Background : Angelica gigas is a monocarpic biennial or short lived perennial plant. A. gigas, also called Dang Gui or Korean Angelica, is a major medicinal herb used in Asian countries such as Korea, Japan and China. In Korea, we are using the roots of A. gigas, but, they are using Angelica sinensis in China and Angelica acutiloba in Japan to obtain many active constituents. The biggest problem in the using of A. gigas would be the confusion with A. acutiloba or A. sinensis. These three plants can't be distinguished by appearance. And the constituent ratios of the three plants are different. This confusion can cause an accident or the pharmaceutical effects do not meet the expectations. In this study, we developed chloroplast SSR markers that can distinguish A. gigas, A. acutiloba and A. sinensis. Methods and Results : We collected A. gigas, A. acutiloba and A. sinensis. and extrated DNA using CTAB method. The DNA was diluted to 10 ng/㎕ and kept -20℃. We designed the primer sets using CLC Main Workbench based on chloroplast DNA SSR region of A. gigas. PCR were performed on the three angelica plant samples (in 5 repeat). Conclusion : We made five primer sets from five SSR regions of A. gigas cpDNA. All of the primer sets amplified the amplicon effectively. Two of the 5 primer sets had polymorphism. We can distinguish A. gigas, A. acutiloba, and A. sinensis using the 2 primer sets

본 연구는 국내에서 유통되고 있는 국화 147품종을 수집하 여 국화 품종의 식별을 위한 SSR 분자표지의 선발 및 이를 이용한 품종별 DNA profile 데이터베이스를 구축하기 위해 수행하였다. 품종식별에 적합한 분자표지를 선정하기 위해 20 개 품종을 대상으로 총 587개의 SSR 분자표지를 검정하여 다형성을 나타내는 27개의 분자표지를 선발하였다. 27개의 분자표지 중 다형성, 재현성, 반복성, 대립유전자 패턴 등을 종합적으로 고려하여 14개의 SSR 분자표지를 데이터베이스 구축에 활용할 마커로 최종 선발하였고 이를 이용하여 국화 147품종에 대한 SSR 분자표지 데이터베이스를 구축하였다. 국화 147 품종을 14개의 SSR 분자표지로 분석한 결과 대립 유전자 수는 79개, 마커별 대립유전자의 분포는 3 ~ 10개로 분포하였고, 분자표지 당 평균 대립 유전자의 수는 5.6개로 나타났다. PIC 값은 0.287 ~ 0.785 범위에 분포하였으며, 평 균값은 0.598로 분석되었다. 국화 147품종에 대한 덴드로그 램을 작성하였을 때 공시품종의 유전적 거리는 0.44 ~ 1.00 범위로 나타났으며, 147품종 중 143품종은 14개 SSR 분자표 지에 의해 식별이 되었으나, 돌연변이 육종법 또는 자연변이 를 통해 육성된 2품종은 원품종과 구분이 되지 않았다. 본 연구에 의해 구축된 국화 품종별 SSR 분자표지 데이터베이스 는 국화 출원품종의 대조품종 선정과 품종보호권 침해 및 종 자분쟁 발생시 유용하게 활용될 수 있을 것으로 사료된다.

Balloon flower (Platycodon grandiflorum A. DC.) is a perennial plant of mainly Campanulaceae family, which have been widely used as a food ingredient and herbal medicine in East Asia. Although demands on related products and yearly cultivation area for balloon flower are increasing, diverse fundamental technologies and molecular breeding studies are not very well supported in Platycodons. In this study, 30 random amplification of polymorphic DNA (RAPD) primers were test in an attempt to explore genetic diversities. In addition, sequences information of the actin gene, a well conserved gene encoding a globular protein that forms microfilaments, was retrieved and analyzed. Two actin homologs were recovered; 3.4 kb fragment is a Pg-actin and 1.4 kb fragment is a Pg-actin homolog with 28.6% similarity. We have confirmed that the Pg-actin gene is configured into 4 exons and 3 introns. A single nucleotide polymorphism (SNP), G↔A, was detected on the intron 3, which served as a target for the CAPS marker development. The marker Pg-Actin-Int3 was applied to 32 balloon flower accessions. Balloon flower DNA sequence information generated in this study is expected to contribute to the analysis and molecular breeding and genetic diversity analysis of balloon flowers.

