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.

Conservation Genetics of Endangered Brasenia schreberi Based on RAPD and AFLP Markers

( Chang Kyun Kim ),( Hye Ryun Na ),( Hong Keun Choi ) 한국식물학회 2008 Journal of Plant Biology Vol.51 No.4

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θB) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (Nem), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoring B. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.

AFLP 표식에 의한 차나무 계통간 유전 분석

류재일(Jae Il Lyu),이선하(Seon Ha Lee),배창휴(Chang Hyu Bae) 한국차학회 2005 한국차학회지 Vol.11 No.3

차 수집종 집단의 분자유전학적 특성을 검토하기 위하여 한국 수집봉 32게통, 대만종 3품종, 중국종 4품중, 일본종 6품종, 총 45 수집계통에 대하여 AFLP분석을 수행하였다. AFLP 분석결과 총 DNA 밴드수 382개 중 87.7%가 다형성을 나타냈다. 총 45 수집계통 간의 유사도지수는 0.05~1.00 범위였고, 이 중 한국수집계통간의 유사도지수는 0.10~0.95로 나타났다. 군집분석 결과 총 45 수집계통이 6개 그룹으로 나눠졌다. 한국수집계통은 제6그룹을 제외한 나머지 그룹에 모두 분포하였으며, 이 중 3, 4, 5그룹은 한국수집종 만으로 구성되었다. 이와 같이 AFLP분석을 통하여 본 연구에서 사용된 한국 수집계통은 높은 유전적 다양성을 가지고 있었으며, 이 수집 차나무집단은 차의 품종육성을 위한 유전자원으로서 활용이 기대된다. Amplified fragment length polymorphism (AFLP) analysis was examined to detect genetic characteristics among 45 accessions of tea(Camellia sinensis var. sinensis (L.) O. Kuntze) from Korea, Japan, China and Taiwan. As a result of AFLP analysis, a total of 382 DNA bands were scored and 335 were polymorphic with a polymorphic rate 87.7%. Genetic similarity shows very different from 0.05 to 1.00 among the 45 accessions and 0.10 to 0.95 among the Korean accessions. By cluster analysis, the 45 accessions were grouped into six. Korean accessions that belonged to group 3.4 and 5 did not have Japanese, Chinese and Taiwanese varieties, The results indicate that the Korean accessions as well as 45 accessions have high genetic diversity, thus the Korean tea accessions serve as basic gene pool in tea breeding.

AFLPs에 의한 Aegilops의 계통발생학적 재평가

박용진,심재욱 韓國作物學會 1997 한국작물학회지 Vol.42 No.6

각 게놈형간의 근연관계 및 배수체종의 게놈분석에 관한 새로운 접근을 시도하고자, Aegilops 19종 및 재배밀(T. aestivum)을 공시하여 AFLP 분석을 실시하여 얻은 결과는 다음과 같다. 1. AFLPs를 이용한 Aegilops종들간 근연관계를 분석한 결과, 7개의 primer 조합에서 총 207개의 다형 band를 조사하였으며 조합당 평균 다형 band수는 29.8개 이었다. 2. 각 게놈간 유연관계로 보아 Ae. heldreichii (Mh ) 는 Ae. comosa (M)와 Ae. uniaristate(N)의 중간위치의 게놈으로 나타났고, UM게놈을 가진 4배체종의 M게놈 공여종으로 판단되었다. 그리고 Ae. squarrosa는 재배밀의 D게놈 공여종임을 확인하였다. 3. 6배체성 Ae. triaristate(UMN)는 4배체성 Ae. triaristate(UM)보다는 Ae. columnaris(UM)와 더 근연인 것으로 나타났다. 그리고 Ae. ventricosa(DN)은 U게놈이 N게놈보다 더 근연인 것으로 나타났다. 4. AFLPs에 의한 군집형성은 5개의 군집으로 구분되었고 이는 기본적으로 Gihara의 Section군과 일치하였고, 다양성분석, 게놈분석 등에 보다 효율적인 것으로 평가되었다. Aegilops genus is known to include the donor species of the Band D genome of the bread wheat(ABD). An effort to establish a better strategy for phylogenetic relationships about Aegilops polyploids by AFLPs(Amplified Fragment Length Polymorphisms) was conducted using the 19 Aegilops sPP. and T. aestivum. The 207 polymorphic bands from the amplified products on the 6% acrylamide denaturing sequencing gels were obtained with the 7 AFLP primer combinations, and used to account for the genetic similarities and cluster analysis using NTSYS program. According to the genome analysis, the Mh -genome of Ae. heldreichii was estimated as an intermediate genome between the M-genome of Ae. comosa and N-genome of Ae. uniaristata and supposed to be incorporated in the establishing process of UM-genome as a possible diploid donor. And Ae. ventricosa(DN) was more close to Ae. umbellulata(U) than Ae. squarrosa(D). The close relationship between Ae. squarrosa and T. aestivum was perceived as a diploid donor of D-genome. As for the polyploid species, hexaploid Ae. triaristata was more closely related to Ae. columnaris rather than tetraploid Ae. triaristata. The clustered groups were, basically same to the previous Gihara's sections based on phenotypes and pairing analysis of interspecific hybrids. AFLP was evaluated as an efficient and powerful method in the genome evaluation of closely related species.

