AFLP Marker를 이용한 한국 재래돼지의 유전적 다양성 및 품종식별

정의룡,김우태,김연수,이정구,한상기 한국동물자원과학회 2001 한국축산학회지 Vol.43 No.6

본 연구는 한국재래돼지의 순수 혈통정립 및 유전자원 보존을 목적으로 AFLP 다형성을 분석하고 이를 유전적 표지인자로 이용하여 재래돼지의 유전적 특성을 분자 수준에서 규명하고 품종 집단내 유전적 변이성과 품종 특이적인 DNA marker를 탐색하며 동시에 타품종들과의 유전적 근연관계를 추정하기 위해 수행하였다. 13종류의 selective primer 조합형을 이용하여 분석한 결과 총 band의 수는 611개로 각 조합형 당 평균 47개의 band가 확인되었으며 이 가운데 다형적 band가 152개로 다형성 수준은 약24.5%였다. 한국 재래돼지의 다형율과 유전적 다양성 값은 각각 29.8%와 2.9로 개량종에 비해서 다소 높은 경향이었다. 한편, 13종류의 primer 조합형 가운데 E35/T38 및 E38/H13 primer 조합형에서 품종간의 차이를 나타내는 DNA marker가 확인되었다. 특히, 재래돼지에서 검출된 E35/T38 primer 조합형의 0.50kb, 0.25kb 및 0.38kb의 DNA band는 개량종과 명확히 구별되어 이들 품종 특이적 DNA band는 한국 재래돼지의 품종식별에 유용한 표지인자로 이용 가능할 것으로 기대된다. 품종간 유전적 근연관계에서 한국 재래돼지는 Hampshire종과 유전적 유사성이 가장 높은 것으로 추정되었다. 본 연구에서 검출한 한국 재래돼지의 AFLP 유전자 지문은 재래돼지 집단의 유전적 변이성 및 타 품종과의 근연관계 분석뿐만 아니라 경제형질과 연관된 marker 개발에 유용한 DNA 표지인자로 활용할 수 있을 것으로 기대된다. For the purpose of genetic conversation and utilization of Korean native pig(KNP) as a valuable animal genetic resource, DNA polymorphisms of AFLP (amplified fragment length polymorphisms) as genetic markers were analyzed in KNP and foreign pig breeds (Landrace, Duroc, Hamphsire and Yorkshire). Using these AFLP markers, the genetic structure and characteristics of KNP population were analyzed at the DNA level and the genetic variability and diversity within and between breed population were evaluated. Breed-specific DNA marker for KNP was screened, and phylogenetic relationship within and among breeds were estimated. A total of 611 AFLP markers were amplified by 13 selective primer combinations, and the average number of bands per primer combination was 47.0. Among them 152 bands were polymorphic (24.5%). The rate of polymorphisms and genetic diversity values of KNP (29.8% and 2.90) was higher than these of other foregin breeds. E35/T38 and E38/H13 primer combinations produced AFLP banding patterns which clearly discriminated between KNP and other foreign pig breeds. Three bands(0.50kb, 0.25kb and 0.20kb) identified in E35/T38 and two bands(0.45kb and 0.38kb) identified in E38/H13 primer combinations were present in all of the KNP examined, but not present in the foreign pig breeds. Therefore, these two primer combinations could be used as breed-specific DNA markers for breed identification of KNP. In comparison of genetic distances among pig breeds, KNP was the most closely related to the Hampshire breed. AFLP fingerprints may be useful for genetic variability and relationships and development of breed-specific DNA markers in pig breeds.