Molecular markers, such as PCR-based and SNP-based markers, are extremely useful for plant genetics and crop breeding. Marker-assisted selection (MAS) has been widely applied in plant breeding to improve crop yield, quality, and tolerance to biotic and abiotic stresses. To develop gene-based (or -specific) molecular markers, three different approaches have been used in Brassica species: Known-gene-based, RNA seq/Exon-based and RNA seq/Intron-based molecular marker development for several years. Using these techniques, molecular markers have been developed to identify flowering time, anthocyanin accumuation and abiotic stresses in B. rapa and B. oleracea. Markers were distributed in exons as well as introns, and coding sequences and untranslated regions (UTRs). All markers developed have been transformed into SNP marker after HRM confirmation. I will discuss efficiency, accuracy, and potential problems and contribution of these markers for Brassica breeding.

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.

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.

Platycodon grandiflorum is a perennial herbal plant belongs to Campanulaceae family. It has very important genetic value as a major plant in Asterids order. The major ingredients are platycosides, terpenoid saponins. In Korean industrial plants market, it was produced 5,633 tons in 2013, and the total amount of production was less than only five species, omija, ginger, raspberry, yam and deodeok. P. grandiflorum is called ‘Gilgyung’ and is used as a fresh vegetable and an ornamental plant. Nowadays, various components of P. grandiflorum were already published. But, genetic research is in the starting stage. In this study, 11 cultivars; 1. MariesⅡ, 2. Hakone double white, 3. Hakone double blue, 4. Fuji white, 5. Fuji pink, 6. Fuji blue, 7. Astra white, 8. Astra pink, 9. Astra blue, 10. Astrasemi double blue, 11. Jangback, were analyzed using 60 Operon Universal RAPD primers. The results were phylogenetically analyzed and related to the morphological characteristics of the cultivars.

The selectable marker-free rice plants containing mcry1Ac insecticidal gene isolated from Bacillus thuringiensis (Bt) were generated using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. The nutritional composition of two lines of transgenic rice plants (RTB5 and RTB11) was compared with that of its non-transgenic counterpart. The results showed that, except for small differences in dietary fiber and some minerals, there was no significant difference between transgenic rice and conventional counterpart variety with respect to their nutrient composition. Most of measured levels of nutrients were within the range of values reported for other commercial cultivars, showing substantial equivalency. Therefore, the insertion of transgenes did not affect the nutritional composition of transgenic RTB5 and RTB11 rice grains.

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of zygosity in resveratrol-enriched transgenic rice plant. This DNA marker will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be use to estimate transgene movement occurred by pollen transfer or seed distribution.

Leaf mold disease in tomato (Solanum lycopersicum) is caused by Cladosporium fulvum, a fungal leaf pathogen. One of effective ways to control leaf mold is to breed disease-resistant tomato cultivars. Cf-4 and Cf-9 resistance (R) genes encode proteins that carry a leucine rich repeat domain and are located in plasma membrane. They trigger hypersensitive response following recognition of corresponding Avr4 and Avr9 proteins of C. fulvum, respectively. Cf-4 and Cf-9 genes are originated from wild tomato species S. habrochaites and S. pimpinellifolium and have been introgressed into commercial tomato cultivars. These two highly homologous orthologs exist as a cluster with four highly homologous paralogs. Due to this reason, development of genetic markers to distinguish these two functional R genes from their orthologs and paralogs is difficult. In this study, we tried to develop single-nucleotide polymorphism (SNP) markers to select tomato cultivars carrying resistant Cf-9 genotype. The genomic sequences of resistant Cf-4 and Cf-9 alleles, susceptible cf-9 alleles, and their paralogs were obtained from the GenBank database, and two functional SNPs causing non-synonymous substitution were found among them. Based on two SNPs, the Cf-9_2-SNP-F/R primer set for high resolution melting (HRM) analysis was developed. HRM analysis with this primer set could successfully distinguish tomato cultivars carrying resistant Cf-9 allele among 30 commercial tomato cultivars, which were characterized with the gene-based marker. These indicate that the SNP marker developed in this study is useful to trace Cf-9 genotype efficiently in marker-assisted selection in tomato.

Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threaten tomato (Solanum lycopersicum) worldwide. So far, eight TSWV resistance genes, Sw1a, Sw1b, sw2, sw3, sw4, Sw-5b, Sw-6, and Sw-7 have been identified and Sw-5b has been incorporated into tomato for prevention of TSWV. The objectives of this research are first to discover single nucleotide polymorphisms (SNPs) in Sw-5 alleles and then to develop SNP markers to distinguish resistant genotypes against TSWV for marker-assisted breeding in tomato. First, DNA sequences of Sw-5b alleles from both resistant and susceptible cultivars amplified using known Sw-5 gene-based marker was analyzed. The single functional SNP (G→A) was detected as non-synonymous substitution because this SNP causes change of arginine (Arg599) to glutamine (Gln599). Next, the primer pair for high resolution melting analysis (HRM) was designed around this SNP. To determine accuracy of this SNP marker to distinguish resistant Sw-5b genotypes against TSWV, genotypes of 32 commercial tomato cultivars were checked. The newly developed SNP marker could select six cultivars carrying resistant Sw-5b genotype, which was 100% correlated with genotypes based on the gene-based marker. These results indicate that the SNP maker developed in this study could be useful for better tracking resistance to TSWV in tomato breeding.

High temperature is one of major environmental stress. Some of molecular markers related heat stress or tolerance have been reported by many researchers. Heat tolerance managing is difficult through the phenotypic selection, so marker assistant selection (MAS) using molecular markers like as RAPD, SSR ect. was tried to selection of useful traits for heat tolerance. Fourteen SSR markers reported by previous research were selected for this research. These markers were linked to important traits including grain filling duration, HIS (Heat susceptibility index) grain filling duration. In this study, we tried to evaluate 14 SSR markers for MAS using 31 useful wheat resources including 24 crossing line from Turkey and six Korean wheat cultivars using 14 SSR markers. The average of the number of alleles and PIC values in this study were 6.14 and 0.63, respectively. Two major clades and six sub clades were grouped by phylogenetic tree using UPGMA program. Six Korean wheat cultivars were distinct from other Turkey resources in the phylogenetic dendrogram. From the results, we expected that these markers were able to adapt to screening wheat genotyping for heat tolerance.

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, whereas that of wheat is suitable for many food products including breads. We have produced marker-free transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using the Agrobacteriummediated co-transformation method. The TaGlu-Bx7-own promoter was inserted into a binary vector for seed-specific expression of the TaGlu-Ax1 gene. Two expression cassettes comprised of separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to the Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring TaGlu-Ax1 or HPTII was infected to rice calli at a 3:1 ratio of TaGlu-Ax1 and HPTII, respectively. Then, among 210 hygromycin-resistant T0 plants, we obtained 20 transgenic lines with both TaGlu-Ax1 and HPTII genes inserted into the rice genome. We reconfirmed integration of the TaGlu-Ax1 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in the rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened at the T1 generation.

The goal of marker-assisted backcrossing is to reduce the number of generations significantly by using genome-based molecular markers. Among other types of molecular markers, SNP (single nucleotide polymorphism) is mostly used in genetic diversity analysis due to its abundance. To develop high-throughput SNP marker for MAB system, we selected 20 Chinese cabbage lines each represent traits as inner leaf color, disease resistance, head type and maturity etc. Then, we sequenced the transcriptomes of 20 lines by using Illumina Hiseq2000. The average transcriptome size was 1.37 Gbase, and the average of short reads mapping rate was about 62.15% (30xcoverage). We identified 13,976 SSR markers and 380,198 SNPs by aligning contigs of 20 Chinese cabbage lines. To develop SNP marker set, we chose 409 SNPs that covers the whole Brassica rapa transcriptome. The filtering criteria were depth, polymorphism, segregation ratio, lack of adjacent SNP and copy number. We positioned the selected SNP markers to the Chinese cabbage linkage map. Clustering dendrogram was produced using SNP marker and three different clusters were generated. The result showed that the genotyping data is partially linked to the phenotyping data. We assume that the developed SNP marker set can be applied in the Chinese cabbage MAB system soon.

Asterales are dicotyledonous flowering plants and are one of the Asterid clade, incuding many species as well as Codonopsis and Platycodon. Here, we have determined the complete chloroplast genome sequences of C. lanceolata and P. grandiflorus by using the targeted denovo assembly method of short reads derived from whole genome resequencing. The total lengths of each chloroplast genome sequence are 156,180 bp for C. lanceolata and 155,453 bp for P. grandiflorus. In their chloroplast genomes, 106 genes (75 protein-coding genes, 4 rRNA genes, 23 tRNA genes, and 4 hypothetical chloroplast open reading frames [ycfs]) exhibited the relatively similar positions. Also, 7 protein-coding genes commonly showed to contain introns in both C. lanceolata and P. grandiflorus chloroplast genome, while psaA gene contain intragenic regions only in P. grandiflorus chloroplast genome. In further analysis, we identified the codon usage bias to A or T and found the different simple sequence repeat (SSR) loci of each chloroplast genome (18 SSR loci of C. lanceolata and 16 SSR loci of P. grandiflorus). In the phylogenetic trees based on 72 protein-coding genes, C. lanceolata is more closely related to P. grandiflorus than the other plant species order Asterales. Also, we found the highest sequence diversities of 12 protein-coding genes in small single copy (SSC) region than in the inverted repeat (IRs) and large single copy (LSC) region, and 3 genes such as rpoC2 (LSC region), ndhB (IRs region), and ndhF (SSC region) showed the highest number of segregating sites in each region. Additionally, we developed the molecular markers for phylogenetic applications of C. lanceolata and P. grandiflorus chloroplast genome.

