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.

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.

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.

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.

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.

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.

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.

Phytophthora capsici an Oomycete pathogen is a major challenge to the pepper (Capsicum spp.) production around the world. Control measures are proved ineffective, so breeding resistant cultivars are the most promising strategy against the pathogen. Resistance against P. capsici is governed by quantitative trait loci (QTL). According to previous studies on QTL detection, the QTL on pepper chromosome 5 is a major contributor to resistance. In this study, to exploit the involvement of this QTL and identify its contributing genes, the F2 population derived from a cross between ECW30R and CM334 was inoculated with a medium virulence P. capsici strain JHAI1-7 zoospores at the 6-8 leaf stage. Composite interval mapping revealed two major QTLs; QTL5-1 from 7 days post inoculation (dpi) and QTL5-2 from 16 dpi on chromosome 5. To characterize and detect interactions of the two QTLs, near isogenic lines (NIL) were constructed by crossing Tean and recombinant inbred line (RIL) derived from a cross between YCM334 and Tean. RILs were screened with P. capsici strain MY-1 and resistant lines were selected. Among the resistance RILs most closely related to Tean were selected using AFLP and SSR genotyping data. These RILs were named as YT39-2 and YT143-2. To develop more advanced NILs, two rounds of marker-assisted backcrossing were done using a high-throughput SNP genotyping system (EPI Fluidigm, USA). Among the NILs derived from YT39-2, YT39-2-64 contains only QTL5-1 whereas YT39-2-61 and YT39-2-69 were identified to have both QTLs. On the other hand, YT143-2-55-7 with the highest Tean genetic background contains QTL5-1 only. In the next step, the 3 different NILs having QTL5-1, QTL5-2 individually and both QTLs will be identified. Furthermore, phenotyping and fine mapping will be done for the analysis of individual and interaction effects of QTLs.

sy-2 (Seychelles-2) is a temperature sensitive natural mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures below 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total 600 individual F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. Fine-mapping of the locus allowed us to position sy-2 to an approximately 170-kb region flanked by markers IN2-1-1 and SNP-3-7 on chromosome 1. Among the approximately 36 hypothetical genes in this region several candidate genes including: HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs) were identified. RT-PCR and sequencing of the hypothetical genes are under way to identify sy-2.

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-D-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UDP-glucose 4-epimerase gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55% of total BrUGE transcripts in most of the tissues examined. All genes showed organ specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4 and -5)were shown to respond considerably against salt, drought and abscisic acid (ABA) treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120 mM Nacl for 2 weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

This study was carried out to determine the amount of lignin, cellulose, and hemicellulose in six kenaf cultivars during different harvesting stages. Three mutant cultivars (Jangdae, Jeokbong and Baekma), two original cultivars (Jinju, C14), and one Chinese cultivar (Auxu) were planted on May 14, 2013. Four harvesting times were made at intervals of 20 days from 15 July to 16 September, 2013. The overall growth characters of mutant cultivar ‘Jeokbong’ such as plant height, stem diameter, flowering time, and dry mass were similar with those of the original variety. The mutant cultivar ‘Baekma’ occurred 10-day late flowering in comparison with the original variety and also displayed higher dry mass than the original variety. Jinju, Auxu and Jangdae, mid-late maturing kenaf cultivars, had high dry weight compared to early maturing cultivars such as Jeokbong, Baekma and C14. In all cultivars, the lignin contents were increased by a late harvest. The Mid-late maturing kenaf cultivars showed high lignin content in comparison with those of the early maturity cultivars. There were no significant differences of cellulose, and hemicellulose content between the cultivars, however cellulose content in stems of these kenaf cultivars were significantly decreased by a late harvest. These results may provide valuable information to assist the parental selection of kenaf breeding.

