큰느타리(Pleurotus eryngii) 품종 판별을 위한 초위성체유래 다중 표지 개발

임착한,김경희,제희정,알리 아스자드,김민근,정완규,이상대,신현열,류재산 한국균학회 2014 韓國菌學會誌 Vol.42 No.2

For development of a method for differentiation of Pleurotus eryngii cultivars, simple sequence repeats (SSR) fromwhole genomic DNA sequence analysis was used for genotyping and two multiplex-SSR primer sets were developed. These SSRprimer sets were employed to distinguish 12 cultivars and strains. Five polymorphic markers were selected based on the genotypingresults. PCR using each primer produced one to four distinct bands ranging in size from 200 to 300 bp. Polymorphism informationcontent (PIC) values of the five markers were in the range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pairgroupmethod with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivarsinto two clusters. Cluster I and II were comprised of four and eight cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 andSSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivars and strains with 21 alleles and a PIC value of0.9090. These results might be useful in providing an efficient method for the identification of P. eryngii cultivars with separatePCR reactions. 큰느타리 품종구분을 위한 마커의 개발을 위하여 큰느타리 전체 유전자 염기서열을 바탕으로 제작한 484개의 SSR 마커를 사용하여 다형성 분석을 실시하였다. 그 결과 각275개의 primer에서 다형성이 관찰되었다. 이 중 품종간에다양한 패턴을 나타내는 5개의 마커를 최종 선발하였다. 이들 마커의 PIC 값은 0.6627에서 0.6848로 나타났고, 평균값은 0.6775였다. 이 결과를 밴드 이미지 인식 방법으로dendrogram을 작성하였다. UPGMA 집괴분석 결과, 큰느타리 품종은 크게 Cluster 1과 Cluster 2로 구분되었다. SSR primer를 이용한 PCR 결과 나타나는 품종별 고유의 DNA 밴드를 품종특이적 마커로 개발하기 위하여, 선발된 마커중에서 SSR312과 SSR366, SSR178과 SSR 277 마커를 조합하여 초위성체 유래 다중 표지 세트를 개발하였다. Multiplex- SSR 마커의 사용을 통해 두번의 PCR 반응만으로 본 연구에서 사용된 12개의 큰느타리 품종을 구분할 수 있었다.

한국 콩 보급품종을 포함한 엘리트품종의 SSR마커에 의한 유전적 다양성과 품종판별

장성진,박수정,박경호,송항림,조용구,정승근,강정훈,김홍식 한국작물학회 2009 Korean journal of crop science Vol.54 No.2

우리나라에서 1994년부터 2007년까지 보급된 콩 20개 보급품종과 6개의 유망품종을 포함한 26개의 엘리트품종들을 SSR마커를 이용하여 유전적 다양성과 유연관계를 분석하고, 품종을 판별한 결과를 요약하면 다음과 같다. 1. SSR마커 15개를 이용하여 분석한 결과 총 201개의 대립인자가 확인되었고, 각 유전좌별로 최소 8개(Satt141)에서 최대 19개(Satt197)의 대립인자가 확인되었으며, 마커당 대립인자수는 평균 13.4개이었다. 2. 15개 SSR마커에 의한 국내 콩 엘리트품종들의 유전적다양성 (PIC값)은 평균 0.874이었고 그 범위는 0.931-0.782이었으며, 마커별로는 Satt197이 0.931로 가장 높았고 Satt141이 0.782로 가장 낮았다. 3. SSR마커를 이용한 유전적거리에 의한 군집분석한 결과, 26개 품종이 3개 그룹으로 분류되었으며, I그룹에 2품종(7.7%), II그룹에 7품종(26.9%), 그리고 III그룹에 17품종(65.4%)이 속하였다. 4. SSR마커에 의하여 분류된 3그룹내의 유전적 다양성은 0.720-0.799으로 평균 0.769이었고, 그룹간의 유전적 다양성은 0.725-0.857으로 평균 0.813이었다. 그룹간이 그룹내보다 유전적 다양성이 더 높았으며, 유연관계는 I그룹은 II그룹 및 III그룹과 유전적거리가 가까웠으며, II그룹과 III그룹간은 서로 유전적거리가 다소 멀었다. 5. 다형성이 높은 5개의 SSR마커 중에서 2개 마커를 이용한 5개조합(Satt197+Sat088 , Satt197+Satt245, Sat088+Sat036 , Sat088+Satt245 , Satt185+Satt245)이 선정되었으며, 이중 어느 조합을 사용하여도 26개 엘리트품종 모두의 판별이 가능하였다. A total of 26 Korean elite soybean cultivars including 21 certified cultivars was assessed to evaluate genetic diversity and to analyze relationship among them based on 15 SSR markers. Fifteen SSR markers generated a total of 201 alleles. Average number of alleles per SSR marker was 13.4 with a range from 8 to 19. Genetic diversity of 26 cultivars estimated by PIC value ranged from 0.782 to 0.931 with an average of 0.874. PIC value of Satt197 was the highest with 0.931 and Satt141 was the lowest with 0.782 among 15 SSR markers. Cluster analysis based on genetic distances classified 26 soybean cultivars into 3 clusters. Cluster I, II and III included 2, 7 and 17 cultivars, respectively. Average genetic diversity within clusters was 0.769 with a range from 0.720 to 0.799. Average genetic diversity between clusters was 0.813 with a range from 0.725 to 0.857. Genetic diversity was higher between clusters than within clusters. Genetic relationship among clusters was near between I and II, and I and III and far between II and III cluster. All of 26 Korean elite soybean cultivars could be identified by using any of 5 combinations of 2 SSR markers with higher PIC value, i.e, Satt197+Sat088 , Satt197+Satt245, Sat088+Sat-036 , Sat088+Satt245 and Satt185+Satt245.

