느타리 품종구분을 위한 마커의 개발을 위하여 곤지7호 의 어버이 일핵 균사중의 하나인 MT07156-97의 전체 유전자 염기서열을 바탕으로 제작한 251개의 SSR 프라이머를 제작하였다. 우선적으로 수한1호, 곤지7호, 흑타리 품종에 다형성 여부를 관찰하여 20개의 SSR을 선발하고, 이를 10개 품종에 적용하였다. 단일의 프라이머로는 일부 품종이 구분되지 않았으므로, 선발된 프라이머 간의 다양한 조합(multiplex 방식)을 적용한 결과 모든 품종을 판별 할 수 있는 분자마커 다형성을 보인 프라이머 "166+115" 조합을 선발하였다. 별도로 프라이머 115와 166가 만들어 낸 산술적인 유전자좌(loci) 31개보다 12개 많은 40개의 유전자좌가 증폭되어 다양한 품종에 특이적인 분자마커를 제공할 수 있었다. 개발된 분자마커는 종균의 품질관리, 품종의 판별, 신품종 보호에 활용될 수 있을 것이다.

Hypsizygus marmoreus is a mushroom with abundant flavor and medicinal properties. However, its application is limited by problems such as long cultivation period, low biological efficiency, and microbiological contamination; therefore, there is a substantial need for development of new cultivars of this species. In this study, 55 strains of H. marmoreus were subjected to inter simple sequence repeat (ISSR) analysis to identify markers for the selection of mother strains for breeding from the collected germplasm. ISSR 13 and 15 were confirmed as polymorphic markers. The three strains (KMCC03106, KMCC03107, and KMCC03108) with white cap color were found to be genetically closely related upon UPGMA analysis of both ISSR 13 and 15. Based on the PCR analysis results for ISSR 15, the collected germplasm were differentiated into three groups according to the strain collection year. Thus, ISSR 15 could be a marker for determining the phylogeny of cap color and genetic variations according to the strain collection year. These results suggest that ISSR markers can be effective tools for the selection of mother strains for breeding of H. marmoreus.

A. bisporus is the fifth most cultivated mushroom in Korea, and approximately 10,757 tons were cultivated in 2015. The genetic diversity of collected strains in Korea and commercial cultivars was analyzed using inter-simple sequence repeat (ISSR) markers. ISSR markers known to be comparable among A. bisporus spp. were selected from various markers. Totally, 16 markers, namely the ISSR markers 807, 808, 810, 811, 834, 835, 836, 841, 842, P3, P8, P17, P22, P30, P38, and P39, were evaluated to discriminate between ASI 1110, 1114, 1115, 1238, 1246, 1365, 1366, and 1369 for selecting suitable markers in 16 markers. The ISSR markers P31, P38 and P39 exhibited various fingerprints that could help classify the strains in species. Using the three markers, genetic relationships among 39 strains, including commercial cultivars, such as SaeA and SaeYeon, were analyzed using the UPGMA method. The results of the analysis of the genetic relationships between commercial cultivars and collected strains in Korea confirmed that the commercial cultivars were different from the collected strains in Korea. These results suggested that the ISSR markers P31, P38, and P30 could be used for selecting the commercial cultivars of A. bisporus.

Background: Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth with radical leaves during the first year and shows reproductive growth with cauline leaves and bolting during the second year. In addition, the shape of the plant varies within the same species. For this reason, there are limitations to classifying the species by visual examination. However, there is not sufficient genetic information or molecular tools to analyze the genetic diversity of the plant. Methods and Results: Approximately 34.59 Gbp of raw data containing 342,487,502 reads was obtained from next generation sequencing (NGS) and these reads were assembled into 357,211 scaffolds. A total of 84,106 simple sequence repeat (SSR) regions were identified and 14,133 primer sets were designed. From the designed primer sets, 95 were randomly selected and were applied to the genomic DNA which was extracted from five plants and pooled. Thirty-nine primer sets showing more than two bands were finally selected as SSR markers, and were used for the genetic relationship analysis. Conclusions: The 39 novel SSR markers developed in this study could be used for the genetic diversity analysis, variety identification, new variety development and molecular breeding of A. triphylla.

