식물이 병원체의 침입을 받으면 식물체내로 단백질을 전달한다. 병원체가 식물체내로 분비하는 단백질 중에서 cell death를 일으키는 단백질들에 대해 관심을 가졌다. 먼저 Pseudoperonospora cubensis 에 감염된 오이 잎에서 발 현되어 병원체 유래의 단백질들을 기존에 알려진 RLXR 도메인을 가진 염기서열에서 프라이머를 디자인 하여 PCR 반응을 통해 cloning 하였다. 이중에서 P. cubensis 접종시 발현이 증가하는 단백질들을 중심으로 Nicotiana benthamiana에 Infection 시켰다 그 결과 Infection된 Effector 들 중에서 2개의 Effctor에서 visible한 cell death를 관찰 할 수 있었다. 사세한 결과가 포스트를 통해서 발표될 것이다.

Downy mildew, caused by P. cubensis, is one of the most devastating diseases in cucumber (Cucumis sativus) worldwide. Due to the variation and mutation of the races of P. cubensis, host resistance in cucumber has been lost in recent years, so the identification of new sources of resistance is one of the most important targets in cucumber breeding programs. Moderate levels of resistance against downy mildew has been identified in different cucumber varieties. In this study, we identified new downy 2mildew resistance QTLs in cucumber using F2 mapping populations originated from the hybridization between breeding lines of cucumber. We used both classical QTL mapping based on SSR markers and GBS (gentyping based sequencing) based QTL mapping. In this presentation, detailed information about downy mildew resistance related QTL will be presented

The aim of the study was to assess the safety of methionine sulfoxide reductase B2(CaMsrB2) protein as toxicity, allergenecity and identity of inserted gene product that transformed rice. Through bioinfomatical research of CaMsrB2, amino acid sequence of CaMsrB2 did not share overall homology with any known or suspected to be allergen or toxin protein. For the biochemical research, CaMsrB2 protein was expressed and purified. Using purified protein, we made a specific antibody. Purified protein was sequenced by Edman degradation methods and confirmed sequence identify.. The amino acid sequences of purified protein were the same as deduced amino acid sequences exclude N-terminal Histidine. And for the internal sequences of CaMsrB2, we performed MALDI-TOF Mass. The results of MALDI-TOF MS was compared Mascot Database and confirmed the sequence coverage was 56%. These results mean bacterially produced CaMsrB2 was the same with inserted gene product. With these purified and identified CaMsrB2 protein, we performed acute toxicity test. Following the OECD guideline 423, 2,000mg/Kg body weight protein were injected as oral administration. After 2 weeks, there did not shown any death and special symptoms.

ORE7 gene incorporated into 3 different promoters including pCKLSL-35S, pCKLSL-TP and pCSENIF was transformed to Korean soybean variety Kwangan using highly efficient soybean transformation system. The gene is known to exhibit a delayed leaf senescence phenotype in Arabidopsis. Fourteen, Fifteen and nine transgenic plants were produced from pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7, respectively. Moreover, transgenic plants were confirmed for gene introduction and their expression using PCR, qRT-PCR and RT-PCR. To identify the transgene insertion events, the analysis of flanking sequence was determined. As a results, T-DNA was integrated intergenically in transgenic line 1 of pCKLSL-35S::ORE7 and line 1 of pCSENIF::ORE7. Currently, flanking sequence analysis with pCKLSL-35S::ORE7, pCKLSL-TP::ORE7 and pCSENIF::ORE7 is carrying out to investigate the stable T-DNA insertions.