Genetic Variation and Polymorphism in Rainbow Trout, Oncorhynchus mykiss Analysed by Amplified Fragment Length Polymorphism

Yoon, Jong-Man,Yoo, Jae-Young,Park, Jae-Il The Korean Society of Fisheries and Aquatic Scienc 2004 韓國養殖學會誌 Vol.17 No.1

The objective of the present study was to analyze genetic distances, variation and characteristics of individuals in rainbow trout, Oncorhynchus mykis using amplified fragment length polymorphism (AFLP) method as molecular genetic technique, to detect AFLP band patterns as genetic markers, and to compare the efficiency of agarosegel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively. Using 9 primer combinations, a total of 141 AFLP bands were produced, 108 bands (82.4%) of which were polymorphic in AGE. In PAGE, a total of 288 bands were detected, and 220 bands (76.4%) were polymorphic. The AFLP fingerprints of AGE were different from those of PAGE. Separation of the fragments with low molecular weight and genetic polymorphisms revealed a distinct pattern in the two gel systems. In the present study, the average bandsharing values of the individuals between two populations apart from the geographic sites in Kangwon-do ranged from 0.084 to 0.738 of AGE and PAGE. The bandsharing values between individuals No.9 and No. 10 showed the highest level within population, whereas the bandsharing values between individuals No.5 and No.7 showed the lowest level. As calculated by bandsharing analysis, an average of genetic difference (mean$\pm$SD) of individuals was approximately 0.590$\pm$0.125 in this population. In AGE, the single linkage dendrogram resulted from two primers (M11+H11 and M13+H11), indicating six genetic groupings composed of group 1 (No.9 and 10), group 2 (No. 1, 4, 5, 7, 10, 11, 16 and 17), group 3 (No. 2, 3, 6, 8, 12, 15 and 16), group 4 (No.9, 14 and 17), group 5 (No. 13, 19, 20 and 21) and group 6 (No. 23). In AGE, the genetic distances among individuals of between-population ranged from 0.108 to 0.392. In AGE, the shortest genetic distance (0.108) displaying significant molecular differences was between individuals No.9 and No. 10. Especially, the genetic distance between individuals No. 23 and the remnants among individuals within population was highest (0.392). Additionally, in the cluster analysis using the PAGE data, the single linkage dendrogram resulted from two primers (M12+H13 and M11+H13), indicating seven genetic groupings composed of group 1 (No. 15), group 2 (No. 14), group 3 (No. 11 and 12), group 4 (No.5, 6, 7, 8, 10 and 13), group 5 (No.1, 2, 3 and 4), group 6 (No.9) and group 7 (No. 16). By comparison with the individuals in PAGE, genetic distance between No. 10 and No. 7 showed the shortest value (0.071), also between No. 16 and No. 14 showed the highest value (0.242). As with the PAGE analysis, genetic differences were certainly apparent with 13 of 16 individuals showing greater than 80% AFLP-based similarity to their closest neighbor. The three individuals (No. 14, No. 15 and No. 16) of rainbow trout between two populations apart from the geographic sites in Kangwon-do formed distinct genetic distances as compared with other individuals. These results indicated that AFLP markers of this fish could be used as genetic information such as species identification, genetic relationship or analysis of genome structure, and selection aids for genetic improvement of economically important traits in fish species.