Amplified Fragment Length Polymorphism (AFLP) DNA Marker를 이용한 한국 재래흑염소육 감별

정의룡 한국축산식품학회 2002 한국축산식품학회지 Vol.22 No.4

본 연구는 AFLP-PCR 유전자 지문분석 기법을 이용하여 우리나라 고유의 동물유전자원으로서 재래흑염소의 품종 및 흑염소육 감별을 위한 품종 특이적 DNA marker를 개발하고자 수행하였다. 흑염소로부터 추출한 genomic DNA를 EcoR I/Hind III 및 Taq I/Hind III 2종류의 제한효소 조합으로 이중 절단한 후 10종류의 two selective primer조합형을 이용하여 분석한 결과 각 printer 조합형당 검출된 AFLP band의 수는 36~74개의 범위로 평균 55.5개였다. 그리고 검출된 총 555개의 band 가운데 polymorphic band의 수는 149 개로 다형성 수준은 약 26.8%로 추정되었다. 재래흑염소 품종 특이적인 AFLP marker를 탐색하고자 육용종 수입흑염소 및 4품종의 유용종 염소와 AFLP 지문양상을 비교 검토한 결과 M13/H13 primer 조합형에서 2.01과 1.26 kb의 2개 band 그리고 E35/H14 primer 조합형에서 1.65 kb의 1개 band가 재래흑염소의 품종 특이적 AFLP marker로 검출되었다. 그리고 E35/H14 primer 조합형에서 수입흑염소의 2.19, 2.03, 0.96 및 0.87 kb band, Saanen종의 2.13 kb band, Nubian종의 2.08 kb band는 각 해당 품종에만 특이적으로 출현하는 품종 특이적 band로 확인되었다. 또한, E35/H13 primer 조합형에서 재래흑염소를 특히, Saanen종과 식별이 가능한 4개의 DNA band가 확인되었다. 따라서, 본 연구에서 AFLP-PCR 기법을 이용하여 검출한 품종 특이적 DNA band들은 우리나라 재래흑염소, 수입흑염소 및 유용종 염소품종들간에 명확히 구별되어 재래종 흑염소 육과 육제품의 품종판별에 매우 유용한 DNA marker로 이용 가능할 것으로 기대된다. This study was carried out to develop the breed-specific DNA markers for breed identification of Korean native goat meat using amplified fragment length polymorphism (AFLP)-PCR techniques. The genomic DNAs of Korean native goat, imported black goat and four dairy goat breeds(Saanen, Alpine, Nubian and Toggenburg) were extracted from muscle tissues or blood. Genomic DNA was digested with a particular combination of two restriction enzymes with 4 base(Mse I and Taq I) and 6 base(EcoR I and Hind III) recognition sites, ligated to restriction specific adapters and amplified using the selective primer combinations. In AFLP profiles of polyacrylamide gels, the number of scorable bands produced per primer combination varied from 36 to 74, with an average of 55.5. A total of 555 bands were produced, 149(26.8%) bands of which were polymorphic. Among the ten primer combinations, two bands with 2.01 and 1.26 kb in M13/H13 primer and one band with 1.65 kb in E35/H14 primer were found to be breed-specific AFLP markers in Korean native goat when DNA bands were compared among the goat breeds. In the E35/H14 primer combination, 2.19, 2.03, 0.96 and 0.87 kb bands detected in imported black goat, 2.13 kb band in Saanen breed and 2.08 kb band in Nubian breed were observed as breed-specific bands showing differences between goat breeds, respectively. The E35/H14 primer combination produced four DNA bands distinguished between Korean native goat and Saanen breed. The is study suggested that the breed specific AFLP bands could be used as DNA markers for the identification of Korean native goat meat from imported black goat and dairy goat meats.

