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.

Differentiation of Pleurotus eryngii is laborious and time-consuming tasks especially in mycelial status. For development of a method for differentiation of P. eryngii cultivars, simple sequence repeats (SSR) from whole genomic DNA sequence analysis was used for genotyping and two multiplex-SSR primer sets were developed. These SSR primer sets were employed to distinguish 12 cultivars and strains. Five polymorphic markers were selected based on the genotypes. PCR with the each primer produced one to four distinct bands ranging in size from 200 to 300 bp. Polymorphism information content (PIC) values of the five markers were in range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pair-group method with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivars into 2 clusters. Cluster I and II comprised of 4 and 8 cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 and SSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivar and strains with 21 allele with a PIC value of 0.9090. These results might be useful to provide an efficient method for the identification of P. eryngii cultivars with separate PCR reactions. (This work was supported by a grant from the Gold Seed Project [Supported by a grant from the IPET (213003-04-3-WTI11), MIFAFF, Republic of Korea.]

본 연구는 ICuGI Database를 활용하여 수박 상용 F1 품종의 순도검정용 EST-SSR 마커를 개발하기 위해 수행되었다. 총 353개 EST-SSR primer set을 선발하여 (주)NH종묘의 수박 F1 품종 7종과 각 품 종의 양친 11종에 대해 검정하였다. 이중 1차 테스트한 96개 primer set 중, ‘오렌지’는 primer WMU0056, ‘흑보’는 WMU0400, ‘신동’은 WMU0056와 WMU0400, ‘새로나’는 WMU0056, WMU0400, WMU0529에 대해 각 품종의 양친들이 다형성이 보였고 F1 개체에서는 이형접합의 유전자형 을 보였다. ‘해동’ F1의 순도검정용 마커를 찾기 위해 추가적인 122개의 primer set에 대해 PCR을 수 행한 결과, WMU0056, WMU0400, WMU0580, WMU1211, WMU4136, WMU448이 순도검정에 적합한 것으로 나타나, WMU0056와 WMU0400이 ‘해동’에서도 유용할 수 있었다. ‘꿀나라’와 ‘황피’의 경우에는 공시된 타 품종들에 비해 양친간 다형성율이 각각 5%와 2%로 매우 낮아 모든 353개 primer set을 테스트하였으며, 그 결과 ‘꿀나라’는 WMU5339, ‘황피’는 WMU7003이 순도검정 마커로 적합 한 것으로 확인되었다. 개발된 마커를 이용하여 실제 농가채종된 4개의 F1 품종의 순도를 검정한 결과, 모두 97.5% 이상의 순도로 확인되었다. 이와 같이 ICuGI Database에 공시된 수박 EST-SSR 마커는 공 우성의 유전자 특이적 마커로서 F1 순도검정에 효과적으로 사용될 수 있었다.

The soybean Kunitz trypsin inhibitor (KTI) protein is responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. Ti locus controls presence or absence of Kunitz trypsin inhibitor protein. Genetic recombination or tight linkage between Ti locus and Satt228 marker that has been identified to be tightly linked to the Ti locus was detected for marker assisted selection (MAS) using two F2 populations of titi genotype in this study. Two F2 populations were developed from the cross of A29 (KTI protein present, TiTi genotype, AA genotype in Satt228 marker) x Gaechuck#1 and Gaechuck#2 (KTI protein absent, titi genotype, BB genotype in Satt228 marker). Among 31 F2 plants derived from A29 x Gaechuck#1, twenty nine F2 plants show BB genotype that indicates no recombination between Satt228 marker and Ti locus. Only 2 F2 plants show AA genotype that indicates recombination between Satt228 marker and Ti locus. Twenty eight F2 plants derived from A29 x Gaechuck#2 show BB genotype that indicates no recombination between Satt228 marker and Ti locus. Expected genetic ratio between Satt228 marker and Ti locus was 3.6 cM in F2 population.