Rose (Rosa Hybrida Hort.) are of a high symbolic value and a great cultural importance in different societies. They are widely used as garden ornamental plants and as cut flowers. For the induction of mutation, gamma-rays are widely used as a mutagen. This study was carried out to establish a system for mutation breeding by irradiation of gamma-ray in rose. The rooted cuttings of five cultivar roses (Lovelydia, Vital, Aqua, Yellowbabe and Haetsal) are grown by in a greenhouse. They were two difference treatment (Before rooting gamma-ray irradiation, After rooting gamma-ray irradiation) were exposed to dose of 70 Gy using a 60Co gamma-irradiator (150 TBq of capacity ; ACEL, Canada) at the Korea Atomic Energy Research Institute. The irradiated plants were planted in a greenhouse, and investigated survival rate, mutation rate, flower buds number, and shoot length were planted after 80days. The two treatments of and growth characters was significantly reduced to 20% to 40% compared with the control. In addition, survival rate and mutation rate were ‘after rooting γ-ray irradiation (37.4~67.3% and 0.5~5.6%)’ higher than ‘before rooting γ-ray irradiation (18.3~50.8% and 0.3~3.4%)’. Mutation types were solid type, chimeric and mosaic petal mutants with various colors were induced from five rose. These results indicate that efficiency of mutation induction in rose by gamma-ray irradiation on petal colors and petal shapes in two difference treatment with rooted cutting system.

Kenaf (Hibiscus cannabinus L.) native to Africa can be used as fiber, food, feedstock and bio plastic. This study was carried out to evaluate the mineral, amino acid and vitamin contents of six selected kenaf cultivars which are enable to produce seed under Korean circumstance. The leaves of three mutant cultivars (Jangdae, Jeokbong and Baekma), two original cultivars (Jinju, C14) and one Chinese cultivar (Auxu) were harvested at flowering time. Mineral components of kenaf leaves, such as calcium, potassium, and mineral, did not showed significant differences among the cultivars. As major amino acids including proline and phenylalanine, significant differences were found in these kenaf cultivars. The Auxu cultivar contained the highest amount of essential amino acid (Phenylalanine, Leucine, Isoleucine, Valine, Methionine and Lysine). The amount of vitamin displayed significant differences such as vitamin E and vitamin K among these cultivars. Especially, Jangdae cultivar contained the highest amount of vitamin E and vitamin K. Thus, these data suggested that Jangdae and Auxu is the most desirable cultivar containing high amount of vitamin and amino acid.

Foxglove aphid, Aulacorthum solani (Kaltenbach), is a Hemipteran insect that infected a wide variety of plants worldwide and caused serious yield losses in crops. The objective of this study was to identify the putative QTL for foxglove aphid resistance in wild soybean, PI 366121, (Glycine soja Sieb. and Zucc.). One hundred and forty one F2-derived F8 recombinant inbred lines developed from a cross of susceptible Williams 82 and resistant PI 366121, were used. The phenotyping of antibiosis and antixenosis was done through choice and no-choice assays with total plant damage (TPD) and primary infestation leaf damage (PLD); a genome-wide molecular linkage map was constructed with 504 single nucleotide polymorphism markers utilizing a GoldenGate assay. Using inclusive composite interval mapping analysis for foxglove aphid resistance, one major candidate QTL on chromosome 7 and 3 minor QTL regions on chromosome 3, 6 and 18 were identified. The major QTL on chromosome 7 showed both antixenosis and antibiosis resistance responses. However, the minor QTLs showed only antixenosis resistance response. The major QTL mapped to a different chromosome than the previously identified foxglove aphid resistance QTL, Raso1, from the cultivar Adams. Also, the responses to the Korea biotype foxglove aphid were different for Raso1, and the gene from PI 366121 against the Korea biotype foxglove aphid were different. Thus the foxglove aphid resistance gene from PI 366121 was determined to be an independent gene to Raso1 and designated to Raso2. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars.

Foxglove aphid, Aulacorthum solani (Kaltenbach), is a Hemipteran insect that infected a wide variety of plants worldwide and caused serious yield losses in crops. The foxglove aphid resistance gene, Raso2 was previously mapped from PI 366121 (Glycine soja Sieb. and Zucc.) to a 26cM marker interval on soybean chromosome 7. The development of additional genetic markers, which are mapped closer to Raso2 were required to accurately position the gene to improve the effectiveness of marker assisted selection. The objective of this study was to narrow down the putative QTL region, which is responsible to foxglove aphid resistance in PI366121 using recently developed high-density 180K Axiom SoyaSNP genotyping array. One hundred and forty one F8-derived F12 recombinant inbred lines developed from a cross of susceptible Williams 82 and resistant PI 366121, were used to generate a fine map of Raso2 interval. The phenotyping of antibiosis and antixenosis was done through choice and no-choice assays with total plant damage (TPD) and primary infestation leaf damage (PLD). The composite interval mapping analysis showed that the physical interval between two flanking makers, which was corresponding to Raso2, was narrowed down to 500kb on the Williams 82 genome assembly (Glyma2.0), instead of 4Mb in the previous report using Goldengate assay. In the Raso2 interval, there are about 60 candidate genes, including 4 of NBS-containing putative R genes. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars.

