Bacterial spot of tomato (Solanum lycopersicum L.) is caused by at least four species of Xanthomonas with multiple physiological races. In this study, we developed a mapping population for association analysis of bacterial spot resistance. For this population, six advanced breeding lines with distinct sources of resistance were first crossed in all combinations and their F1 hybrids were intercrossed. The 1,100 segregating progeny from these crosses were evaluated in the field against T1 strains. Based on this individual evaluation, we selected 5% of the most resistant and 5% of the most susceptible progeny for evaluation as plots in two subsequent replicate field trials inoculated with T1 and T3 strains. A total of 461 markers across 12 chromosomes were used for genotyping these selections. Of these markers, an optimized subset of 384 SNPs was derived from the 7,720 SNP Infinium array developed by the Solanaceae Coordinated Agricultural Project (SolCAP). For association analysis to detect known resistance loci and additional novel loci, we used the mixed models with correction for population structure, and found that accounting for kinship appeared to be sufficient. Detection of known loci was not improved by adding a correction for structure using either a Q matrix from model-based clustering or covariate matrix from Principal Component Analysis. Both single-point and haplotype analyses identified strong associations in the region of the genome known to carry Rx-3 (chromosome 5) and Rx-4/Xv3 (chromosome 11). Additional QTL associated with resistance were detected on chromosomes 1, 3, 4, 6 and 7 for T1 resistance and chromosomes 2, 4, and 6 for T3 resistance. Haplotype analysis improved our ability to trace the origin of positive alleles. These results demonstrate that both known and novel associations can be identified using complex breeding populations that have experienced directional selection.

sequence and more than fifty thousand proteins have been obtained to date. Transcription factors (TFs) are important regulators involved in plant development and physiological processes and the AP2/ERF protein family contains TFs that also plays a crucial role as well and response to biotic and abiotic stress conditions in plants. However, no detailed expression profile of AP2/ERF-like genes is available for B. oleracea. In the present study, 226 AP2/ERF TFs were identified from B. oleracea based on the available genome sequence. Based on sequence similarity, the AP2/ERF superfamily was classified into five groups (DREB, ERF, AP2, RAV and Soloist) and 15 subgroups. The identification, classification, phylogenetic construction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction network were then predicted and analyzed. AP2/ERF TFs expression levels exhibited differences in response to varying abiotic stresses based on expressed sequence tags (ESTs). BoCBF1a, 1b, 2, 3 and 4, which were highly conserved in Arabidopsis and B. rapa CBF/DREB genes families were well characterized. Expression analysis enabled elucidation of the molecular and genetic level expression patterns of cold tolerance (CT) and susceptible lines (CS) of cabbage and indicated that all BoCBF genes responded to abiotic stresses. Comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes and BoCBF family genes in B. oleracea is required to elucidate AP2/ERF, which will provide rich resources and opportunities to understand abiotic stress tolerance in crops.

Sequence diversity was accumulated through evolution and breeding process. A set of 595 PCR-based novel insertion/deletion (InDel) markers was designed in order to widen the genetic basis for national rice breeding programs. The markers were generated by analyzing of 40 Korean cultivars and published genome sequences of rice(Oryza sativa L. spp japonica). We selected 112 markers spread across all rice chromosomes among the 595 InDel markers, and they showed polymorphic between rice cultivars, which are 284 Korean japonica and Tongil varieties. Due to their simplicity in design and robustness in genotyping, these InDel markers have been routinely used in quantitative trait loci (QTL) mapping studies and marker assisted selection programs for rice. Moreover, the PCR amplification type of InDel markers was converged to digital code, 0 or 1 and then finally represented as one- and two dimensional bar-code system, which could easily differentiate genetically highly homologous japonica rice cultivars. The developed InDel markers uniquely discriminated among each of the Korean cultivars. Therefore, the systems we developed may be valuable tools in discrimination from cultivars

