Watermelon (Citrullus lanatus) is one of the most economically important cucurbitaceous crop over the world. Anthracnose disease caused by Colletotrichum orbiculare, result to severe damage to cucurbits worldwide. Anthracnose is a typical plant disease which significantly affecting the yield of cucurbit crop. Pathogeneis-related (PR) proteins are well-known plant defense proteins against pathogens. Therefore, we observed PR genes expression patterns when watermelon got anthracnose disease. We did RT-PCR experiment to evaluate differences of PR genes expression pattern among Au-Producer(R), one of the representative of resistance watermelon varieties against anthracnose, and 920533(S), one of the representative of susceptible watermelon varieties against anthracnose. As a result, there were differences of the expression of several PR genes between the R and S watermelon. Analysis of the function of these genes is expected to perform in the future.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained GPD, UtrCSP, BrTSR15 and BrTSR53 genes overexpression constructs under the control of the constitutive CaMV 35S promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also FSTs analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 95 FSTs, among which 37 (38.9%) were integrated into genic regions and 58 (61.1%) into intergenic regions. About 27 homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S::BrTSR15 and 35S::BrTSR53 transgenic plants were displayed higher levels of transgene protein than WT plants. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

Citrus canker caused by Xanthomonas citri pv. citri is one of economically important diseases in the citrus industry. The devastating bacterial disease results in unattractive quality and a significant reduction in fruit production. Citrus growers and industry in Korea has been struggling with the serious disease causing the prohibition of export market. Korea also became the top import market for oranges. The development of markers linked to citrus canker resistance is strongly needed. In this study, we investigated molecular markers between ‘Kiyomi’ (Citrus unshiu ｘ C. sinensis), a resistant cultivar, and Natsudaidai (C. natsudaidai), a susceptible cultivar. To develop markers, we focused on structural variation (copy number variation, CNV, and presence/absence variation, PAV). It has been well documented that CNV and PAV of defense-related genes are associated with resistant cultivars. Using a read depth approach following next-generation sequencing, we performed genome-wide analysis of CNV and PAV in two varieties. As a result, 633 genes showing at least two times difference between the mapping reads from two varieties and 61 genes showing presence of the mapping reads in only either one of them were screened. Visual inspection using the Integrative Genomics Viewer (IGV) was performed and experimental validation is being investigated. Interestingly, one of PAV candidates showed polymorphism in ‘Kiyomi’ and ‘Natsudaidai’ as well as other resistant and susceptible cultivars. Our results suggest a necessity for the detection of structural variation and indicate that the candidates may be useful for molecular breeding for citrus canker resistance and understanding disease resistance mechanism.

The PepMoV has been considered the most frequently detected potyvirus. When it co-infects with CMV or PMMoV, it gives severe impact to total pepper harvest in Korea. Since F1 hybrid that resistant to PepMoV has not been developed, we have developed transgenic peppers using Agrobacterium-mediated transformation with a Hc-Pro gene of the PepMoV. A large number of GM peppers were tested for resistance to the PepMoV, and after consequent self-crossing up to T4 generation, a highly tolerant pepper to PepMoV called T20 was selected. So far, BC4F1 lines have been selected by back-crossing with 4 elite lines through a breeding program. Very recently, based on molecular analysis, we have selected another event, #10-2, which is also resistant to PepMoV. Horticultural difference was investigated for both GM lines, #T20 and 10-2, and no significance was found comparing to non-GM lines.

The correct development of male gametophytes (pollen grains) in flowering plants is essential for proliferate in gamete production. Here we have taken a map-based cloning approach using Arabidopsis male gametophytic mutant, named gemini pollen3 (gem3) to identify and characterize key gene that is expressed gametophytically for the completion of microgametogenesis focusing on genes which control cell division and cell fate determination. Previously reported gem1 and gem2 mutants with similar characteristics to gem3 that are disturbed at asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. However, gem3 was mapped to a different genetic locus, and pollen developmental analysis revealed that gem3 exert an effect at meiosis and mitosis causing complete sterility. We also discovered that gem3 homozygous lines produce aberrant pollen grains, arising from incomplete cytokinesis during male meiosis with sporophytic phenotypes of twisted-shape leaves, large flowers. This mutation shows reduced genetic transmission of gem3 allele through male gametophyte. In previous results, the gem3 locus was confirmed by mapping to the region located on chromosome 5. To further confirm strong candidate gene, we performed sequencing and genetic complementation analysis. Currently, we are performing functional studies of the gem3 gene for the better understanding of molecular mechanisms that control asymmetric division at meiosis and mitosis during pollen development.

