Hyun A. Kim

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Genome Sequence of the Plant Growth-Promoting Rhizobacterium Bacillus sp. Strain JS

Volatile and nonvolatile compounds emitted from the plant growth-promoting rhizobacterium Bacillus sp. strain JS enhance the growth of tobacco and lettuce. Here, we report the high-quality genome sequence of this bacterium. Its 4.1-Mb genome reveals a number of genes whose products are possibly involved in promotion of plant growth or antibiosis.

High-throughput sequencing and de novo assembly of Brassica oleracea var. Capitata L. for transcriptome analysis.

Background The cabbage, Brassica oleracea var. capitata L., has a distinguishable phenotype within the genus Brassica. Despite the economic and genetic importance of cabbage, there is little genomic data for cabbage, and most studies of Brassica are focused on other species or other B. oleracea subspecies. The lack of genomic data for cabbage, a non-model organism, hinders research on its molecular biology. Hence, the construction of reliable transcriptomic data based on high-throughput sequencing technologies is needed to enhance our understanding of cabbage and provide genomic information for future work. Methodology/Principal Findings We constructed cDNAs from total RNA isolated from the roots, leaves, flowers, seedlings, and calcium-limited seedling tissues of two cabbage genotypes: 102043 and 107140. We sequenced a total of six different samples using the Illumina HiSeq platform, producing 40.5 Gbp of sequence data comprising 401,454,986 short reads. We assembled 205,046 transcripts (≥ 200 bp) using the Velvet and Oases assembler and predicted 53,562 loci from the transcripts. We annotated 35,274 of the loci with 55,916 plant peptides in the Phytozome database. The average length of the annotated loci was 1,419 bp. We confirmed the reliability of the sequencing assembly using reverse-transcriptase PCR to identify tissue-specific gene candidates among the annotated loci. Conclusion Our study provides valuable transcriptome sequence data for B. oleracea var. capitata L., offering a new resource for studying B. oleracea and closely related species. Our transcriptomic sequences will enhance the quality of gene annotation and functional analysis of the cabbage genome and serve as a material basis for future genomic research on cabbage. The sequencing data from this study can be used to develop molecular markers and to identify the extreme differences among the phenotypes of different species in the genus Brassica.

Screening of tissue-specific genes and promoters in tomato by comparing genome wide expression profiles of Arabidopsis orthologues.

Constitutive overexpression of transgenes occasionally interferes with normal growth and developmental processes in plants. Thus, the development of tissue-specific promoters that drive transgene expression has become agriculturally important. To identify tomato tissue-specific promoters, tissue-specific genes were screened using a series of in silico-based and experimental procedures, including genome-wide orthologue searches of tomato and Arabidopsis databases, isolation of tissue-specific candidates using an Arabidopsis microarray database, and validation of tissue specificity by reverse transcription-polymerase chain reaction (RT-PCR) analysis and promoter assay. Using these procedures, we found 311 tissue-specific candidate genes and validated 10 tissuespecific genes by RT-PCR. Among these identified genes, histochemical analysis of five isolated promoter::GUS transgenic tomato and Arabidopsis plants revealed that their promoters have different but distinct tissue-specific activities in anther, fruit, and root, respectively. Therefore, it appears these in silico-based screening approaches in addition to the identification of new tissue-specific genes and promoters will be helpful for the further development of tailored crop development.

Microarray Analysis of bacterial blight resistance 1 mutant rice infected with Xanthomonas oryzae pv. oryzae

We analyzed the transcriptional profile of the Xoo infected bbr1 mutant using a commercial rice gene chip containing 51,279 transcripts. Microarray revealed 92 genes with increased levels of expression and 22 genes with decreased levels of expression in bbr1. Some of the differentially expressed genes were validated by qRT-PCR. Higher expression of defense-related genes and AP2 domain containing transcription factors along with lower expression of reactive oxygen scavenging enzymes may be responsible for defense signaling in the bbr1 upon Xoo infection. The putative target genes of AP2 domain containing transcription factors also showed differential gene expression during Xoo infection, some of which encoded bacterial pathogen resistance-related protein. Induction of AP2 domain containing transcription factors along with up-regulation of their putative target genes during Xoo infection may inhibit pathogen spread in the bbr1. This observation supports the hypothesis that AP2 domain containing transcription factors is involved in the regulation of differentially expressed genes in bbr1.

