Beom-Seok Park

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. Here we construct a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis, which includes several important dietary legumes in Asia, and to enable a better understanding of the evolution of leguminous species. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (V. reflexo-pilosa var. glabra) provides genomic evidence of a recent allopolyploid event. The species tree is constructed using de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.

Abstract Adzuki bean ( Vigna angularis var. angularis ) is a dietary legume crop in East Asia. The presumed progenitor ( Vigna angularis var. nipponensis ) is widely found in East Asia, suggesting speciation and domestication in these temperate climate regions. Here, we report a draft genome sequence of adzuki bean. The genome assembly covers 75% of the estimated genome and was mapped to 11 pseudo-chromosomes. Gene prediction revealed 26,857 high confidence protein-coding genes evidenced by RNAseq of different tissues. Comparative gene expression analysis with V. radiata showed that the tissue specificity of orthologous genes was highly conserved. Additional re-sequencing of wild adzuki bean, V. angularis var. nipponensis and V. nepalensis , was performed to analyze the variations between cultivated and wild adzuki bean. The determined divergence time of adzuki bean and the wild species predated archaeology-based domestication time. The present genome assembly will accelerate the genomics-assisted breeding of adzuki bean.

Abstract Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred ∼0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.

Abstract Most agronomical traits exhibit quantitative variation, which is controlled by multiple genes and are environmentally dependent. To study the genetic variation of flowering time in Brassica napus, a DH population and its derived reconstructed F2 population were planted in 11 field environments. The flowering time varied greatly with environments; 60% of the phenotypic variation was attributed to genetic effects. Five to 18 QTL at a statistically significant level (SL-QTL) were detected in each environment and, on average, two new SL-QTL were discovered with each added environment. Another type of QTL, micro-real QTL (MR-QTL), was detected repeatedly from at least 2 of the 11 environments; resulting in a total of 36 SL-QTL and 6 MR-QTL. Sixty-three interacting pairs of loci were found; 50% of them were involved in QTL. Hundreds of floral transition genes in Arabidopsis were aligned with the linkage map of B. napus by in silico mapping; 28% of them aligned with QTL regions and 9% were consistent with interacting loci. One locus, BnFLC10, in N10 and a QTL cluster in N16 were specific to spring- and winter-cropped environments respectively. The number of QTL, interacting loci, and aligned functional genes revealed a complex genetic network controlling flowering time in B. napus.

Brassica rapa is one of the most economically important vegetable crops worldwide. Owing to its agronomic importance and phylogenetic position, B. rapa provides a crucial reference to understand polyploidy-related crop genome evolution. The high degree of sequence identity and remarkably conserved genome structure between Arabidopsis and Brassica genomes enables comparative tiling sequencing using Arabidopsis sequences as references to select the counterpart regions in B. rapa, which is a strong challenge of structural and comparative crop genomics. We assembled 65.8 megabase-pairs of non-redundant euchromatic sequence of B. rapa and compared this sequence to the Arabidopsis genome to investigate chromosomal relationships, macrosynteny blocks, and microsynteny within blocks. The triplicated B. rapa genome contains only approximately twice the number of genes as in Arabidopsis because of genome shrinkage. Genome comparisons suggest that B. rapa has a distinct organization of ancestral genome blocks as a result of recent whole genome triplication followed by a unique diploidization process. A lack of the most recent whole genome duplication (3R) event in the B. rapa genome, atypical of other Brassica genomes, may account for the emergence of B. rapa from the Brassica progenitor around 8 million years ago. This work demonstrates the potential of using comparative tiling sequencing for genome analysis of crop species. Based on a comparative analysis of the B. rapa sequences and the Arabidopsis genome, it appears that polyploidy and chromosomal diploidization are ongoing processes that collectively stabilize the B. rapa genome and facilitate its evolution.

Abstract A detailed genetic linkage map of Brassica rapa has been constructed containing 545 sequence-tagged loci covering 1287 cM, with an average mapping interval of 2.4 cM. The loci were identified using a combination of 520 RFLP and 25 PCR-based markers. RFLP probes were derived from 359 B. rapa EST clones and amplification products of 11 B. rapa and 26 Arabidopsis. Including 21 SSR markers provided anchors to previously published linkage maps for B. rapa and B. napus and is followed as the referenced mapping of R1–R10. The sequence-tagged markers allowed interpretation of the pattern of chromosome duplications within the B. rapa genome and comparison with Arabidopsis. A total of 62 EST markers showing a single RFLP band were mapped through 10 linkage groups, indicating that these can be valuable anchoring markers for chromosome-based genome sequencing of B. rapa. Other RFLP probes gave rise to 2–5 loci, inferring that B. rapa genome duplication is a general phenomenon through 10 chromosomes. The map includes five loci of FLC paralogues, which represent the previously reported BrFLC-1, -2, -3, and -5 and additionally identified BrFLC3 paralogues derived from local segmental duplication on R3.

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate‐derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa , we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B. rapa . Glucosinolate genes in B. rapa share 72–94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers. The exon/intron split pattern of B. rapa is almost identical to that of Arabidopsis , although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B. rapa , whereas only three were found in Arabidopsis . The expression of those paralogs was not tissue‐specific but varied greatly depending on B. rapa tissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation in B. rapa appears to be more complex than that of Arabidopsis . With the isolation and further characterization of the endogenous genes, health‐beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.

In view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing.We identified simple sequence repeat (SSR) motifs in the B. rapa genome with the sequences of seed BACs used for the BrGSP. By testing 749 amplicons containing SSR motifs, we identified polymorphisms that enabled the anchoring of 188 BACs onto the B. rapa reference linkage map consisting of 719 loci in the 10 linkage groups with an average distance of 1.6 cM between adjacent loci. The anchored BAC sequences enabled the identification of 30 blocks of conserved synteny, totaling 534.9 cM in length, between the genomes of B. rapa and Arabidopsis thaliana. Most of these were consistent with previously reported duplication and rearrangement events that differentiate these genomes. However, we were able to identify the collinear regions for seven additional previously uncharacterized sections of the A genome. Integration of the linkage map with the B. rapa cytogenetic map was accomplished by FISH with probes representing 20 BAC clones, along with probes for rDNA and centromeric repeat sequences. This integration enabled unambiguous alignment and orientation of the maps representing the 10 B. rapa chromosomes.We developed a second generation reference linkage map for B. rapa, which was aligned unambiguously to the B. rapa cytogenetic map. Furthermore, using our data, we confirmed and extended the comparative genome analysis between B. rapa and A. thaliana. This work will serve as a basis for integrating the genetic, physical, and chromosome maps of the BrGSP, as well as for studies on polyploidization, speciation, and genome duplication in the genus Brassica.

