Molecular genetic markers have been widely used as powerful tools for analyzing the genome. In particular, SSR markers have practical applications in breeding systems because they can be used in high-throughput analyses for genetic mapping, heritable diversity testing, purity analysis, and marker-assisted selection. Currently, due to technical advances in DNA sequencing, large sequence databases are available for large-scale SSR mining and marker development. Here, we describe an automated method, the SSR Finder program, for SSR discovery in the onion sequence database, and primer design for amplifying the detected SSRs. A total of 1,049 SSR primers were obtained for genetic purity testing, and 100 SSR sets were analyzed in 14 bulb onion breeding lines using clustering analysis. A total of 20 selected markers from screening of all 1,049 SSR primers, were finally applied for genetic purity testing in three breeding lines, NW1, NW9, and NW10. The initial tests showed that 15%, 71%, and 97% of individuals within NW1, NW9, and NW10, respectively, were genetically homogeneous. These markers produced using the SSR Finder will be useful for investigating the genetic purity of onion breeding lines.

탈모방지 및 발모효과를 가지는 소재를 개발하기 위해 한의학에서 전통적으로 사용되는 23가지 한방소재를 선정하여 한방복합처방단을 개발하였다. 한방복합처방단을 구성하는 한방소재들은 예로부터 전통적으로 발모 및 탈모방지, 흰머리방지, 염증 치료 및 혈액순환개선 효과를 가진 것으로 알려져 있는 당귀, 보골지, 측백엽, 한련초, 구기자, 복분자, 상백피, 숙지황, 여정실, 적하수오, 흑지마, 고삼, 백지, 익모초, 단삼, 도인, 몰약, 감국, 유향, 인삼, 천궁,합환피, 현호색 등이다. 또한, 한방복합처방단의 발모효과를 확인하기 위해 in vitro와 in vivo 평가모델을 이용하여 모발성장 및 촉진에 미치는 영향을 실험하였다. In vitro 상에서는 모유두세포, 각질형성세포 및 섬유아세포의 증식을 확인하였다. 또한, 흰머리방지 효과와 관련하여 멜라노마 세포에서의 멜라닌 합성능력을 확인하였다. 한방복합처방단의 in vitro 상에서의 육모효과는 C57BL/6 마우스를 이용한 in vivo 상에서도 확인하였다. 연구 결과 한방복합처방단은 50 μg/mL의 농도에서 모유두 세포의 증식을 175 %까지, 섬유아세포인 NIH3T3 세포의 증식은 120 %까지 증가시켰으며, 20 μg/mL의 농도에서 각질형성세포인 HaCaT 세포의 증식을 133 %까지 증가시켰다. 멜라닌 합성의 경우, 50 μg/mL의 농도에서 154 %까지 증가시켰다. 또한, C57BL/6 마우스를 이용한 육모효과에 있어서는 한방복합처방단 처리 4주 후 98 % 이상의 육모효과를 나타내는 것을 확인하였다. 이상의 결과로 볼 때 본 연구에서 개발한 한방복합처방단은 모발의 성장 촉진에 유용하게 활용될 수 있는 처방으로 사료된다.

Translationally controlled tumor protein (TCTP) is one of important immune regulator. TCTP has been implicated in cellular processes including the cell growth, cell cycle progression, apoptosis regulation and the protection of cells against various stress condition. In this study, we cloned and characterized TCTP from rock bream (Oplegnathus fasciatus), which is an economically important species in the Korea aquaculture industry. The Full-length of rock bream TCTP (RbTCTP) cDNA was 1041 bp and contained an open reading frame (ORF) of 513 bp, which encoded 170 amino acid sequence. The 5' untranslated region (UTR) was 90 bp while the 3' UTR was 538 bp, containing a polyadenylation signal (ATTAAA). The identity of amino acid sequence was 76%, 75% and 74% in tilapia, orange-spotted grouper and Japanese seaperch, respectively. The positions of microtuble-binding region, Ca⁺ binding region and TCTP signature regions in RbTCTP were similar with those of other fish species and mammalian. The RbTCTP mRNA was expressed highest in the muscle. Expression of TCTP mRNA were significantly variable according to injection of red seabream iridovirus (RSIV), Streptococcosis (S. iniae) and Edwardsiella tarda (E. tarda).