분자육종법과 관행육종법을 활용한 고식미계통 선발효율성 비교분석

서정필,조영찬,원용재,이정희,안억근,전재범,이점식,김명기,정응기,김보경 한국육종학회 2014 한국육종학회지 Vol.46 No.3

지금까지 밥맛특성이 우수한 품종을 육성하려는 노력이 부단히 진행되고 있으나, 저세대에서 미질을 평가하는 기준과 지표가 미흡한 실정이다. 고식미 품종육성과정 중 저세대에서 고식미계통을 선발하기에는 어려운 실정이다. 본 연구에서는 DNA분석에 의한 식미회귀식값을 자포니카 간의 교잡에서 유래된 저세대 계통선발에 적용하여 선발효율성을 관행육종법과 비교분석하였다. 9개 교배모본들을 군집분석하여 그룹간에 식미회귀식값과 밥의 윤기치의 연관분석을 실시한 결과, 유의성이 있었다. 분자육종법과 관행육종법으로 계통을 선발한 결과 선발집단규모는 각각 34.4%, 38.6%로 비슷하였고, 분자육종법과 관행육종법을 병행하였을 때는 19.5%로 집단규모가 상당히 줄었다. 밥의 윤기치와 식미회귀식값은 집단간에는 차이가 있었으나, 육종방법에 따라서는 차이가 없었다. 식미회귀식값은 13개 DNA마커를 가지고 값을 구하게 되는데, 본 실험에 사용된 교배조합에서 양친 간에 다형성을 보이는 DNA마커는 3~7개로 상당히 적어, 식미회귀식값을 구하여 표현형을 설명하기에는 부족한 것으로 생각된다. 식미회귀식값에 의한 고식미계통선발을 하려면 교배조합별로 DNA마커조합과 식미회귀식값을 다시 산출해서 사용을 하거나, 식미회귀식에 활용되는 모든 DNA마커에 다형성을 보이는 교배조합을 선정해서 적용한다면 어느 정도 효과가 있을 것으로 생각된다. 본 실험에 활용된 식미연관 13개 DNA마커에 의한 식미회귀식값은 모든 조합의 육종집단에서 선발에 활용하기는 어려울 것으로 생각된다. This paper compares selection efficiency for high palatability breeding lines using marker-assisted selection and conventional selection methods in rice. A total 4 cross combinations of japonica cultivars were selected using marker-assisted selection with a set of 13 DNA markers associated with grain quality and conventional selection methods in F3 and F5 generations assessing palatability using the Toyo taste meter. The multiple regression value with a set of 13 DNA markers was utilized as the marker-assisted selection index. The number of polymorphic markers among 13 DNA markers ranged from 3 to 7 between the parental cultivars. Among these cross combinations, there was no significant difference between marker-assisted selection and conventional selection methods for selection of lines with high palatability. This demonstrates that marker-assisted selection by marker-based regression value might not be a good method for selection to apply the all breeding populations for high palatability line selection. While each method allowed equally effective selection of high palatability lines, the regression analysis using polymorphic markers will need to be re-calculated for each cross combination.

식물에서 분자 마커의 동향

허만규(Man Kyu Huh) 한국생명과학회 2015 생명과학회지 Vol.25 No.7

분자 마커는 유기체에서 다른 유기체와 분자적 수준에서 식별하는 마커이다. 유전적 분석을 위한 분자 마커의 발달은 식물 유전학, 다양한 구조와 가능을 이해하는데 기여하였다. DNA 마커는 임의유전자 증폭에서 다형성을 탐지하는 기법이나 방법(예를 들면 서든 블로팅, 핵산 교잡법, PCR을 이용한 중합효소 연쇄 증폭 반응, DNA 서열화)으로 RFLP, AFLP, RAPD, SSR, SNP 등을 이용하였고 현재에도 이용하고 있다. 최근 기능성 유전자를 이용한 기능성 마커가 각광을 받고 있다. 기능성 마커는 다형성 서열에서 유래한 것으로 표현형 변이를 내포하고 있다. 이런 개념에서 출발한 기능성 마커는 모든 유전자를 타깃으로 할 수 있으나 식물에서는 P450, 튜블린 형성 유전자의 다형성(TBPs), 전이요소 마커(TEMs), 병원균 저항성 유전자 마커(RGMs), RNA를 기반으로 한 마커(RBMs) 등이 널리 이용되고 있다. 본 연구는 Poczai 등의 총설을 기반으로 구성하였다. 식물에서 이런 분자 마커의 이용은 식물의 분화, 진화, 생리적 기능성 유전자의 변화 등 생물학 전반에 관한 정보 획득에 도움을 될 것이다. A molecular marker is a molecule contained within a sample taken from an organism or other matter. The development of molecular techniques for genetic analysis has led to a great contribution to our knowledge of plant genetics and our understanding of the structure and behavior of various genomes in plants. Recently, functional molecular markers have been developed to detect the presence of major genes from the analysis of pedigreed data in absence of molecular information. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods such as RFLP, AFLP, RAPD, SSR, SNP, etc. A new class of very useful DNA markers called genic molecular markers utilizing the ever-increasing archives of gene sequence information being accumulated under the EST sequencing projects on a large number of plant species. Functional markers are derived from polymorphic sequences, and are more likely to be involved in phenotypic trait variation. Based on this conceptual framework, the marker systems discussed below are all (gene)-targeted markers, which have the potential to become functional. These markers being part of the cDNA/EST-sequences, are expected to represent the functional component of the genome i.e., gene(s), in contrast to all other random DNA based markers that are developed/generated from the anonymous genomic DNA sequences/domains irrespective of their genic content/information. Especially I sited Poczai et al’ reviews, advances in plant gene-targeted and functional markers. Their reviews may be some useful information to study molecular markers in plants.