Rice germplasm collections continue to grow in number and size around the world. Since maintaining and screening such massive resources remains challenging, it is important to establish practical methods to manage them. A core collection, by definition, refers to a subset of the entire population that preserves the majority of genetic diversity, enhancing the efficiency of germplasm utilization.Here, we report whole-genome resequencing of the 137 rice mini core collection or Korean rice core set (KRICE_CORE) that represents 25,604 rice germplasms deposited in the Korean genebank of the Rural Development Administration (RDA). We implemented the Illumina HiSeq 2000 and 2500 platform to produce short reads and then assembled those with 9.8 depths using Nipponbare as a reference. Comparisons of the sequences with the reference genome yielded more than 15 million (M) single nucleotide polymorphisms (SNPs) and 1.3 M INDELs. Phylogenetic and population analyses using 2,046,529 high-quality SNPs successfully assigned rice accessions to the relevant rice subgroups, suggesting that these SNPs capture evolutionary signatures that have accumulated in rice subpopulations. Furthermore, genome-wide association studies (GWAS) for four exemplary agronomic traits in the KRIC_CORE manifest the utility of KRICE_CORE; that is, identifying previously defined genes or novel genetic factors that potentially regulate important phenotypes.This study provides strong evidence that the size of KRICE_CORE is small but contains high genetic and functional diversity across the genome. Thus, our resequencing results will be useful for future breeding, as well as functional and evolutionary studies, in the post-genomic era.

Summary Ginseng ( P anax ginseng ) is a famous medicinal herb, but the composition and structure of its genome are largely unknown. Here we characterized the major repeat components and inspected their distribution in the ginseng genome. By analyzing three repeat‐rich bacterial artificial chromosome (BAC) sequences from ginseng, we identified complex insertion patterns of 34 long terminal repeat retrotransposons (LTR‐RTs) and 11 LTR‐RT derivatives accounting for more than 80% of the BAC sequences. The LTR‐RTs were classified into three Ty3 / gypsy ( PgDel , PgTat and PgAthila ) and two Ty1 / Copia ( PgTork and PgOryco ) families. Mapping of 30‐Gbp Illumina whole‐genome shotgun reads to the BAC sequences revealed that these five LTR‐RT families occupy at least 34% of the ginseng genome. The Ty3 / Gypsy families were predominant, comprising 74 and 33% of the BAC sequences and the genome, respectively. In particular, the PgDel family accounted for 29% of the genome and presumably played major roles in enlargement of the size of the ginseng genome. Fluorescence in situ hybridization (FISH) revealed that the PgDel1 elements are distributed throughout the chromosomes along dispersed heterochromatic regions except for ribosomal DNA blocks. The intensity of the PgDel2 FISH signals was biased toward 24 out of 48 chromosomes. Unique gene probes showed two pairs of signals with different locations, one pair in subtelomeric regions on PgDel2 ‐rich chromosomes and the other in interstitial regions on PgDel2 ‐poor chromosomes, demonstrating allotetraploidy in ginseng. Our findings promote understanding of the evolution of the ginseng genome and of that of related species in the Araliaceae.

Expressed sequence tag (EST)-based markers are preferred because they reflect transcribed portions of the genome. We report the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers derived from transcriptome sequences in cabbage, and their utility for map construction. Transcriptome sequences were obtained from two cabbage parental lines, C1184 and C1234, which are susceptible and resistant to black rot disease, respectively, using the 454 platform. A total of 92,255 and 127,522 reads were generated and clustered into 34,688 and 40,947 unigenes, respectively. We identified 2,405 SSR motifs from the unigenes of the black rot-resistant parent C1234. Trinucleotide motifs were the most abundant (66.15%) among the repeat motifs. In addition, 1,167 SNPs were detected between the two parental lines. A total of 937 EST-based SSR and 97 SNP-based dCAPS markers were designed and used for detection of polymorphism between parents. Using an F2 population, we built a genetic map comprising 265 loci, and consisting of 98 EST-based SSRs, 21 SNP-based dCAPS, 55 IBP markers derived from B. rapa genome sequence and 91 public SSRs, distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The parental lines used in this study are elite breeding lines with little genetic diversity; therefore, the markers that mapped in our genetic map will have broad spectrum utility. This genetic map provides additional genetic information to the existing B. oleracea map. Moreover, the new set of EST-based SSR and dCAPS markers developed herein is a valuable resource for genetic studies and will facilitate cabbage breeding. Additionally, this study demonstrates the usefulness of NGS transcriptomes for the development of genetic maps even with little genetic diversity in the mapping population.

