Plants have evolved a set of protecting mechanisms against pathogens, which include secondary metabolites and induced defense responses to pathogen attack. The biological role of purine alkaloids including caffeine is largely unknown. It has been proposed that caffeine confers a resistance against pathogenic bacteria and herbivores. We, in this study, tested direct effects on the growth of rice pathogenic microbes, Xanthomonas oryzae pv. oryzae (Xoo) causing a bacterial leaf blight and Magnaporthe grisea (M. grisea) causing a rice blast. Cell growth of Xoo and M. grisea were significantly retarded in presence of high concentration (2mM) of caffeine. Exogenous caffeine (5mM) induced resistance of wild type rice (cv. Dongjin, susceptible to Xoo and M. grisea) against those pathogens. These results indicated that caffeine enhanced the basal resistance to infection with Xoo. In addition, expression of pathogenesis-related (PR) genes was tested in the caffeine treated rice to elucidate the acquired resistance by caffeine, resulted in induction of PR genes including OsPR1a and OsPrb1. We have generated a transgenic rice producing caffeine by introduction of three N- methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) identified from coffee plant. The transgenic rice successfully expressed the three genes, synthesized caffeine up to 5ug/g and showed enhanced resistance to Xoo. We also observed that transcripts of PR genes such as the OsPR1a and OsPrb1 encoding PR-1 type pathogenesis-related protein increased in the caffeine-producing rice. These result showed that caffeine is likely to act a powerful factor to increase level of rice defense as a natural and non-harmful metabolite.

With the rapid development of sequencing technologies, next-generation sequencing is widely utilized for molecular breeding in several crops including rice. We performed whole genome resequencing of ten Korean rice accessions including six cultivars and four mutant lines. In total, 2,448 million raw reads were generated with over 58x coverage of Nipponbare genome. We mapped the reads from each of the ten accessions onto genomic sequence of japonica rice cultivar, Nipponbare. We detected 3,144,016 SNPs, which estimated to be one per 2.2kb on average. We found SNPs in genes that have been reported to be involved in rice flowering time regulation and bacterial blight resistance among ten rice accessions. Unmapped region against Nipponbare genome occupied about 1 ~ 2% in each accession. Over 50% of the unmapped region were found in the repeat region. The minimum length of gap in all accessions were 1bp and the maximum length of gap was 45,967bp in Ilpum. We also identified 3,497 possible gene loss events within these unmapped regions. The frequency of gene loss in each chromosome ranged from 33 on chromosome 5 to 913 on chromosome 11. The genetic variations we detected among ten rice accessions will provide invaluable resources for identification of genes associated with diverse traits of agronomical importance for molecular breeding.

In order to find new genetic sources of rice salt tolerance, we did screening with about 10,000 rice mutant lines created by Ac/Ds insertional mutagenesis. First, we raised rice seedlings with media soil on 0.7% NaCl solution and selected 71 putative salt tolerant lines and analyzed their Ds insertion sites. We tested their salt tolerance by growing seedlings on MS medium containing, 0 mM, 150 mM, and 250 mM NaCl. Also, their seedling salt tolerance were evaluated by growing on Yoshida nutritional solution containing 0.6% NaCl. Finally, we selected eight mutant lines showing increased seedling salt tolerance compared with wild type variety, Dongjin, repeatedly. We grow them in rice field and investigated their agronomic traits such as heading time, culm length, panicle length, and panicles per hill. Among them two lines which were named Salt10 and Salt23 and showed favorable agronomic characteristics were crossed with Dongjin for further genetic analysis and mapping the causative gene variation.

Progress in next-generation sequencing technologies have enabled discovery of massive amount of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertion-deletion (InDels), which are an invaluable resource to analyze genetic diversity in a population. We performed whole-genome resequencing of ten Korean rice accessions including six cultivars and four mutant lines. A total of 2,448 million raw reads was generated with 58-fold coverage and uniquely mapped to 87.5% of the Nipponbare as a reference genome. We identified 3,240,025 DNA polymorphisms including 2,867,878 SNPs, 151,845 insertions and 220,302 deletions between the Korean rice accessions and Nipponbare. We observed that in ten Korean rice accessions, the frequency of potential SNPs was estimated to be one per 2.1kb on Nipponbare (382Mb). According to annotation of DNA polymorphisms, 634,617 SNPs were found in gene region, and only 169,738 SNPs were occurred in coding region. Altogether, 86,251 non-synonymous SNPs were located on 76,891 genes. We also examined the cultivar-specific SNPs to select candidate SNPs which would have possibility of being associated with unique phenotype or agronomical trait of each cultivar. It was estimated that the portion of cultivar specific SNPs is 1~12% of the total SNPs. These DNA polymorphisms obtained from our result will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.

In plants, the Dof (DNA binding with One Finger) proteins are plant-specific transcription factors with a particular class of zinc-finger DNA-binding domain. The Dof genes have been predicted 30 different Dof genes in the rice Oryza sativa genome by phylogenetic analysis. The mostly Dof proteins contain a conserved region of 50 amino acids with a C2-C2 zinc finger motifs that binds a cis-regulatory element sequence 5’-T/AAAAG-3’. We found that a member of the DOF transcription factor family, Dof1 gene of rice, was expressed to wound from Ds insertion mutant population. Sequencing of the flanking regions of the transposon insertion site indicated that the gene-trap had been inserted near the front of the second exon of OsDof1 gene in chromosome 7. Genomic southern analysis revealed that mutant line contained a single copy of Ds gene trap. The Ds tagged rice mutant line, OsDof1::Ds, wound-inducible GUS expression was identified. To analyze the cis-acting elements, we constructed fusion genes with the OsDof1 promoter fused to the β-glucuronidase (GUS) reporter gene and transformed Arabidopsis and rice plants with these constructs. Wound-induced GUS expression was observed in the leaves of transgenic OsDof1::GUS rice and Arabidospsis plants. These results showed that, OsDof1 protein might be involved in stress responses and growth regulation in plant, might plays a role as a transcription regulator in stress response signal transduction pathways of plant.

Advances in genome sequencing technologies have aided discovery of millions of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertion-deletion (InDels), which are an invaluable resource to analyze genetic diversity in a population. We performed whole-genome resequencing of ten Korean rice accessions including six cultivars and four mutant lines. A total of 2,447 million raw reads were generated with over 58x coverage and detected 3,240,025 DNA polymorphisms between the Korean rice accessions and Nipponbare as reference genome. We observed that in ten Korean rice accessions, the frequency of potential SNPs was estimated to be one per 2.1kb on Nipponbare (382Mb). Potential SNPs were classified into two types, homozygous SNP and heterozygous SNP, which approximately 87% of the total was homozygous SNPs from ten accessions and heterozygous SNPs accounted for 13%. According to annotation of DNA polymorphisms, 634,620 SNPs were found in gene region, and only 169,738 SNPs were occurred in coding region. Altogether, 86,251 non-synonymous SNPs were located on 76,891 genes. We also examined genes which had at least one SNP in all ten accessions. It was estimated that the total of 290 genes had one or more non-synonymous SNPs and 25 genes had only synonymous SNPs. These genes were functionally classified based on gene ontology (GO). These DNA polymorphisms obtained from our result will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.