Background : Lycium ruthenicum Murr. was known as black wolfberry. It belongs to Solanaceae. In order to elucidate biological activities and functional substances of Lycium ruthenicum Murr., the methanol extract of Lycium ruthenicum Murr. harvested from Qaidam Besin. area was prepared and was studied. Methods and Results : Antioxidant, anticancer, anti-inflammatory and antidiabetic activities of the methanol extract of Lycium ruthenicum Murr. were examined using diverse in vitro bioassays and functional substances were analyzed by HPLC and LC-MS/MS. The methanol extract of Lycium ruthenicum Murr. showed strong DPPH and ABTS radical scavenging activities and also showed strong SOD-like activity. To examine the effects of the methanol extract of Lycium ruthenicum Murr. for the expression of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), RT-PCR was performed. SOD expression was strongly increased by the methanol extract of Lycium ruthenicum Murr. CAT expression was also increased with dose-dependent manner. The methanol extract of Lycium ruthenicum Murr. showed inhibitory effects against α-glucosidase and α-amylase, and also inhibited NO production. Anticancer effects of the methanol extract of Lycium ruthenicum Murr. assay were examined with five human cancer cell lines. The potent anticancer activity exhibited in HepG2 hepatoma cells. As a result of main component analysis by HPLC, chlorogenic acid and rutin were identified as the major flavonoids of Lycium ruthenicum Murr.. In addition, by LC-MS/MS, petunidine-3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside was determined as a major anthocyanin of Lycium ruthenicum Murr.. Conclusion : Lycium ruthenicum Murr. showed various biological activities and could be used as a food and a functional material to enhance human health. Further study to identify an unknown flavonoids of Lycium ruthenicum Murr. would be needed.

Safflower (Carthamus tinctorius L.) seeds have long been clinically used in Korea to promote bone formation and prevent osteoporosis. In addition, the safflower buds (SB) were found to have more useful functional ingredients than safflower seed. Thus, we investigated the preventive effects of SB diet in ovariectomized (OVX) rats. The rats were divided into five groups; sham operated group, OVX alone group, OVX plus 17β-estradiol (E2 10 ㎍/㎏, i.p.) and OVX plus SB diet feeding group (0.3% or 1%). Feeding of SB diet (0.3% or 3%) to OVX rats markedly increased trabecular formation in femur compared to OVX rats. Feeding of SB diet (0.3% or 3%) to OVX rats also decreased TRAP activity compared to OVX rats. These results suggest that SB diets have bone sparing effects by the decrease of osteoclast activity. We also observed that OVX rats fed with SB diet (0.3% or 3%) exhibited the decrease of calcium and phosphorus in serum compared to OVX-induced rats. Therefore, SB may be beneficial for the patients of osteoporosis, especially in postmenopausal women.

The microbial composition in Nuruk, a Korean cereal fermentation starter, is a critical factor for the quality and organoleptic properties of traditional alcoholic beverages. This study was aimed at monitoring the compositional change and enzyme activity of culturable lactic acid bacteria (LAB) in two types of Nuruk fermented at different temperatures. All culturable LAB were isolated at various time points (0, 3, 6, 10, 20, and 30 days) and identified by 16S rRNA sequencing. In traditional Nuruk type A (TN-A), which was fermented at 36℃, the population of total culturable LAB during the fermentation period was between 104 and 105 log CFU/mL. On the other hand, the LAB population in traditional Nuruk type B (TN-B) fermented at 45℃ (primary fermentation for 10 days) and 35℃ (secondary fermentation for 20 days) was 102 log CFU/mL; however, these bacteria could not be detected after 6 days. Major LAB strains were identified in both Nuruk types: (1) from the MRS-culture of TN-A, Pediococcus pentosaceus at 3-30 days; (2) from MRS-culture of TN-B, P. pentosaceus at 3 days and Enterococcus hirae at 6 days. The protease activities of the dominant LAB isolated from the TN-A and TN-B cultures were within the ranges of 0.64~1.03 mg/mL and 0.74~0.81 mg/mL (tyrosine content), respectively, whereas the α-amylase activities were 0.75~0.98 mg/mL and 0.78~0.79 mg/mL (amylose content), respectively.