Human embryonic stem cells (hESCs) are promising cell source because of their unique self-renewal and pluripotency. Although hESC-derived cardiac cells are currently generated worldwide, cryopreservation of these cells is still limited due to low rate of post-thaw survival. Cryopreservation of hESC-derived cardiac cells is critical in that their long-term storage can accelerate their use in regenerative medicine. However, to date, there are few reports on efficient cryopreservation and post-thaw survival of hESC-derived cardiac cells. In this study, we evaluated the effects of ginsenoside, which is known to improve survival of rat embryonic cardiomyocytes against myocardial ischemia injury in diabetic rats (Wu et al., 2011), on the survival of hESC-derived cardiac cells after thawing. We induced differentiation into cardiac cells using our previously reported method (Kim et al., 2011). Differentiated, pre-beating stage cardiac cells were cryopreserved using either mass cryopreservation or vitrification. To evaluate the effects of ginsenoside (Re, Rb), we compared three sets: pre- and post-thaw treatment, pre- or post-thaw treatment only. The survival of post-thaw cardiac cells were evaluated using Trypan-blue and Annexin V staining. In addition, the three groups were treated with ROCK inhibitor Y-27632, and compared with non-treatment groups. The effect of ginsenoside was significant in post-thaw treatment group, i.e, thawed cells expressed cardiac specific genes and showed specific functionality such as spontaneous beating. Taken together, we demonstrated favorable effects of ginsenoside on the survival of hESC-derived cardiac cells after cryopreservation and thawing. These results suggest a possible application of well-known cardioprotectant ginsenoside in cell-based tissue engineering using hESC-derived cardiac cells.

Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

The purposes of this research project are to identify quantitative trait loci (QTLs) associated with yield-related traits using a recombinant inbred line (RIL) population derived from a cross between a high-yield soybean genotype SS0404-T5-76 and Daewonkong and to develop high-yield soybean and lodging-resistant sprout soybean cultivars. For development of DNA markers and identification of functional sequence variations, firstly, whole genome of five soybean genotypes, Sinpaldalkong 2, SS2-2, Pungsanamulkong, SS0404-T5-76 and Daewongkong, were sequenced using Illumina Hi-Seq technology. SS2-2 is a EMS-induced mutant of Sinpadalkong 2. SS0404-T5-76 showing high-yield is a F8 RIL derived from a cross of Pungsanamulkong x SS2-2. Daewonkong is a elite cultivar with high-protein. Furthermore, to construct a genetic linkage map, we are advancing F4 lines of SS0404-T5-76 x Daewonkong by single seed-descent. Secondly, we developed high-protein and high-yield soybean lines and lodging-resistant sprout lines. Area-adaptability tests of these promising lines are performing in three different locations including Jeju, Naju, and Suwon. Based on the results of area adaptability tests, we are planing to conduct cultivar registration of the promising soybean lines.

R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain. Some soybean NBS-LRR genes have also been reported to function in disease resistance. A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS-LRR genes have disease resistance functions in the soybean genome. The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.

The rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early responsible genes in rice in response to M. oryzae, we analyzed transcriptomics analysis using 300 K tilling microarray chip. The quality of RNA samples was initially validated by 4 defense related genes and phytoalexins measurement using RT-PCR and HPLC, respectively, which are well known defense markers. We determined that accumulation of 608 genes showed statistically significant changes in the level of transcription (>2 fold change, P<0.05). Among them, 261 genes were more up-regulated in incompatible interaction than that of compatible one. We further analyzed GO enrichment analysis of the 41 and 231 which were 2 fold up-regulated genes at 12h and 48h in incompatible interaction, respectively, using Rice Oligo nucleotide Array Database (http://ricearray.org). Furthermore, MapMan analysis (http://mapman.gabipd.org/) revealed that 21 and 85 genes including 18 receptor-like genes which were more induced in incompatible interaction compared to compatible interaction were found to be involved in biotic stress. Thus, this study suggests that early inducible genes including receptor-like protein kinases in incompatible interaction may play a key role in disease resistance against M. oryzae attacks.

Next generation sequencing (NGS) approaches can also be useful tool for characterization of organelle genomes. We generated chloroplast (CP) genome sequences of two Korean ginseng cultivars, Chunpoong and Yunpoong, based on reference-guided assembly using whole genome NGS data. We used 0.5x of P. ginseng genome NGS reads to assemble CP genome. Of the NGS reads used, about 6% were mapped to the reference CP genome with mean coverage of 94x due to high copy number of CP genome in plant cell. CP genomes of the two cultivars were predicted to be 156,248 bp and 156,355 bp in length and showed about 0.1% differences at nucleotide level, compared to reference CP genome sequenced from P. ginseng (Acc.no. NC_006290), whereas difference between CP genomes of the two cultivars is very rare. In this study, we developed the molecular marker to perform taxon identification and also to elucidate phylogenetic relationship among Korean ginseng cultivars. Now, we are analyzing the CP genomes of other P. ginseng cultivars together with other Panax species including American ginseng and Panax related species.