The depletion of stratospheric ozone has resulted in increased amount of ultraviolet-B radiation (UV-B: 280-320 nm) reaching the Earth’s surface and could cause significant biological effect in plants. In this study, putative quantitative trait loci (QTL), which is responsible to UV-B resistance in soybean, was identified using recently developed high-density 180K Axiom SoyaSNP genotyping array. A population of 115 recombinant inbred lines (RILs) derived from a cross between susceptible Keunolkong and resistant Iksan 10 was analyzed. A total 8,970 polymorphic SNP markers were used to construct linkage map. The both parents and RILs were grown with supplemental UV-B radiation in a greenhouse condition. Three categories of UV-B induced morphological damage, degree of leaf chlorosis, leaf shape change, and total plant damage were evaluated. Using composite interval mapping analysis, one major QTL associated with all of the phenotypic traits was detected on 7.7cM of soybean chromosome 7 with 22 of LOD score accounting for about 60% of phenotypic variance. Also, the allele from Iksan 10 were responsible for the UV-B resistance. Thus, the UV-B resistance QTL on chromosome 7 from Iksan 10 was designated to qUVBR1, corresponding to 30kb on the Williams 82 genome assembly (Glyma2.0) including 7 candidate genes. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars. In addition, these results provided useful information not only for marker-assisted selection for UV-B resistance soybean, but also for the future identification of putative candidate genes, responsible for UV-B resistance in soybean.

A simple in-situ biomethane system to upgrade biogas was developed by using differential solubility of biogas which normally contains 35-45% carbon dioxide (CO2) and 55-65% methane (CH4) by volume. The biomethane system consists of mesophilic plug-flow sorghum digester coupled with a leachate recycle loop to an external CO2 stripper. The leachate produced in the mesophilic plug-flow digester flows to the stripper where dissolved CO2 is removed. Then the leachate that CO2 was completely stripped out is recycled back to the plug-flow reactor, resulting in absorbing CO2 and enriched CH4 contents in digester offgas from the mesophilic plug-flow digester. Offgas CH4 contents was correlated well with leachate recycle rates and alkalinity. To maintain a biogas methane content over 95%, 3 volume of leachate recycle per volume of reactor per day(3 v/v-d) and at the reactor alkalinity of 4 g/L as CaCO3 was required. Even at an intermittent stripping ratio up to 3 hours stripping(N2 sweep gas 700 ml/min) and 1 hour no-stripping, the offgas methane content over 95% was achieved. It thus resulted in a 25% reduction in the total energy and sweep gas consumption. The TVS removal efficiency of the biomethane system was 80 percent which corresponded to 96% of the control reactor. The leachate recycle rates directly affected methane productivity that appeared to be 0.71 v/v-d at 3 volume of leachate recycle per volume of reactor per day(3v/v-d) and at the reactor alkalinity of 4 g/L as CaCO3.

Efforts were made to identify the optimum operational condition of Semi-continuously Fed and Mixed Reactor(SCFMR) to treat the dairy cow manure and saw dust mixture. Step-wise increase in organic loading rates (OLRs) and decrease in hydraulic retention times (HRTs) were utilized until the biogas volume became significantly decreased in SCFMR at mesophilic temperature (35℃). The optimum operating condition of the SCFMR fed with TS 15% dairy cow manure and saw dust mixture was found to be at HRTs of 30 days and its corresponding OLRs of 4.27 kgVS/m³-day. The optimum ranges of biogas and methane production rates were 1.47 volume of biogas per volume of reactor per day(v/v-d) and 1.14 v/v-d, respectively. This result was due to the high alkalinity concentration of SCFMR fed with the original substrate, dairy cow manure and saw dust mixture whose alkalinity was more than 10,000 mg/L as CaCO3. The parameters for the reactor stability, the ratios of volatile acids and alkalinity concentrations (V/A) and the ratio of propionic acid and acetic acid concentrations (P/A) appeared to be 0.07-0.09 and 0.38-0.43, respectively, that were greatly stable in operation. The Total Volatile Solids(TVS) removal efficiency based on the biogas production was 45.2% at the optimum HRTs. Free ammonia toxicity was not experienced at above 160 mg/L due to the acclimation of high concentration of ammonia by the high reactor TS content above 9.0%.