세계화로 인해 교역 및 여행객의 증가로 외래 병해충의 유입이 증가하고 있고 이들의 유입 및 확산 경로 규명을 위하여 분자 유전학적 분석 방법이 이용되고 있는 실정이다. 본 연구는 외래 해충인 Reticulitermes kanmonensis의 유전적 특성을 분석하기 위한 18개의 초위성체마커(microsatellite)를 이용하여 총 16개 지역(전주, 김제1, 김제2, 군산1, 군산2, 서천, 완주1, 완주2, 완주3, 완주4, 익산1, 익산2, 논산, 일본1, 일본2, 일본3)의 흰개미 244개체를 분석하였다. 집단간 대립 유전자 패턴(allelic patterns)분석결과 일본3 지역의 유전적 다양성이 높게 나타났고 완주2 지역은 다른 지역에 비해 비교적 낮게 나타났다. 집단 간 유전적 거리(Genetic differentiation)는 평균적으로 0.163정도의 유전적 다향성 비율을 나타냈고, 서천과 군산2 지역이 0.048로 낮게, 서천과 일본2 지역이 0.442로 높게 나타나 서천과 군산2 집단이 유전적으로 가깝게 나타나고, 반면 서천과 일본2 집단과는 유전적으로 멀게 나타났다. STRUTURE 분석결과는 클러스터 집단 수가 6(k=6)일때 이상적인 집단 패턴을 나타내었고, 유전적 구조는 A그룹(전주, 김제1, 완주2), B그룹(서천, 군산1, 군산2), C그룹(완주1, 김제2), D그룹(논산, 익산1, 익산2), E그룹(김제1, 완주2, 완주3), F그룹(일본1, 일본2, 일본3)의 6가지의 패턴이 지역별로 분리되어 나타났다.

외래 병해충의 유입이 증가 하고 있고 이들의 유입 및 확산 경로 규명을 위하여 분자 유전학적 분석 방법이 이용되고 있는 실정이며, 본 연구는 외래 해충인 Reticulitermes kanmonensis의 유전적 특성을 분석하기 위한 초위성체마 커(microsatellite)를 문헌을 통해 탐색하였고, NGS(Next-Gen Sequencing) 기술을 통해 새로운 초위성체마커를 개발하였 다. 문헌을 통해 Reticulitermes속에서 개발된 마커29개와 NGS를 통해 선발된 25개의 마커를 탐색 하여, 이중 증폭 및 유용성이 있는 18개의 마커를 선정하였다. 선정된 단일 마커들을 이용하여 6개의 multiplex PCR set 및 증폭 조건을 수립하여 집단유전학 분석에 활용하고자 한다.

For preliminary molecular typing, PCR-based fingerprinting using random amplified polymorphic DNA (RAPD) is the method of choice. In this study, 14 bacterial strains were isolated from different Korean food sources, identified using 16S rRNA gene sequencing, and characterized through RAPD-PCR. Two PCR primers (239 and KAY3) generated a total of 130 RAPD bands, 14 distinct PCR profiles, 10 polymorphic bands, one monomorphic band, and four unique bands. Dendrogram-based analysis with primer 239 showed that all 14 strains could be divided into seven clades out of which clade VII had the maximum of seven. In contrast, dendrogram analysis with the primer KAY3 divided the 14 L. citreum strains into four clades out of which clade IV consisted of a maximum of 10 strains out of 14. This research identified and characterized bacterial populations associated with different Korean foods. The proposed RAPD-PCR method, based on sequence amplification, could easily identify and discriminate the lactic acid bacteria species at the strain-specific level and could be used as a highly reliable genomic fingerprinting tool.