Background: Panax ginseng C. A. Meyer is wood-cultivated ginseng (WCG) in Korea which depends on an artificial forest growth method. To produce this type of ginseng, various P. ginseng cultivars can be used. To obtain a WCG similar to wild ginseng (WG), this method is usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. Recently, the WCG industry is suffering a problem in that Panax notoginseng (Burk.) F. H. Chen or Panax quinquefolium L. are being sold as WCG Korean market; These morphological similarities have created confusion among customers. Methods and Results: WCG samples were collected from five areas in Korea. After polymerase chain reaction (PCR) amplification using the primer pair labeled with fluorescence dye (FAM, NED, PET, or VIC), fragment analysis were performed. PCR products were separated by capillary electrophoresis with an ABI 3730 DNA analyzer. From the results, WCG cultivated in Korea showed very diverse genetic background. Conclusions: In this study, we tried to develop a method to discriminate between WCG, P. notoginseng or P. quinquefolium using simple sequence repeat (SSR) markers. Furthermore, we analyzed the genetic diversity of WCG collected from five cultivation areas in Korea.

Background : Panax ginseng C.A. Meyer is a perennial herb belongs to the family Araliaceae. Wild-cultivated ginseng (WCG) is a specific type of ginseng in Korea which cultivated on artificial forest cultivation method. To obtain a WCG which is similar to wild ginseng (WG), this method usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. WCG is very expensive because it is difficult to cultivate. However, systematic cultivation method have not yet been developed compared to high added value. Furthermore, very high price of WCG caused the problem that Panax notoginseng or Panax quinquefolium are sold as WCG in Korean market. In this study, we analyzed the genetic diversity of WCG collected from five areas in Korea using SSR markers. Methods and Results : WCG samples were collected from five areas in Korea (Bucheon, Cheongju, Hoengseong, Judeok and Ulsan). DNA extraction was performed using CTAB method. SSR markers were collected from the published papers. After test PCR using the markers, one of the primer pair was labeled with fluorescence dye (FAM, NED, PET, or VIC) and GeneScan analysis were performed. DNA amplification was conducted using T-100 Thermal Cycler (Bio-Rad). PCR products were separated by capillary electrophoresis on the ABI 3730 DNA analyzer (Applied Biosystems). Conclusion : Eight SSR markers were collected from the published literature and used for the analysis. From the 8 tested SSR markers, 7 SSR markers showed polymorphism between varieties. GenScan analysis were performed using the selected SSR markers to analyze the phylogenetic relationship of WCG. From the results, WCG cultivated in Korea showed that they have a very diverse genetic background.

Background : Codonopsis lanceolata is a perennial plant of Campanulaceae with characteristic flavor and aroma and this plant has saponin, flavonoid, and inulin, which are reported to have physiological activity and antioxidant activity. In contrast, breeding or study of C. lanceolata varieties had not been done for a long time. Genetic polymorphism and phylogenetic relationship analysis of the plants by region of the crops can help the collection of genetic backgroud data for variety development. Methods and Results : In this study, we collected 26 C. lanceolata lines (95 individual plants) from 26 regions in Korea. We genotyped the collected lines using SSR markers developed in the previous study and analyzed the population structure based on the results. Population structures were analyzed using model-based STRUCTURE software (version 2.3.4) using the following parameters: Number of clusters (K) set = 1 to 12; Number of Iterations = 5; Length of Burning Period = 100,000; Number of MCMC (Markov Chain Monte Carlo) Reps after Burnin = 100,000. As a result, Of the 26 collections, were genetically grouped into 6 or 7 groups. Conclusion : The 26 C. lanceolata collections (95 individual plants) were genetically grouped but not grouped by collected regions. These results suggest that C. lanceolata has diverse genetic backgrounds and this data could be used as a basis for genetic polymorphism analysis of Codonopsis species.

Background : Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth by radical leaf until 1 year after sowing and shows reproductive growth during the second year and there is a characteristic of bolting by turning into cauline leaf. In addition, the phenotypes of plants varies even though they are belonging to the same species. For this reason, there is a limit for the classification of the species by the method of visual examination. Methods and Results : Simple sequence repeat (SSR) markers were developed based on the genomic sequence of A. triphylla using next generation sequencing to prepare the basis of molecular breeding and analyze the genetic diversity. Ninety-five primer sets including tri-, tetra- and penta-nucleotide motif types were randomly selected and they were applied to mixed genomic DNA and finally 39 primer sets showing from two to four bands were selected and used for genetic relationship analysis. Conclusions : Using the next generation sequencing, 39 polymorphic SSR markers were developed.