Shaggy-like kinases (SKs), also known as Glycogen synthase kinase 3 (GSK3) proteins, play many important roles in cellular signaling in animals, fungi and amoebae. In particular, SKs participate in key developmental signaling pathways and also regulate the cytoskeleton. SKs -encoding genes are also present in all land plants and in algae, raising questions about possible ancestral functions in eukaryotes. Unlike in animals and Dictyostelium, land plant SKs are encoded by relatively large multi-gene families whose members share high sequence similarity. Along with the studied 10 ASKs (Arabidopsis shaggy-like kinases) indicate that plant SK proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this study, 18 BrSKs are identified from Chinese cabbage, and they are classified into four groups according to the classification of Arabidopsis. The characterization, classification, gene structure and phylogenetic construction of BrSK proteins are performed. Distribution mapping shows that BrSKs are absented in A02 and A10 chromosome. 8 orthologous gene pairs are shared by Chinese cabbage and Arabidopsis. The expression patterns of BrSK genes exhibit differences in five tissues based on RNA-seq data in public data base. Specially, BrSKβ-1 and BrSKβ-2 show floral buds specifically expressed, which indicate that BrSKβ may play a key role during flower or pollen development. We deomonatrated that suppresion of Arabdiopsis orthology of BrSKβ impaired the late pollen in Arabidopsis plants. Taken together, our analyses provided insights into the characterization of the BrSK genes in Chinese cabbage, providing foundation of further functional studies of those genes. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ008118), Rural Development Administration, Republic of Korea]

Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by the promoter. Therefore, it is important to determine the binding motifs of transcription factors to understand the networks associated with embryogenesis. Here, we used a protein-binding microarray (PBM) to determine the binding motif of OsSMF1, which is a basic leucine zipper transcription factor that is involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage proteins (SSPs). In addition, OsSMF1 (also known as OsbZIP58) functions as a key regulator of starch synthesis in the rice seed. Quadruple 9-mer-based PBM (Q9-PBM) and electrophoretic mobility shift assay (EMSA) experiments revealed that OsSMF1 binds to the ACGT (CCACGT(C/G)), GCN4 (TGA(G/C)TCA), and GCN4-like (GGATGAC) motifs with Kd values of 0.3353 μM, 0.6458 μM, and 1.117 μM, respectively. We also identified 60 putative OsSMF1 target genes using a combination of data from expression microarrays and RiceArrayNet (RAN) analysis. Of these OsSMF1 target genes, 20, 22, and 17 genes contained ACGT, GCN4, and GCN4-like motifs within the 2-kb promoter region, respectively. In addition to known target genes, we also identified 35 potential OsSMF1 target genes that have not been previously described in immature seeds. We also confirmed that OsSMF1 directly regulates Os03g0168500 (thioredoxin-related protein), RPBF, NAC6, and two hypothetical proteins (Os12g0621600 and Os11g0582400) in vivo. This study suggests that OsSMF1 functions in a wide range of seed development processes with specific binding affinities for three DNA binding motifs

Salt toxicity is the major factor limiting crop productivity in saline soils. Rice is an important staple food crop of nearly half of the world population and is well known to be a salt sensitive crop. The completion and enhanced annotations of rice genome sequence has provided the opportunity to study functional genomics of rice. With the rapid development of the biotechnology techniques, we can use more accurate and reliable methods to study the mechanism and function in different stress conditions. In present study, 295 rice accessions of diverse origin were re-sequenced and used for genome-wide association study (GWAS) with several germination-related traits, including germination percentage (GP), germination energy (GE), germination rate (GR), germination index (GI), salt tolerance index (STI) in salt tolerant germination stage. Phenotyping of the rice accessions were carried out at 200mM NaCl to screen salt tolerance levels. GWAS was applied to detect the associated genes related to salt tolerance in rice germination stage. Variations and haplotypes of the associated genes were detected and correlation between the phenotypes and genotypes were validated using qRT-PCR.