SSR 마커에 의한 한국 콩 품종의 판별

김성훈,정종욱,문중경,우선희,조용구,정승근,김홍식 韓國作物學會 2006 Korean journal of crop science Vol.51 No.7

SSR 마커를 이용하여 우리나라 콩의 품종판별 기술을 확립하기 위하여 1913년부터 2002년까지 국내에서 육성된 콩 91개 품종에 대하여 5개의 SSR마커(Sat043, Sat036, Sat022, Sat088 및 Satt045)를 이용하여 판별하였다. 판별용으로 이용된 SSR 5개 마커의 총 대립인자수는 64개이었고, 범위는 10~15 개이었으며, 평균 대립인자 수는 12.8개이었다. PIC값은 0.790(Satt045)~0.905(Sat043) 의 범위이었으며, 평균 PIC값은 0.857이었다. SSR 마커 5개의 조합으로 5단계의 판별을 통하여 총 91품종 중에서 82품종이 구별되어 약 90%가 판별되었다. 판별 1단계에서 Sat043으로 판별하였을때 부석의 1품종이, 2단계의 Sat036으로는 호장콩 등 34품종이, 3단계의 Sat022로는 단경콩 등 29품종이, 4단계의 Sat088로는 신팔달콩2호 등 12품종이, 5단계의 Satt045로는 새별콩 등 6품종이 판별되었다. 서로 간에 판별되지 않은 품종들은 형태적 특성에 의하여 구별이 가능하였다. The objective of this study was to develop a technique for the cultivar discrimination using SSR markers in soybean. A total of 91 soybean cultivars developed from 1913 to 2002 in Korea were evaluated by five polymorphic SSR markers (Sat043, Sat036, Sat022, Sat088 and Satt045). Five SSR markers generated a total of 64 alleles and the number of alleles for each SSR marker ranged from 10 to 15 with average of 12.8. Polymorphic information contents (PIC) by five markers of 91 cultivars were ranged from 0.790 to 0.905 with average of 0.857. A total of 82 cultivars (90%) among 91 soybean cultivars could be individually discriminated by combination of five SSR markers through five step analysis. A cultivar, Buseok, by Sat043 at the first step, 34 cultivars including Hojangkong by Sat036 at the second step, 29 cultivars including Dankyeongkong by Sat022 at the third step, 12 cultivars including Sinpaldalkong 2 by Sat088 at the fourth step, and 6 cultivars including Saebyeolkong by Satt045 at the fifth step were discriminated. Soybean cultivars which were not discriminated by SSR markers could be discriminated by morphological characteristics

Characterization and development of EST-SSR markers in sweet potato ( <i>Ipomoea batatas</i> (L.) Lam)

Kim, Jin-Hee,Kim, Jun-Hoi,Jo, Won-Sam,Ham, Jeong-Gwan,Chung, Il Kyung,Kim, Kyung-Min Springer Berlin Heidelberg 2016 3 Biotech Vol.6 No.2

