• Title/Summary/Keyword: RAPD method

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Characterization of intraspecific hybrids by di-mon crossing in Pleurotus eryngii (Di-Mon 교잡법에 의한 큰느타리버섯 교잡주의 특성)

  • Shin, Pyung-Gyun;Yoo, Young-Bok;Kong, Won-Sik;You, Chang-Hyun;Oh, Se-Jong
    • Journal of Mushroom
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    • v.2 no.2
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    • pp.109-113
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    • 2004
  • Pleurotus eryngii is not edible and medicinal mushrooms indigenous to Korea. To improvement of strain suitable to the geographic setting of Korea, we are mated with 22 dikaryons and 47 monokaryons isolated from Pleurotus eryngii ASI 2547 by Di-Mon mating. 19 strains forming fruit body obtained from clamped 253 bred strains. 7 excellent strains are selected from 19 bred strains by various morphological features of fruit body. Among the selected 7 strains, H6 strain were identified into ASI 2547-like recombinant hybrids with URP uniprimer by RAPD analysis. This suggested that Di-Mon crossing is one of rapid and easy breeding method for strain improvement with molecular techniques.

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Genotyping and Molecular Characterization of Carbapenem-resistant Acinetobacter baumannii Strains Isolated from Intensive Care Unit Patients

  • Abozahra, Rania;Abdelhamid, Sarah M.;Elsheredy, Amel G.;Abdulwahab, Kawther E.;Baraka, Kholoud
    • Microbiology and Biotechnology Letters
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    • v.49 no.2
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    • pp.239-248
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    • 2021
  • The emergence of multidrug-resistant Acinetobacter baumannii has partly increased treatment failure and patient mortality. Class D β-lactamases is an important mechanism of resistance to beta-lactam antibiotics in this species. This study aimed to investigate the relationship between the presence oxacillinase gene and genetic fingerprints of A. baumannii isolates from the intensive care unit of an Egyptian tertiary care hospital. One hundred and twenty A. baumannii clinical isolates were collected. Multiplex PCR was performed to detect genes encoding oxacillinases (OXA-23, OXA-24, OXA-51, OXA-58 and OXA-143). Molecular typing of all collected isolates was performed using random amplified polymorphic DNA (RAPD)-PCR assay. Out of 120 examined isolates, 92, 88 and 84% were resistant to ertapenem, imipenem and meropenem, respectively. The species-specific, commonly present OXA-51 gene was found in all isolates while OXA-23 showed a high prevalence of 88% of isolates. OXA-24 and OXA-143 genes were detected in 3% and 1% of isolates, respectively. No OXA-58 gene was detected. Five clusters consisting of 19 genotypes were detected using RAPD-PCR. Genotype A was the most prevalent, it was observed in 62% of the isolates followed by genotype B (12%). These results revealed that genotypes A and B are common in the hospital. Results also demonstrate that RAPD-PCR is a rapid and reliable method for studying the clonal similarity among A. baumannii isolated from different clinical specimens.

Molecular Genetic Classification of Phytophthora Species and P. infestans-specific Marker Selection by RAPD Fingerprinting (Phytophthora species의 분자유전학적 분류 및 RAPD fingerprinting을 이용한 P. infestans-specific 분자마커의 선발)