Background : Atractylodes japonica koidz (AJ) is a perennial herb that belongs to Atractylodes genus. The dried rhizome of AJ is known as ‘Baek-chul’. The ‘Baek-chul’ is used as important traditional medicine in north-east Asia. It is considered to be effective for the treatment of stomach disorder, virus, diuresis, inflammation, arthritis. AJ is heavily depend on import from china and only few studies have been carried out. In this study, we develop SSR marker to build a foundation of breeding, to analyze genetic diversity and to construct core collection. Methods and Results : AJ resources was collected from each different place. To find simple sequence repeat (SSR) marker, we sequenced genomic DNA of AJ resources using Illumina HiSeq 2000 System. As a result of next generation sequencing (NGS), we obtained putative SSR loci. From these SSR primers, 553 SSR primer sets were designed successfully and confirmed polymorphism by in silico analysis. Nucleotide motifs ranged from tri- to penta-. Among these, 48 primer were tested in 4 individuals by capillary electrophoresis. Finally, selected 28 SSR marker were showed clear band and polymorphism by Electrophoresis. Conclusion : In this study, we developed 28 polymorphic SSR marker using NGS, and it could be used for analyzing genetic diversity of A. japonica. These marker would be useful for breeding of new cultivar in the future.

Background : Several members of the genus Clausena have a great potential as a candidate for the identification of new drug lead molecules, but lack of their genomic information can be a hindrance for the verification of the genetic background for future use. To broaden and delve into the genomic features of this genus, Clausena excavata, an important medicinal plant in many Asian countries, was used for RNA-seq analysis. Methods and Results : A ten ㎍ of the total RNA was used for mRNA isolation using oligo-dT beads and random sheared mRNAs were used to prepare a cDNA library for a illumina hiseq 2500 analysis. In total, 17,580,456 trimmed clear reads from the illumina hiseq 2500 were used for de novo assembly using three assemblers, CLC genomics workbench, velvet-oases, and Trinity. A total of 16,638 non-redundant unigenes with an average length of 755 bp were generated by the assembly. The functional categorization of the identified unigenes by a gene ontology (GO) term resulted in 2,305 genes in the cellular component, 5,577 in the biological processes, and 8,056 in the molecular functions, respectively. The top sub-category in biological processes was the metabolic process with 4,374 genes. Among annotated genes, 3,006 were mapped to 123 metabolic pathways by KEGG metabolic pathway analysis tool. The search for simple sequence repeats (SSRs) resulted in 845 SSRs from 749 SSR-containing unigenes and the most abundant SSR motifs was AAG/CTT with 179 occurrences. Twelve SSR markers were tested for cross transferability among five Clausena species; eight of them exhibited polymorphism. Conclusion : In the present study, genomic resources of the genus Clausena were enriched through RNA-seq and SSR markers, which will serve as valuable resources for genomic/genetic studies of the genus Clausena and close relatives.

Background: In the herbal medicinal industry, Angelica gigas Nakai, Angelica sinensis (Oliv.) Diels. and Angelica acutiloba (Siebold & Zucc.) Kitag. are often confused, because the roots of the three species can not be distinguished by their appearance. This confusion can cause serious side effects. In this study, we determined the origins of Angelica roots distributed in the Korean market using the simple sequence repeat (SSR) markers developed based on the A. gigas chloroplast DNA sequence. Methods and Results: We collected twenty seven A. gigas and three A. acutiloba samples from the Seoul, Daegu, and Cheongju herbal medicinal markets. Fifty sections of one collection were mixed and ground to make a powder, which was used for DNA extraction using the cetyl trimethylammonium bromide (CTAB) method. Chloroplast based SSR markers were applied to the DNA for the determination of the species. In addition, polymorphism was found in eight samples. The phylogenetic analysis showed that the A. gigas roots collected from herbal medicinal markets were clearly discriminated from A. sinensis and A. acutiloba even though they were grouped into four clusters. Conclusions: This study showed that chloroplast based SSR markers would help the discrimination of Angelica roots in the Korean herbal medicinal industry and the markers are useful to prevent confusion between Angelica roots.

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, it is easy to be confused with other species of the same genus. Simple sequence repeat (SSR) marker is a powerful tool for distinguish specific species. In addition, there are many studies that show species-specific polymorphisms in chloroplasts SSR. In this study, we developed chloroplast SSR markers that can distinguish C. lanceolata from 6 Codonopsis species. Methods and Results : We collected 6 Codonopsis species include C. lanceolata. and extrated DNA using CTAB method. The DNA was diluted to 10 ng/㎕ and kept at –20℃. We designed the primer sets using CLC Main Workbench based on chloroplast DNA SSR region of C. lanceolata. PCR was performed using three independent plants for each species. Conclusion : We designed six primer sets from six SSR regions of C. lanceolata cpDNA. All of the primer sets amplified the amplicon effectively. Two of the 6 primer sets had polymorphism. We could distinguish C. lanceolata from 6 Codonopsis species using two primer sets.

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.