<P>In this study, a cDNA library was constructed from the total RNA of sweet potato leaves. A total of 789 copies of the cDNA were cloned in <I>Escherichia coli</I> by employing the pGEM-T Easy vector. Sequencing was carried out by Solgent Co. (Korea). As many as 579 expressed sequence tag–simple sequence repeat (EST-SSR) markers were designed (73.38%) from the known cDNA nucleotide base sequences. The lengths of the developed EST-SSR markers ranged from 100 to 499 bp (average length 238 bp). Their motif sequence types were varied, with most being dinucleotides and pentanucleotides, and the most commonly found motifs were CAGAAT (29.0%) and TCT (2.8%). Based on these SSR-containing sequences, 619 pairs of high-quality SSR primers were designed using WebSat and Primer3web. The total number of primers designed was 144. Polymorphism was evident in 82 EST-SSR markers among 20 Korean sweet potato cultivars tested and in 90 EST-SSR markers in the two parents of a mapping population, Yeseumi and Annobeny. In this study, the hexaploid sweet potato (2<I>n</I> = 6<I>x</I> = 90) EST-SSR markers were developed in the absence of full-sequence data. Moreover, by acting as a molecular tag for particular traits, the EST-SSR marker can also simultaneously identify information about the corresponding gene. These EST-SSR markers will allow the molecular analysis of sweet potato to be done more efficiently. Thus, we can develop high-quality sweet potato while overcoming the challenges from climate change and other unfavorable conditions.</P>

Evaluation of Genetic Diversity among Soybean Genotypes Using SSR and SNP

P. Tanya,P. Srinives,T. Toojinda,A. Vanavichit,Bo-Keun Ha,Jeong-Suk Bae,Jung-Kyung Moon,Suk-Ha Lee 韓國作物學會 2001 Korean journal of crop science Vol.46 No.4

Two different types of molecular markers, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP), were used to measure genetic diversity among five Korean, eight Thai, and three wild soybeans. For SSR analysis, a total of 20 markers were surveyed to detect polymorphisms. For SNP analysis, four primers were designed from consensus sequence regions on disease resistance protein homolog genes, and used to amplify the genomic region. The PCR products were sequenced. A number of polymorphic SSR and SNP bands were scored on all genotypes and their genetic similarity was measured. Clustering analysis was performed independently on both types of markers. Clustering based on SSR markers separated the genotypes into three main groups originated from Korea, Thailand, and wild soybeans. On the other hand, two main groups were classified using SNP analysis. It seemed that SSR was more informative than SNP in this study. This may be due to the fact that SNP was surveyed on the smaller genomic region than SSR. Grouping based on the combined data of both markers revealed similar results to that of SNP rather than that of SSR. This might be due to the fact that more loci from SNP were considered to measure genetic relatedness than those from the SSR.

큰느타리(Pleurotus eryngii) 품종 판별을 위한 초위성체 유래 다중 표지 개발

임착한 ( Chak Han Im ),김경희 ( Kyung Hee Kim ),제희정 ( Hee Jeong Je ),알리아스자드 ( Asjad Ali ),김민근 ( Min Keun Kim ),정완규 ( Wan Kyu Joung ),이상대 ( Sang Dae Lee ),신현열 ( Hyunyeol Shin ),류재산 ( Jae San Ryu ) 한국균학회 2014 Mycobiology Vol.42 No.2

For development of a method for differentiation of Pleurotus 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 genotyping results. PCR using 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 the range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pairgroup method with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivars into two clusters. Cluster I and II were comprised of four and eight cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 and SSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivars and strains with 21 alleles and a PIC value of 0.9090. These results might be useful in providing an efficient method for the identification of P. eryngii cultivars with separate PCR reactions.

SSR 표식에 의한 차나무 계통간 유전 분석

류재일(Jae-Il Lyu),이선하(Seonha Lee),김길자(Gil-Ja Kim),박인진(In-Gin Park),배창휴(Chang-Hyu Bae) 한국차학회 2005 한국차학회지 Vol.11 No.2

