Three insect pests internally feed pome fruits in Korea. These include oriental fruit moth (Grapholita molesta), plum fruit moth (Grapholita dimorpha) and peach fruit moth (Carposina sasakii). Molecular markers discriminating these three species were developed using PCR-RFLP technique. Mitochondrial (mt) genomes were analyzed to locate polymorphic loci. Six mtDNA regions (CO-Ⅰ, CO-Ⅱ, CB, 16SrRNA-12SrRNA, ND3, ND4) of G. dimorpha were cloned and sequenced. These six sequences of G. dimorpha were aligned with those of C. sasakii and G. molesta to determine polymorphic restriction sites. Predicted PCR-RFLP markers were confirmed with known insect samples. With the validated PCR-RFLP markers, field male adults collected in traps baited with rubber sept lures impregnated with different ratios of major sex pheromone components of G. molesta were analyzed.

Acquisition of a reference Tetranychusstrains that are completely susceptible to acaricides and retain identical genetic backgrounds to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish both completely susceptible and acaricide-resistant strains for this purpose, I collected Tetranychus mite populations from various regions in South Korea including both heavily cultivated and remote regions. Suitability as a susceptible or resistant reference strain was tested by determining species identity as Tetranychus urticae along with baseline susceptibility to common acaricides. The majority of mite populations collected from cultivated areas belonged to a monophyletic group of the previously reported green-type T. urticae as determined by mtCOI- and ITS2-based phylogenetic analysis. These strains showed relatively reduced levels of susceptibility to the acaricides tested, suggestive of the development of resistance. Among them, the AbaR strain was classified as a minor group in the T. urticae complex. The UD strain, originally collected from a remote island region, was found to be susceptible to the acaricides tested and generated an independent phylogenetic branch. The UD strain was also considered a minor group in the T. urticae complex. Phenotypic analysis based on morphological characters confirmed that both the AbaR and UD strains were statistically undistinguishable from the major green-type T. urticae. Taken together, I propose that the UD strain be used as a susceptible reference strain as it provides baseline susceptibility to acaricides and a wild-type genetic background for the resistance studies.

Genetic variation within and among 12 populations of whip grass in south China were investigated using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP). High genetic diversity was found in whipgrass by two molecular makers (PPB=86.21%, I=0.357 based on ISSR; PPB=82.21%, I=0.352 based on SRAP). However, there was relatively low genetic diversity at population levels. A high degree of genetic differentiation among populations was detected based on different measures and different molecular markers. We also found that SRAP markers were more efficient than ISSR markers in this species. Based on these findings, sampling strategy was proposed for successfully utilizing the genetic resource of this 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.

An unidentified moth was captured in sex pheromone traps of the oriental fruit moth, Grapholita molesta, especially at spring season in apple orchards and their vicinity. Though the captured males were similar in appearance to G. molesta males, they were easily distinguished by a marked difference in body size. Their occurrence pattern was also similar to that of overwintering G. molesta population from April to May, at which more males were captured in the pheromone traps installed in the vicinity of apple orchards than within apple orchards. After May, they were no longer captured in the pheromone traps. To investigate any larval damage due to this unidentified moth, molecular markers needed to be developed. Four PCR-RFLP markers originated from cytochrome b region of mitochondrial DNA could distinguish this unidentified moth from G. molesta.