Heterosis is very important for breeding hybrid cultivars and is intensively used to increase the productivity of crop plants. But the molecular basis of heterosis is still unrevealed to the scientists. This study selected 51 heterosis associated genes of Arabidopsis of different family on the basis of their high differential expression in a hybrid compared to its midparent value and identified their orthologues in Brassica oleracea. Then the selected B. oleracea genes were characterized based on their structural properties, recognized functions and expression patterns in a cabbage hybrid progeny (Cabbage-36) of crosses between Cabbage-34 and Cabbage-35 accessions. Among these genes, a good number were found to express highly in the hybrid then the midparent value and better parent in some cases. Moreover, these highly expressed genes are mostly related to the yield contributing characters. Cotyledon and young leaf sizes of these three genotypes also well correlated with gene expression. Thus, it can be said that, the identified genes might be associated with the mechanism of heterosis of B. oleracea hybrid and provide a foundation for the exploration of gene regulatory networks associated with the specification of the phenomenon heterosis in the plant life cycle. Subsequently, these genes would be useful resources for molecular hybrid breeding in Brassica crops as well.

The most important factor in breeding program is to obtain the value-added genetic line. Generally, breeders develop genetic sources using several methods such as segregation-breeding, cross-breeding, backcross-breeding, mutation induction, tissue culture and so on. Here, we present one classical way but very valuable method called cell fusion or protoplast fusion to create genetic sources for the breeding practice. The method we developed was the asymmetric somatic-hybridization of protoplast isolated from carrots. This is rather to transfer the nucleus from the high quality F1 hybrid to other mediocre line to produce a new carrot line. Since the breeding a carrot line for higher quality and purity takes a long time, therefore this nuclear transfer technology is very beneficial to generate a new line that could be useful to breed elite varieties. We had obtained around 200 fused carrots (cybrids), 12 cybrids were self pollinated and produced seeds. Selected progenies (C3) have been evaluated for horticultural characteristics and we have found new genetic lines that show better phenotypes.

An important worldwide plant source of dietary protein and oil, modern breeding and improvement of soybean is suffered from a narrow cultivated germplasm relative to other crop species likely because of underuse of wild soybeans as breeding resources. SNP genotyping array is regarded as a promising tool for dissecting wild and cultivated germplasms to find important adaptive genes by high-density genetic mapping and genome-wide association studies (GWAS). Here, we present the establishment of a large soybean SNP array and its use for diversity analysis and high density linkage mapping. More than 4 million high-quality SNPs identified from 16 high-depth and 31 low-depth soybean genome resequencing data were used to select 180,961 SNPs for the AxiomÒ SoyaSNP array. Our validation analysis for a set of 222 diverse soybean lines showed that a total of 171,161markers were of good quality for genotyping. Of the converted SNPs, 82.6% 82.6% SNPs had a marker spacing of less than 9 kb and 17.4% SNPs greater than 9 kb with the 297 inter-SNP spacings of >100 kb and with 812 kb of the largest spacing, thereby suggesting that our array is likely suitable for GWAS of soybean germplasm. This array is being used to construct high-density genetic map in populations generated from intermatings of two cultivated and two wild soybeans, with an objective to confirm large structural variations of chromosomes using the ultra-high-density maps