돼지 품종별 specific DNA marker에 관한 연구

김철욱 진주산업대학교 농업기술연구소 1998 農業技術硏究所報 Vol.11 No.-

본 연구에서는 종돈의 순수성을 식별할 수 있는 DNA marker를 개발하기 위해 최근에 많이 이용되고 있는 PCR-RAPD 분석법을 수행하였다. 우선 대한양돈협회 제 2 종돈 능력검정소에서 검정합격된 Duroc, Landrace, 그리고 Yorkshire종으로부터 EDTA-coated tube에 혈액을 채취하였다. 채취한 혈액은 Wizard genomic DNA purification kits와 phenol solution을 이용한 분리방법으로 순수한 genomic DNA를 분리할 수 있었다. 그리고 최적 PCR 조건을 맞춘 후, 50개 primer pairs를 이용하여 DNA sample의 PCR 분석을 수행하여 그 결과를 2% agarose gel 전기영동으로 확인하였다. 대부분의 primer pairs의 경우에는 polymorphism을 확인할 수 없었으며, 50개 primer pairs중 2개에서 Duroc에 8개, Landracedp 9개, 그리고 Yokshire에 7개의 polymorphic band를 확보할 수 있었다. 품종별 polymorphic band는 여러번의 반복적인 PCR test를 거쳐서 재현성있게 나타났으므로 품종의 순수성을 판별할 DNA marker로서 활용할 수 있을 것이다. This study was carried out to develop DNA markers that potentially can be used for identification of major pig breeds, using PCR-RAPD that are widely used for this kind of purposes. Blood samples were collected in EDTA coated tubes from Duroc, Landrace, and Yorshire pigs that had passed the test of the Korea Swine Association Second Tesitng Station. Genomic DNA was extracted from blood using a commercial genomic DNA purification kit and phenol solutions. After estabilishment of optimizing PCR condition, PCR amplification of the DNA samples was performed using 50 primer pairs, followed by 1.2% agarose gel electrophoresis. Twelve primer pairs out of 50 that had been tested yielded 8, 9 and 7, breed-specific polymorphic bands for Duroc, Landrace and Yorkshire, respectively. These polymorphic bands, as DNA markers, should be useful for determination of genetic purity line of each of above pig breeds.

DNA markers in chicken for breed discrimination

Md. Rashedul Hoque(라세둘),Seung-Hwan Lee(이승환),Jun-Heon Lee(이준헌) 충남대학교 농업과학연구소 2012 농업과학연구 Vol.39 No.2

There is an emerging interest in using DNA markers for breed identification in animals. This article reviews the breed identification markers in chicken, mainly developed in Chungnam National University, with particular emphasis on the mitochondrial DNA markers and the nuclear DNA markers including the SNPs in MHC region and the plumage color related MC1R markers. This information would be very useful for an appropriate conservation breeding program as well as for the establishment of molecular markers for chicken breed identifications.

Utilization of DNA Marker-Assisted Selection in Korean Native Animals

Yeo, Jung-Sou,Kim, Jae-Woo,Chang, Tae-Kyung,Park, Young-Ae,Nam, Doo-Hyun 영남대학교 약품개발연구소 2000 영남대학교 약품개발연구소 연구업적집 Vol.10 No.-

The recent progress of DNA technologies including DNA fingerprinting(DFP) and random amplified DNA polymorphism(RAPD) analysis make it possible to identify the spe-cific genetic traits of animals and to analyze the genetic diversity and relatedness between or within species or populations. Using those techniques, some efforts to identify and develop the specific DNA markers based on DNA polymorphism, which are related with economic traits for Korean native animals, Hanwoo(Korean native cattle), Korean native pig and Ko-rean native chichen, have been made in Korea for recent a few years. The developed specific DNA markers successfully characterize the Korean native animals as the unique Korean ge-netic sources, distinctively from other imported breeds. Some of these DNA markers have been related to some important economic traits for domestic animals, for example, growth rate and marbling for Hanwoo, growth rate and back fat thickness for native pig, and growth rate, egg weight and egg productivity for native chichen. This means those markers can be used in important marker-assisted selection(MAS) of Korean native domestic animals and further contribute to genetically improve and breed them.