A complete genome sequence provides unlimited information in the sequenced organism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequencing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa . We have selected 48 seed BACs on chromosome 1 using EST genetic markers and FISH analyses. Among them, 30 BAC clones have been sequenced and 18 are on the way. Comparative genome analyses of the EST sequences and sequenced BAC clones from Brassica chromosome 1 revealed their homeologous partner regions on the Arabidopsis genome and a syntenic comparative map between Brassica chromosome 1 and Arabidopsis chromosomes. In silico chromosome walking and clone validation have been successfully applied to extending sequence contigs based on the comparative map and BAC end sequences. In addition, we have defined the (peri)centromeric heterochromatin blocks with centromeric tandem repeats, rDNA and centromeric retrotransposons. In-depth sequence analyses of five homeologous BAC clones and an Arabidopsis chromosomal region reveal overall co-linearity, with 82% sequence similarity. The data indicate that the Brassica genome has undergone triplication and subsequent gene losses after the divergence of Arabidopsis and Brassica . Based on in-depth comparative genome analyses, we propose a comparative genomics approach for conquering the Brassica genome. In 2005 we intend to construct an integrated physical map, including sequence information from 500 BAC clones and integration of fingerprinting data and end sequence data of more than 100 000 BAC clones. The sequences have been submitted to GenBank with accession numbers: 10 204 BAC ends of the KBrH library (CW978640–CW988843); KBrH138P04, AC155338; KBrH117N09, AC155337; KBrH097M21, AC155348; KBrH093K03, AC155347; KBrH081N08, AC155346; KBrH080L24, AC155345; KBrH077A05, AC155343; KBrH020D15, AC155340; KBrH015H17, AC155339; KBrH001H24, AC155335; KBrH080A08, AC155344; KBrH004D11, AC155341; KBrH117M18, AC146875; KBrH052O08, AC155342.

The genus Brassica includes the most extensively cultivated vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. A physical map of the B. rapa genome is a fundamental tool for analysis of Brassica "A" genome structure. Integration of a physical map with an existing genetic map by linking genetic markers and BAC clones in the sequencing pipeline provides a crucial resource for the ongoing genome sequencing effort and assembly of whole genome sequences.A genome-wide physical map of the B. rapa genome was constructed by the capillary electrophoresis-based fingerprinting of 67,468 Bacterial Artificial Chromosome (BAC) clones using the five restriction enzyme SNaPshot technique. The clones were assembled into contigs by means of FPC v8.5.3. After contig validation and manual editing, the resulting contig assembly consists of 1,428 contigs and is estimated to span 717 Mb in physical length. This map provides 242 anchored contigs on 10 linkage groups to be served as seed points from which to continue bidirectional chromosome extension for genome sequencing.The map reported here is the first physical map for Brassica "A" genome based on the High Information Content Fingerprinting (HICF) technique. This physical map will serve as a fundamental genomic resource for accelerating genome sequencing, assembly of BAC sequences, and comparative genomics between Brassica genomes. The current build of the B. rapa physical map is available at the B. rapa Genome Project website for the user community.

Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.

Glucosinolates (GSLs) are secondary metabolites that have anticarcinogenic activity and play defense roles in plants of the Brassicaceae family. MYB28 is known as a transcription factor that regulates aliphatic GSL biosynthesis in Arabidopsis thaliana. Brassicaceae plants have three orthologous copies of AtMYB28 derived from recent genome triplication. These BrMYB28 genes have a high level of sequence homology, with 81-87% similarities in the coding DNA sequence compared to Arabidopsis. Overexpression of three paralogous BrMYB28 genes in transgenic Chinese cabbage increased the total GSL content in all T1 generation plants and in two inbred lines of homozygous T2 plants. The highest total GSL contents were detected in homozygous T2 lines overexpressing BrMYB28.1, which showed an approximate fivefold increase compared to that of nontransgenic plants. The homozygous T2 lines with overexpressed BrMYB28.1 also showed an increased content of aliphatic, indolic, and aromatic GSLs compared to that of nontransgenic plants. Furthermore, all of the three BrMYB28 genes were identified as negative regulators of BrAOP2 and positive regulators of BrGSL-OH in the homozygous T2 lines. These data indicate the regulatory mechanism of GSL biosynthesis in B. rapa is unlike that in A. thaliana. Our results will provide useful information for elucidating the regulatory mechanism of GSL biosynthesis in polyploid plants.

Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium <i>Synechocystis</i> sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria <i>Nostoc punctiforme</i> ATCC 29133 and <i>Anabaena</i> sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, <i>Microcoleus</i> IPPAS B353 (<i>Microcoleus</i> B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant <i>Escherichia coli</i> indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: 10 GAFs contained PCB chromophores, whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of <i>Microcoleus</i> B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

Genome wide transcription analysis in response to stresses is essential to provide the basis of effective engineering strategies to improve stress tolerance in crop plants. In order to perform transcriptome analysis in Brassica rapa, we constructed a B. rapa oligo microarray, KBGP-24K, using sequence information from approximately 24,000 unigenes and analyzed cold (4°C), salt (250 mM NaCl), and drought (air-dry) treated B. rapa plants. Among the B. rapa unigenes represented on the microarray, 417 (1.7%), 202 (0.8%), and 738 (3.1%) were identified as responsive genes that were differently expressed 5-fold or more at least once during a 48-h treatment with cold, salt, and drought, respectively. These results were confirmed by RT-PCR analysis. In the abiotic stress responsive genes identified, we found 56 transcription factor genes and 60 commonly responsive genes. It suggests that various transcriptional regulatory mechanisms and common signaling pathway are working together under the abiotic stresses in B. rapa. In conclusion, our new developed 24K oligo microarray will be a useful tool for transcriptome profiling and this work will provide valuable insight in the response to abiotic stress in B. rapa.

Brassica rapa , which is closely related to Arabidopsis thaliana , is an important crop and a model plant for studying genome evolution via polyploidization. We report the current understanding of the genome structure of B. rapa and efforts for the whole-genome sequencing of the species. The tribe Brassicaceae , which comprises ca. 240 species, descended from a common hexaploid ancestor with a basic genome similar to that of Arabidopsis . Chromosome rearrangements, including fusions and/or fissions, resulted in the present-day “diploid” Brassica species with variation in chromosome number and phenotype. Triplicated genomic segments of B. rapa are collinear to those of A. thaliana with InDels. The genome triplication has led to an approximately 1.7-fold increase in the B. rapa gene number compared to that of A. thaliana . Repetitive DNA of B. rapa has also been extensively amplified and has diverged from that of A. thaliana . For its whole-genome sequencing, the Brassica rapa Genome Sequencing Project ( Br GSP) consortium has developed suitable genomic resources and constructed genetic and physical maps. Ten chromosomes of B. rapa are being allocated to Br GSP consortium participants, and each chromosome will be sequenced by a BAC-by-BAC approach. Genome sequencing of B. rapa will offer a new perspective for plant biology and evolution in the context of polyploidization.