AFLP와 RAPD 방법을 이용한 꼬리진달래(Rhododendron micranthum) 수집종의 유전적 변이 분석

김진홍,박철호,김남수,이명숙,이재선,이주경,김남희 한국자원식물학회 2004 한국자원식물학회지 Vol.17 No.3

Rhododendron micranthum is an endangered species in Korea. In order to develop the strategy of gene diversity conservation, estimation of the amount of genetic diversity, the genetic variation and relationship in the native populations of Rh. micranthum was performed on the basis of AFLP and RAPD analysis. Analysis of 56 accessions derived from 6 populations of Rh. micranthum with four AFLP primer combinations and ten RAPD primers detected a total of 33 polymorphic AFLP fragments and 15 polymorphic RAPD fragments, respectively. By UPGMA cluster analysis with molecular markers, the 56 accessions were grouped into three major clusters at 73.3% genetic similarity; group I consists of most accessions of populations I, II, IV, V and Vl, group II consists of 7 accessions of population III, and group III consists of only two accessions of population IV. The geographic locations of the most accessions derived from six populations were not related to their position in the UPGMA cluster analysis, except for several accessions of populations III and IV. The genetic similarity of among six populations measured by AFLP and RAPD markers ranged from 0.66 to 0.99. Among them, population VI showed the highest GS with means of 0.87, while population I showed the lowest GS with means of 0.78. This result will be useful for designing the strategy of conservation in the native populations of Rh.micranthum.

AFLP를 이용한 무당벌레 초시 색상패턴변이의 연구

박초롱,조창욱,윤규식,강민아,권혜리,강은진,서미자,유용만,윤영남 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

무당벌레(Harmonia axyridis)는 전 세계에 걸쳐 다양하게 분포하고 있으며, 여러 종류의 진딧물을 섭식하는 진딧물의 포식자로 널리 알려지면서 진딧물 의 생물적 방제 프로그램에 유용하게 사용되고 있다. 무당벌레는 같은 종 내 에서도 변이가 매우 심하여 다양한 초시 색상패턴을 나타내는데, 이러한 종이 나 개체간의 분자적 요인의 차이를 밝혀내는 Fingerprinting 방법으로는 RAPD, RFLP, AFLP방법 등이 있으며, 이 중에서 AFLP는 높은 재현성과 정확성을 나 타냄으로 가장 널리 사용되고 있다. 따라서 본 연구에서는 AFLP 분석을 통해 무당벌레의 초시 색상패턴에 따른 유전적 다양성을 알아보고자 하였다. 2007년부터 2009년까지 채집된 월동개체군 중에서 검은색 바탕에 2개의 붉 은색 점을 가진 conspicua 개체와, 검은색 바탕에 4개의 붉은색 점을 가진 spectabilis 개체, 노란색 바탕에 19개의 검은색 점을 가지고 있는 succinea1 개 체, 그리고 노란색 바탕에 검은색 점이 전혀 없는 succinea2 개체를 가지고 실 험을 실시한 결과, 8개의 AFLP primer조합을 통해 총 822개의 밴드가 확인되 었고, 그 중 37개의 밴드 특정 색상패턴에서만 확인되어, 특이적 밴드를 통해 무당벌레의 색상변이에 있어 유전적 다양성을 추측할 수 있었다.

Genetic Analysis of Garlic(Allium sativum L.)Cultivars Using AFLP

Lee, Mi-Kyung,Lim, Yong Pyo,Bang, Jae-Wook 한국유전학회 2002 Genes & Genomics Vol.24 No.1

Eleven cultivars of garlic (Allium sativum L.) were analyzed with the amplified fragment length polymorphism (AFLP) approach. Total number of AFLP products from ten selective primer combinations was 1,026. Of these, the two kinds of band that affect clustering, specific and polymorphic bands, make up 84.5%. The number of polymorphic fragments by individual primer combinations greatly varied from 168 to 10 and the average polymorphic fragments per primer combinations was 54.2. Distance matrix and dendrogram were generated using simple matching coefficients and neighbor joining cluster analysis. The lowest total character difference was 24, shared by cultivars Sandong-beogu (AS5) and Sandong-taeahn (AS6). This study resulted in five distinct groups from eleven cultivars. The largest group included three subgroups such as Sandong cultivars (AS5, AS6 and AS7), three Seosan cultivars ((AS8, AS9 and As10), and cultivar Seocheon (AS11). The cultivars Namdo (AS2), Daeseo (AS3) and Seocheon-Jan (AS4), and cultivar Elephant (AS1) made up its own group, respectively. These data showed that culivars of Sandong and Seosan were clustered by high value of bootstrap. This study demonstrated that AFLP method can be used reliably to examine the genetic polymorphism and genetic relationships in different cultivars of garlic.

