Chang-Kug Kim

Improved PCR for identification of Pseudomonas aeruginosa

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41 × 103 copies/μl of cloned amplified target DNA using purified DNA, or 2.7 × 102 colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.

Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers

The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for specific detection of Pectobacterium wasabiae using a primer pair based on the YD repeat protein gene for amplification of a 140-bp DNA fragment from infected wasabi (Wasabia japonica), a member of the crucifer family. The soft rot caused by P. wasabiae is an emerging disease that is present in many wasabi-producing areas. However, specific and reliable methods for identifying the pathogen are not available. Therefore, a qPCR primer set for accurate diagnosis of P. wasabiae was developed from publically available genome sequences. A SYBR Green qPCR primer set was designed based on a YD repeat protein gene of P. wasabiae WPP163 because it is known that this gene is structurally diverse among species, pathovars, or subspecies. The specificity of the primer set was evaluated using genomic DNA from 5 isolates of P. wasabiae, 5 different species of Pectobacterium, and 16 other pathogenic reference bacteria. The primer set used in the PCR assay successfully amplified a 140-bp amplicon for all five P. wasabiae strains. No amplification was obtained from 29 other pathogenic bacteria. The assay was also able to detect at least two genomic DNA, or 3 CFU per reaction, when using calibrated cell suspension.

A database for allergenic proteins and tools for allergenicity prediction.

The AllergenPro database has developed a web-based system that will provide information about allergen in microbes, animals and plants. The database has three major parts and functions:(i) database list; (ii) allergen search; and (iii) allergenicity prediction. The database contains 2,434 allergens related information readily available in the database such as on allergens in rice microbes (712 records), animals (617 records) and plants (1,105 records). Furthermore, this database provides bioinformatics tools for allergenicity prediction. Users can search for specific allergens by various methods and can run tools for allergenicity prediction using three different methods. Availability The database is available for free at http://www.niab.go.kr/nabic/

The complete chloroplast genome of two Brassica species, Brassica nigra and B. Oleracea

Abstract The two Brassica species, Brassica nigra and Brassica oleracea, are important agronomic crops. The chloroplast genome sequences were generated by de novo assembly using whole genome next-generation sequences. The chloroplast genomes of B. nigra and B. oleracea were 153 633 bp and 153 366 bp in size, respectively, and showed conserved typical chloroplast structure. The both chloroplast genomes contained a total of 114 genes including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis revealed that B. oleracea is closely related to B. rapa and B. napus but B. nigra is more diverse than the neighbor species Raphanus sativus.

NABIC: A New Access Portal to Search, Visualize, and Share Agricultural Genomics Data

The National Agricultural Biotechnology Information Center developed an access portal to search, visualize, and share agricultural genomics data with a focus on South Korean information and resources. The portal features an agricultural biotechnology database containing a wide range of omics data from public and proprietary sources. We collected 28.4 TB of data from 162 agricultural organisms, with 10 types of omics data comprising next-generation sequencing sequence read archive, genome, gene, nucleotide, DNA chip, expressed sequence tag, interactome, protein structure, molecular marker, and single-nucleotide polymorphism datasets. Our genomic resources contain information on five animals, seven plants, and one fungus, which is accessed through a genome browser. We also developed a data submission and analysis system as a web service, with easy-to-use functions and cutting-edge algorithms, including those for handling next-generation sequencing data.

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

Identification and characterization of flowering repressor-related genes in Chinese cabbage

In this study, we used a five-step process to identify those genes most likely involved in flowering repression in the Chinese cabbage (Brassica rapa). We tested 6,275 candidate genes with 300K microarrays, which included specific gene expression profiles of FLOWERING LOCUS C (FLC) mutants and normal cultivars during five cold vernalization stages. From that, we identified 289 transcription factor genes and 59 pathway network genes associated with floweringrelated metabolism. Then we compared the 348 genes to 1,287 genes from Gene Ontology and Clusters of Orthologous Groups analyses, which use similar orthologs to categorize conserved genes. Those analyses revealed 10 hypothetical genes for B. rapa, which we verified by reverse transcription-polymerase chain reaction. The final selected genes most likely play regulatory roles in either B. rapa flowering time control or flowering repression during vernalization. While these final genes require further characterization and validation, our study illustrates the usefulness of a multi-layered screening method after initially identifying genes from microarrays.

Current status of Ac/Ds mediated gene tagging systems for study of rice functional genomics in Korea

Abstract Rice is the staple food of more than 50% of the worlds population. Cultivated rice has the AA genome (di-ploid, 2n=24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized trans-posable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos (Hiro-chika. 1997) have been used to generate gene-tagged popu-lations. To date T-DNA and maize transposable element systems has been utilized as main insertional mutagens in rice. A main drawback of a T-DNA scheme is that Agro-bacteria-mediated transformation in rice requires extensive facilities, time, and labor. In contrast, the Ac/Ds system offers the advantage of generating new mutants by secondary trans-position from a single tagged gene. Revertants can be uti-lized to correlate phenotype with genotype. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of inser-tionally mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been launched by collaborative works from 2001 in Korea.

An integrated database for the enhanced identification of silkworm gene resources.

The National Academy of Agricultural Science (NAAS) has developed a web-based database to provide characterization information in silkworm. The silkworm database has four major function menus: variety searching, characterization viewing, general information and photo gallery. It provides 321 silkworm varieties characterization information for six different regions namely, Korean, Japanese, Chinese, European, Tropical and nonclassified group. Additionally, the database provides 1,132 photo images regarding life cycle of various silkworm varieties. A specific characterization information table provides accession number, variety, strain and larval marking, blood color, cocoon color, cocoon shape, egg colors, remarks and image table provides photos which consist of shape and color in the different stages of larval, egg and cocoon stages. Availability The database is available for free at http://www.naas.go.kr/silkworm/english/

Whole-Genome Resequencing and Transcriptomic Analysis to Identify Genes Involved in Leaf-Color Diversity in Ornamental Rice Plants

Rice field art is a large-scale art form in which people design rice fields using various kinds of ornamental rice plants with different leaf colors. Leaf color-related genes play an important role in the study of chlorophyll biosynthesis, chloroplast structure and function, and anthocyanin biosynthesis. Despite the role of different metabolites in the traditional relationship between leaf and color, comprehensive color-specific metabolite studies of ornamental rice have been limited. We performed whole-genome resequencing and transcriptomic analysis of regulatory patterns and genetic diversity among different rice cultivars to discover new genetic mechanisms that promote enhanced levels of various leaf colors. We resequenced the genomes of 10 rice leaf-color accessions to an average of 40× reads depth and >95% coverage and performed 30 RNA-seq experiments using the 10 rice accessions sampled at three developmental stages. The sequencing results yielded a total of 1,814 × 106 reads and identified an average of 713,114 SNPs per rice accession. Based on our analysis of the DNA variation and gene expression, we selected 47 candidate genes. We used an integrated analysis of the whole-genome resequencing data and the RNA-seq data to divide the candidate genes into two groups: genes related to macronutrient (i.e., magnesium and sulfur) transport and genes related to flavonoid pathways, including anthocyanidin biosynthesis. We verified the candidate genes with quantitative real-time PCR using transgenic T-DNA insertion mutants. Our study demonstrates the potential of integrated screening methods combined with genetic-variation and transcriptomic data to isolate genes involved in complex biosynthetic networks and pathways.