Identification of Coupling and Repulsion Phase DNA Marker Associated With an Allele of a Gene Conferring Host Plant Resistance to Pigeonpea sterility mosaic virus (PPSMV) in Pigeonpea (Cajanus cajan L. Millsp.)

Daspute, Abhijit,Fakrudin, B. The Korean Society of Plant Pathology 2015 Plant Pathology Journal Vol.31 No.1

Pigeonpea Sterility Mosaic Disease (PSMD) is an important foliar disease caused by Pigeonpea sterility mosaic virus (PPSMV) which is transmitted by eriophyid mites (Aceria cajani Channabasavanna). In present study, a F2 mapping population comprising 325 individuals was developed by crossing PSMD susceptible genotype (Gullyal white) and PSMD resistant genotype (BSMR 736). We identified a set of 32 out of 300 short decamer random DNA markers that showed polymorphism between Gullyal white and BSMR 736 parents. Among them, eleven DNA markers showed polymorphism including coupling and repulsion phase type of polymorphism across the parents. Bulked Segregant Analysis (BSA), revealed that the DNA marker, IABTPPN7, produced a single coupling phase marker (IABTPPN $7_{414}$) and a repulsion phase marker (IABTPPN $7_{983}$) co-segregating with PSMD reaction. Screening of 325 F2 population using IABTPPN7 revealed that the repulsion phase marker, IABTPPN $7_{983}$, was co-segregating with the PSMD responsive SV1 at a distance of 23.9 cM for Bidar PPSMV isolate. On the other hand, the coupling phase marker IABTPPN $7_{414}$ did not show any linkage with PSMD resistance. Additionally, single marker analysis both IABTPPN $7_{983}$ (P<0.0001) and IABTPPN $7_{414}$ (P<0.0001) recorded a significant association with the PSMD resistance and explained a phenotypic variance of 31 and 36% respectively in $F_2$ population. The repulsion phase marker, IABTPPN7983, could be of use in Marker-Assisted Selection (MAS) in the PPSMV resistance breeding programmes of pigeonpea.

고인골 유전자 증폭 효율이 높은 PCR 반응 조건

김경용(Kyung-Yong Kim),우지영(Ji-Young Woo),김기정(Kijeong Kim) 대한체질인류학회 2008 해부·생물인류학 (Anat Biol Anthropol) Vol.21 No.2

고인골의 시료 및 핵산의 양적 제한 때문에 표적핵산의 PCR 증폭이 고인골 핵산 분석에 필수적으로 요구되는 전제 조건이다. 현대인의 신선한 핵산과는 달리, 사후 보존에 불리한 환경 속에 오랜 시간 동안 방치되어 온 고인골 시료로부터의 PCR 증폭은 성공적이지 못한 경우가 흔하다. 따라서 고인골 DNA에 적합한 PCR 증폭방법의 시행이 요구된다. 그러나 고대 시료에 적합한 향상된 PCR 증폭 기법에 대한 체계적인 연구가 시행된 보고가 없다. 이 연구에서는 한국과 몽골의 약 500년에서 3300년 된 PCR 증폭이 어려운 8개 고인골 검체들로부터 광범위한 PCR 조건들을 체계적으로 시도하고 PCR 증폭의 성공율을 비교하였다. PCR 조건들의 증폭율을 비교하기 위한 표적 핵산으로서는 카피수가 많은 사립체 핵산과 세포내 단일 카피 DNA로서 현대 한국인에서 흔한 Y 염색체 일배체 그룹으로 알려진 O 일배체 그룹 표지(M175) 핵산을 이용하였고, 성공적인 증폭산물의 형성의 검증은 증폭산물들의 염기서열을 분석을 통하여 시행하였다. 이 연구를 통하여 고인골에서 핵산을 증폭하기 위한 최적의 PCR 조건을 확립하였고, 결정된 방법은 검사한 모든 샘플에서 사립체 핵산을 성공적으로 증폭시켰고, M175 Y 염색체 일배체 표지 핵산의 증폭은 검사된 시료수의 50% 성공율을 나타냈지만 시도된 조건들에서 가장 높은 성공율을 나타내었다. 이 결과는 성공적인 고인골 핵산분석을 위한 최적의 PCR 조건으로 분자 유전학적 인류학 연구에 유용하게 쓰일 수 있음을 시사한다. The ancient bone DNA analysis essentially requires PCR amplification of the targeting genes of study due to the limitation of the ancient bone sample and DNA amounts. In contrast to the fresh living human DNA, it is common to face failing in amplifying the poorly preserved ancient DNA after death. Therefore, the optimized PCR methods appropriate for ancient DNA are required. However, there is no report to date that a systemic investigation of enhanced PCR amplification methods suitable for ancient samples has been conducted. Approximately 500~3,300-year-old Korean and Mongolian ancient bones that are resistant to PCR were selected and an extensive number of PCR conditions were systematically investigated for the comparison of PCR success rates. For the PCR analysis, a mitochondrial DNA fragment as a multicopy DNA and a M175 Y chromosome biallelic marker DNA fragment as a single copy DNA that is the marker of the prevalent Y haplogroup (haplogroup O) in Korea were targeted. The identity of the amplified products were confirmed by DNA sequencing. Through this study, we established the optimized PCR conditions for the highly successful amplification of ancient bone DNAs. This estabilished method allowed for the successful amplification of mitochondrial DNAs from all the ancient bone samples tested and the amplification by 50% success rates in the amplification of M175 Y chromosome biallelic marker DNA but with the highest success rates. These results demonstrate that the optimized PCR condition will be useful for the promising ancient DNA analysis in the fields of molecular genetic anthropological studies.