Background : Codonopsis lanceolata is a flowering perennial climber. The roots are used as medicinal materials or vegetables. C. lanceolata is distributed in India and East Asia such as China, Japan as well as Korea. Recently, demand for C. lanceolata is increasing as a healthy food. In South Korea, this plant is widely cultivated in Gangwon-do province. Although, C. lanceolata is one of the most important medicinal plants in Korea, an elite, inbred line or a variety has not been developed yet. Simple sequence repeat (SSR) marker is a powerful tool for analysis of genetic relationships. In addition, it is a useful tool for studying the non-reference plant genome, due to its even distribution throughout the genome, as well as its high polymorphism between individuals. Methods and Results : We constructed microsatellite-enrichment libraries using C. lanceolata genomic DNA, and obtained a total of 226 non-redundant contig sequences. Routine PCR was performed using gDNA as templates for the polymorphic markers screening. Finally, total 15 polymorphic SSR markers based on C. lanceolata genomic sequences were successfully developed. These markers were applied to 53 C. lanceolata collected from Korea. 103 alleles of the 15 SSR markers ranged from 3 to 19 alleles at each locus, with an average of 6.87. The average of observed heterozygosity and genetic diversity were 0.42 and 0.62, respectively. The average of polymorphism information content (PIC) value was 0.57. The genetic distance value ranged from 0.73 to 0.93, and there was no observed distinct group according to the collecting areas. Conclusion : We developed 15 novel SSR markers from C. lanceolata genomic sequences for further genetic studies. Also, we concluded that the lineage of C. lanceolata collected in Korea has not been established systematically.

Background : In the herbal medicine market, Angelica gigas, Angelica sinensis, and Angelica acutiloba are all called "Danggui" and used confusingly. We aimed to assess the genetic diversity and relationships among 14 Angelica species collected from different global seed companies. Toward this aim we developed DNA markers to differentiate the Angelica species. Methods and Results : A total of 14 Angelica species, A. gigas, A. acutiloba, A. sinensis, A. pachycarpa, A. hendersonii, A. arguta, A. keiskei, A. atropurpurea, A. dahurica, A. genuflexa, A. tenuissima, A. archangelica, A. taiwaniana, and A. hispanica were collected. The genetic diversity of all 14 species was analyzed by using five chloroplast DNA-based simple sequence repeat (SSR) markers and employing the DNA fragment analysis method. Each primer amplified 3 - 12 bands, with an average of 6.6 bands. Based on the genetic diversity analysis, these species were classified into specific species groups. The cluster dendrogram showed that the similarity coefficients ranged from 0.77 to 1.00. Conclusions : These findings could be used for further research on cultivar development by using molecular breeding techniques and for conservation of the genetic diversity of Angelica species. The analysis of polymorphic SSRs could provide an important experimental tool for examining a range of issues in plant genetics.

Background: In the herbal medicine market, Angelica gigas, Angelica sinensis, and Angelica acutiloba are all called "Danggui" and used confusingly. We aimed to assess the genetic diversity and relationships among 14 Angelica species collected from different global seed companies. Toward this aim we developed DNA markers to differentiate the Angelica species. Methods and Results: A total of 14 Angelica species, A. gigas, A. acutiloba, A. sinensis, A. pachycarpa, A. hendersonii, A. arguta, A. keiskei, A. atropurpurea, A. dahurica, A. genuflexa, A. tenuissima, A. archangelica, A. taiwaniana, and A. hispanica were collected. The genetic diversity of all 14 species was analyzed by using five chloroplast DNA-based simple sequence repeat (SSR) markers and employing the DNA fragment analysis method. Each primer amplified 3 - 12 bands, with an average of 6.6 bands. Based on the genetic diversity analysis, these species were classified into specific species groups. The cluster dendrogram showed that the similarity coefficients ranged from 0.77 to 1.00. Conclusions: These findings could be used for further research on cultivar development by using molecular breeding techniques and for conservation of the genetic diversity of Angelica species. The analysis of polymorphic SSRs could provide an important experimental tool for examining a range of issues in plant genetics.