The genus Vicia comprises 166 annual or perennial species distributed mainly in Europe, Asia, and North America, also extending to the temperate regions of South America and tropical. However, utilization of SSR markers have not been investigated extensively in Vicia species as compared to other crop species. Here, we have assessed the potential for transferability (cross-species amplification) of cDNA microsatellites markers developed from common vetch (Vicia sativa subsp. sativa). In total, 226 alleles were detected in 36 microsatellite loci. The number of alleles per marker ranged from one to 20, with an average of 6.3. The gene diversity and polymorphism information content value averaged 0.540 and 0.503, with a range of 0–0.85 and 0–0.84 respectively. For transferability of the SSRs, amplification was carried out with selected from two to 8 accessions of 22 different Vicia species. For individual species, the successful amplification rate ranged from 32.6% in V. ervilia to 81.9% in V. sativa subsp. nigra, with average of 48.8%. As the rate of successful amplification of microsatellite markers generally correlates with genetic distance, these SSR markers are potentially useful in the analysis of genetic relationships between or within Vicia species.

최근 흑토마토는 기능성 토마토로써 소비자에게 각광을 받고 있으며 다양한 품종이 개발되고 있다. 흑토마토 형 질 결정 유전자를 기반으로 흑토마토 판별용 분자표지 셋트를 개발하였으며, 이를 국내 시판 토마토 품종에 적용 시켜본 결과, 대부분의 흑토마토를 정확하게 판별해 낼 수 있음을 확인할 수 있었다. 토마토 반신위조병 (Verticillium wilt)은 Verticillium dahliae와 Verticillium alboatrum에 의한 곰팡이병으로 전형적인 토양전염성 병해이 다. 토마토 황화 잎말림 바이러스(TYLCV; Tomato yellow leaf curl virus)는 담배가루이를 매개로 전염되며, 최근 기 후온난화와 더불어 미국, 남미, 유럽, 동남아시아, 일본, 중국 등지로 확산되고 있어 다양한 TYLCV에 대한 안정적 저항성을 지닌 품종의 육성이 요구된다. 토마토 반신위조병에 대한 저항성 유전자인 Ve1은 extracellular leucine-rich repeat (eLRR) receptor-like protein으로써 cell surface receptor를, TYLCV 저항성 Ty-1은 RNA-dependent RNA polymerase 를 각기 암호화하고 있음이 기존에 확인된 바 있다. Ve1와 Ty-1에 대한 연관 분자표지는 각기 개발 되어 분자육종에 적극 활용 중에 있으나, 본 연구에서는 유전자 염기서열 내의 다형성에 기반을 둔 functional marker의 개발을 진행하였다. 저항성 Ve1과 이병성 ve1의 염기서열 상의 다형성 중, 이병성 ve1에서 premature stop codon을형성하여 저항성 여부를 직접적으로 결정하는 다형성을 기반으로 cleaved amplified polymorphic sequence (CAPS) 분자표지를 개발하였다. 저항성 Ty-1과 이병성 ty-1의 염기서열 상의 7개의 SNP를 비교분석하여, 단백질 서열 상의 차이를 유도하는 다형성을 기반으로 CAPS 분자표지로 제작하였다. TYLCV 저항성 중 열성유전양상을 보인다고 알려져 있는 ty5저항성에 대해서도 유전자 기반 분자표지를 제작하였다. 본 연구에서 개발된 흑토마토, Ve1, Ty-1, ty5 에 대한 기능성 분자표지는 과색판별 및 병저항성 육종의 효율 및 정확도 향상에 크게 기여할 것으로 기대된다.

Sorghum (Sorghum bicolor (L.) Moench) has been cultivated for cereal grain which has been traditionally used for steaming with rice in Korea. Various Korean sorghum varieties have been developed and distributed for farmers and consumers to meet their needs. Korean sorghum grains have been mostly sold at higher price in the market than sorghum grains imported from abroad. However, no varietal identification method was established to support fair trade in the cereal market. The objective of this study is to develop the identification method of Korean sorghum varieties using a multiplexed fingerprinting platform of SSR markers. One marker for the waxy allele and nine SSR markers were carefully selected based on their product sizes for the multiplexing. A robust multiplexed combination was revealed from serially designed experiments for the optimization of multiplex PCR. Five varieties and two elite breeding lines could be separated with their unique fingerprinting pattern from other sorghum individuals collected over the world. The platform separated most of individuals tested in this study, remaining three genotypes contained two or three identical individuals. The technique may be applied to detect closely-related individuals including full sibling progeny