In this study, antioxidant and anti-inflammatory activities of water, methanol, and ethanol extracts obtained from Allium hookeri root were evaluated. The ethanol extract of A. hookeri was found to possess the strongest reducing power and also exhibited dominant effects on scavenging of nitrites, DPPH radicals, and superoxide radicals. The water extract showed more efficient DPPH and hydroxyl radical-scavenging activities than those of the methanol extract. Furthermore, the inhibitory activity against nitric oxide (NO) production in RAW 264.7 macrophages was evaluated to elucidate the anti-inflammatory properties of the extracts. Results indicated that all the extracts of A. hookeri exerted inhibitory activities against NO production, especially the ethanol extract (IC5029.13μg/mL). Total phenolic and total flavonoid contents were found to be abundant in the ethanol extract, with values of 24.96 mg gallic acid equivalent/g extract and 4.27 mg rutin equivalent/g extract, respectively. Total thiosulfinate content was determined for the first time and a high amount was present in the ethanol extract (14.2 μM/g extract). These results suggest that A. hookeri root has antioxidant and anti-inflammatory properties and could be used as a natural source for the development of pharmaceutical agents or functional foods.

Environment-friendly growth enhancers for rice are being promoted to reverse the negative impact of intensive chemical-based and conventional rice farming on yield sustainability and environmental problems. Several rhizosphere microorganisms and pyroligneous acids (PA) had demonstrated beneficial influence on growth, yield and grain quality of rice. Since most of the previous study had evaluated the effect of PGPR and PA on paddy rice singly, the effect of combined application of these on the growth and yield of paddy rice and on some soil chemical properties were determined. A four factorial pot experiment was conducted to evaluate the effect of PGPR, PA in combination with fertilizers and on different soil types. There were 54 treatment combinations including the control with three replications under complete randomized design. Plant growth parameters were evaluated using standard procedures during tillering and heading stages. Rice yield and some soil chemical properties were determined at harvest. Results showed that inoculation of Bacillus licheniformis and Fusarium fujikuroi enhanced plant growth by increasing the plant height which could be ascribe to its ability to promote IAA and GA production in plants. Inoculation of Rhizobium phaseoli enhanced chlorophyll content indicative to its ability to improve the N nutrition. However, these plant growth benefits during the vegetative stage were override by the fertilizer application effect especially during the maturity stage and grain yield. High fertilization rates on coarse-textured soil without nutrient loss resulted to high available nutrients and consequently high yield. Wood vinegar application however improved nutrient availability in soil which could be beneficial for improving soil quality. Further evaluation is necessary to fully assess the potential benefits that could be derived from inoculation of these organisms and wood vinegar application in different soil environment especially under different field conditions.

본 연구는 rutin 및 rutin 대사체인 DHPAA, HPAA, DHT, HVA가 버섯 유래 tyrosinase 활성과 마우스 피부표피 세포주인 JB6 P+ 세포의 증식에 미치는 영향에 대해 살펴보았다. HVA가 버섯유래 티로시나제 저해활성이 가장 우수한 것으로 관찰되었으며, DHPAA, HPAA, rutin, DHT 순서로 저해효능을 나타내었다. DHT를 제외한 rutin 및 rutin 대사체들 모두 100 μM 까지 세포 독성이 관찰되지 않은 것으로 관찰되었으며, DHT는 50 또는 100 μM에서 세포증식을 저해하였으나 그 이하의 농도에서는 세포독성을 유도하지 않았다. 따라서 본 연구결과, rutin 대사체 모두 세포 독성이 없는 수준에서 버섯 유래 티로시나제 활성을 효과적으로 억제하였으므로, 앞으로 미백제제로 활용될 수 있을 것으로 기대된다.

Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.

Plant breeding requires genetic diversity of useful traits for crop improvement. EMS-induced mutation is practiced to generate mutations at loci regulating economically important traits and/or to knock out the genes to elucidate their functions. The present study was aimed to induce mutations in a Korean local land race Capsicum annuum ‘Yuwol-cho’. This accession is pungent and also has advantage to mature early. A total of about 1,500 M2 families were screened and three non-pungent mutants were identified and crossed with wild type ‘Yuwol-cho’. After phenotyping of F2 population for pungency, MutMap approach will be used to identify the genes controlling the pungency in mutants. In addition to this, another C. annuum accession “Micro-Pep” was used to develop a mutant population. Micro-Pep is a small, pungent pepper generally used as ornamental purpose. Having compact growth habit, and small size, it has advantage to handle and utilize easily in mutation study and molecular research. On the basis of preliminary experiment 1.3% of mutagen was used for treatment of pepper seeds and 30% less germination percentage was observed in EMS treated seeds in comparison to control seeds. A total of 4,674 M1 plants are grown under greenhouse condition and M2 population will be studied for characterization of phenotypic variation including fruit color and pungency. Newly constructed mutant populations will be valuable assets for identification of functional genes and molecular breeding of pepper.