Phomopsis seed decay (PSD), primarily caused by Phomopsis longicolla, is a major contributor to poor soybean seed quality and significant yield loss, particularly in early maturing soybean genotypes. However, it is not yet known whether PSD resistance is associated with early maturity. This study was conducted to identify quantitative trait loci (QTLs) for resistance to PSD and maturity time using a recombinant inbred line (RIL) population derived from a cross between the PSD-resistant Taekwangkong and the PSD-susceptible SS2-2. Based on a genetic linkage map incorporating 117 simple sequence repeat markers, QTL analysis revealed two and three QTLs conferring PSD resistance and maturity time, respectively, in the RIL population. Two QTLs (PSD-6-1 and PSD-10-2) for PSD resistance were identified in the intervals of Satt100-Satt460 and Sat_038-Satt243 on chromosomes (Chrs) 6 and 10, respectively. These QTLs do not overlap with any previously reported loci for PSD resistance in other soybean genotypes. Two QTLs explained phenotypic variances in PSD resistance of 46.3% and 14.1%, respectively. Among three QTLs for maturity time, two (Mat-6-2 and Mat-10-3) were located at positions similar to the PSD resistance QTLs. The identification of the QTLs linked to both PSD resistance and maturity time indicates a biological correlation between these two traits. The newly identified QTLs for resistance to PSD associated with maturity time in Taekwangkong will help improve soybean resistance to P. longicolla.

Recent release of whole genome draft sequences in legume species have led comparative genome studies among legume plants including Glycine max, G. soja, Cajanus cajan and Medicago truncatula. The majority of comparative genomic researches have been conducted based on synteny of coding sequences and coding sequence variations may be one of major forces for speciation and evolution. However, non-coding sequences have been also reported to be important phenotypic regulators. Especially, since short sequence motifs in the promoter regions are highly conserved, they are suggested to be another resources of interests in comparative studies. In this study, we predicted the conserved short sequence motifs by BLASTN algorithm using dicot promoter database from Softberry (http://www.softberry.com). A total of 37,396 conserved short sequence motifs were identified onto 2 kb upstreams of 46,367 high confident gene model of G. max (cv. Williams 82). Meanwhile, whole genome of 7 soybean landraces (G. max) and 7 wild soybean genotypes (G. soja) were sequenced at low depth of less than ten using Illumina Hiseq 2000. Among these genotypes, nucleotide variations were identified in predicted conserved regulatory motifs by mapping of short reads to the reference genome sequence using the Samtools program (http://samtools.sourceforge.net/). Fifteen and two genes, which have SNPs in regulatory motifs and no SNP in coding sequence, were identified by comparisons of inter-species and intra-species, respectively. qRT-PCR experiments are in progress for investigating differences of these 17 genes expressions at transcriptional level.

As soybean (Glycine max) is known for its high nutritional value of oil and protein, soybean has been domesticated and cultivated by one specific character trait based on human selection. Importantly, tracing back in time where G. max and G. soja, the undomesticated ancestor of G. max have diverged plays an important role in studying of genetic diversity and in investigating the common ancestor of soybean. In this study, we sequenced 6 G. max and 6 G. soja using Illumina’s Hiseq 2000 with a low coverage sequencing technology to estimate the divergence of times between genotypes and populations. A total of the 12 genotypes were sequenced at the average depth of 6.5 and resulted 892.5 Mb and 903.3 MB consensus sequences with the coverage of 91.54% and 92.65% for G. max and G. soja, respectively. The whole genome SNP analysis showed that G. max had lower frequency levels of polymorphism (~0.1%) than G. soja (~0.25%). And, a high number of SNPs located in introns were found among 6 G. soja genotypes as SNPs were approximately twice than those found in 6 G max. The number of SNPs in G. max intronic regions was 53,134, whereas a total of 133,329 SNPs were discovered in G. soja introns. Almost an equal number of SNPs were discovered in 5’ UTR and exon regions; however, different numbers of SNP in CDS and 3′ UTR were identified. By the rate of nonsynonymous change, divergence of time between G. soja and G. max would be investigated.