차 수집종 집단의 분자유전학적 특성을 검토하기 위하여 한국 수집종 32계통, 대만종 3품종, 중국종 4품종, 일본종 6품종, 총 45 수집계통에 대하여 SSR분석을 수행하였다. SSR 분석결과 총 DNA 밴드수 92개 중 87%가 다형성을 나타냈다. 80개의 다형성 DNA 밴드 중 27개가 계통 특이적으로 나타났으며, 이 DNA 밴드는 동정을 통해 차나무 개체식별에 활용될 수 있을 것이다. 총 45 수집계통 간의 유사도지수는 0.10~1.00 범위였고, 한국수집계통간의 유사도지수는 0.10~0.97로 나타났다. 군집분석 결과 총 45 수집계통이 7개 그룹으로 나눠졌다. 한국수집계통은 제4그룹을 제외한 나머지 그룹에 모두 분포하였으며, 이 중 3, 5, 6, 7그룹은 한국 수집종만으로 구성되었다. 이와 같이 SSR 분석을 통하여 본 연구에서 사용된 한국 수집계통은 높은 유전적 다양성을 가지고 있음을 확인할 수 있었으며, 이 수집 차나무집단은 차의 품종육성을 위한 유전자원으로 활용될 수 있을 것으로 기대된다. Simple sequence repeat (SSR) analysis was examined to detect genetic characteristics at molecular level among 45 accessions of tea (Camellia sinensis var. sinensis) from Korea, Japan, China and Taiwan. As a result of SSR analysis, a total of 92 DNA bands were detected and 80 were polymorphic with a polymorphic rate 87%. Out of 80 polymorphic bands, 27 DNA bands showed line specific. The DNA bands might be used as molecular marker to detected tea at plant level. Genetic similarity shows very different from 0.10 to 1.00 among the 45 accessions and 0.10 to 0.97 among the Korean accessions. By cluster analysis, the 45 accessions revealed seven groups. The Korean accessions belonged to all groups except group 4. Out of the Korean accessions group 3, 5, 6 and 7 did not have Japanese, Chinese and Taiwanese varieties. The results indicate that the Korean accessions showed high genetic diversity by analyzing SSR markers, thus the Korean accessions will be used as basic gene pool for tea breeding.

Distribution and Frequency of SSR Motifs in the Chrysanthemum SSR-enriched Library through 454 Pyrosequencing Technology

Kyaw Thu Moe,Sang-Bog Ra,Gi-An Lee,Myung-Chul Lee,Ha-Seung Park,Dong-Chan Kim,Cheol-Hwi Lee,Hyun-Gu Choi,Nak-Beom Jeon,Byung-Jun Choi,Ji-Youn Jung,Kyu-Min Lee,Yong-Jin Park 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.5

국화과(Compositae)는 현화식물 중 세계에서 가장 넓게 분포하고, 쌍자엽식물 중 가장 진화된 식물분류군이며, 우리나라에는 약 300여종이 존재하는 것으로 알려져 있다. 구절초, 감국, 쑥, 쑥갓, 개미취, 참취, 곰취 등 국화과 식물들은 예로부터 민간에서 약용 및 식용 소재로써 다양하게 사용되어왔다. 본 연구는 국화 및 국화근연종 유용유전자원 선발을 통하여 육종 소재를 확대하고, 중간모본 및 신품종 육성기반을 구축하고자 DNA 마커시스템의 개발을 위해 수행되었다. 1. 화단국인 Smileball(Dendranthema grandiflorum) 품종을 사용하여 SSR-enriched library를 작성하였고, GS FLX 분석을 통해 18.83Mbp의 염기서열 결과를 얻었으며, read의 평균 길이는 280.06bp로 나타났다. 2. 단순반복염기서열(SSR) 부위를 포함하는 26,780개 clones 중 di-nucleotide motifs가 16,375개(61.5%)로 우세하였고, trinucleotide motifs(6,616개, 24.8%), tetra-nucleotide motifs(1,674개, 6.3%), penta-nucleotide motifs(1,283개, 4.8%), hexa-nucleotide motifs(693개, 2.6%) 순으로 나타났다. 3. 얻어진 di-nucleotide motifs들 중에서는, AC/CA class가 93.5%로 대부분이었고, tri-nucleotide motifs에서는 AAC class가 50.5%, tetra-nucleotide motifs는 ACGT class가 43.6%이고, pentanucleotide motif에서는 AACGT class 27.2%이며, hexa-nucleotide motif에서는 ACGATG class 21.8%였다. 4. 얻어진 염기서열 결과를 토대로 다양한 motif를 갖는 100개의 SSR 마커를 제작하였고, 차후 이를 활용하여 국화 유전자원의 다형성 및 유전자형 분석을 통해 분자유전학적 다양성 및 집단의 구조분석이 가능하고, 국화의 분자육종기반 구축을 위한 유용한 도구가 될 것 이다. Chrysanthemums, often called mums or chrysanths, belong to the genus Chrysanthemum, which includes about 30 species of perennial flowering plants in the family Asteraceae. We extracted DNA from Dendranthema grandiflorum (‘Smileball’) to construct a simple sequence repeat (SSR)-enriched library, using a modified biotin-streptavidin capture method. GS FLX (Genome Sequencer FLX System which provides the flexibility to perform the broad range of applications) sequencing (at the 1/8 run specification) resulted in 18.83 mega base pairs (Mbp) with an average read length of 280.06 bp. Sequence analyses of all SSR-containing clones revealed a predominance of di-nucleotide motifs (16,375, 61.5%) followed by tri-nucleotide motifs (6,616, 24.8%), tetra-nucleotide motifs (1,674, 6.3%), pentanucleotide motifs (1,283, 4.8%), and hexa-nucleotide motifs (693, 2.6%). Among the di-nucleotide motifs, the AC/CA class was the most frequently identified (93.5% of all di-nucleotide types), followed by the GA/AG class (6.1%), the AT/TA class (0.4%), and the CG/GC class (0.03%). When we analyzed the distribution of different repeat motifs and their respective numbers of repeats, regardless of the motif class, of 100 SSR markers, we found a higher number of di-nucleotide motifs with 70 to 80 repeats; we also found two di-nucleotide motifs with 83 and 89 repeats, respectively, but their product lengths were within optimum size (297 and 300 bp). In future work, we will screen for polymorphisms of possible primer pairs. The results will provide a useful tool for assessing molecular diversity and investigating the population structure among and within Chrysanthemum species.