Capsinoids, low-pungent compounds, have the same biological effects as capsaicinoids such as anticancer and anti-obesity. A precursor of capsinoids, vanillyl alcohol, is known to be produced by mutations in the p-aminotransferase (p-AMT) gene. In the previous study, SNU11-001 (C. chinense) containing high levels of capsinoids was found in germplasm collections of Seoul National University. We found that this collection has a unique mutation in the p-AMT gene. In order to develop a cultivar containing high capsinoids contents, marker-assisted foreground and background selection were performed in this study. Backcrossing is an effective breeding method for introducing useful traits to an elite cultivar. Compared to conventional backcrossing, marker-assisted backcrossing (MABC) is extremely useful for recovery of a recurrent parent’s genetic background. To obtain background selection markers, a total of 412 single nucleotide polymorphism (SNP) markers was screened to obtain polymorphic SNP markers between ‘Takanotsume (C. annuum)’ and ‘SNU11-001’. Of the 412 SNP markers, 96 polymorphic SNP markers evenly distributed in pepper genome were finally selected. Plants carrying the pAmt/pamt genotype were selected from a BC1F1 population using SCAR markers derived from the unique p-AMT mutation of SNU11-001. BC1F1 plants carrying the pAmt/pamt genotype were subjected to background selection. Multiple genotype analysis was done using Fluidigm platform (BioMark). Once we obtain plants carrying most similar genetic background to recurrent parent, capsinoids contents will be measured and another round of MABC be done to obtain plants containing high levels of capsinoids.

Radiation technologies have been widely used in mutation breeding of crops. Although gamma-radiation has been predominantly applied in radiation breeding, ion beam irradiation is recently emerging as a methodology highly effective in induction of mutations that are useful for plant breeding. Therefore, we investigated the biological effects caused by irradiation of two types of ion beams, which were proton and ion beams, respectively, in pepper. In the evaluation of survival rate, LD50 values were between 300 and 350 Gy, 30 and 40 Gy in irradiation of proton and carbon beams, respectively, while LD50 for gamma-ray was determined to be between 50 and 100 Gy. Growth traits including shoot length, root length, and root width were also examined in pepper seedlings according to does of proton and carbon ion beams to estimate biological effectiveness of each radiation. The result was applied in the construction of pepper mutant population which will be used to develop pepper breeding materials containing novel characteristics.

The Alfin-like transcription factor family is one of the important gene families in eukaryotic plants. They are involved in many biological processes, such as lignocellulosic wall biosynthesis, meristem development, metabolite transport, and responses to biotic and abiotic stresses. But the regulatory mechanism of these genes involved in stresses responses is still unrevealed. In this study, we identified a total of 16 Alfin-like genes from Brassica rapa database. The 16 putative Alfin-like proteins were divided into four groups (group I-IV) based on structural and phylogenetic analyses. Accordingly, this study analyzed stress resistance-related functions of all B. rapa Alfin-like (BrAL) genes through a homology study with existing biotic and abiotic stress resistance-related Alfin-like genes of other plant species and found a high degree of similarity with them. Subsequently, these genes were further investigated by real-time quantative PCR under cold, salt and drought stresses and after infection with Fusarium oxysporum f. sp. conglutinans in B. rapa. These genes showed an organ specific expression and all genes differentially expressed in Chiifu compared to Kenshin under cold stress. Ten and seven BrALs responded highly in Kenshin compared to Chiifu under salt and drought stresses respectively. In addition, six BrAL genes showed responsive expression after Fusarium oxysporum f. sp. conglutinans infection in B. rapa. Interestingly, four BrAL genes showed responses against both biotic and abiotic stress factors. Thus, our result provides a useful reference data set as the basis for functional analysis and utilization in the resistance molecular breeding of B. rapa.

