major environmental factors determining plant productivity that due to their high magnitude of impact and wide distribution. The regulatory circuits include stress sensors, signaling pathways comprising a network of protein-protein reactions, transcription factors and promoters, and finally the output proteins or metabolites. Plant receptor-like kinases (RLKs) are transmembrane proteins family, are predicted to be major components of the signaling pathways that allow plants to respond to diverse environmental and development condition. Subfamily of Catharanthus roseus RLK1-like kinases (CrRLK1Ls) is a novel type of RLK, was identified in Arabidopsis with 17 members carrying a putative extracellular carbonhydrate-binding malectin-like domain. To study the function of CrRLK1Ls subfamily in rice which is a most widely consumed staple food, we produced the phylogenomic data with the integration of microarray-based anatomical and stress expression profiling data to the context of rice CrRLK1Ls family phylogenic tree. The expression profiling data are based on a large number of public microarray data such as 1150 Affymetrix arrays and 209 Agilent 44K arrays. Chromosomal localization of CrRLK1Ls reveals that three of 16 genes were tandem duplicated. Subsequently, we identified 7 genes that showed circadian regulation pattern and three genes of them simultaneously response to drought stress: two were down-regulated and one was up-regulated. Functional gene network development mediated by these stress responsible genes might be an useful foundation to explain the molecular mechanism of stress response mediated by this gene family.

Backcrossing is a plant breeding method most commonly used to incorporate one or a few genes into an adapted or elite variety. To facilitate MAB (marker-assisted backcrossing) in a practice breeding program, we developed a SNP database and a program for providing selected markers for background selection from genome-wide SNPs of seven tomato accessions downloaded from NCBI-SRA. We identified 425,935 SNPs among 21 parental combinations with data from seven transcriptomes and developed a SNP database. To select the optimized number of markers for background selection, we divided 12 chromosomes according to physical length and genetic length. Initially, each chromosome was equally divided into five blocks according to physical length, and three SNPs were positioned per block. Additionally, we applied the genetic distance calculated from the recombination rate because the frequency of recombination can vary greatly among chromosomal regions. When considering genetic distance, each chromosome was divided into fifteen blocks unequally and one marker composed of EXPEN-2000 was positioned per block. The program for background selection was designed to be simple and easy to use, and it is available at http://tgsol.seeders.co.kr/ index.php/tg/mab. When the user selects the parental combination, the program provides selected markers with primer information. The value of this program for tomato breeding will further increase if more accession numbers are added to the database.

As the market demand on functionality rice has been increasing, embryo rice in which embryo residue remains even after milling has come to comsumers’ attention because rice embryo contains several functionality components. Consequently, development of rice varieties for higher rate of embryo adhesion to grains after milling has become one of the breeding objectives for quality improvement. In this study, we observed embryo dent of 49 commercial varieties and analyzed the relationship between embryo dent and grain size and shape. Embryo dent of rice grains varied 0.27 (Keunnun)~0.59 (Daerip 1) mm. Varieties Jinbu, Jinbo, Heugseol, Obong, Unkwang, and Cheongnam showed relatively deeper embryo dent, suggesting that they will be applicable in breeding for embryo rice. Embryo dent was correlated positively with grain width (r=0.53**) and grain size(r=0.34*), and negatively with grain width/length ratio (r= -0.38**). Strategies for breeding embryo rice were discussed in relation to embryo dent, grain size and shape.

Rice blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive rice diseases worldwide. Resistance to rice blast pathogen mostly shows a quantitative trait controlled by several genes. A total of 13 major blast resistance (R) genes were reported in a number of Korean rice varieties using molecular markers. The Pi-ta gene, which locates near to the centromere of chromosome 12, was haplotyping using 1790 accessions including cultivated and wild varieties in previous research. However, the genetic variations of other R genes in rice still not clear. Three R genes, Pi9, Pia, and Pib on chromosome 6, 11 and 2 respectively, were resequenced among 84 accessions of rice core set. Different types of halotype among the 84 accessions were detected. Some new SNPs and InDels found in exon part of R genes were expected to result into amino acid changes following analysis of the genetic code variations, and the germplam in this rice core set which are resistance to blast were explored. We are expecting to develop the new functional markers and incorporate of resistance genes into existing rice cultivars and finally these apply outcomes in breeding rice resistance to blast diseases.

Preharvest sprouting (PHS) not only causes reduction of grain yield, but also affects the quality of grains, resulting into significant economic losses. PHS is governed by multiple genes. Little is known about the large genetic variation of preharvest sprouting in rice. In the present study, genetic variations of four PHS genes, OsVP1, Osaba1, Alpha-amylase3D and OsGA20ox1 were studied by using whole-genome resequencing data of 84 accessions of rice core set. A total of haplotype groups; 27, 29, 6 and 14, for OsVP1, Osaba1, Alpha-amylase3D and OsGA20ox1, respectively, were detected among the 84 accessions. Some new SNPs and InDels were found in exon part of PHS related genes were expected to result in amino acid changes following analysis of the genetic code variations, and the germplasm or varieties which are resistant to preharvest sprouting were explored. Based on this step, phenotyping for PHS is ongoing, and the association mapping of PHS will be conducted by using SNPs resulted from the haplotyping data. The present results will be ultimately useful to the molecular breeding for the development of PHS resistant rice cultivars.