A phylogeny of the tribe Aphidini (Hemiptera: Aphididae) was reconstructed using three molecular gene fragments: partial mitochondrial tRNA-leucine + cytochrome oxidase II (tRNA/COII), partial mitochondrial 12S + tRNA-valine + 16S (12S/16S), and nuclear elongation factor-1 alpha (EF1α) for a total of 41 species consisting of 37 representative species in Aphidini and 4 outgroup species in the tribe Macrosiphini. Also, to compare with the molecular analyses, a second phylogenetic reconstruction was performed using 41 morphological characters for equally weighted analysis. Minimum evolution, maximum parsimony, maximum likelihood, Bayesian phylogenetic analyses, and Bremer support test were performed for the phylogenetic construction. As our phylogenetic results were compared with the classical taxonomy based on morphological characteristics and biological data, we newly confirmed the following phylogenetic relationships within Aphidini: (1) each monophyletic subtribe of Aphidina and Rhopalosphina was supported by significant P values in the combined analysis, (2) contrary to the classical taxonomic position, Cryptosiphum should be moved to Macrosiphini because it is more closely related to Lipaphis and Brevicoryne in the myzine group of Macrosiphini, (3) The genus Toxoptera was possibly considered as a non-monophyletic group because three species were separately positioned in Aphidina, (4) In Rhopalosiphina, the genus Melanaphis was relatively divergent from three genera, Rhopalosiphum, Schizaphis and Hyalopterus, clustered in a strongly supported clade, (5) in the relationships among four the Aphis species-groups, which are accepted by most aphid taxonomists, most species except for some morphologically distinct species were separated to the two main lineages; the fabae-group was confirmed to be closely related with the spiraecola-group and craccivora-group, but the gossypii-group diverged as a monophyly from those three group.

Oriental fruit moth, Grapholita molesta, is a serious pest on apples. To control this pest in an environmentally friendly method, mating disruption strategy using sex pheromone has been developed. Area-wide application of mating disruption has been needed to be effective, with little understanding on how much size of apple cultivating area should be treated in one time application of the mating disruption technique. On this matter, we needed to determine a minimal mating active zone of G. molesta that should be applied with mating disrupters to be effective. Molecular markers to discriminate a specific population should be developed to trace population migration for reproductive behaviors. Here we developed two effective molecular markers using random amplified polymorphic DNA (RAPD) technique. Different field populations of G. molesta, based on locations and seasons, were analyzed with these markers. In a specific location, G. molesta populations varied in genetic composition with different seasons. Different local populations showed differential variation according to their relative distances among apple orchards. In overall, genetic variation among different populations became lessen with progression of seasons.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) sequence of common buckwheat, Fagopyrum esculentum and tartary buckwheat, F. tataricum. The diversity of the nucleotides and haplotypes, Tajima’s D, and Fu’s Fs was analyzed and compared among the varieties of common buckwheat and tartary buckwheat. The diversity of nucleotides and haplotypes indicated that the buckwheat populations had undergone rapid population expansion but D and Fs did not support their expansion statistically. The phylogenetic analysis of ITS sequences did not clearly establish the phylogenetic relationships between the varieties of common buckwheat. The In/Del sequence of ITS-1 region could, therefore, be used as a DNA marker to distinguish raw or manufactured products derived from common buckwheat and tartary buckwheat.

Background : Kalopanacis Cortex (海桐皮) is listed in「The Korea Pharmacopoeia (KP)」as the original plant of Kalopanax septemlobus (Thunb.) Koidz. However, the dried cortex of Erythrina eariegata L (刺桐) is an adulterant, one of the most indiscriminately used herbal medicines because of its similar morphologic. Due to the morphological similarities of the dried cortex of this plant to those of K. septemlobus which is used as a substitute herbal for E. eariegata, distinguish these two species is extremely difficult. Meanwhile, K. septemlobus is a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, it is conducted to develop molecular markers to distinguishing these K. septemlobus with E. eariegata by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of K. septemlobus, E. eariegata was analyzed from four barcode regions of chloroplast DNA (psbA-trnH, rbcL, matK, atpH-atpF) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcode region. The present study aimed to analyze the percent of variable sites were provided the highest ITS2 (2.3%) followed by rbcL (8.2%), in oder to develop a species-specific primer that can distinguish K. septemlobus form E. eariegata. Conclusion : The INDEL markers were developed based on the divergence of each sequence, and it is possible now to identify the two species of K. septemlobus with just a single performance of PCR. This will not only prevent misused of the plant, but also to maintain the quality of the herbal medicine as well as to verify and guarantee safety for public health.