The MethioninesulfoxidereductaseB2(MsrB2) gene catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine and is known to provide tolerance to biotic and abiotic environmental stresses. There have yet to be any reports that MsrB2 enhances drought tolerance. Two drought-tolerant transgenic rice lines, L-8 (single copy) and L-23 (two copy), expressing the Capsicum annuum MsrB2 (CaMsrB2) gene were selected for stress tolerance phenotyping under drought stress conditions. CaMsrB2 enhanced relative water content (RWC), maintained substantial quantum yield (Fv/Fm ratio), and subsequently improved photosynthetic pigments. Interestingly, L-23, carrying two-copy T-DNA insertion, showed greater drought tolerance through more effective stomatal regulation, carotenoid concentration, and osmotic potential than the wild type. High-tech infrared technology (FLIR SC620) was used for the selection of stress-tolerant physiotypes. Later, the IR results were correlated with other tested physiological parameters. The IR images, average plant temperature, and physiological parameters of the treated plants were discussed in detail.

Steroidal glycoalkaloids which serve the plant defense, are toxic secondary metabolites present in the plants of solanaceae family. The upper safe limit of glycoalkaloids for human consumption is 20mg/KG FW, excess of which may cause severe health disorders. Several factors like drought, high temperature, light exposure, and wounding increase tuber glycoalkaloid content. Among these, drought is an important factor which causes a rapid increase in potato glycoalkaloid content. Glycoalkaloid biosynthetic genes and their expression pattern need to be characterized to regulate the glycoalkaloid accumulation. Three key genes SGT1, SGT2 and SGT3 are demonstrated to be directly participated in the biosynthetic pathway for glycoalkaloid formation. Present study was focused on the study of expression pattern of key genes in GA pathway under drought stress in two different potato cultivars Atlantic and Haryoung which are low and high glycoalkaloid accumulating respectively. Drought stress was imposed by withholding water to the plants grown in pots and control plants kept irrigated. Expression analysis of SGT1, SGT2 and SGT3 was done from the leaf and tuber sample of three time intervals i.e 5, 10 and 20 days after imposing stress. Variation in the expression level of genes was observed in leaf and tuber where the fold increase in expression over control was higher in tuber sample compared to leaf. Expression levels also varied in leaf and tuber among two cultivars. However, expression of SGT1, SGT2 and SGT3 is significant indicating the involvement of these genes in glycoalkaloid accumulation under drought stress.

Common buckwheat (F. esculentum) and tartaryan buckwheat, also called as bitter buckwheat (F. tartarycum) grain and leaves (buds) are used in various dietary preparations and as leafy vegetable. The cultivated area of buckwheat is increased based on its nutritional value. Particularly bitter buckwheat is a rich source of rutin compared to common buckwheat which helps in reducing intra-vascular cholesterol, high blood pressure, diabetes and is also reported to have a crucial role in pharmaceutical research. With this functional characteristics of bitter buckwheat, the cultivation is now highly increased. But a few genetic and genomic research of tartari buckwheat are published until now. Here we described the complete full chloroplast genome sequence with NGS. Tartary buckwheat complete chloroplast genome is composed of a total sequence length of 159,272 bp which is 327 bp lesser than common buckwheat genome of 159,599 bp. Large single copy region (LSC) is comprised of 84,398 bp in tartary and 84,888 bp in common buckwheat whereas small single copy region (SSC) is 13,292 bp and 13,343 bp and the size of inverted repeat region (IR) is 61,532 bp and 61,368 bp in tartary and common buckwheat respectively. Total RNA bases were 11942 and 11950 and overall GC-content in tartary and common buckwheat is almost similar which is 37.9% and 38% with a GC skew of -0.016 and 0.02 respectively. Total repeat bases accounted for 1,056 bp and 804 bp with an average repeat length of 48 bp and 45 bp and the length of an average intergenic distance was 495 bp and 502 bp in tartary and common buckwheat respectively. F. tarataticum cp genome has a total of 104 genes including 82 protein coding genes, 29 transfer RNA genes and 4 ribosomal RNA genes. Protein coding genes include photosynthesis related genes majorly in addition to transcription and translation related genes. LSC region has 62 protein coding genes and 22 tRNA genes whereas SSC region contains 11 protein coding genes and one tRNA gene. The nucleotide and amino acid sequences of protein coding genes in LSC, SSC and inverted repeat regions in F.tartaricum and F.esculentum are highly similar with a total average identity of 98.8 and 98.3% respectively.