For preliminary molecular typing, PCR-based fingerprinting using random amplified polymorphic DNA (RAPD) is the method of choice. In this study, 14 bacterial strains were isolated from different Korean food sources, identified using 16S rRNA gene sequencing, and characterized through RAPD-PCR. Two PCR primers (239 and KAY3) generated a total of 130 RAPD bands, 14 distinct PCR profiles, 10 polymorphic bands, one monomorphic band, and four unique bands. Dendrogram-based analysis with primer 239 showed that all 14 strains could be divided into seven clades out of which clade VII had the maximum of seven. In contrast, dendrogram analysis with the primer KAY3 divided the 14 L. citreum strains into four clades out of which clade IV consisted of a maximum of 10 strains out of 14. This research identified and characterized bacterial populations associated with different Korean foods. The proposed RAPD-PCR method, based on sequence amplification, could easily identify and discriminate the lactic acid bacteria species at the strain-specific level and could be used as a highly reliable genomic fingerprinting tool.

Recently, a rapid movement of agricultural products with an extensive international trading and climate change have led to an increased attention to nematodes as having regulatory significance. With the increase of global dispersal of pests, new diagnosis methods are required for a rapid and reliable species and/or biotype identification to restrict introduction of the pests. Recently, novel molecular diagnostic techniques provide clues to solve taxonomic problems associated with conventional species identification. In this articles, we proposed various molecular diagnostic techniques to complement the limitation of morphological taxonomy.

Two closely related species, the soybean podworm, Matsumuraeses phaseoli, and the podborer, M. falcana, gives differential economic damages on crops. It is difficult to discriminate these potential sympatric species by morphological characters. The goal of this study was to develop a discriminating molecular marker based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A partial genomic fragment (500 bp) of mitochondrial cytochrome oxidaseⅠ (mtCOI) was sequenced in both species, in which restriction site by Rsa I was selected as a dichotomous marker. PCR-RFLP in the mtCOI region clearly discriminated both species.

Local and seasonal populations of the oriental fruit moth, Grapholita molesta , were monitored with sex pheromone trapping and RAPD (random amplified polymorphic DNA) molecular marker to analyze their movement in apple orchards. To detect their movements among farms, pheromone traps were placed at regions between apple farms (‘outside-farms’) as well as within-farms (‘inside-farms’). Four seasonal adult peaks were evident in apple-cultivating fields from April to October in both trappings of inside- or outside-farms. After overwintering generation, populations of inside-farms were significantly reduced with frequent insecticide applications, compared to populations of outside-farms. Within apple farms, G. molesta tended to be unevenly distributed because of significant sublocal preference. Active movements of local and seasonal populations of G. molesta were supported by gene flow analysis using RAPD marker. Monitoring data using sex pheromone and seasonal reduction in initial genetic differentiation detected in the overwintering populations suggest that there must be significant movement of G. molesta among different orchards in apple-cultivating areas.

과피와 과육의 다양한 색은 복숭아 분류에 가장 널리 사용되는 상업적 기준 중 하나이다. 새로운 적색 과육 품종을 육성하기 위해서는 많은 교배 조합과 세대가 진전되어야 한다. 따라서 육종 효율을 높이기 위해서는 목적 형질을 가진 개체에 적용할 조기 선발 분자표지를 개발할 필요가 있다. 과육색이 다르게 발현되는 복숭아 품종의 유전자 발현을 비교하기 위해 2개의 cDNA library를 제작하였다. 적색 과육 품종인 ‘조생혈도’와 백색 과육 품종인 ‘미백도’의 유전자 발현 차이를 보기 위해 차세대 염기서열 분석(NGS) 기술을 사용하였고, 두 품종으로부터 얻은 EST의 염기서열을 결정하고 기존에 보고된 유전자와의 상동성을 분석하였다. ‘조생혈도’와 ‘미백도’의 EST database로부터 72쌍의 SNP 분자표지를 선발하였고, 적색 과육 품종 8개와 백색 과육 품종 24개를 구분할 수 있는 SNP 분자표지를 HRM 방법으로 분석하였다. 본 연구에서는 복숭아 EST database를 기반으로 HRM 분석 방법을 이용하여 복숭아 품종의 적육계와 백육계를 구분할 수 있는 효율적인 SNP 분자표지를 개발하였다. 이러한 SNP 분자표지는 복숭아 육종에 유용하게 사용할 수 있으며, 복숭아 품종의 다양한 색 변화에 관한 분자 기작 연구에 좋은 참고자료가 될 수 있을 것이다.