Vigna is a genus of flowering plants in the legume family, and about 100 species belong to this genus, including azuki bean, blackgram and mungbean which are well-known in Asia. Among the species in Vigna genus, Vigna reflexo-pilosa has its own unique characteristic form as allotetraploid (2n=4x=44), whereas other Vigna species exist as a diploid form (2n=2x=22). In this study, we de novo assembled V. reflexo-pilosa genome and 22 accessions of 18 Vigna species’ transcriptome using NGS platform and calculated the Ks values of synteny blocks within V. reflexo-pilosa to determine the whole-genome duplication event existence. Two WGD events had occurred on V. reflexo-pilosa unlike V. radiata which only occurred once. Also, we tried to find out which progenitor had contributed to formation of V. reflexo-pilosa using transcriptome assemble results of 22 Vigna accessions. Ks value calculation between the transcriptome assemble results and predicted gene sets of V. reflexo-pilosa has been excuted. V. trinervia showed 2 peaks (0.0075, 0.0495) on its Ks value bin distribution and confirmed as A-type genome donor. Based on the peak value, we considered the predicted genes of V. reflexo-pilosa in Ks value range from 0.033 to 0.072 as B-type genes, inherited from other progenitor. Re-calculating the Ks values of 4,796 predicted genes in B-type with the transcriptome assemble results of 22 Vigna accessions, we were able to find 6 Vigna species, V. umbelleta(CIAT34386), V. umbelleta(2004T2), V. nakashimae, V. nepalensis, V. riukiuensis, V. minima as candidate B-type genome donor.

레스베라트롤(3, 5, 4′-trihydroxytibence)은 포도, 와인, 땅콩 등에서 발견되는 항산화 물질로 과일에서 레스베라트 롤과 3-β-mono-D-glucoside가 결합된 피세이드 형태로 발견되며, 항암, 고지혈증, 항혈전작용등 다양한 약리효과 를 가지는 것으로 밝혀졌다. 익산 526호를 25℃와 30℃ 암조건에서 1, 3, 5일간 수분 95%이상 유지하여 발아시켰다. 발아처리 전과 비교하였을 때, 25℃에서 발아처리 후 레스베라트롤 생합성 유전자의 발현은 1.7 ~ 2.5배 증가하였 으며, 레스베라트롤 및 피세이드함량은 각각 1.1 ~ 1.6배, 1.1 ~ 3.8배 증가하였다. 또한, 30℃에서 레스베라트롤 생 합성 유전자의 발현은 2.7 ~ 14.0배 증가하였으며, 레스베라트롤 및 피세이드함량은 각각 1.2 ~ 2.3배, 2.0 ~ 10.5배 증가하였다. 30 ℃에서 5일간 발아시켰을 때, 레스베라트롤 생합성 유전자의 발현수준이 가장 높았으며, 레스베라 트롤 및 피세이드 함량 또한 가장 높은 것으로 나타났다. 발아과정을 통한 레스베스베라트롤 생합성 유전자 발현 증가는 레스베라트롤 및 피세이드 함량 증가와 밀접한 상관관계가 있는 것으로 생각된다. 따라서 발아처리는 익 산 526호의 이용확대를 위한 레스베라트롤 및 피세이드 함량 증진을 위해 효과적인 방법이라고 생각된다.

Apple is a typical climacteric fruit, whose loss of firmness during storage is associated with internal levels of ethylene. MdACS1 (1-aminocyclopropane-1-carboxylate synthase) and MdACO1 (1-aminocyclopropane-1-carboxylate oxidase) genes are characterized well as functional markers for the ethylene production of ripening apple fruit. Presence of two alleles for each gene are commonly reported in cultivated apples. MdACS1-1/1, 1-1/2 and 1-2/2 generally induce high, medium, and low ethylene production, respectively. Homozygosity of MdACO1-1 resulted in low levels of ethylene production than MdACO1-1/2 and 1-2/2. It was reported that cultivars homozygous for MdACS1-2 and MdACO1-1 had superior shelf-life with lowest ethylene production. In this study, genotypes of 42 apple cultivars including Korean-developed ones at MdACS1 and MdACO1 loci were determined. Polymorphisms were detected by PCR and were separated by electrophoresis in a 2.0% agarose gel. Of PCR products of MdACS1, the fragrament of 490 bp was corresponded to the MdACS1-1 allele and 640 bp corresponded to the MdACS1-2 allele. ACO1-1 and ACO1-2 alleles had the fragment size of 525 and 587 bp, respectively. The 42 cultivars could be grouped into three classes to each gene, MdACS1-1/1, MdACS1-1/2 and MdACS1-2/2; MdACO1-1/1, MdACO1-1/2 and MdACO1-2/2.