The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level.We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events.Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions of genes in B. rapa based on the locations of orthologues in A. thaliana can be misleading. Our results will be of relevance to a wide range of plants that have polyploid genomes, many of which are being considered according to a paradigm of comprising conserved synteny blocks with respect to sequenced, related genomes.

The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREBs) are important proteins in involved in responses to abiotic stress in plants. We identified ten BrDREB1 genes belonging to the CBF/DREB1 gene family in the Brassica rapa whole genome sequence, whereas six genes are found in the Arabidopsis thaliana genome. The deduced amino acid sequences of the B. rapa genes showed conserved motifs shared with other known plant CBF/DREB1s. Comparative analysis revealed that nine of the BrDREB1 genes were derived from the recent genome triplication in the tribe Brassiceae and the other one was translocated. The nine genes were located in seven of the 12 macrosyntenic blocks that are triplicated counterparts of four Arabidopsis macrosyntenic blocks harboring six CBF/DREB1 genes: one gene on each of three blocks and three tandemly arrayed genes on another block. We inspected the expression patterns of eight BrDREB1 genes by RT-PCR and microarray database searches. All eight genes were highly up-regulated during cold (4 °C) treatment, and some of them were also responsive to salt (250 mM NaCl), drought (air drying), and ABA (100 μM) treatment. Microarray data for plant developmental stages revealed that BrDREB1C2 was highly expressed during a period of cold treatment for vernalization, similar to abiotic stress-inducible genes homologous to Bn28a, Bn47, Bn115, and BoRS1, but almost opposite of BrFLC genes. Taken together, the number of BrDREB1 genes increased to 10 by genome triplication and reorganization, providing additional functions in B. rapa abiotic stress responses and development, as distinct from their Arabidopsis homologs.

Early detection of appendicitis has increased due to development of computed tomography and ultrasonography, yet we are frequently meeting complicated appendicitis, including perforation, abscess and a gangrenous appendicitis due to delayed diagnosis. For that reason, we want to evaluate predictive factors for the complicated appendicitis.A total of 128 patients with appendicitis, after 13 patients with a duration of under 12 hours and 15 patients with pathological non-appendicitis were excluded from 156 patients, who visited Kwangju Christian Hospital from November 2008 to November 2010 were retrospectively reviewed.There were 62 patients (48.3%) with simple appendicitis and 66 patients (51.7%) with complicated appendicitis. In univariate analysis, age (P < 0.001), C-reactive protein (P < 0.001) and the diameter of the appendix (P = 0.006), were found to be significant. Multivariate analysis demonstrated that C-reactive protein was an independent predictor for complicated appendicitis (odds ratio, 1.371; 95% confidence interval, 1.155 to 1.628; P < 0.001). The cut-off value of C-reactive protein was set at 7.05 mg/dL by using receiver operating characteristic curve (0.805; sensitivity, 57.6%; specificity, 98.3%).This study suggests that if C-reactive protein is above 7.05 mg/dL, immediate and proper management should be performed due to a high probability of complicated appendicitis, especially in young children or elderly patients who frequently present with vague symptoms.

ABSTRACT Pectobacterium carotovorum is a plant-pathogenic enterobacterium responsible for soft rot in various commercially important plants. Here we report the complete genome sequence and automatic annotation of strain PCC21.

Miniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome.We identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs.A Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species.

Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5'-flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with peri-centromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa.

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.

Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-meAiateA transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glucobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.

Abstract Background Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana , provides means to explore their genomic complexity. Results A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5% (shown to contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana , a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity. Conclusions A high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes. All the physical mapping data is freely shared at a WebFPC site ( http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/ ; Temporarily password-protected: account: pgml; password: 123qwe123.

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.

xBrassicoraphanus line BB#5, a new synthetic intergeneric hybrid between Brassica rapa L. ssp.pekinensis and Raphanus sativus L. var.rafiphera induced by N-methyl-N-nitroso-urethane mutagenesis in microspore culture, shows high seed fertility and morphological uniformity.Dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes and genomic in situ hybridization (GISH) using B. rapa genomic DNA probe were carried out to analyze the chromosome composition and the meiosis pairing pattern compared to its parental lines.The somatic chromosome complement is 2n = 38, which consists of 17 metacentric and two submetacentric chromosomes with lengths of 2.18 to 5.01 µm.FISH karyotype analysis showed five and eight pairs of 5S and 45S rDNA loci.GISH meiosis pairing analysis showed that 19 complete bivalents were most frequent and accounted for 42% of the 100 pollen mother cells examined.Based on chromosome number, size, morphology, rDNA distribution, and meiosis pairing pattern, both parental genomes of B. rapa and R. sativus appear to exist in xBrassicoraphanus line BB#5, demonstrating its genome integrity.Such stable chromosome constitutions and meiotic pairing patterns in somatic and meiotic cells are very rare in natural and synthetic intergeneric hybrids.Chromosomal studies and genetic and phenotypic changes in allopolyploids are discussed.The results presented herein will be useful for further genomic study of xBrassicoraphanus lines and their improvement as promising new breeding varieties.

Circulating tumor DNA (ctDNA) can be utilized to identify the presence of cancer as well as minimal residual disease. Quantification of ctDNA using plasma-based tests can be a useful cancer management tool to assess prognosis; however, some methodologies also require tumor tissue for analysis or are limited to tumor types that tend to have higher amounts of associated ctDNA. Here we demonstrate the feasibility of using a tumor-uninformed genome-wide methylome enrichment platform to quantify ctDNA in plasma and predict prognosis in RCC. Biobanked pre-treatment samples from individuals with newly diagnosed stage I-IV RCC (University Health Network, Ontario Tumor Biobank) were analyzed with a bisulfite-free, non-degradative genome-wide DNA methylation enrichment platform using 5-10 ng of cell-free DNA isolated from plasma. ctDNA was quantified from average normalized counts across informative regions. Events were defined as cancer recurrence or progression. A ctDNA quantity threshold was set such that 95% of samples without an event fell below the threshold (i.e., 95% specificity). Time to event was compared for samples with ctDNA quantities above the threshold to those below the threshold. The cohort included 151 samples [64 stage I, 2 stage II, 23 stage III, 15 stage IV, 47 with unknown or incomplete stage information]. There was a median follow-up of 15.4 months and a total of 21 events. Samples with ctDNA quantities above the threshold were significantly more likely to recur or progress than those below the threshold [hazard ratio 13.28 (95% CI 5.47, 32.26), log-rank P<0.001]. This analysis demonstrates the feasibility of using a blood-based, tumor-uninformed genome-wide methylome enrichment platform for ctDNA quantification and prognostic prediction in renal cancer. This is a promising demonstration of prognostic performance in a cancer type that is typically difficult to detect due to low amounts of ctDNA. Additional evaluation in post-treatment and longitudinal samples will further test the performance of this platform.