The newly developed varieties, Jayoung (violet flesh color) and Hongyoung (red flesh color) that harboring various anthocyanins and flavonoids in flesh colored potato are highly increase their interesting not only for food but also functional characteristics such as anti-inflammatory effects. Up to date, most of the molecular markers developed in potato are linked to disease resistance including late blight and PVY, nematode. A few markers linked to economically important functional materials such as anthocyanin biosynthesis are published. With the low cost and high throughput of NGS (Next Generation Sequencing) technology, numerous molecular markers are highly increased in may crops. Among the molecular markers, SNPs (Single nucleotide Polymorphisms) are most useful markers owing to their large numbers in inter and intra varieties in potato. Here we reported SNPs discovery from transcriptome sequencing data acquired from colored flesh potato cultivars, Jayoung and Hongyoung with white flesh color Atlantic. Total RNA was isolated from shoot in tuber after breaking dormancy about 2cm length. Short read sequence data were obtained form Illumina Hiseq2000 and the raw dat set were trimmed with Q socore over 20. Sequencing data were align to reference genome (Solanum tuberosum v4.03, http://potatogeomics.plantbiolgy.msu.edu). About 70% of sequence read were mapped int to reference genome. 139,050, 140,976 and 146,429 total SNPs were discovered in Hongyoung, Jayoung and Atlantic, respectively. All SNPs are mapped into the psedomolecules in reference genome by chromosome. SNPs are also analyzed with homozygous and heterozygous SNPs and genic and intergenic region. SNPs are compared with Potato Infinium 8K Chip data. SNPs found in candidate genes of anthocyanin biosynthesis were discovered. These SNPs information of flesh colored potato will be further analyzed for the allele mining for anthocyanin syhthesis and control region

AtRabG3b and CaMsrB2 genes incorporated into pPZP vetor were transformed to Korean soybean cultivar Kwangan using highly efficient transformation system. AtRabG3b gene plays a positive role in xylem development in Arabidopsis and 64 transgenic plants were produced. CaMsrB2 gene is known to confer drought tolerance in rice and 63 transgenic plants were produced. As a result of PPT leaf painting assay, about 20% of transformation efficiency was observed from 2 times of inoculation. These transgenic plants were confirmed for gene introduction using PCR. Currently, the copy number and the gene expression is investigating using qRT-PCR and RT-PCR. Moreover, 62 lines and 53 lines of T1 seeds from AtRabG3b and CaMsrB2, respectively, were sown in GMO field.

Iron is an essential mineral found in every cell of the human body to make the oxygen-carrying proteins hemoglobin and myoglobin. More than 2 billion people face Fe deficiency. Rice can be a potentially valuable source to supplement that mineral since it is staple food for two-thirds of the world’s population. To bring the nutritional level of the milled product up to that of the whole grain (brown), rice should be enriched with thiamin, niacin and iron. Thus we searched a possible allelic source from Heuristic or core rice set, which is derived from a total 24,368 rice germplasms, to increase the mineral content in rice varieties. The concentration and distribution of Fe in 137 accessions of core set of brown rice grain flour sample were determined by ICP-OES. The range of the concentration and distribution of Fe in 137 core accessions of brown rice grain were wide, from 0.088mg/L to 1.205mg/L, with mean 0.292mg/L. To examine Fe related genes, whole-genome resequencing data of 137 accessions of rice core set were analyzed by Genome Wide Associations Study (GWAS). Our result suggests that Fe determined by ICP-OES facilitates the evaluation of the differences in Fe composition for future rice breeding program