In plants, eukaryotic translation elongation factor 1B (eEF1B) is composed of three subunits, eEF1Bα, eEF1Bβ and eEF1B γ. Two subunits are nucleotide exchange subunits (eEF1Bα and eEF1Bβ) and one is a structural protein (eEF1Bγ). In the previous study, eEF1B was identified as a common host factor for several RNA viruses. To test which subunit of eEF1B is essential for Potato virus X (PVX) replication, the virus-induced gene silencing (VIGS) for eEF1Bα, β or γ was performed in Nicotiana benthamiana and green fluorescent protein (GFP)-tagged PVX was inoculated. PVX-GFP accumulation was decreased when eEF1Bβ or γ subunit was silenced, whereas eEF1Bα had no effect on PVX-GFP accumulation in inoculated leaves. Targeting induced local lesions in genome (TILLING) was performed using a Capsicum annuum EMS population to test whether mutations in eEF1Bβ subunit affect virus infection in pepper. We obtained 81 eEF1Bβ mutant lines consisted of 16,759 individuals. These mutant lines are being tested to validate the function of eEF1B β in PVX replication.

Chloroplasts are plant-specific organelles, which have their own genome. Most of the plant chloroplast genomes (CP genome) are highly conserved in terms of its gene contents and genome structures, and they exist in cells with abundant copy numbers. Because of numerous copy numbers, the complete chloroplast sequence assembly pipeline with small amount of whole genome resequencing data, produced by NGS technique, was established in our laboratory. From 14 accessions of cabbage (Brassica oleracea L.) resequencing data produced by Illumina Hi-seq 2000, CP genomes were assembled and compared to each other. 18 sequence variance regions were detected, and 6 HRM(High Resolution Melting curves) markers were developed. Approximately 1 Gb of whole genome sequencing data of 10 Brassica rapa and 2 Brassica napus were also obtained from Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science. With these resequencing data, all CP genomes from these accessions were assembled. Total 27 complete CP genomes of B.oleracea, B.rapa, B.napus, and brassico-raphanus which is a novel allotetraploid species between B.rapa and Raphanus sativus, were compared in sequence level. Phylogenetic analysis based on the comparison revealed that B.rapa could be the maternal species when rapeseeds and brassico-raphanus became allotetraploid species. Additionally, CP genome of B.napus cv.M083 is closer to B.rapa accessions than the other B.napus accessions, thus B.napus could have several different origins.

본 연구는 강원도농업기술원 옥수수연구소에서 새로운 기능성 색소옥수수 품종을 개발하기 위해 육성한 총 12개 의 색소옥수수 계통들과 찰옥수수 및 일반옥수수 계통들에 대하여 SSR 분자마커를 이용하여 유전적 다양성, 집단 구조 및 association mapping 분석을 실시하였다. 그 결과 분석에 이용된 300개의 SSR primer들은 12개의 옥수수 자 식계통들에서 총 1,331개의 대립단편을 나타내었으며, 각 SSR primer 당 증폭된 대립단편의 수는 2개(umc1515, umc2249, umc1158, umc1659, phi102228, umc2262, umc1058, nc004, phi092, umc2308, umc1314, umc1178, umc1352a, umc2092, phi057, umc1139, umc1473, umc2338, umc2093, umc1107, umc1054)에서 10개(mmc0111)의 범위로 나타났 으며, 평균 대립단편 수는 4.44개였다. 집단구조 분석결과, 12개의 옥수수 자식계통들은 groups I, II, III, admixed group으로 구분되었다. 2개의 자식계통(10S4015, 10S4026)은 group I에 포함되었고, Group II는 총 4개의 자식계통 (Mo17, B14A, HW7, HW3)이 포함되었다. 그리고 4개의 자식계통(11CS4117, 11CS4124, 11CS7014, HW9)은 Group III에 포함되었으며, 2개의 자식계통(10S4032, KW7)은 admixed group에 포함되었다. 더욱이 본 연구에서는 색소 및 비색소옥수수 자식계통들에서 분석에 이용된 300개 SSR 마커와 4개의 양적 형질(간장, 착수고, 간경, 수술길이) 과의 연관성을 분석하기 위해서 population structure(Q) 값을 이용하여 Q GLM 분석을 실시하였다. 0.01의 유의수준 에서, Q GLM 분석을 이용하여 총 17개의 SSR 마커가 4개의 형질과 association을 확인하였다. 본 연구에서 12개의 색소 및 비색소옥수수 자식계통들에 대한 유전적 다양성, 집단구조 및 association mapping 분석의 결과는 앞으로 강원도농업기술원 옥수수연구소에서 기능성 색소옥수수 품종개발을 위한 계통 육성 및 교배조합 구성 등에 유용 한 정보를 제공할 것으로 기대한다.

Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil (Lissorhoptrus oryzophilus Kuschel). In this study, we estimated the insert number of foreign genes, and analyzed the flanking sequences of T-DNA in rice genome. As a result, The T-DNA of Btt12R 3-1-1-1 line was inserted in exon region of rice chromosome 10 and Btt12R 6-1-1-1 line was inserted in two copies of foreign gene. Btt12R 9-1-1-1 line was analyzed at only left border flanking sequence. The T-DNA of Btt12R 13-1-1-1 line was inserted one copy of foreign gene between position 24,516,607~24,516,636 of rice chromosome 5 and 30bp known genomic sequences were deleted. The Btt12R 13-1-1-1 line confirmed to be inserted in intergenic region having not any expressed gene and no any deletion/addition of T-DNA sequence. From these results, we demonstrated that the molecular data of rice water weevil resistant Bt rice could be acceptable to conduct the biosafety and environment risk assessment for GM crop commercialization

A root serves as an essential organ in plant growth by up-taking nutrients and water from soil and supporting the rest of a plant body. Root apical growth and system architecture have been extensively studied because they strongly affect overall plant growth and yields. Some plant species also utilize roots as storage organs. Many of them, including sweet potatoes (Ipomoea batatas), cassava (Manihot esculenta), and radish (Raphanus sativus) are important crops, however their root development has remained elusive. In this study, we characterized radial root growth in the radish and found that it is very similar to the secondary growth in stems. We identified well established cambium zones in the actively growing radish roots. Cell proliferation activities in the cambium zones positively correlated with root growth rates and final yields. Through a comparative analysis with Arabidopsis root expression data, we selected some putative transcription factors whose expression is highly enriched in the cambia and validated their expression in various stages of radish roots. By comparing their expression in two inbred lines with distinctive radial root growth, we identified transcription factors that are involved in morphological differences. More importantly, our investigation suggests that the differences in the root growth of two radish inbred lines are from changes in cytokinin responses. These findings together highlight that radish could serve as an excellent system for studying root crops and that transcriptional regulation and cytokinin signaling are indispensable for the secondary root growth.

Comparative analysis is a typically useful tool for translating genomic information from one species to another. However, currently available softwares are relatively difficult to directly use for researchers that are not familiar with use of bioinformatic tools. Therefore, we intended to develop a new platforms and/or interface through which one can use in more comfortable way, based on the concept of interactive comparative analysis. Towards this direction, we, firstly, constructed relational database to store the information on abiotic stress genes identified from multiple plant species using various resources, such as the TAIR (http://www.arabidopsis.org), gene expression profiles and relevant literatures, and linked with comparative analysis interface. For purposes of comparative analysis and identification of synteny blocks, cross-species orthologous genes were determined using a combination of tBlastX and BlastP homology searches. We adapted and developed a Circos-like format to present resulting comparative maps. Users can readily choose analysis parameters, for example individual genes and specific chromosomes for chosen species, in the pane of analysis DB, which is useful feature to avoid complexity of comparative genomic analysis. This DB-associated comparative analysis tool, developed in this study, will be able to provide customer-friendly interface for comparative analysis and extend its utility across a broader range of plant genomes.