Genetic diversity assessment of lily genotypes native to Korea based on simple sequence repeat markers

Shipra Kumari,Young-Sun Kim,Bashistha Kumar Kanth,Ji-Young Jang,Geung-Joo Lee 한국식물생명공학회 2019 JOURNAL OF PLANT BIOTECHNOLOGY Vol.46 No.3

Molecular characterization of different genotypes reveals accurate information about the degree of genetic diversity that helps to develop a proper breeding program. In this study, a total of 30 EST-based simple sequence repeat (EST-SSR) markers derived from trumpet lily (Lilium longiflorum) were used across 11 native lily species for their genetic relationship. Among these 30 markers, 24 SSR markers that showed polymorphism were used for evaluation of diversity spectrum. The allelic number at per locus ranged from 1 at SSR2 locus to 34 alleles at SSR15 locus, with an average of 11.25 alleles across 24 loci observed. The polymorphic information content, PIC, values ranged from 0.0523 for SSR9 to 0.9919 for SSR2 in all 24 loci with an average of 0.3827. The allelic frequency at every locus ranged from 0.81% at SSR2 locus to 99.6% at SSR14 locus. The pairwise genetic dissimilarity coefficient revealed the highest genetic distance with a value of 81.7% was in between L. dauricum and L. amabile. A relatively closer genetic distance was found between L. lancifolium and L. dauricum, L. maximowiczii and L. concolor, L. maximowiczii and L. distichum (Jeju), L. tsingtauense and L. callosum, L. cernuum and L. distichum (Jeju ecotype), of which dissimilarity coefficient was 50.0%. The molecular fingerprinting based on microsatellite marker could serve boldly to recognize genetically distant accessions and to sort morphologically close as well as duplicate accessions.

Genetic diversity assessment of lily genotypes native to Korea based on simple sequence repeat markers

이긍주,Shipra Kumari,Bashistha Kumar Kanth,김영선,장지영 한국식물생명공학회 2019 JOURNAL OF PLANT BIOTECHNOLOGY Vol.46 No.3

Molecular characterization of different genotypes reveals accurate information about the degree of genetic diversity that helps to develop a proper breeding program. In this study, a total of 30 EST-based simple sequence repeat (EST-SSR) markers derived from trumpet lily (Lilium longiflorum) were used across 11 native lily species for their genetic relationship. Among these 30 markers, 24 SSR markers that showed polymorphism were used for evaluation of diversity spectrum. The allelic number at per locus ranged from 1 at SSR2 locus to 34 alleles at SSR15 locus, with an average of 11.25 alleles across 24 loci observed. The polymorphic information content, PIC, values ranged from 0.0523 for SSR9 to 0.9919 for SSR2 in all 24 loci with an average of 0.3827. The allelic frequency at every locus ranged from 0.81% at SSR2 locus to 99.6% at SSR14 locus. The pairwise genetic dissimilarity coefficient revealed the highest genetic distance with a value of 81.7% was in between L. dauricum and L. amabile. A relatively closer genetic distance was found between L. lancifolium and L. dauricum, L. maximowiczii and L. concolor, L. maximowiczii and L. distichum (Jeju), L. tsingtauense and L. callosum, L. cernuum and L. distichum (Jeju ecotype), of which dissimilarity coefficient was 50.0%. The molecular fingerprinting based on microsatellite marker could serve boldly to recognize genetically distant accessions and to sort morphologically close as well as duplicate accessions.