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.

Microsatellite SSR markers were developed and utilized to reveal the genetic diversity of 32 strains of Flammulina velutipes collected in Korea, China, and Japan. From SSR-enriched library, 490 white colonies were randomly selected and sequenced. In the 490 sequenced clones, 85 clones (17.35%) were redundant. Among the remaining 405 unique clones, 201 clones (49.6%) contained microsatellite sequences. As a result, 12 primer pairs produced reproducible polymorphic bands within diverse 4 strains and these selected markers were further characterized in 32 Flammulina velutipes strains. A total of 34 alleles were detected using the 12 markers, with an average of 3.42 alleles and the number of alleles ranged from two to seven per locus. The major allele frequency ranged from 0.42 (GB-FV-127) to 0.98 (GB-FV-166), and values for observed (HO) and expected (HE) heterozygosity ranged from 0.00 to 0.94 (mean = 0.18) and from 0.03 to 0.67 (mean = 0.32), respectively. SSR loci amplified with GB-FV-127 markers gave the highest polymorphism information content (PIC) of 0.61 and mean allele number of five, while for loci amplified with GB-FV-166 markers these values were the lowest, namely 0.03 and two. The mean PIC value (0.29) observed in the present study with average number of alleles (3.42). The genetic relationships among 32 Flammulina velutipes strains based on SSR data were generated by UPGMA cluster analysis. In conclusion, we succeeded in developing 12 polymorphic SSRs markers from SSR-enriched library of Flammulina velutipes. These SSRs are presently being used for phylogenic analysis and evaluation of genetic variations. In future, these SSR markers will be used in clarifying taxonomic relationships among the Flammulina velutipes.

Simple sequence repeats (SSR), also referred to “microsatellites” consist of tandemly repeated short DNA sequence motifs and have been applied in various marker-based studies. SSRs were isolated and characterized from ‘Heuktari’ and ‘Miso’, which are major oyster mushroom cultivars in Korea, by genome sequencing and bioinformatic analysis. The genome sizes of ‘Heuktari’ and ‘Miso’ were estimated to be 40.8 and 40.3 Mb, respectively, which are larger than those of other P. ostreatus species (PC9 and PC10) and smaller than those of P. eryngii (KNR2312P5). In total, 949 and 968 SSRs were found in the ‘Heuktari’ and ‘Miso’ genomes, respectively. Comparative analysis of five mushrooms including P. ostreatus var. florida (PC9 and PC15) and P. eryngii revealed that the number of SSRs in ‘Heuktari’ and ‘Miso’ were the highest among them. All mushrooms studied showed similar SSR distribution patterns. Tri-, hexa-, and octanucleotide motifs accounted for the top three fractions of all SSRs.

Twenty Inter simple sequence repeat (ISSR) primers were used to assess genetic diversity of 64 Agaricus strainsincluding 45 A. bisporus strains and other 19 Agaricus spp. ISSR primers, (GA)T, (AG)YC, (GA)C and (CTC) amplified PCRpolymorphic bands between the Agaricus species or within A. bisporus strains. PCR polymorphic bands were inputted forUPGMA cluster analysis. The varieties, Saea, Saedo, Saejeong and Saeyeon that have recently been developed in Korea wereinvolved in the same group with closely genetic relationship of coefficient similarity over 0.92, whereas, other Korean strains weregenetically related to A. bisporus strains that were introduced from USA, Eroupe and Chinese. Furthermore, ISSR-PCRpolymorphism could potentially be used to identify homokaryon isolates.

Twenty Inter simple sequence repeat (ISSR) and 30 SSR primers were used to assess genetic diversity of 64 Agaricus strains including 45 A. bisporus strains and other 19 Agaricus spp. Of them, four ISSR primers, (GA)₈T, (AG)₈YC, (GA)₈C and (CTC)₆and seven SSR markers produced PCR polymorphic bands between the Agaricus species or within A. bisporus strains. PCR polymorphic bands were inputted for UPGMA cluster analysis. Forty five strains of A. bisporus are genetically clustered into 6 groups, showing coefficient similarity from 0.75 to 0.9 among them. The varieties, Saea, saedo, Saejeong and Saeyeon that have recently been developed in Korea were involved in the same group with closely genetic relationship of coefficient similarity over 0.96, whereas, other strains were genetically related to A. bisporus strains that were introduced from USA, Eroupe and Chinese.

Geographic clines in genetic polymorphisms are widely believed as an evidence of climate change. We hypothesized green peach aphid, Myzus persicae Sulzer, one of the major insect pests in highland chinese cabbage cultivation, may also have some interactions with climate change. As the first step, we tried to find the available markers from six local strains (five collected at different heights in Hoengseong and Pyeongchang area and one from laboratory). A strain from Jeju island was used as an out-group. Although there was no significant difference in sequences of partial ribosomal RNA fragment and mitochondrial cytochrome oxidase I, and esterase isozyme pattern, we found four inter-simple sequence repeat (ISSR) markers in 22 used ISSR primers (+AGA, +CCA, +CGA, CGA+). These primers can be used as good markers to trace the M. persicae gene flow because they showed specific bands according to local strains.

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.