고추의 역병 저항성과 연관된 분자표지의 효용성 검정

이원필(Won Phil Lee),이준대(Jundae Lee),한정헌(Jung-Heon Han),강병철(Byoung-Cheorl Kang),윤재복(Jae Bok Yoon) 한국원예학회 2012 원예과학기술지 Vol.30 No.1

Phytophthora root rot has been causing a serious yield loss in pepper production. Since 2004, the year in which commercial cultivars resistant to the disease were firstly commercialized, it has been necessary to introduce the resistance into domestic pepper cultivars for dried red pepper. Therefore, developing molecular markers linked to the resistance is required for an accurate selection of resistant plants and increasing breeding efficiency. Until now, several markers associated with the major dominant gene resistant to Phytophthora root rot have been reported but they have some serious limitations for their usage. In this study, we aimed to develop molecular markers linked to the major dominant gene that can be used for almost of all genetic resources resistant to Phytophthora root rot. Two segregating F₂ populations derived from a ‘Subicho’ × ‘CM334’ combination and a commercial cultivar ‘Dokyacheongcheong’ were used to develop molecular markers associated with the resistance. After screening 1,024 AFLP primer combinations with bulked segregant analysis, three AFLP (AFLP1, AFLP2, and AFLP3) markers were identified and converted into three CAPS markers (M1-CAPS, M2-CAPS, and M3-CAPS), respectively. Among them, M3-CAPS marker was further studied in ten resistants, fourteen susceptibles, five hybrids and 53 commercial cultivars. As a result, M3-CAPS marker was more fitted to identify Phytophthora resistance than previously reported P5-SNAP and Phyto5.2-SCAR markers. The result indicated that the M3-CAPS marker will be useful for resistance breeding to Phytophthora root rot in chili pepper.

DNA 마커를 이용한 야생 복숭아 유전자원의 유전적 다양성 분석

조강희,남은영,배경미,신일섭,김세희,김현란,정경호 한국육종학회 2012 한국육종학회지 Vol.44 No.4

본 연구는 복숭아 유전자원의 유전적 다양성을 분석하여 육종의 기초 자료로 활용하기 위하여 재배 품종 및 국내에서 수집한 야생 복숭아 64점을 대상으로 SRAP, AFLP와 RAPD 분석을 수행하였다. 19종과 20종의 primer조합을 이용한 SRAP와 AFLP분석에서 각각 143개(26.7%)와 193개(27.2%)의 다형성 밴드를 획득하였고, 평균 다형성 밴드 수는 7.53개와 9.65개였다. RAPD분석에서 52종의 선발 primer를 이용하여 201개(45.9%)의 다형성 밴드를 얻었으며, 평균 다형성 밴드 수는 3.90개였다. SRAP, AFLP와 RAPD분석에서 획득된 537개의 다형성 밴드를 이용하여 UPGMA(비가중 평균결합) 방식으로 유사도 및 집괴분석을 수행한 결과 유전적 유사도 0.751을 기준으로 8개의 그룹으로 분류되었다. 복숭아 유전자원 간 유전적 유사도 값은 0.370~0.978의 범위로 평균 유전적 유사도는 0.737이었다. 가장 높은 유사도 값(0.978)을 나타낸 유전자원은 PH065와 PH066 간이었고 가장 낮은 유사도 값(0.370)을 나타낸 유전자원은 Prunus davidiana와 PH020간이었다. 본 연구에서는 이용된 64종의 복숭아 유전자원은 집괴분석 결과 유전자원들이 수집된 지역은 다르지만 유전적 다양성은 낮은 것으로 추정되었다. In this study, sequence-related amplified polymorphism (SRAP), amplified fragment length polymorphism (AFLP), and randomly amplified polymorphic DNA (RAPD) analyses were used to assess genetic diversity among 64 peach germplasms including commercial cultivars and wild accessions. Nineteen SRAP primer combinations were detected a total of 143 polymorphic bands (26.7%) with an average 7.53. In AFLP analysis, twenty primer combinations were generated a total of 193 polymorphic bands (27.2%) with an average 9.65. Fifty-two RAPD primers were detected a total of 201 polymorphic bands (45.9%) with an average of 3.90. By UPGMA (unweighted pair-group method arithmetic average) cluster analysis with 537 polymorphic bands, the peach germplasms were classified eight groups by similarity index of 0.751. Genetic similarity values among the tested peach germplasms ranged from 0.370 to 0.978, and the average similarity value was 0.737. The similarity index was the highest (0.978) between PH065 and PH066, and the lowest (0.370) between Prunus davidiana and PH020. The genetic diversity among the 64 peach germplasms revealed by cluster analysis was low, despite they were collected from different regions.