Susceptible Vitis vinifera responds to Xylella infection with a massive redirection of gene transcription. This transcriptional response is characterized by increased transcripts for phenlypropanoid and flavonoid biosynthesis, ethylene production, adaptation to oxidative stress, and homologs of pathogenesis related (PR) proteins, and decreased transcripts for genes related to photosynthesis. In addition, the results suggest that susceptible genotypes respond to Xylella infection by induction of limited, but inadequate, defense response. We also compared the transcriptional and physiological response of plants treated by pathogen infection, low or moderate water deficit, or a combination of pathogen infection and water deficit. Although the transcriptional response of plants to Xylella infection was distinct from the response of healthy plants to moderate water stress, we observed synergy between water stress and disease, such that water stressed plants exhibit a stronger transcriptional response to the pathogen. This interaction was mirrored at the physiological level for aspects of water relations and photosynthesis, and in terms of the severity of disease symptoms and pathogen colonization, providing a molecular correlation of the classical concept with the disease triangle.

Rice mesocotyl is the region between the coleoptile node and point of union of the culm with the root. The mesocotyl is one of the important factor contributing to rice seedling emergence from soil in direct seedling. Several quantitative trait loci (QTLs) for mesocotyl elongation of rice had been reported in few studies. However, association mapping of mesocotyl elongation QTL was not conducted. For that reason, we detected QTLs for mesocotyl elongation in agar and soil conditions and confirmed the potentials of QTLs using chromosome substitution lines (CSSLs). Backcross inbred line (BILs) and chromosome segment substitution lines (CSSLs) derived from a cross between Kasalath and Nipponbare were employed to detect QTLs for mesocotyl elongation in rice. A total of 12 QTLs for mesocotyl elongation were detected on chromosome 1, 3, 6, 7, 9 and 12 using 98 BILs in agar and soil conditions. Two QTLs, qMel-1 and qMel-3 were consistently detected in both conditions. For substitution mapping of qMel-1 and qMel-3, across was made between 2 CSSLs, CSSL-6 and CSSL-15. Our results showed that the qMel-1 was located between two markers RM5448 and RM5310 on chromosome1 and the qMel-3 was located between RM15859-RM15974 on chromosome3. To understand factors controlling mesocotyl elongation, cell expansion and division of rice mesocotyl were investigated. Moreover, microarray analysis was conducted to select candidate genes using near-isogenic lines for two QTLs. 194 genes were up- and down regulated in rice mesocotyl.

The legume family is the third largest group, including approximately 650 genera and 18,000 species, in the flowering plants and the second important crops to the Poaceae in the agricultural economy. Comparative analysis is a useful tool to understand cross-species genomic structure and alterations during organism’s evolutionary history. In this study, we constructed a composite comparative map of ten legume species, including Medicago truncatula, Medicago sativa, Lens culinaris, Pisum sativum, Lotus japonicus, Cicer arietinum, Vicia faba L, Vigna radiata, Phaseolus vulgaris and Glycine max. Of these species, M. truncatula, which is a representative model system, played a central role to develop the cross-genome amplifiable PCR gene markers for the purpose of transferring them to other related legume species. A total of 140 cross-species core markers were employed to analyze genomic colinearity across this broad array of legume species. The comparative map demonstrates a diverse array of evolutionary events, such as duplications, inversions and reciprocal translocations. It is anticipated that resulting maps would provide a broader insights into the lineage-specific genomic organization of these glalegoid/phaseoloid legumes, which are two clades containing almost all crop legumes of economic importance, and can further used for the molecular breeding through translating genomic information into other orphan legumes.