De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

Expression of Dengue virus EIII domain-coding gene in maize as an edible vaccine candidate

Plant-based vaccines possess some advantages over other types of vaccine biotechnology such as safety, low cost of mass vaccination programs, and wider use of vaccines for medicine. This study was undertaken to develop the transgenic maize as edible vaccine candidates for humans. The immature embryos of HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vectors (V662 or V663). The vectors carrying nptII gene as selection marker and scEDIII (V662) or wCTB-scEDIII (V663) target gene, which code EIII proteins inhibite viral adsorption by cells. In total, 721 maize immature embryos were transformed and twenty-two putative transgenic plants were regenerated after 12 weeks selection regime. Of them, two- and six-plants were proved to be integrated with scEDIII and wCTB-scEDIII genes, respectively, by Southern blot analysis. However, only one plant (V662-29-3864) can express the gene of interest confirmed by Northern blot analysis. These results demonstrated that this plant could be used as a candidated source of the vaccine production.

The development of transgenic maize expressing Actinobacillus pleuropneumoniae ApxIIA gene using Agrobacterium

To develop edible vaccines for swine, the embryogenic calli (type II) derived from HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector pMYV611, 613, 616, and V621, 622 and 623 respectively. Six of those vectors carry nptII gene which confers resistance to paromomycin and apxIIA gene producing ApxII toxin which is generated in various serum types of A. pleuropneumoniae as a target gene. The 4,120 callus clones for pMYV611, 5,959 callus clones for pMYV613, 7,581 callus clones for pMYV616, 52,329 callus clones for V621, 48,948 callus clones for V622, and 56,188 callus clones for V623 were inoculated. The frequency of positive response clone was confirmed into range of 2.3% - 4.4% for each vectors by NPTII ELISA kit assay, and the selected callus clones of them were finally 3 callus clones from pMYV611 (0.07%), 4 callus clones from pMYV613 (0.07%), 2 callus clones from pMYV616 (0.03%), 51 callus clones from V621 (0.1%), 72 callus clones from V622 (0.15 %), and 102 callus clones from V623 (0.18%) respectively. From the selected callus clones of each binary vector, the integration of the apxIIA gene into maize genome was detected from 2 plants of pMYV613 and 2 plants of V623 by Southern blot analysis.

SlPMEI, a pollen-specific gene in tomato

Kim, W. B., Lim, C. J., Jang, H. A., Yi, S. Y., Oh, S.-K., Lee, H. Y., Kim, H. A., Park, Y.-I. and Kwon, S.-Y. 2014. SlPMEI, a pollen-specific gene in tomato. Can. J. Plant Sci. 94: 73-83. Pectin is one of the main components of plant cell walls, and its biosynthesis is controlled by pectin methylesterase (PME). Pectin methylesterase inhibitors (PMEIs) are key regulators of PME. We report here the cloning and characterization of a novel Solanum lycopersicum L. PMEI gene, SlPMEI. RT-PCR studies of leaf, seed, fruit, flower, and flower organs confirmed that SlPMEI is expressed specifically in pollen. Promoter analysis of SlPMEI revealed pollen-specific cis-acting elements (pollen lat52 and g10). In addition, SlPMEI is expressed independently of abiotic stress, pathogen exposure, and growth stage in tomato, and a histochemical analysis of promoter activity revealed pollen-specific expression in both Arabidopsis and tomato. Under the microscope, we observed pollen-specific GUS expression in the stamen of transgenic tomato plant. These results indicate that the promoter of SlPMEI has strong pollen-specific activity, and could therefore be useful for development of industrially and agronomically important transgenic plants.

Development of transgenic cucumber expressing TPSP gene and morphological alterations

Abstract To develop transgenic cucumber tolerant to abi-otic stress, a cotyledonary-node explants were co-cultivated with Agrobacterium tumefaciens (EHA101) carrying TPSP gene (pHC30-TPSP). After transfer to fresh medium every two week for eight weeks, putative transgenic plants were selected when shoots grown a length greater than 3 cm from the cotyledonary-node explants on selection medium supple-mented with 5 mgl -1 phospinotricin as selectable agent. The confirmation of transgenic cucumber was based on the North-ern blot analysis. Thirty four shoots (5.2%) with resistance to phospinotricin were obtained from 660 explants inoculated. Of them, transformants were only confirmed from 11 plants (1.7%). Transgenic cucumber expressing TPSP gene was more synthesized at 3.8 times amounts of trehalose (0.014 mg g fresh wt -1 ) than non-transformants (0.0037 mg g fresh wt -1 ). However, all of transgenic plants showed abnormal morphology, including stunted growth ( height 150 cm) under the same growth envi-ronment. These results lead us to speculate that the over-production of trehalose was toxic for cucumber, even though that had known for rice as non-toxic.