Background : In the KHP (the Korea Herbal Pharmacopoeia), the Cuscutae Semen (菟絲子) is defined as the seed of the Cuscuta chinensis Lamark (family: Convolvulaceae). Using authentic raw herbal materials is fundamental to herbal medicine quality and Cuscutae Semen is widely distributed in many asian countries. Due to having tiny bodies of seeds, it is extremely difficult to differentiate them from adulterants and closely related species by morphologic characteristics, leading to serious safety problems. For this reason, there was conducted to develop molecular markers to distinguishing these Cuscuta chinensis with Cuscuta japonica and Cuscuta pentagona by using conventional polymerase chain reaction (PCR). Method and Results : In this study we developed a clearly and efficient method to identify Cuscutae Semen on the market. These samples (C. chinensis, C. japonica and C. pentagona) were analyzed from two barcode regions of chloroplast DNA (rbcL, psbA-trnH) and nuclear ribosomal DNA (ITS2). Based on genetic distance, the precent of variable sites were provided the highest psbA-trnH value (38.7%), followed by ITS2 (23.4%), rbcL (9.9%), in order to develop a specific primer that can distinguish C. chinensis, C. japonica and C. pentagona. Conclusion : From the above results, DNA barcoding was proved to be a successful tool for authentication the three species of Cuscutae semen. The adoption of DNA barcoding as an authentication tool by food safety agencies can safeguard the interests of both consumers and traders.

Background : Chrysanthemi Indici Flos (甘菊) is listed in 「The Korea Herbal Pharmacopoeia (KHP)」as the original plant of Chrysanthemum indicum L. C. indicum was one of the most representative medicinal plants in Asteraceae, Dried flowers of this plant have been valid chemical composition such as flavonoids, phenylpropanoids, terpenoids, and polysaccharides, possessing broad spectrum antibacterial, antiviral, antihypertensive and anti-oxidation functions. Meanwhile, C. indicum was a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, there was conducted to develop molecular markers to distinguishing these C. indicum with C. morifolium, C. zawadskii var. latilobum and Aster spathulifolius by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of Chrysanthemi Indici Flos, these samples (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius) were analyzed from five barcoding regions of chloroplast DNA (rbcL, matK, rpoB, atpF-atpH) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcoding region. Based on genetic distance, the percent of variable sites were provided the highest ITS2 value (56.9%), followed by atpF-atpH (48.18%), matK (27.2%), psbK (8.2%), and rbcL (2.9%). Comparative analysis based on the complete genome sequence of the petL-petG region INDEL (insertion/deletion) that the gene annotations were registered to the GenBank (accession number: JN-867592.1, NC-020092.1, MF-034027.1, NF-279514.1). Conclusion : From the above results, we may suggest that the petL-petG region INDEL analysis were conducted for molecular authentication of four plants (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius). The findings of results indicated that petL-petG region might be established INDEL analysis systems and hence were proved to be an effective tools for molecular evaluation and comparison of “Chrysanthemi Indici Flos” with other plants.

Background : The Codonopsis genus belongs to the Campanulaceae, and it is recorded that there are four species of Codonopsis genus in Korea, such as Codonopsis lanceolata, Codonopsis pilosula, Codonopsis minima, and Codonopsis ussuriensis. C. lanceolata has been proved to be safety and efficacy, and has been widely used for medicinal and edible purposes for a long time in East Asian countries including Korea, China and Japan. However, little genetic research has been done. Methods and Results : Ten species of Codonopsis plants were collected and DNA was extracted using CTAB (cetyl trimethylammonium bromide) method. The extracted DNA was diluted to 5 ng/㎕ for the PCR (polymerase chain reaction) process. C. lanceolata genome was used to develop molecular markers by searching insertion and deletion regions (InDel) in the chloroplast sequence. The developed markers were applied to 4 individuals per Codonopsis species. PCR amplification was carried out using a denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec and extension at 72℃ for 30 sec, repeated for 35 total cycles. The PCR products were separated in a 4% agarose gell at 100 V for 40 min. Conclusion : Using the molecular markers developed in this study, genetic diversity of Codonopsis genus was tested, and at the same time, a specific molecular marker was developed to differentiate C. lanceolata from the Codonopsis plants.