Induced Mutagenesis in Jatropha curcas L. Using Ethyl Methanesulphonate (EMS) and Assessment of DNA Polymorphism through RAPD Markers

Dharman Dhakshanamoorthy,Radhakrishnan Selvaraj,Alagappan Chidambaram 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.3

Ethyl methanesulphonate (EMS) has been employed in a number of genotoxic studies in plants as a model alkylated agent that readily reacts with DNA-producing alkylated nucleotides. Therefore, the present study was aimed at assessing DNA polymorphism induced by different concentrations (control, 1, 2, 3, and 4%) of EMS through a Randomly Amplified Polymorphic DNA (RAPD)marker analysis. The improved agronomic traits such as germination, flowering, maturity, seed traits, and oil content were recorded in 1% EMS-treated plants, while the corresponding parameters reduced significantly (P > 0.05) in 4% EMS-treated plants as compared to the control. Among 25 random primers used, 19 primers produced polymorphic bands. The number of amplicons varied from 1 to 8 with an average of 3.68 bands, of which 2.12 were polymorphic. The highest polymorphic bands (6) and percentage of polymorphism (85.71) were produced by the primer OPAK-20. In a dendrogram constructed based on Jaccard’s coefficient similarity,the treated plants and control were grouped into three clusters: (a) control and 2 and 3% concentrations of EMS-treated plants merged together; (b) 1% concentration of EMS-treated plants clustered alone; (c) 4%concentration of EMS-treated plants also clustered alone. We conclude that the effect of EMS could change the pattern of germination, flowering, seed yield, and oil content of J. curcas. DNA polymorphism detected by RAPD marker analysis offered a useful biomarker assay for the evaluation of effects of chemical mutagens Ethyl methanesulphonate (EMS) has been employed in a number of genotoxic studies in plants as a model alkylated agent that readily reacts with DNA-producing alkylated nucleotides. Therefore, the present study was aimed at assessing DNA polymorphism induced by different concentrations (control, 1, 2, 3, and 4%) of EMS through a Randomly Amplified Polymorphic DNA (RAPD)marker analysis. The improved agronomic traits such as germination, flowering, maturity, seed traits, and oil content were recorded in 1% EMS-treated plants, while the corresponding parameters reduced significantly (P > 0.05) in 4% EMS-treated plants as compared to the control. Among 25 random primers used, 19 primers produced polymorphic bands. The number of amplicons varied from 1 to 8 with an average of 3.68 bands, of which 2.12 were polymorphic. The highest polymorphic bands (6) and percentage of polymorphism (85.71) were produced by the primer OPAK-20. In a dendrogram constructed based on Jaccard’s coefficient similarity,the treated plants and control were grouped into three clusters: (a) control and 2 and 3% concentrations of EMS-treated plants merged together; (b) 1% concentration of EMS-treated plants clustered alone; (c) 4%concentration of EMS-treated plants also clustered alone. We conclude that the effect of EMS could change the pattern of germination, flowering, seed yield, and oil content of J. curcas. DNA polymorphism detected by RAPD marker analysis offered a useful biomarker assay for the evaluation of effects of chemical mutagens