Superjami is a new rice breed resulted from crossing ‘C3GHi (has high amount of Cyanidin 3-glucoside, and was developed from a cross between ‘Heugjinjubyeo’ and ‘Suweon 425’) and ‘Daeribbyeo 1’. Superjami has 10.9 times higher C3G content compared with ‘Heugjinjubyeo’. It also contains the highest essential amino acids of all kinds (except tryptophan content). This study was done to investigate the effects of extracts from superjami bran on the in-vitro antioxidant metabolism, in-vivo antioxidant metabolism and bone metabolism on menopause- induced condition in experimental rats. Overall, extract from superjami bran was confirmed of improving antioxidant and bone metabolism which can be considered as a good dietary supplement.

GWAS (Genome-wide association study) provides a useful to associate phenotypic variation to genetic variation. It has emerged as a powerful approach for identifying genes underlying complex diseases or morphological traits at an unprecedented rate. Despite benefits, there are only a few examples applied in crop plants due to lack of effective genotyping techniques and well prepared resources for developing high density haplotype maps. In this study, 350 core accessions selected from almost 5,000 Capsicum accessions were used for GWAS. We are planning to construct a high-density haplotype map using GBS platform and perform GWAS for various agronomic traits including fruit traits and metabolites related to pungency to identify genes controlling the traits. These results will not only provide a list of candidate loci but also a powerful tools for finding genetic variants that can be directly used for crop improvement and deciphering the genetic architecture of complex traits.

The ovariectomized Sprague-Dawley female rats were randomly assigned to Sham-Control, OVX-Control, OVX-Superjami (extract) groups. The results showed that the activity of glucokinase to keep the blood sugar constant is increased by increasing insulin secretion from pancreatic β- cells and the homeostatic regulation of glucose. Meanwhile the glyconeogenesis which is involved in the actions of the enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase showed that the glucose level is decreased. It was confirmed that these enzymes regulate the carbohydrate metabolism. On the other hand, results of the measurement of the lipid metabolism in the fat tissue and liver tissue, effect of β-oxidation enzymes and carnitine palmitoyl transferase which is involved in fatty acid oxidation for energy generation is increased. Moreover, the activity of fatty acid synthase, glucose-6-phosphate dehydrogenase and malic enzyme have been reduced, therefore, it was confirmed that these enzymes regulate the lipid metabolism.

Pepper (Capsicum spp.) germplasm shows diverse phenotypic variations including fruit size, color, pungency, and many other horticultural traits. Traditional markers including SSR, AFLP, and RFLP have been used to construct genetic maps using biparental populations. However to assess the genetic diversity of large number of germplasm, a robust and rapid marker development and genotyping approach is needed. We used six pepper accessions including C. annuum, C. chinense, C. baccatum and C. frutescens and performed genotyping-by-sequencing (GBS). To select the most appropriate condition, eight different 2 bp selective nucleotides were used to make GBS libraries. Selective nucleotide ‘OO’ showed the largest number of reads in all samples, and 11,026 to 47,957 high-quality SNPs were called in six accessions. When C. annuum ‘CM334’ genome sequence was used as a reference, C. annuum showed the smallest number of SNPs, while C. baccatum which was known to be a different Capsicum clade showed the largest number of SNPs. Pepper core collection chosen to represent the genetic diversity of whole germplasm will be genotyped by high-density SNPs developed from GBS. We will perform genome-wide association study (GWAS) using genetic and phenotypic variation to identify the functional genetic loci controlling horticultural traits.