Total of fifty accessions of Brassica rapa with various morphological characteristics were used for production of double haploid plants though microspore culture in Brassica rapa.Among them, only 30 accessions induced embryos from microspores.The highest efficiency of embryo induction of 1.194 per bud was obtained from IT135449 of turnip type, while 3 accessions of sarson (winter oil) type did not generate embryo.The effect of heat shock periods for embryogenesis was also investigated with 4 accessions (IT135449; Turnip type, IT199710; Chinese cabbage type, IT212886; Pak choi type, IT218043; Summer oil type).The high productions of embryos were observed in IT135449, IT199710 and IT212886 when microspores were pre-cultured to 32˚C for 2 days.In IT218043, high embryogenesis was observed at the 3 days of heat shock treatment.The optimal condition of shoot regeneration for IT199710 was observed in MS medium supplemented with NAA 0.5 mg•L -1 and BAP 1 mg•L -1 .In contrast, the IT135449 and IT212886 were observed high regeneration frequency in MS medium without plant growth regulators.All the plantlets regenerated from microspore-derived embryos have been successfully transplanted to soil, and bud self-pollinated seeds were produced from doubled haploid plants.This indicated that double-haploid genotype was likely generated naturally during embryogenesis process.

The authors introduce a minimally invasive muscle sparing transmuscular microdiscectomy (MSTM) to treat herniated lumbar disc disease. Its results are compared with conventional subperiosteal microdiscectomy (CSM) to validate the effectiveness.Muscle sparing transmuscular microdiscectomy, which involves muscle dissection approach using the natural fat cleavage plane between the multifidus to expose the interlaminar space, was performed in 23 patients to treat a single level unilateral lumbar radiculopathy. The creatine phosphokinase (CPK)-MM serum levels were measured on admission and at 1, 3, and 5 days postoperatively. Postoperative pain was evaluated using a 10-point visual analogue scale (VAS) and recorded on admission and at 1, 3, and 5 days postoperatively. The results were compared to those from the conventional subperiosteal microdiscectomy (43 patients).The CPK-MM levels were significantly lower in the serum of the MSTM group compared to the CSM group on postoperative days three and five (p = 0.03 and p = 0.02, respectively). The clinical scales for back pain using VAS were significantly lower in the MSTM group than in the CSM group on postoperative days three (p = 0.04). The mean VAS scores for leg pain in both groups showed no significant differences during the early postoperative period.Muscle sparing transmuscular microdiscectomy is a minimally invasive surgical option to treat lumbar radiculopathy due to herniated disc. The approach affected minimal injury to posterior lumbar supporting structures with alleviated postoperative back pain.

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.

The sequencing of the Brassica rapa genome has enabled better understanding of its structure and evolution, and created numerous opportunities for exploration of genome function and breeding applications. Nevertheless, the currently available completed genome sequences are estimated to cover only about 60 % of the genome, while the remaining 40 % is unassembled mainly due to the highly repetitive nature of this portion of the genome. Elucidation of the nature and distribution of repeat elements in the context of the entire genome would enhance our understanding of their role in genome structure, function, and evolution. In this chapter, we review the genomic distribution, characterization and evolutionary implications of currently identified repeat elements comprising the ‘hidden’ portion of the B. rapa genome. Low-coverage whole-genome sequence (WGS) was used to survey the major genomic repeats and their proportion in the B. rapa genome. Coupling this with molecular cytogenetics, we characterized the abundance and genomic distribution of seven major repeats, namely centromeric tandem repeats 1 and 2, centromeric retrotransposons, pericentromeric retrotransposons, 5S rDNA, 45S rDNA, and subtelomeric tandem repeats. These repeats accounted for approximately 20 % of the B. rapa genome, which is much more than the <1 % covered by repeats in the currently available genome assembly. We also compared their distributions among different B. rapa accessions and in the close relative Brassica oleracea, for better understanding of the plasticity of the Brassica genomes.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosenory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years.

The cDNA clone of Brassica rapa WRKY7 (BrWRKY7) was obtained from EST collection in Brassica genomics team and its DNA sequence was determined.The cDNA clone is 1,037 bp long in nucleotides and encodes an open reading frame of 307 amino acids.Based on a phylogenetic tree, BrWRKY7 belongs to group IId.BrWRKY7 was induced by wound and SA.It was also induced by pathogen attack such as Xanthomonas campestris pv.campestris (Xcc), suggesting that this BrWRKY may play an essential role in defense response of chinese cabbages.

In a previous study, we cloned 27 discrete genome segments of Cotesia plutellae bracovirus (CpBV) and provided the complete nucleotide sequences and annotation. Seven putative coding regions were predicted from one of the largest segments, CpBV-S30. The activity of promoters associated with six predicted ORFs from this segment were investigated using both transient and baculovirus expression assays with enhanced green fluorescent protein as a reporter gene. CpBV promoters showed activity earlier than the polyhedrin promoter and the activity of some of these promoters was superior to that of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter in the baculovirus expression assays. The promoter of ORF3004 showed the highest level of activity in insect cells, exhibiting 24 % of the activity obtained with the AcMNPV polyhedrin promoter in Sf9 cells. In Spodoptera exigua larvae, the ORF3006 promoter showed the highest activity, with about 35 % of the activity measured with the polyhedrin promoter. In addition, analysis of the ORF3006 promoter revealed that the region between -382 and -422 from the translation start point was critical for activity of this promoter. These results suggest that the CpBV-S30 promoters characterized here could be useful tools in a variety of biotechnological applications, such as gene expression analyses and insecticide development.

Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA sequences in the specific region of the chromosomes.As well as for the integrated physical mapping, FISH karyotype analysis has to be preceded.Karyotype of Raphanus sativus 'Wonkyo 10039' was analyzed by a dual-color FISH technique; using various repetitive DNA probes, including 5S rDNA, 45S rDNA, and centromere retrotransposon.The length of the somatic metaphase chromosome ranged from 1.35 to 2.06 µm with a total length of 15.29 µm.The chromosome complements comprised of eight pairs of metacentrics and one pair of submetacentric.Bleached DAPI Band analysis revealed a heterochromatin region, covering 28.6% to 50.4% each chromosomes.5S and 45S rDNA sequences were located on two and three pairs of chromosomes, respectively.The centromere retrotransposon of Brassica (CRB) is a major component in Brassica related species that has been maintained as a common centromere component.CRB signals were detected on the centromere and pericentromeric region of R. sativus 'Wonkyo 10039' and three basic Brassica species (B.rapa, B. nigra, and B. oleracea).These results will provide a valuable background for physical mapping and elucidation of the evolutionary relationship among the Brassica related species.

In Brassica campestris, self-incompatibility (SI) can be overcome by CO2 gas treatment. Previously, we reported inhibition of the SI in the Brassica campestris line, 734, by CO2 gas. In this study, we used a cDNA microarray comprising 1,184 unique cDNA ESTs from a pistil-specific cDNA library of the Brassica line to examine the expression of the pistil genes in response to CO2. Microarray analysis revealed that expression of 2% of the pistil genes was altered by exposure to CO2 gas. Surprisingly, the SLG gene of the susceptible line was one of the down-regulated genes. Transmission electron microscopic analysis showed that the CO2 caused morphological changes in the papillary cell. Our results demonstrate that the overcoming of SI by CO2 gas involves reduced expression of the SLG gene in Brassica.

Glutathione S-transferases (GSTs) are multifunctional proteins encoded by a large gene family divided into Phi, Tau, Theta, Zeta, Lambda and DHAR classes on the basis of sequence identity. The Phi(F) and Tau(U) classes are plant-specific and ubiquitous. Their roles have been defined as herbicide detoxification and responses to biotic and abiotic stresses. Fifty-two members of the GST super-family were identified in the Arabidopsis thaliana genome, 13 members of which belong to the Phi class of GSTs (AtGSTFs). Based on the sequence similarities of AtGSTFs, 11 BAC clones were identified from Brassica rapa. Seven unique sequences of ORFs designated the Phi class candidates of GST derived from B. rapa (BrGSTFs) were detected from these 11 BAC clones by blast search and sequence alignment. Some of BrGSTFs were present in the same BAC clones indicating that BrGSTFs could also be clustered as usual in plant. They were mapped on B. rapa linkage group 2, 3, 9 and 10 and their nucleotide and amino acid sequences were highly similar to those of AtGSTFs. In addition, in silico analysis of BrGSTFs using Korea Brassica Genome Project 24K oligochip and microarray database for cold, salt and drought stresses revealed 15 unigenes to be highly similar to AtGSTFs and six of these were identical to one of BrGSTFs identified in the BAC clones indicating their expression. The sequences of BrGSTFs and unigenes identified in this study will facilitate further studies to apply GST genes to medical and agriculture purposes.

본 연구에서는 감정 어휘 분류와 체계의 이론적 배경을 검토하여 한국과 영어 문화권 감정 어휘 번역의 가능성과 한계를 논의하고자 하였다. 영어 감정 어휘를 이해하기 위해서는 감정 어휘의 사전적 의미뿐만 아니라 감정 어휘 간의 의미론적 관계를 이해할 필요가 있다. 영어 감정 어휘가 우리말 감정 어휘와 정확히 일치하지 않을 뿐만 아니라 영어사전의 뜻만으로는 미표한 의미 차이를 전달할 수 있다고 기대하기 어렵기 때문이다. 어떤 어휘든 그 어휘가 사용되는 맥락에 따라 의미가 상이할 수 있지만 감정 어휘의 뉘앙스는 개인의 심리적 특성과 문화적 상황에 따라 의미가 크게 달라질 수 있다. 감정 어휘의 의미관계와 기능성을 고려한 개념사전을 편찬한다면 이러한 문화적 차이에 따른 감정어를 용이하게 접근할 수 있을 것으로 기대된다. 특히 영미권과 한국의 문학과 문화를 비교하려는 연구자들에게 작품을 깊이 있게 이해하고 문화적 정서를 공감하는 데 도움이 될 것이다. 본 연구는 감정개념 사전 편찬의 필요성을 논의하기 위한 예비적 고찰이다. 감정 개념 사전을 구축하기 위해 한국과 영어권의 감정어를 살펴보고 영어의 감정 어휘 분류체계가 한국의 감정 어휘로 해석될 수 있는 가능성에 대해 논의하고자 한다.

Microplastics can cause physical, chemical and biological problems for organisms in water. In this study, we investigated the level of microplastic pollution in freshwater, brackish water, and seawater in the downstream of Nakdong river in Korea. After density separation and organic matter removal, the collected water was checked for the quantity, size, type, shape, and color of microplastics in the water using an FTIR microscope and a digital microscope. The amount of microplastics was found to be relatively high in seawater, with an average of 9.5 MPs/L in fresh water, 13.5 MPs/L in brackish water, and 20.5 MPs/L in sea water. Microplastic sizes above 50 ㎛ and below 100 ㎛ were most widely distributed in freshwater, brackish water, and seawater. The proportion of microplastics above 50 ㎛ and below 100 ㎛ was confirmed to be freshwater (38.6 %), brackish water (47.8 %), and seawater (65.3 %). Microplastic types were abundant in the following order: PP>PE>PET>PVC>Nylon in freshwater, brackish water, and seawater. The form of microplastics was confirmed in the following order: Fiber>Film>Fragment>Sphere in freshwater, brackish water, and seawater. The main colors of microplastics are blue and black, while yellow, white, and transparent microplastics were also found. Through this study, it was confirmed that microplastics can flow into freshwater, brackish water, and seawater due to various causes such as nearby industrial areas, roads, and trails. And it was confirmed that the level of microplastic pollution gradually increases from land to sea. The results of this study can be used as research data on microplastics in freshwater, brackish water, and seawater, and are expected to be helpful in preventing microplastic pollution in surface waters.