Korean soybean variety Kwangan was transformed with coat protein (CP), helper component-proteinase (HC-Pro), and ABRE binding factor 3 (ABF3) genes using highly efficient soybean transformation system. Among these genes, CP and HC-Pro were transformed using RNAi technology. Transgenic plants with CP were confirmed for gene introduction and their expression using PCR, real-time PCR, RT-PCR, Southern blot, and Northern blot. To investigate the response of viral infection with CP, T1 plants were inoculated with SMV-infected leaves and confirmed the existence of mosaic symptom in both leaves and seeds. Two transgenic lines with CP were highly resistant to SMV with clear leaves and seeds while SMV-susceptible lines showed mosaic symptom with seed mottling. The transcript levels of T1 plants with CP were also determined by northern blot, suggesting that SMV-resistant T1 plants did not show viral RNA expression whereas SMV-susceptible T1 plants showed viral RNA expression. Currently, the response of viral infection with HC-Pro is investigating to produce SMV-resistant soybean transgenic plants, and the physiological experiment with ABF3 is also carrying out to produce drought-tolerant soybean transgenic plants.

The amount of genetic variability of a species is essential for its survival and adaptation in different environments, and studies of genetic diversity using molecular markers are necessary to understand the genetic structure of a population and to orientate effective strategies of germplasm conservation. The aim of current study was to determine the SSR markers that can be used rapidly and reliably to evaluated the pepper of Bulgaria landraces, and applied the markers to assement of introduce genetic diversity of the pepper germplasm. We used 22 polymorphic microsatellite markers to analysis of genetic diversity within 61 pepper collection of Bulgaria landraces germplasm, all SSR primers pairs produced 80 polymorphic and reproducible amplification fragments. An average value of polymorphic information contents (PIC) were 0.334 with a range of 0.061 to 0.63. The mean values of observed (HO) and expected heterozygosity (HE) were 0.383 and 0.154, respectively, indicating a considerable amount of polymorphism within this collection. A genetic distance-based phylogeny grouped into three distinct groups, which was the landrace, moderate and wilde type, genetic distance (GD) value was 0.540. An average day of flowering time was 53 days with a range of 45 to 60 days. The everage od fruit length and width were 9.38cm with a range 2.1 to 23.6cm, and 3.51cm with a range 0.6 to 8.9cm, respectively. Molecular data were complemented with morphological measurements according to the descriptor list for the pepper collection of Bulgaria landraces germplasm.

Cereal seeds, sorghum, foxtail millet, hog millet, adlay, and corn are traditionally used as health assistant as well as energy supplying food in Korea. While beneficial phytochemicals to human have revealed in cereals, the information on peptides from cereals is far less accumulated than major reserve protein. Here, we analyzed peptide profiles using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) in cereal seeds for construction of peptide information and attempted to develop peptide biomarkers for cereal identification. To optimize the analysis condition of SELDI-TOF MS, the effect of dilution factor on binding affinity to protein chips was tested using CM10 and Q10 arrays. Peptide clusters were significantly different at the level of 0.01 p-value. Peak spectra were the most stable in 1:50 of dilution factor in both chip arrays. Numbers of detected peak of 5 cereal seeds were 131 in CM10 and 74 in Q10 array. Each cereal was grouped as a cluster and well discriminated into different cluster in the level of 0.01 p-value. Numbers of potentially identified peptide biomarkers are 11, 13, 9, 5 and 12 in sorghum, foxtail millet, hog millet, adlay and corn, respectively. This study demonstrates that each cereal seed have own distinguishable specific peptides although their function are not identified yet in this study. In addition, the proteomic profiling using SELDI-TOF MS techniques could be a useful and powerful tool to discover peptide biomarker for discrimination and assess crop species, especially under 20 kDa.

Sorghum is the fifth most important cereal in the world as one of the staple food. For the use of natural dye, we have done some researches about sorghum red pigments extracted from stalk and leaves on its physiochemical properties, extracting methods and applications. The researches involved maximum extraction of sorghum pigment and analysis of its processing condition. Total polyphenol and tannin contents were measured by varieties and different plant parts. The stabilities of pigment by irradiation and heat treatment for processing were measured by colorimeter and thermal gravimetric analysis (TGA). In addition, hybrid nano-silica composites with sorghum pigment were made by combining with polyvinyl alcohol, polyvinyl acetate and sodium silicate. Water silica hybrids with sorghum pigment were performed by emulsion treatment. Nano-silica particles were identified and measured their size to be about 200 ~ 400 nm by SEM analysis.

Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important vegetables and widely cultivated in Asia countries including Korea and China. Recently, whole genome sequence and full-length cDNA information of this species became available, which are encouraging genetic studies of this species to characterize agricultural important traits. Orange-colored (Or) cultivar of Chinese cabbage has inner leaves in orange, whereas other cultivars generally cultivated have yellow (Ye)- or white-colored inner leaves. In this study, we investigated phenotypes and carotenoid biosynthesis genes related to color variation in the Or cultivar. Firstly we compared the carotenoid content and composition between the Or and Ye cultivars by HPLC analysis. The inner leaves of Or cultivar contained approximately 9-fold high β-carotene content, whereas content of both lutein and violaxanthin was decreased to less than 30%, compared to Ye cultivar. Or cultivar was segregated with ratio of 3:1 in F2 population derived from crossing between Or and Ye inbred lines, indicating that Or phenotype is controlled by single recessive gene. To identify this gene, we investigated the expression of several genes involved in carotenoid biosynthesis by RT-PCR analysis. Among genes tested, two encoding putative carotenoid isomerase (CRTISO) and phytoene desaturase (PDS) were identified to show different expression between Or and Ye cultivars. Through further analysis of genomic DNA regions of these two genes, we could expect that several mutations such as InDel and base-substitution occurred and then affected expression of these genes in Or cultivar. In this presentation, I will introduce more detailed results for Or cultivars.

A new cultivars Dendranthema grandiflourm ‘Dream River’ was developed at Gyeonggi-do Agricultural Research ＆ Extension Services(GARES),Korea in 2010. The cultivar ‘Dream River’ was crossed in 2007 between ‘Geumsu’, a spray cultivar with yellow single type, and ‘Crangball’, a spray cultivar with pink single type. Trials were conducted from 2008 to 2011 for evaluations and selection of this variety, including a shading culture in summer and a retarding culture in autumn. The natural flowering time of ‘Dream River’ was late October, and year-round flowering is possible by shading or lighting treatment. After the test of specific characters from 2009 to 2011, it was finally selected and named. The cultivar has an single type flowers with ivory petals and a green flower center. The diameter of flower is 56.0mm. Numbers of flowrs per stem and petals per flower are 15.2 and 26.5, respectively. Days to flowering under the short day treatment is about 53 days in the four season. The cultivar ‘Dream River’ has ivory petal with 7.5weeks to flowering under the short day treatment.

A new rose variety, ‘Venus Berry’ was selected from the progenies of a cross between ‘Boy Friend’ and ‘GSR10315’ by rose breeding team of the Gyeonggi-Do Agricultural Research and Extension Services(GARES) in 2011. ‘Venus Berry’ was crossed in 2007 and seedlings were produced. After the test of specific characters from 2008 to 2011, it was finally selected and named. ‘Venus Berry’ was developed because of distinctive characters such as growth uniformity and high yielding potential. The petal of flower is so thick and has no scratch. A standard type with large sized flower, it has light pink(Red Purple Group 69C) color flower. ‘Venus Berry’ takes 45 days from pruning to blooming and cut flower productivity was 194.1 stems/m2 in a year. The length of cut flower was long with 65.5 cm. It has 10.2 cm in flower diameter and 43.6 in petal numbers per flower. Vase life of the this cultivar could be as long as 12 days.

A new rose variety, ‘Love Letter’ was selected from the progenies of a cross between ‘Red Giant’ and ‘Ensemble’ by rose breeding team of the Gyeonggi-Do Agricultural Research and Extension Services(GARES) in 2011. ‘Love Letter’ was crossed in 2007 and seedlings were produced. After the test of specific characters from 2008 to 2011, it was finally selected and named. ‘Love Letter’ was developed because of distinctive characters such as growth uniformity and high yielding potential. A standard type with large sized flower, It has red(Red Group 46A) color flower. ‘Love Letter’ takes 43 days from pruning to blooming and cut flower productivity was 152 stems/m2 in a year. The stems of cut flower have no thorn and the length was long with 70.5 cm. It has 9.3 cm in flower diameter and 32.4 in petal numbers per flower. Vase life of the this cultivar could be as long as 12 days.