Preharvest sprouting resistance (PHS) causes the reduction of grain yield and also affects the quality of grains, resulting significant economic losses. PHS and its related traits were evaluated and observed in wide range among the 137 diverse rice accessions. To mine the associated signals for PHS resistance, genome wide association study (GWAS) was performed using phenotype data and whole genome resequencing data of 137 diverse rice accessions. This study not only could detect the previously identified dormancy and PHS associated genes but also explore the new candidate genes associated with the PHS and related traits. An example of them is seed dormancy 4 (Sdr4) gene which was found to be associated with germination % at day 14 (D14). This study provided the potential associated candidate genes which might be very useful to improve the PHS resistance in future rice breeding.

One of the biotic stresses in rice production is rice blast disease caused by Magnaporthe oryzae, which is one of the most destructive fungal diseases in rice. We outlined an approach towards genome wide association study for the blast disease resistance in rice. In total, 295 rice accessions including 137 Heuristic Set accessions (HS) and 158 Korean Bred varieties (KB) were screened for the rice blast disease resistance. Firstly, Magnaporthe oryzae were inoculated to the rice seedlings of two weeks after germinations. Then, evaluation of the disease symptoms and checking the crossing point (CP) value were conducted one week after inoculation. To quantify the CP value, real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to Magnaporthe oryzae HYDROPHOBIN class 1 (MHP1) which is an indispensable unigene encoding HYDROPHOBIN for normal virulence expression. Based on these CP values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template’s copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. These steps are still being undertaken, and after the complete process of disease resistance phenotyping for the whole population containing 295 accessions, GWAS will be performed to examine the associated genes involving in blast resistance mechanism using the whole genome resequencing data of 295 accessions. This approach would be a useful technique for identifying genetic loci responsible for natural variation in rice blast disease resistance and ultimately, new R genes which can improve the blast resistance in rice.

Genetic resources play a great role in crop breeding because of containing a broad array of useful genes. Currently, the harder are rice breeders trying to develop new rice cultivars with the improved traits, they are more often handicapped by the limited availability of germplasm resources. Thus, a desirable core or heuristic (HS) set of germplasm with maximum genetic diversity can be usefully exploited to breakthrough the present and future challenges of the rice breeding. As such we previously developed the rice HS sets of 166 diverse accessions out of a total 24,368 rice germplasms. Here, we report a large-scale analysis of the patterns of genome-wide genetic variations accumulated in the HS as well as Korean rice over the time. We characterized a total of about 11.8 millions of single nucleotide polymorphisms (SNPs) across the rice genome from resequencing a total of 295 rice genomes including 137 HS and 158 KB rice sets, with an average of approximately 10x depth and > 90% coverage. Using about 460,000 high-quality SNPs (HQSNPs), we specified the population structure, confirming our HS set covers all the rice sub-populations. We further traced the relative nucleotide variabilities of HQSNPs and found the level of the diversity was dynamically changing across the KB genome, which reveals the selection history of KB lines in the past and present. In addition, the results of our genome wide association study (GWAS) suggests that our HS can be also a good reservoir of valuable alleles, pinpointing those alleles underlying the important rice agronomical traits. Overall, the resequencing of our HS set re-illuminates the past, present of the germplasm utilization, which will support the Korean rice breeding in the future.