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.

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.

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.

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.

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.

Next generation sequencing technologies have proven to be a rapid and cost-effective means to assemble and characterize gene content and identify molecular markers in various organisms. Pepper (Capsicum annuum L., Solanaceae), is a major staple vegetable crop, which is economically important and has worldwide distribution. High-throughput transcriptome profiling of two pepper cultivars, Mandarin and Blackcluster using 454 GS-FLX pyrosequencing yielded 279,221 and 316,357 sequenced reads with a total 120.44 and 142.54 Mb of sequence data (average read length of 431 and 450 nucleotides). These reads resulted from 17,525 and 16,341 ‘isogroups’ and were assembled into 19,388 and 18,057 isotigs, and 22,217 and 13,153 singletons for both the cultivars, respectively. Assembled sequences were annotated functionally based on homology to genes in multiple public databases. Detailed sequence variants analysis identified a total of 9,701 and 12,741 potential SNPs for both cultivars, which eventually resulted in 1,025 and 1,059 genotype specific SNPs, for both the varieties, respectively. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies.

Amplified fragment length polymorphism (AFLP) is one of molecular marker technique based on DNA and is extremely useful in detection of high polymorphism between closely related genotypes like Korean wheat cultivars. Six Korean wheat cultivar specific marker sets have been developed from inter simple sequence repeat (ISSR) analysis and we can identify the 13 Koran wheat cultivars form other cultivars using six that (Son et al., 2013). We used four combinations of primer sets in our AFLP analysis for developing additional cultivar specific markers in Korean wheat. Twenty-one of the AFLP bands were isolated from ACG/M-CAC primer combination and 19 bands were isolated from E-AGC/M-CTG primer combination, respectively. We used forty bands to design sequence characterized amplified region (SCAR) primer pairs for Korean wheat cultivar identification. Only one of 40 amplified primer pairs, C2, were able to use for wheat cultivar identification. The DNA band of 215bp length was amplified by C2 primer pairs in ten cultivars, Eunpa, Olgeuru, Gobun, Saeol, Milsung, Sinmichal, Jokyung, Sugang, Goso, and Joah. Then C2 primer was applied to these primer sets as newly SCAR marker, six cultivars are identifying from other cultivars, additionally. Finally, to use the C2 and six primer sets, 19 Korean wheat cultivars are identified.

To select genes associated with the high-temperature tolerance from Brassica, two transcriptomic analyses have been used: microarray and RNA Seq. Using two contrasting inbred lines of B. rapa, Chiifu and Kenshin, version 3 microarray (135 K microarray) was conducted to RNA samples extracted from series of 45℃-treated leaves and 29 genes were selected for genomic DNA cloning of cabbage. Of 29 genes, 8 genes contain 40 SNPs, 11 SSRs and 23 In-Del markers that distinguish high-temperature tolerant and susceptible cabbages, BN1 and BN2. These 8 genes include a unknown gene, AP2, SMP, FBD, SKP2B, IAA16, HSP21 and OLI2-2. We also selected 16 cabbage genes from RNA Seq analysis using two inbred lines, BN1 and BN2: 5 genes for BN1-high expression, 5 genes for BN1-specific expression, 5 genes for BN2-specific expression, and BoCaMB. Using RNA sequences, genomic DNAs corresponding to 16 genes have been clones and analyzed to find out molecular markers. Markers were further transformed into PCR-based marker and confirmed with additional cabbage genetic lines. We are currently transforming PCR-makers into SNP markers. To examine function of high-temperature tolerant genes, we also transformed 5 genes into Arabidopsis plants. We will describe detailed methods and results in a poster. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ009085), Rural Development Administration, Republic of Korea]