For efficient introgression of the downy mildew resistance gene from a resistant cultivar into domestic breeding lines, molecular markers used for marker-assisted backcrossing (MAB) were developed in onion (Allium cepa L.). The resistance gene (Pd) was originally introgressed from a wild species, A. roylei, by interspecific hybridization, and the resistant gene was known to be positioned at the end of chromosome 3. Therefore, cDNA sequences of loci located at the ends of chromosome 3 of two linkage maps were obtained from a transcriptome database. Primer pairs were designed on exon sequences of eight loci. Among them, the PCR products of the i25255 locus showed length polymorphism between A. roylei and onions, and both large and small-sized PCR products were observed in the resistant cultivar. Sequence analysis showed that a 67-bp indel existed in the intron sequences. Based on this indel polymorphism, a simple PCR marker, designated DMR1, was developed. Analysis of diverse onion accessions showed that no accessions contained the A. roylei-specific marker genotype except for the resistant cultivar. These results indicated that the DMR1 marker was successfully tagging the A. roylei fragment harboring the downy mildew resistance gene, and the resistant cultivar was heterozygous for the resistance gene. After further analysis of multiple loci positioned at chromosome 3, a range of the A. roylei fragment introgressed in the resistant cultivar was determined in two linkage maps. On the basis of the range of the A. roylei fragment, three molecular markers used for recombinant selection in MAB were also developed.

Cucumber is a typical monoecious plant with individual male and female flowers, and sex expression in cucumber is mainly determined by three major genes: F/f, M/m and A/a. Gynoecy plays an important role in cucumber hybrid breeding and use of gynoecious lines as maternal parent ensures high productivity. The purpose of this study is to identify a co-dominant molecular marker linked to F locus to distinguish homozygous and heterozygous gynoecious plants for cucumber breeding programme. Firstly, we analyzed the sequence polymorphism of 5 gynoecious and 5 monoecious inbred lines to detect polymorphism to develop the marker linked to F locus. A pair of specific primer based on insertion/deletion polymorphism on branched-chain amino acid transaminase (BCAT) gene was designed and examined the polymorphism in the parents, F1 and F2 segregating population derived from gynoecious (WJEF11) and monoecious (WNEF8) inbred lines. The result showed that the specific fragment amplified with Cs-Female-F/Cs-Female-R, was identified as a co-dominant marker and co-segregated with sex phenotype in F2 population. Furthermore, we present a new linkage map for F locus using Indel markers. This is the first report for the development of F locus specific co-dominant marker which can distinguish homozygous and heterozygous gynoecious and it could be used in marker-assisted selection in cucumber breeding.

Magnoliae Flos (Sini in Korean) is one of the most important oriental medicinal plants. In the Korean Herbal Pharmacopeia, the bud of the all species in Manolia denudate and Manolia genus were regarded as the botanical sources for ‘Sini’. Most the dried bud of Manolia denudata, Manolia biondii and Manolia sprengeri were used as ‘Xin-yi’ in China. Therefore, the purpose of this study was to determine and compare the ‘Magnolia’ species, four species including Manolia denudata, M. biondii, M. liliiflora and M. Kobus were analysis of sequencing data revealed DNA polymorphisms. The based on tRNA coding leucine/phenylalanine (trnL-F) and NADH-plastoquinone oxidoreductase subunit 5 (ndhF) sequences in chloroplast DNA. For the identification of ‘Magnolia’ species, polymerase chain reaction (PCR) analysis of chloroplast DNA regions such as ndhF have proven an appropriate method. A single nucleotide polymorphism (SNP) has been identified between genuine “Sini” and their fraudulent and misuse. Specific PCR primers were designed from this polymorphic site within the sequence data, and were used to detect true plants via multiplex PCR.