본 연구는 감 수집종의 분류 및 품종 육종을 위하여 EST-SSR 마커를 개발해 유전적 유연관계를 분석하고, 형태적 유연관계를 비교 분석하여 DNA 마커의 효율성을 극대화한 연구 결과이다. 경북농업기술원 상주감시험장에서 수집한 42품종을 대상으로 6가지의 양적형질(과실크기, 과고, 과경, 과경굵기, 과경길이, 종자크기)과 19가지의 질적형질(횡단면, 종단면, 골의 정도, 얕은 동심원 균열, 옆모양, 정부열과, 세로홈, 꽃받침 끝 주름, 배꼽 홈길이, 꽃받침 쪽의 홈, 꽃받침 크기)을 사용하여 형태적 유연관계를 분석하였다. 유전적 유연관계를 분석하기 위해 수집한 감에서 cDNA library를 만들어 sequence를 분석한 후, PCR을 통해 얻은 polymorphism이 인정되는 25개의 primer set에서 16개의 EST-SSR primer set를 선발하였다. 수집한 감 42품종의 형태적 유연관계와 개발한 14개의 EST-SSR 마커를 이용하여 유전적인 유연관계를 분석한 결과 형태적 유연관계에서는 여러 그룹이 형성되었지만 coefficient가 0.02 이하로 형성되어 형태적 특성을 사용해 분류하기는 어려웠다. 유전적 유연관계는 coefficient 0.77에서 3개 그룹으로 분류되어, 상주수수감과 상주수꽃감, 밀양반시와 밀양고동시, 영동반시와 영동수시는 각각 같은 그룹으로 분류되었다. 형태적 분석과 유전적 분석의 상관관계를 조사한 결과 형태적 분석의 유사도 거리와 유전적 분석의 유사도 거리 간의 값이 -0.03으로 유의성이 매우 낮게 나왔다. 본 실험에서 얻어진 분자마커는 (EST-SSR 마커) 국내 감육종 효율 증진뿐만 아니라 우수형질을 도입하는데 유용하게 이용할 수 있을 것으로 기대된다.

Molecular genetic markers have been widely used as powerful tools for analyzing the genome. In particular, SSR markers have practical applications in breeding systems because they can be used in high-throughput analyses for genetic mapping, heritable diversity testing, purity analysis, and marker-assisted selection. Currently, due to technical advances in DNA sequencing, large sequence databases are available for large-scale SSR mining and marker development. Here, we describe an automated method, the SSR Finder program, for SSR discovery in the onion sequence database, and primer design for amplifying the detected SSRs. A total of 1,049 SSR primers were obtained for genetic purity testing, and 100 SSR sets were analyzed in 14 bulb onion breeding lines using clustering analysis. A total of 20 selected markers from screening of all 1,049 SSR primers, were finally applied for genetic purity testing in three breeding lines, NW1, NW9, and NW10. The initial tests showed that 15%, 71%, and 97% of individuals within NW1, NW9, and NW10, respectively, were genetically homogeneous. These markers produced using the SSR Finder will be useful for investigating the genetic purity of onion breeding lines.

In this study, Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) analyses were used to clarify the genetic polymorphisms among Korean ginseng cultivars and breeding lines and to classify them into distinct genetic groups. Polymorphic and reproducible bands were produced by 14 primers out of total 30 primers used in this study. Fourteen EST-SSR loci generated a total of 123 bands. Amplified PCR products showed the highly reproducible banding patterns at 110~920 bp. The number of amplified bands for each EST-SSR primers ranged from 2 to 19 with a mean of 8.8 bands. P26 and P35 primers showed 13 and 12 banding patterns, respectively. The number of alleles for each EST-SSR locus ranged from 1.67 to 2.00 with a mean of 1.878 alleles. P34 and P60 primers showed the highest and the lowest genetic polymorphism, respectively. Cluster analysis based on genetic similarity estimated by EST-SSR markers classified Korean cultivars and breeding lines into 4 groups. Group included Gopoong and Chunpoong and 9 breeding lines (55%), group included 2 breeding lines (10%), group included 3 breeding lines (15%), group included Gumpoong and 3 breeding lines (20%). Consequently, the EST-SSR marker developed in this study may prove useful for the evaluation of genetic diversity and differentiation of Korean ginseng cultivars and breeding lines.