Capsicum annuum ‘Bukang’ is a resistant variety to Cucumber mosaic virus isolate-P0 (CMV-P0), CMV-P1 can overcome the CMV resistance of ‘Bukang’ due to mutations in Helicase (Hel) domain of CMV RNA1. To identify host factors involved in CMV-P1 infection, a yeast two-hybrid system derived from C. annuum ‘Bukang’ cDNA library was used. A total of 156 potential clones interacting with the CMV-P1 RNA helicase domain were isolated. These clones were confirmed by β-galactosidase filter lift assay, PCR screening and sequence analysis. Then, we narrowed the ten candidate host genes which are related to virus infection, replication or virus movement. To elucidate functions of these candidate genes, each gene was silenced by virus induced gene silencing in Nicotiana benthamiana. The silenced plants were then inoculated with green fluorescent protein (GFP) tagged CMV-P1. Virus accumulations in silenced plants were assessed by monitoring GFP fluorescence and enzyme-linked immunosorbent assay (ELISA). Among ten genes, silencing of formate dehydrogenase (FDH) or calreticulin-3 (CRT3) resulted in weak GFP signals of CMV-P1 in the inoculated or upper leaves. These results suggested that FDH and CRT3 are essential for CMV infection in plants. The importance of FDH and CRT3 in CMV-P1 accumulation was also validated by the accumulation level of CMV coat protein confirmed by ELISA. Altogether, these results demonstrate that FDH and CRT3 are required for CMV-P1 infection in plants.

Carotenoids are vital pigments responsible for yellow, orange and red color in plants. In Capsicum, capsanthin-capsorubin synthase (CCS), phytoene synthase (PSY), β-Carotene hydroxylase (CRTZ-2) and lycopene β-cyclase (LCYB) were identified to be involved in the carotenoids synthesis pathway. Previously molecular markers based on the CCS and PSY genes have been developed to distinguish fruit colors in pepper. However these markers can distinguish fruit colors of limited pepper genotypes. Therefore, there is need of developing additional markers for accurate prediction of fruit colors using molecular markers. In this study carotenoids contents of 16 pepper accessions were analyzed and the CCS, PSY, CRTZ-2, LCYB genes were sequenced to identify the genes affecting the fruit color. Among all the analyzed carotenoids, capsanthin was accumulated in much higher amount in red and orange fruits (1100-2500 mAU·min and 30-500 mAU·min respectively) while violaxanthin (20-1200 mAU·min) was accumulated more in yellow fruits. Sequence analysis revealed that deletions and two frame shift mutations in CCS gene for yellow accessions. Frame shift mutations of the PSY gene were detected in two orange accessions. These results show that mutations in CCS and PSY genes affect the fruit colors of pepper, and markers can be developed using mutations of these genes.

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse pepper production. In contrast to most epiphytic powdery mildew species, L. taurica is an endophytic fungus which colonizes in the mesophyll tissues of the leaf. In the genus Capsicum, several studies have been conducted to identify resistance sources to L. taurica. In previous studies, five quantitative trait loci (QTLs) for powdery mildew resistance have been identified. An F2 population derived from self-pollination of the commercial cultivar Capsicum annuum ‘PM Singang’ was used for genetic analysis of powdery mildew resistance. Resistance of the F2 plants was tested under the natural environmental conditions. Sporulation intensity on infected leaves was used as a disease scale to assign resistance levels to plants, where 0-5% is Resistant, 6-15% Moderate resistant and 16-100% Susceptible. A total of 83 F2 plants were evaluated for resistance. The results showed that 59 plants were resistant, 10 susceptible and 14 moderately resistant. If we consider MR as S, segregation ratio fitted to a single dominant resistance gene model. In the future study, closely linked molecular marker will be developed and tested to locate this gene. The developed marker will be used to identify the powdery mildew resistance gene.