Background : Astragalus membranaceus is one of the most widely used traditional medicinal herbs in Korea. Studies on the genomic of A. membranaceus resources have not been carried out so far. The present study was carried out to discriminate A. membranaceus based on genetic diversity using genomic simple sequence repeat (SSR) markers. Methods and Results : We collected 5 A. membranaceus lines: Asung, Poongsung, Am-Jecheon, Am-Sancheong, and Am-China. One hundred mg of fresh leaves were used for genomic DNA extraction using the DNeasy plant DNA isolation kit (Qiagen GmbH, Hilden, Germany). 450,449 contigs were searched for 147,766 SSR candidate loci in this study using the MicroSAtellite identification tool (MISA). We selected 949 A. membranaceus genomic SSR markers that were showed variation for the five collections in silico screening with CLC genomics workbench program. The genetic diversity of all A. membranaceus resources was analyzed using 17 SSR markers employing the DNA fragment analysis method. Based on the genetic diversity analysis, these lines were classified into four distinct groups. Conclusion : These findings could be used for further research on cultivar development using molecular breeding techniques and for conservation of the genetic diversity of A. membranaceus. Furthermore, the markers could be used for marker-assisted selection for crop breeding.

Background : Codonopsis lanceolata is used as a natural medicine or vegetables. It originates in East Asia such as Korea, Japan and China. C. lanceolata roots contain various chemical compounds including saponins like Panax ginseng. Although C. lanceolata are cultivated in different regions of South Korea, no variety has been developed. Therefore, it is necessary to develop discriminating methods such as molecular markers in C. lanceolata species. Methods and Results : To find simple sequence repeat (SSR) markers, we sequenced C. lanceolata genomic DNA using Illumina HiSeq 2000 System. A total of 250,455 putative SSR loci were obtained, and 26,334 non-redundant primers were designed to amplify these SSRs. Di-nucleotied repeats were the most abundant SSR reapeats, accounting for 89.53% (23,578) of primer designed SSRs. Tri-nucleotide, tetra-nucleotide and penta-nucleotide accounted for 8.44% (2,223), 1.3%, (348) 0.2% (55), respectively. Tri-, tetra-, and penta-nucleotide (total of 2,626 SSRs) were investigated in silico to identify polymorphism between individuals. Consequently, 573 SSRs showed polymorphism. Forty genomic SSR markers were tested in 16 C. lanceolata plants for determination of PCR amplification and polymorphism. From these primers, 27 (67.5%) amplified products and the average polymorphism information content (PIC) value was 0.52. Conclusion : We development 27 SSR markers from C. lanceolata using NGS, and it could be used for breeding of new varieties in the future.

Ramie (Boehmeria nivea L.) is a hardy perennial herbaceous plant of the Urticaceae family and has been grown as a fiber crop in several countries including Korea for many centuries. Ramie leaves also have been traditionally used as a major ingredient of a type of rice cake called ‘Song-pyun’ in the Southwest area of Korea, especially Yeong-Gwang province. Despite its economic importance, the molecular genetics of ramie have not been studied in detail yet. Genetic resources of ramie were widely collected from domestic local sites by Bioenergy Crop Research Center (RDA) and Yeong-Gwang Agricultural Technology Center. For the systematic and efficient management of the genetic resources, we developed SSR (simple sequence repeat) markers of ramie. To do this, we generated microsatellite-enriched genomic DNA libraries using magnetic bead hybridization selection method. 247 non-redundant contigs containing SSR motif were generated using nucleotide sequences of 376 clones from the libraries. Primer sets were designed from the flanking sequences of the repeat motif. Finally, we selected 10 SSR markers, possibly showing polymorphism among the genetic resources. Results on the genotype analysis of the ramie genetic resources using the SSR markers will be presented.