Background : Cudrania tricuspidata Bureau is a widely used medicinal perennial woody plant. Obtaining information about the genetic diversity of plant populations is highly important for conservation and germplasm utilization. In this study, we developed single nucleotide polymorphism (SNP) markers derived from chloroplast genomic sequences to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the chloroplast TrnL-F intergenic region. Methods and Results : SNPs were identified based on the results of nucleotide sequence for the intergenic region of TrnL-TrnF gene (chloroplast). Molecular markers were designed for those SNPs with additional mutations on the second base from SNPs for amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). HRM pattern analyses were performed using the Mx3005P QPCR System (Agilent Technologies, CA, USA). Conclusion : We collected 12 individual lines of C. tricuspidata from various region in South Korea and China. Based on the nucleotide sequence in the trnL-trnF intergenic region of these lines, six SNPs and a deletion of 12 bps were identified and 12 individual lines were able to be grouped in one Korean ecotype and two different ecotypes of chinese lines, chinese line 1 and 2. The SNP markers developed in this study are useful for rapidly identifying these specific C. tricuspidata ecotypes collected from different regions.

Background : The advancement of next-generation sequencing technology dramatically reduces the cost for sequencing and it contributes to create a new research environment that utilizes large amount of genome sequences to answer many biological questions. With this new research trend, reference genome sequences of several major crops have been released to the research community and utilized in various researches in agriculture. Coupled with molecular breeding technology, NGS based genome research will possibly allow selecting a new plant material possessing useful traits in early stage and efficiently developing a superior cultivar. Methods and Results : The objectives of this research are to collecting various genetic variations (SNPs, indels and TE mediated variations) in major and minor crops, to develop molecular markers using NGS based genomic data (resequencing, GBS, transcriptome), and to develop a visualization tools to enhance the utility of the NGS data. Currently major analysis pipelines have been developed to detect SNPs, indel and polymophic SSRs using whole genome and transcriptome data, and a pipeline for identification of MITE insertion polymorphism is under development. In addition to that, for orphan crop, we also implemented an efficient and robust method to assemble a complete chloroplast, mitochondria and 45S rDNA using low coverage whole genome data in order to develop an inter- and intra-specific molecular barcode markers. Conclusion : NGS provide a new level of researches in many crop plants. Large amount of genomic information provides an opportunity to understand domestication and genetic variations, and to develop a better crop for future.

Background : Medicinal crop has been used in the traditional Asian medicinal methods. From ancient times, various kinds of medicinal crop are being cultivated in East Aisa including Korea, China and Japan. In Korea, they used a variety of medicinal plants in folk medicine and oriental medicine since ancient times. Molecular markers can be widely used in a variety of settings such as effective-loci estimation, genetic-diversity characterization, allelic-effect studies, gene-flow studies, quantitative-trait locus (QTL) mapping, and evolutionary studies. The genetic analyses of crops require large numbers of useful molecular markers for genetic or QTL identification, comparative mapping and breeding. Studying the genetic diversity and genetic relationship of crops can assist breeders. Crop genetics within a breeding program enable the economic and effective cultivar development. We tried to develop a variety of molecular markers from Angelica gigas genomic sequences for genetic studies and breeding. Methods and Results : A. gigas resources cultivated in Republic of Korea were collected. Fresh leaves were ground with liquid nitrogen and gDNA was extracted using a DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). We sequenced the whole genomes of five A. gigas accessions using Illumina HiSeq 2500 platform and identified genomic Simple Sequence Repeat (SSR) and InDel markers. DNA amplification was conducted using the PCR system (Bio-Rad T-100 Thermal Cycler). PCR products were separated by capillary electrophoresis on the ABI 3730 DNA analyzer (Applied Biosystems) and Fragment analyzer automated CE system (Advanced Analytical Technologies, Ankeny, IA, USA). Conclusion : We developed novel SSR and InDel markers from A. gigas genomic sequences for further genetic studies and breeding.