This study was carried out to develop expressed sequence tag-Simple sequence repeat (EST-SSR) markers of brown plant hopper resistance gene originated from a rice cultivar ‘Cheongcheong’ and sensitive rice cultivar ‘Nakdong’. Total RNA extracted from the leaves of ‘Cheongcheong’ and ‘Nakdong’ were used to synthesize a cDNA library. As a result of analyzing the cDNA library, EST-SSR sites were found and the EST-SSR primer sets were developed. This study enables to provide effective marker assisted selection (MAS) methods on the selection of white-backed planthopper resistance gene originated from a rice cultivar more simply, quickly and precisely. Furthermore, using this marker’s advantage of deriving from cDNA, it is possible to identity the white-backed planthopper resistance gene.

A set of nine Korean rice germplasm (KRG) along with the six indica lines were screened under irrigated non-stress and drought stress situations at IRRI in dry season (DS) 2011. The experiment received mild to moderate drought stress. Under irrigated situation, among all lines, IRRI119 yielded highest followed by PSBRc80 and PSBRc14. Among nine KRG, Hanareumbyeo yielded highest followed by Gayabyeo. Yield of Hanareumbyeo was similar to high yielding indica lines. Under drought, PSBRc14 provided the highest yield among indica lines and Hanarembyeo provided highest yield among nine KRG. Among nine KRG, Hanarembyeo provided the highest yield both under irrigated non-stress and drought stress situation. Parental polymorphism was performed with 125 SSR markers taking six KRG and three drought tolerant donors and polymorphic markers and japonica lines background specific markers were identified. The polymorphic markers in the region of three QTLs (DTY1.1, DTY2.2, DTY3.1) will be used for foreground genotyping for QTL introgression and background specific markers will be used for background genotyping. Sixteen rice germplasm could be separated into two main groups, japonica and indica groups by cluster analysis. The japonica and indica groups also classified as two subgroups, respectively. Based on results of screening of japonica lines under irrigated non-stress and drought stress situations, two KRG- Hanarembyeo and Jinmibyeo were selected for introgression of three QTLs (DTY1.1, DTY2.2 and DTY3.1) associated with grain yield under drought stress.

For the development of SSR marker system in Vicia villosa Roth, an enriched library was constructed by using a modified biotin-streptavidin capture method and the selected clones were sequenced with GS-FLX(454). Of 37,794 sequenced reads, we found that 8,474 reads (22.4%) were redundant, leaving 29,320 unique ones (77.6%). Among the unique clones, 17,174 reads (58.6%) were having microsatellite repeating motifs. Sequence analysis of all SSR-containing reads revealed a predominance of the di-nucleotide SSRs (62.5%). The tri-nucleotide and the tetra-nucleotide SSRs were 5.7% and 22.5%, respectively. As the di-nucleotide type, the AG/GA class of repeat motif was most frequently identified (55.0% of the total di-nucleotide SSRs), followed by the CT/TC class (19.5%), and the TA/AT class (12.1%). Among the tri-nucleotide SSRs, the AGT/GTA/TAG class of repeat motifs was predominant (22.2%), followed by the ACT/CTA/TAC class (17.8%). Among the tetra-nucleotide SSRs, the CTTT/TTTC/TTCT/TCTT class of repeat motifs was predominant (31.2%), followed by the AAAG/AAGA/AGAA/ GAAA class (19.9%). Finally, we designed 779 primer pairs from the flanking sequences of SSR containing reads. We are undertaking the analysis of polymorphisms using the diverse collected accessions of Vicia villosa Roth now. This newly developed SSR marker set shall provide a very useful tool for implementing molecular diversity assessment and population structure studies of Vicia villosa Roth onward.

본 실험은 SSR marker를 이용하여 밀 자원의 근연관계 및 유전자연관지도 작성의 기초 자료가 되는 anchor marker를 탐색하고자 수행되었다. 실험재료는 금강밀 등 41개 국내육성 품종 및 계통과 Genaro81 등 외국품종 7개 등 총 48개 자원을 사용하였고, SSR marker는 밀의 21개 염색체에서 각각의 short arm에 존재하는 3개와 long arm에 존재하는 5개 SSR marker를 선발하여 총 168개의 SSR primer set을 구성하였다. 이 중 108개의 SSR marker에 대한 PCR 실험결과 92개에서 다형성을 보였으며, 국내외 밀 자원은 SSR marker에 따라 1~7개의 loci에서 유전자다형성을 보였다. 선발된 SSR marker들은 국내외 밀 자원의 분류 및 mapping 집단의 anchor marker로서 이용될 수 있을 것이다.