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 putative-aminotransferase (pAMT) gene. In the previous study, ‘SNU11-001’ (Capsicum chinense) containing high levels of capsinoids was identified in germplasm collections of Capsicum. This collection has a unique mutation in the pAMT gene that can cause dysfunction of this gene. In order to develop pepper varieties containing high capsinoids contents, marker-assisted foreground and background selections were performed during backcross breeding. Compared to the conventional backcrossing, marker-assisted backcrossing (MABC) is extremely useful for recovery of a recurrent parent’s genetic background. For foreground selection, plants carrying the pAMT/pamt genotype were selected from a BC1F1 and BC2F1 populations using SCAR markers derived from the unique pAMT mutation of ‘SNU11-001’. To obtain background selection markers, a total of 412 single nucleotide polymorphism (SNP) markers was screened on ‘Shinghong’ parental lines and ‘SNU11-001’ to obtain polymorphic SNP markers. Of the 412 SNP markers, 144 and 204 polymorphic SNP markers evenly distributed in pepper genome were finally selected. BC1F1 and BC2F1 plants carrying the pAMT/pamt genotype were subjected to background selection using the selected marker sets. Multiple genotype analysis was done using a high-throughput genotyping system (EP1TM, Fluidigm®, USA). As a result, one BC1F1 plant 84% similar to the recurrent parent and several BC2F1 plants more than 96% recovery rate of the recurrent parent were selected. Genetic backgrounds of the selected BC2F1 plants were evaluated by the genotype-by-sequencing (GBS) method in order to confirm the background selection results using the SNP marker set. GBS results showed that recovery rate and positions of introgressed segments were well matched between two methods demonstrating MABC can be successfully done with a couple hundred SNP markers.

The transposable element is a DNA sequence that can be changed its position within the genome, sometimes it can create or reverse mutations and altering the cell's genome size. Target region amplification polymorphism (TRAP) is a rapid and efficient PCR-based marker technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. TE-TRAP is a new marker system which used terminal inverted repeat (TIR) instead of targeted candidate gene sequences. Sorghum holds a good potential plant organism for transposon tagging due to its small genome size, low amount of repetitive DNA and co-linearity with other cereal genomes, which allows the use of information derived from sorghum in other cereal grasses. IS2868 of sorghum accession was treated Gamma irradiation on seed. To define availability and utilization of TE-TRAP, twenty-one accessions were used to evaluate the genetic diversity and underlying relationships. One-thousand thirty-three TE-TRAP markers were amplified by thirty-one primer combination. Altogether, 712 (62.8%) markers were observed polymorphic segregation, whereas 421 (37.2%) showed monomorphic patterns. To estimate genetic differentiation of population by various gamma radiation doses, the analysis of molecular variance (AMOVA) was performed using 4 to 5 different radiation doses population of M1 sorghum individuals. This study and marker system will provide valuable information to assist radiation mutation breeding.

Root-knot nematode, Meloidogyne incognita is a virulent pest of solanaceaous crops worldwide. The M. incognita resistance gene Me7 derived from Capsicum annuum CM334, is located on chromosome 9. In the present study, an F2 population derived from a cross between ECW03R and CM334 was used to locate the Me7 gene. An F2 population was inoculated using approximately 1,000 second-stage juveniles per individual plant. Phenotype screening was done 45 days after inoculation by using gall index system. The phenotype study of 503 F2 individual showed 391 resistant and 112 susceptible plants. The 3:1 phenotypic ratio confirmed that resistance phenotype is controlled by a single dominant gene. Previously reported two markers were tested to reveal the linkage of markers to phenotype. Two markers, CAPS_F4R4 and SCAR_PM6a were located at 4.3 and 2.7 cM from the resistance gene, respectively. Additional SNP markers were developed using CM334 reference genome information to narrow down the position of the gene, but no closer markers could be developed due to errors of DNA sequence assembly. The closest marker was positioned on telomere of the chromosome 9 long arm, where tens of other NB-LRR genes are clustered. NB-LRR genes are being used as candidates to identify the Me7 gene.

Space has many distinguishable characteristics from earth such as strong cosmic radiation, microgravity, supervaccum and weak magnetic field. For this reason, space environments can be used an efficient mutagen for plant breeding nowadays. To identify the affected genes by condition in space with outer space, Brachypodium seeds were placed in the Russia Segment (RS) Biorisk module of International Space Station (ISS). Brachypodium distachyon is a model system for temperature grass, because they represent the characteristics for annual winter grass. Seeds and organs of plants carried by satellite or spacecraft to space can be genetically mutated by exposing space environment. We performed a duplicated RNA sequencing to profile the differentially expressed genes. As a results, about 700 genes were upregulated and 250 genes were downregulated by cosmic environments, respectively. In the molecular function category, protein kinase and transcription activity related genes were upregulated. Among the many transcription factors (TFs), stress related TFs such as ERF, NAC and WRKY were differentially expressed in space exposed samples. In the future, their expression will be identified by using qRT_PCR.