The karyotypes of three Brassica species, B. rapa (AA, 2n=20), B. nigra (BB, 2n=16), and B. oleracea (CC, 2n=18), were constructed based on fluorescence in situ hybridization (FISH), using various DNA probes, including 5S rDNA, 45S rDNA, 176 bp centromere satellite repeats, and Cot-100 DNA. The lengths of the somatic metaphase chromosomes ranged from 2.05 to 4.21 ㎛ in B. rapa, 2.37 to 3.13 ㎛ in B. nigra, and 2.94 to 4.44 ㎛ in B. oleracea. The karyotypes were composed of eight pairs of metacentrics and two pairs of submetacentrics in B. rapa, five pairs of metacentrics and three pairs of submetacentrics in B. nigra, and six pairs of metacentrics and three pairs of submetacentrics in B. oleracea. Ten 45S rDNA loci were detected in B. rapa, whereas six were detected in B. nigra, and four were detected in B. oleracea. In addition, six 5S rDNA loci were detected in B. rapa, whereas two 5S rDNA loci were detected in both B. nigra and B. oleracea. Both CentBr1 and CentBr2 originated from the B. rapa centromere sequence were detected in nine and five pairs of chromosomes in B. oleracea, respectively; however, they were not observed in B. nigra. Cot-100Br signals were detected on the centromeric regions of all chromosomes in the three Brassica species; however, BDB signals appeared in the centromere region of B. rapa and B. oleracea, but not in B. nigra.

This study examined the effects of cardiopulmonary physiotherapy on the cardiopulmonary function, metabolism, inflammatory markers, and quality of life in patients with coronary artery disease who underwent percutaneous coronary intervention (PCI).

Cucurbita moschata, an economically and nutritionally important vegetable in Korea, is seriously damaged in fruit quality and productivity by powdery mildew fungus (Podosphaera xanthii). To understand genetic bases of the powdery mildew disease resistance, an F2 population was developed from a cross between TG201 (C. moschata, susceptible, female parent) and TG10 (resistant, male parent) heirloom from Hongikbio Inc. Randomly selected 204 F2 individuals were assayed against powdery mildew pathogen, and construction of genetic map and QTL analysis were conducted using SNPs derived from genotyping-by-sequencing (GBS) method. The single major QTL was located in 6.9 ‑ 7.3 Mb region of TG10 chromosome 3. Within the 400 kb region, we investigated DNA insertion and deletion (indel) variations between TG201 and TG10 genome, and successfully designed seven sets of indel PCR markers. The functional validity of the powdery mildew resistance markers was confirmed with F2 individuals, breeding lines, and commercial varieties. From genomic sequence comparison on the basis of the breeding history, we also could clarify that the powdery mildew resistance region was introduced from C. okeechobeensis subsp. martinezii to C. moschata. Moreover, whole genome assemblies of two heirloom parents and genome-wide SNP/indel information were produced and are expected to be utilized as a genome resource for study of genus Cucurbita. The results and information of this study will be useful for genetics researches and breeding practices of C. moschata.

The genus Brassica includes the most extensively cultivated dicotyledonous vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. Among the Brassica crops, Brassica rapa has been an ideal model for genomic studies on the Brassica species. B. rapa (AA genome) has a relatively compact diploid genome (529 Mb), compared to Brassica nigra (BB genome, 632 Mb) and Brassica oleracea (CC genome, 696 Mb). There is also a large collection of cultivars and a broad array of available genomic resources including five large-insert bacterial artificial chromosome (BAC) libraries providing 53-fold genome coverage, end sequences of approximately 146,000 BAC clones, >150,000 ESTs from 33 cDNA libraries, successful shotgun sequencing of 886 euchromatic region-tiling BACs, and a BAC-based physical map. These genomic resources provided fundamental basis of the genome sequencing project and contributed to successful assembly of the whole genome sequences.

Angiomyoma (Vascular leiomyoma) is a benign tumor composed of smooth muscle cell and vascular endothelium. It rarely occurs in the nose and the paranasal sinus. To date, only 2 cases from inferior turbinate have beed reported in domesticity. We experienced a case of angiomyoma originating in the left side of the inferior turbinate in a 80-year-old woman who complained of intermittent epistaxis, and we report this case with literature review. (J Clinical Otolaryngol 2011;22:98–101)

Lipoma of the head and neck region rarely occurs, if any, on the posterior triangle of neck. We have experienced a lipoma on Eustachian tube, which caused recurrent middle ear effusion. A 42-year-old female patient was presented with right aural fullness and hearing disturbance. She had a history of several cases of right middle ear effusion in the past and insertions of ventilation tubes to relieve each of them. A yellowish round mass was found on the pharyngeal orifice of the right Eustachian tube by fiberoptic nasopharyngoscopy. It was removed via endoscopic endonasal approach and there is no evidence of recurrence along about 3 months after the surgery. This would be a good case for the importance of nasopharyngeal examination in patient with middle ear effusion. (Korean J Otorhinolaryngol-Head Neck Surg 2008;51:950-3) KEY WORDS：Lipoma·Eustachian tube·Middle ear effusion.