Assessing genetic diversity, population structure, and linkage disequilibrium is important in identifying potential parental lines for breeding programs. In this study, we assessed the genetic and phenotypic variation of 174 normal maize (Zea mays) inbred lines and made association analyses with respect to nine agronomical traits, using 150 simple sequence repeats (SSR). From population structure analysis, the lines were divided into three groups. Association analysis was done with a mixed linear model and a general linear model. Twenty one marker-trait associations involving 19 SSR markers were observed using the mixed model, with a significance level of P<0.01. All of these associations, as well as 120 additional marker-trait associations involving 77 SSR markers, were observed with the general model. Two significant marker-trait associations (SMTAs) were detected at P ≤ 0.0001. In the mixed linear model, one locus was associated with water content, two loci were associated with 100-kernel weight, setted ear length, ear thickness and stem thickness; three loci were associated with ear height, four loci were associated with total kernel weight and five loci were associated with plant height. These results should prove useful to breeders in the selection of parental lines and markers.

본 연구는 국내외에서 육성 및 수집된 장미품종에 대하여 SSR 마커를 이용하여 품종식별 연구를 수행하였다. 장미 품종식별에 적합한 SSR 마커를 선정하기 위하여 112개의 SSR 마커를 12개의 주요품종을 대상으로 분석하였다. 최종 선발된 22개의 SSR 마커를 대상으로 69품종을 이용하여 분석하였을 때 대립유전자의 수는 2～10개의 분포를 나타내었으며 총 114개의 대립유전자가 분석되었다. PIC 값은 0.211～ 0.813의 범위에 속하였으며 평균값은 0.621로 나타났다. SSR 마커를 이용하여 작성된 장미 69품종의 품종간 유전적 거리는 0.41～0.87의 범위로 나타났고, 공시 품종 모두 SSR 마커의 유전자형에 의해 구분되었다. 본 연구결과는 장미 품종의 식별과 유전적 다양성 연구를 위한 기초 자료로 유용하게 활용될 수 있을 것으로 기대된다.

총 22개의 감 SSR primer set을 사용하여 주요 재배품종 17품종, 수집한 단감 변이개체 13종 등, 총 30개의 유전적 연관성을 분석하였다. 획득한 469개의 다형성 밴드를 이용하여 UPGMA 방식으로 유사도 및 집괴분석을 수행한 결과, 30개의 품종 및 수집 계통들은 크게 2개의 그룹(cluster)으로 나뉘어졌으며, 제 1 cluster는 다시 3개의 subcluster로, 제 2 cluster는 다시 2개의 subcluster를 형성하였다. 이는 수집 계통들이 기존 대조품종들과 서로 동일군으로 분포되고 있어 형태적으로 유사할 수 있고 근연되어 있지만, 유전자적 수준에서는 다른 조성을 가지고 있음을 보여준다. 평균 유사도의 값은 0.164 이였고 품종간 가장 높은 유사도 값(0.31)을 나타낸 것은 ‘차랑’과 ‘전천차랑’이였고, 가장 낮은 유사도 값(0.02)를 나타낸 것은 Ⅱ 그룹과 비교하여 Ⅰ 그룹에 속하는 ‘Rojo Brillante’였다. 본 연구에 사용된 22 SSR primer set을 통해, ‘차랑’과 ‘전천차랑’을 제외한 대조품종과 수집 계통간의 구별이 가능하여 향후 신품종 개발시 품종보호를 위한 특이적 마커로 효율적으로 사용될 수 있을 것으로 판단된다.

The SSR markers were generally used to analysis the plant genetic diversity, but it have been rarely reported in case of castor bean. We constructed the microsatellite-enriched genomic library and sequenced totally 775 clones to obtain the microsatellite sequence information of the castor bean. Among the sequenced clones, one hundred fifty clones (19.3%) were redundant and four hundred twenty (67.2%) were found to contain microsatellite sequences within the remaining 625 unique clones. A total of 237 primer pairs were designed based on the sequenced microsatellite clones information and evaluated for polymorphism in ten castor bean accessions. Twenty-eight SSR markers produced reproducible polymorphic bands and were further characterized using a diverse set of 25 castor bean accessions. The majority of unique SSRs revealed dinucleotide motives (84%) on the other hand the ratio of trinucleotide motives was 15%. A microsatellite enriched library from the Ricinus communis L. was mainly consisted of [(AG), (GA)/(CT), (TC)] and [(CTT)/(AAG)] microsatellite motifs. The length of dinucleotide SSRs ranged from 4 to 50 repeats with an average 12.4, and that of trinucleotide SSRs from 4 to 56 with an average of 7.35 repeats. These newly developed microsatellite markers will be useful for breeding system and classification of Ricinus communis L.