Radish is one of the most widely consumed vegetable crops in Korea. Root is the major part of radish supplied to the market, thus the size, shape, and quality of radish roots are main targets of breeding programs. Despite of the importance of this crop, the molecular breeding of radish is still in the rudimentary stage. In Golden Seed Project, we aim to establish the molecular breeding program of radish using genome-wide approaches. To this end, we selected inbred lines that have distinctive root traits such as yield, shape, disease resistance, and texture. Single nucleotide variation (SNV) among these lines will be identified based on the low coverage genome sequencing data. These SNVs can be used for finding genomic regions associated with root traits from segregating mapping populations which are also in the middle of development. Korean radish roots are harvested after being grown for only nine weeks. During that period, root biomass reaches to more than two kilograms. While investigating the root growth of radish inbred lines, we found that cytokinin contributes as a key growth regulator that promotes radial growth of radish roots. A difference in growth rates of two distinctive inbred lines was explained by the difference in response to cytokinin. Genes responsive to cytokinin are highly enriched in the cambium, the meristematic cell population that drives radial growth. For comprehensive understanding of genes that affect yields of radish roots, we turned to developing a tissue specific transcriptome data using laser capture microdissection. We expect that the compendium of genomics-based data will help establishing molecular breeding of radish at a fast track.

Bacterial wilt (BW) caused by Ralstonia solanacearum is one of the most common soil-borne vascular diseases of many solanaceous crops such as pepper and tomato. This study aimed to develop molecular markers closely linked to bacterial wilt resistance genes using a 150 F8 recombinant inbred line (RIL) population obtained from a cross of ‘YCM334’ x ‘Taean’. For pathogen inoculations, R. solanacearum isolate WR-1 was cultured on NB medium at 28℃ for 48 h and a bacterial suspension was adjusted to 1 x 107 to 1 X 108 CFU/mL (A 600 = 0.3 to 0.4). Each RIL and the parents were sown in a 72-cell plastic tray filled with sterilized soil, and the seedlings were inoculated at the 6 to 8 leaf stage using soil-drenching (3 to 5 ml/ plant) inoculation methods with 3 replications. After 10 days post inoculation (dpi), each line was evaluated visually for occurrence of bacterial wilt ranging from 1 (most resistant) to 5 (most susceptible). Two candidate R-response genes, AT4G14130 and AT3G23730, were selected to find SNPs between YCM334 and Taean. In previous transcriptome analysis, these two genes were reported as significantly differentially expressed in Capsicum annuum L. root inoculated with R. solanacearum, which were up-regulated in a resistant genotype. Once the synteny of the gene locations between Arabidopsis and pepper was documented, the sequences on pepper chromosome 12 were obtained from pepper. v.1.55 (http://solgenomics.net). SNP markers associated with resistance to BW will be mapped using pepper RIL population.

Low temperature germinability (LTG) is an important trait for breeding of varieties for use in direct-seeding rice production systems. Although rice (Oryza sativa L.) is generally sensitive to low temperatures, genetic variation for LTG exists and several quantitative trait loci (QTLs) have been reported. The objective of this study was to develop and employ high-efficiency molecular markers for evaluation of LTG in rice germplasm. A panel of japonica rice accessions (n=180) from temperate regions in Asia was evaluated for LTG and genotyped with markers from regions previously reported to harbor other LTG QTLs. ANOVA revealed that four markers on chromosome 2, 4, and 11 from previously reported QTLs showed highly significant value (p < 1.0e-04) and their R2 ranged 0.083 (qLTG11-1) to 0.190 (qLTG4b-1). An association analysis was conducted using SNP data generated by sequencing of the panel. Eight SNP markers were found to be significantly associated with LTG using general and mixed linear models. Three SNP-based CAPS and dCAPS markers from these results were developed and showed higher accuracy in predicting sensitive LTG germplasm. These new LTG markers will be useful for molecular evaluation of germplasm, particularly to identify sensitive or weak LTG accessions.