본 연구에서는 토마토 MAB에 활용하고자 토마토 7 품종의 genome-wide SNPs 데이터베이스를 구축하고, MAB를 위한 분자마커 선발 프로그램을 개발하였다. 토마토 전사체 데이터를 NCBI-SRA에서 다운로드 하여 in silico 분석으로 SNP를 추출하였다. 전사체 데이터에서 추출된 SNP를 재료로 7 품종의 토마토 계통을 이용해 총 21개 교배조합별 SNP 분자마커를 선발하였고, primer가 이용 가능한 마커를 이용하여 데이터베이스를 구축하였다. 마커를 선발하기에 앞서 염색체의 분획으로 두 가지 방법을 사용하였는데, 물리적 거리에 따른 분획과 유전거리에 따른 분획 방법이다. 물리적 거리를 이용한 분획은 각 염색체를 동일한 크기의 5개의 구획으로 나누고, 한 구획 당 교배조합별 차이를 보이는 3개의 SNP를 선발하였다. 교배조합이 바뀔 때마다 이용 가능한 SNP가 자동으로 primer 정보와 함께 제공되도록 하였다. 유전거리를 반영한 분획 방법은 각 염색체의 유전적 거리를 측정하여 물리적 거리에 차등을 두어 염색체 구획을 설정하였다. 즉 재조합이 자주 일어나는 염색체 양끝 말단 부분은 구획을 조밀하게 나누어 MAB 마커 또한 많이 할당하여 자세히 조사하도록 구성하였다. 유전거리에 따른 마커 선발에는 1,924개의 tomato- EXPEN 2000 map 분자마커와 SNP 마커를 이용하였다. 교배조합별로 이용할 수 있는 마커를 12개 염색체 상에 그래픽적으로 제공함으로써 사용자가 쉽게 이해하고 이용할 수 있는 MAB 위한 마커 선발 프로그램을 개발하였다. 이러한 토마토 MAB용 분자마커를 제공하는 프로그램은 실제적인 여교잡 선발 육종에 적용하여 분자마커의 활용을 높이고, 육종효율을 증진시킬 것이다.

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.

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.

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.

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.

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 downregulated 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.

Abiotic stresses such as extreme temperatures frequently limit the plant growth and productivity of major crop species. Two Chinese cabbage DH lines that have different geographic origins, in that Chiifu is from temperate regions, while Kenshin is from subtropical and tropical regions have been expected to show the specific response to high or low temperature. To find the temperature response genes between Chiifu and Kenshin, we analyzed transcriptomic profiling from light-chilling (6h at 4°C) and high temperature (6h at 38°C) treated plants using the KBGP-24K chip. Distribution of genes classified by PI (probe intensity) values showed remarkable difference between Chiifu and Kenshin. The number of genes up- and down-regulated gens by both temperatures were 135 and 79 genes, respectively. These genes may be temperature stress-related genes. Genes involved in the response to stress were changed by light-chilling stress. Chiifu specifically up-regulated genes upon light chilling-stress belong to cold acclimation proteins, calcium binding proteins, cell wall biogenesis proteins and lipoxygenase. On the other hand, Kenshin specifically up-regulated genes by heat-shock treatment include heat-shock proteins, phosphatases, protein folding and phosphorylation-associated ones. Further study on these specific genes function may provide insight to adaptation of Chinese cabbage and clue to develop molecular markers.

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.

Genome-wide association study (GWAS) is a very powerful method to identify the natural allelic variation present in crop plants causing variation to economically important traits. The recent advances in high throughput genotyping and sequencing technology supplemented greatly to GWAS. Taking this advantage, we selected a total of 382 Chinese cabbage inbred lines for GWAS study. The selected inbred lines are being sequenced using next generation sequencing technology to develop genome wide gene specific single nucleotide polymorphism markers. The morphological and quality traits data were taken from field grown inbred lines. The phenotype and genotype association study will be done with more environmental grown data’s and developed SNP. At the end of this project, gene specific SNP markers will be developed for Chinese cabbage breeding for morphological and quality traits.

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.

Miniature inverted- repeat transposable elements are expected to play vital role in evolution of genes and genome of major eukaryotic organisms. However, there have been little reports on MITEs in B. rapa, a polyploidy model genome. We identified 13 novel MITE families in B. rapa genome by computational approach. Out of 13 MITEs families three, eight and two were classified under stowaway-like, tourist-like and hAT super families based on their unique structural characteristics. We characterized the members of 13 MITE families from the available 256 Mbp from whole genome draft sequences of B. rapa. We found ech MITE has high copiy number ranges from 14 to 977 which are distributed randomly along all the chromosomes. We also found more than 40% of the MITE members were associated with genes and gene rich regions. Furthermore, the polymorphism due to insertion and non-insertion of MITEs analysis suggest that MITEs are active in the genome. As, such the newly identified MITEs will provide a foundation for the further analysis of roles of MITEs in gene and genome evolution.