Melatonin plays pleiotropic roles in both animals and plants. Among them, the possible role of melatonin in the innate immune response in plants was emerging recently. As an initial study, we employed Arabidopsis to see whether melatonin is involved in the defense system against a virulent bacterial pathogen Psudomonas syringae DC3000. It was obviously observed that melatonin application of 10 μm concentration onto Arabidopsis and tobacco leaves induced various pathogenesis-related (PR) genes as well as a series of defense genes activated by salicylic acid (SA) and ethylene (ET), two key factors involved in the plant defense response compared to the mock-treated Arabidopsis and tobacco leaves, respectively. The induction of these defense-related genes in the melatonin treated Arabidopsis was well matched with an increase in resistance against pathogenic bacterium by suppressing its multiplication with about 10 fold relative over the mock-treated Arabidopsis. Furthermore, melatonin induced PR genes were almost completely or partially suppressed in npr1, ein2, and mpk6 Arabidopsis mutants indicative of SA and ET dependency of melatonin in plant defense signaling. These results suggest that melatonin may play a novel defense signaling molecule in plant-pathogen interaction

With the advent of next generation sequencers (NGS) that provide sequencing at a substantially lower cost, the development SNPs at the level of whole genomes can be done in a single laboratory. However, genome structural variation including large insertions and deletions, and chromosomal reciprocal translocations has not yet been focused due to the limitation of re-sequencing methods as genome structures rely to that of a known reference genome. For an improved detection of the structural variations after deep re-sequencing of the Glycine soja accession CS-14, we de novo assembled the whole paired-end reads (W-contigs). After the de novo assembly, the paired-end reads that did not match the reference genome of Williams 82 were retrieved and de novo assembled them (U-contigs). We then predicted structural variation candidates. For predicting the function of the structural variation candidates, we compared those structural variation candidates with SwissProt DB using BLASTX. Most of them were matched with transposable element related proteins or stress tolerance related proteins (Table 1). We designed 24 primers for all candidates and tested in CS-14 and Williams 82 for validation. As a result, the DNA polymorphism was observed between CS-14 and Williams 82 in the three primer sets, CS14IC10, CS14IC12 and CS14IC15, with the expected size of the PCR product . For further validation, we sequenced the DNA band amplified by CS14IC15, and its sequences were aligned well against the Williams 82 and CS-14 contig. Especially, IC15R-CS14 was aligned in the predicted insertion region, consequently, this sequenced region would indicate structural variation. The other primer sets did not amplified either because they were designed for an amplifying long genomic region or because of the fragmented template DNA

Watermelon (Citrullus lanatus) is one of the most economically important cucurbitaceous crop over the world. Screening of proper reference genes was needed to reverse transcription quantitative real-time PCR (qRT-PCR), and it is bausic step of many researches including gene expression analysis. However, the reference genes on watermelon has not yet been reported systematically. Therefore, eight candidates of reference genes were selected with reference to Arabidopsis or cucumber papers. They are β-Actin, elongation factor 1-α, glyceraldehy-3-phosphate-dehydrogenase, NADP-isocitrate dehydrogenase, leunig, polypyrimidine tract-binding protein1, ubiquitin-conjugating eznyme E2, and 18S ribosomal RNA. The expression levels of genes were evaluated by qRT-PCR under biotic stress (Colletotrichum orbiculare treatment), plant hormone treatment (100 μM ABA), and abiotic stresses such as drought, cold (4℃), salt (250 mM NaCl) stresses. We founded appropriate reference genes which did not induce or reduce gene expression levels under broad spectrum of stresses by qRT-PCR analysis. These results may provide proper information for the use of appropriate reference genes for gene expression studies in watermelon qRT-PCR analysis.

Plastochron and phyllotaxy are important traits to determine plant architecture. A plastochron 1-6 mutant was derived from japonica rice cultivar Koshihikari treated with ethylmethane sulfonate (EMS). The plastochron 1-6 mutant showed reduced plant height, shorter internode length, smaller and more leaves than Koshihikari, its wild type. In addition, the mutant has abnormal panicle, abnormal seed and upper node tillers. The F1 plants between mutant and Koshihikari were normal. In F2 population, segregation ratio between the wild type and mutant was fitted to 3:1. This genetic analysis indicated that plastochron 1-6 is controlled by a single recessive gene. Bulked segregant analysis revealed that the gene was located on chromosome 10. Through sequencing analysis, we found that plastochron 1-6 mutant had a single nucleotide transition occurred in the first exon of LOC_Os10g26340 (encoding P450 CYP78A11). This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008125), Rural Development Administration, Republic of Korea.