List of KRICE_CORE accessions used for whole-genome resequencing. (XLSX 15 kb)

HQSNPs of KRICE_CORE from chromosomes 7â 12. Individual HQSNPs (minor allele frequency &gt;0.05) from chromosomes 7â 12 of 137 KRICE_CORE accessions are included in the zip file chr7_12_KRICE_CORE_HQSNP.zip (TAB delimited txt file format). (ZIP 17471 kb)

Sequencing and mapping statistics for individual KRICE_CORE accessions. (XLSX 36 kb)

High-quality SNPs (HQSNPs) in KRICE_CORE from chromosomes 1â 6. Individual HQSNPs (minor allele frequency &gt;0.05) from chromosomes 1â 6 of 137 KRICE_CORE accessions are included in the zip file chr1_6_KRICE_CORE_HQSNP.zip (TAB delimited txt file format). (ZIP 24253 kb)

Brassica rapa is an A genome model species for Brassica crop genetics, genomics, and breeding. With the completion of sequencing the B. rapa genome, functional analysis of the genome is forthcoming issue. The expressed sequence tags are fundamental resources supporting annotation and functional analysis of the genome including identification of tissue-specific genes and promoters. As of July 2011, 147,217 ESTs from 39 cDNA libraries of B. rapa are reported in the public database. However, little information can be retrieved from the sequences due to lack of organized databases. To leverage the sequence information and to maximize the use of publicly-available EST collections, the Brassica rapa tissue-specific EST database (BrTED) is developed. BrTED includes sequence information of 23,962 unigenes assembled by StackPack program. The unigene set is used as a query unit for various analyses such as BLAST against TAIR gene model, functional annotation using MIPS and UniProt, gene ontology analysis, and prediction of tissue-specific unigene sets based on statistics test. The database is composed of two main units, EST sequence processing and information retrieving unit and tissue-specific expression profile analysis unit. Information and data in both units are tightly inter-connected to each other using a web based browsing system. RT-PCR evaluation of 29 selected unigene sets successfully amplified amplicons from the target tissues of B. rapa. BrTED provided here allows the user to identify and analyze the expression of genes of interest and aid efforts to interpret the B. rapa genome through functional genomics. In addition, it can be used as a public resource in providing reference information to study the genus Brassica and other closely related crop crucifer plants.

유전자총(Particle bombardment) 방법에 의해 snowdrop lectin (Galanthus nivalis agglutinin; GNA) 유전자가 도입된 해충저항성 형질전환 벼를 개발하였다. GNA 유전자가 도입된 많은 형질전환 식물체가 재분화되었으며, 형질전환 벼의 GNA 유전자의 integration, expression 및 inheritance는 Southern 및 western analysis분석 방법에 의해 증명되었다. 벼 genome 내 도입 유전자 수는 one 혹은 five copies이었다. 형질전환 <TEX>$R_1$</TEX>과 <TEX>$R_2$</TEX> 식물체의 GNA 단백질 발현 분석 결과, 함량은 총 단백질 중 0.01%부터 2.0%까지 포함하고 있었다. 형질전환 식물체 중 GNA 과발현 형질전환 벼를 이용한 생물 검정 결과, 대조구와 비교하여 벼의 주요 해충인 벼멸구(Nilaparvata lugens St<TEX>${\aa}$</TEX>l)에 저항성을 나타내었다. 위의 실험 결과를 통해, 해충저항성 유전자인 GNA가 도입된 형질전환 벼는 해충의 증식을 억제하는데 이용될 수 있을 것이다. Transgenic rice plants with increased resistance to rice brown planthopper (Nilaparvata lugens St<TEX>${\aa}$</TEX>l) were generated by particle bombardment-mediated transformation of plants with a gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under control of the rice Rubisco small subunit (rbcS) promoter.. A large number of transgenic rice plants containing the GNA gene were generated. The integration, expression, and inheritance of this gene in the <TEX>$R_1$</TEX> and <TEX>$R_2$</TEX> generations were demonstrated by Southern and western blot analyses. The plants contained one to five copies of the transgene. The GNA protein comprised approximately 0.01-2.0% of total soluble protein in the <TEX>$R_1$</TEX> and <TEX>$R_2$</TEX> transgenic plants. Insect bioassays and feeding studies showed that the GNA protein expressed in the <TEX>$R_2$</TEX> transgenic rice plants reduced the survival of brown planthoppers. The introduction of GNA into rice plants therefore can help to control insect pests.

In the bilateral tele-operation system, time delay may be a critical problem. Even if system modeling error or time delay occurs, when applied to wave transformation system, the system's stability can be achieved. Using the characteristic b which is an important parameter of wave transformation, the system can display robust performance for time delay. However, since assuming and that the time delay was fixed developing a theory, a stability cannot be guaranteed about the time-varying delay. Therefore, In the paper, Therefore, in this paper, we studied for the method that controls this by applying the fuzzy algorithm which surveyed the timevarying delay characteristics and can adjust the b according to it adaptively.

In this technical note, we concerned the problem of stochastic stability, stabilization and H ∞ -control of state delay systems. Some sufficient conditions are derived for this class of robust feedback stabilization of state delay systems. The fundamental point in the analysis is an LMI characterization of state delay that stabilize the closed loop system in the mean square sense.

Background and Objectives:Compensatory hypertrophy of the inferior turbinate is frequently observed in case of nasal septal deviation. In case of surgical treatment, some advocate that septoplasty must be accompanied by reduction of inferior turbinate. So, the authors analyzed the structural change of the inferior turbinate in nasal septal deviation using the CT scan. Subjects and Method:The CT films of 154 patients who had undergone septoplasty were reviewed retrospectively. All subjects had only C- shaped septal deviation and had no abnormality. Inferior turbinate was divided equally into three portions as anterior, middle and posterior portions. The thickness of mucosa and bone of the inferior turbinate and the degree of inferior conchal bone mediali- zation were measured. Results:In the anterior and middle portion, the degree of medialization at concave side of inferior con- chal bone was significantly increased compared to the convex side. In addition, the thickness of bone and mucosa at the concave side was significantly thicker than those of the convex side (p<0.05). However, no significant differences were found in the posterior portion. Conclusion:In the nasal septal deviation, the degree of medialization of inferior conchal bone and the thickness of bone and mucosa at the concave side were significantly greater than those of the convex side in the anterior and