  • Kim, Kyoung-Su;Shin, Whan-Sung;Kim, Hee-Jong;Woo, Su-Jin;Ham, Young-Il;Shin, Kwan-Yong;Lee, Jeong-Oon;Kim, Byung-Sup;Shim, Jae-Ouk;Lee, Min-Woong;Lee, Youn-Su
    • The Korean Journal of Mycology
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    • v.27 no.6 s.93
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    • pp.394-398
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    • 1999
  • Taxonomic and genetic analysis of Phytophthora species belonging to six different morphological groups (GI, GII, GIII, GIV, GV, GVI) was conducted using RAPD method. Amplified fragments ranged $0.3{\sim}3.2$ kb in their molecular weights. Among total of 145 bands, there were 109 polymorphic bands. Seven isolates of P. infestans showed high similarities of $0.92{\sim}0.99$, and P. infestans isolate 3 from potato showed similarities of $0.93{\sim}0.95$ compared with other P. infestans. Among isolates of P. capsici, similarities of $0.77{\sim}0.86$ were observed and they were grouped in 80% level. P. cinnamomi and P. cryptogea isolates which belonging to group GVI showed very similar RAPD fingerprinting pattern. Primers OPA-04, OPA-17, OPA-18, OPA-19, and OPB-12 showed high level of differences among the tested isolates in major bands and molecular weights. The similarity between the isolates was 0.67. P. megasperma and P. sojae in group GV showed similarity of 0.65. These two isolates showed big differences in single major band in reactions with primers OPA-08, OPA-17, and OPA-19. Phytophthora-specific and P. infestans-specific molecular markers were also selected with one of the random primers tested. In reaction with primer OPA-20, all the genus Phytophthora showed common band at 600 bp, and all the P. infestans isolates showed specific band at 680 bp. These markers can be useful for identification of Phytophthora speices or P. infestans. As a result, P. infestans isolated from tomato and/or potato can easily be differentiated from other Phytophthora species with this primer.

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A Simple Method for Extraction of High Molecular Weight DNA fromPorphyra Tenera (Rhodophyta) Using Diatomaceous Earth

  • Kim, Tae-Hoon;Hwang, Mi-Sook;Song, Ju-Dong;Oh, Min-Hyuk;Moon, Yong-Hwan;Chung, Ik-Kyo;Rhew, Tae-Hyoung;Lee, Choon-Hwan
    • ALGAE
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    • v.21 no.2
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    • pp.261-266
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    • 2006
  • The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.

Analysis of Genetic Diversity in Soybean Varieties Using RAPD Markers (사료작물로 이용이 가능한 한국 재배콩의 RAPD 표지인자에 의한 유전적 다양성 분석)

  • Lee, Sung-Kyu;Kim, Bum-Jun
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.18 no.4
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    • pp.277-284
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    • 1998
  • Random amplified polymorphic DNA (RAPD) analysis was used to detect the genetic diversity of soybean (Glycine max (L.) Merr.) varieties and field bean (Glycine soza Sieb. and Zucc.) Five soybean varieties and one field bean were analysed with random primers using the polymerase chain reaction (PCR). Nine primers of a total twenty random primer were selected to amplify DNA segments. A total of 74 PCR products were amplified and 67.6% of which were polymorphic. The size of DNA molecule is ranged 0.13~2.0Kb and typically generated four to eight major bands. Specific genetic marker were revealed in primer sequence 5'-CAG GCC CIT C-3', 5'-TGC TCT GCC C-3' and 5'-GTC CAC ACG G-3', respectively. Genetic similarity between each of the varieties were calculated from the pair-wise comparisons of amplification products and a dendrogram was constructed by an unweighted pair-group method with arithmethical means (UPGMA). The results indicate that intervarietal relationships of soybean have a narrow genetic base and between the varieties, Hwanggum-kong and Seckryang-bootkong is more closely related than the rest of varieties, and field bean is related quite distant.

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Identification and Characteristics of a Purple, Non-Sulfur Bacterium, Rhodobacter sp. EGH-24 from Korea Coast (한국 해안으로부터 Purple, Non-Sulfur Photosynthetic Bacterium, Rhodobacter sp. EGH-24의 분리 및 특성)

  • 차미선;김기한;조순자;이나은;이정은;이재동;박재림;이상준
    • Journal of Environmental Science International
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    • v.12 no.12
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    • pp.1293-1301
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    • 2003
  • A species of facultative photo-organotrophic, purple, non-sulfur bacterium was isolated from the 47 point at west and south coast of Korea in September 2001. Separated 13 samples of changes with red color under 28-32$^{\circ}C$, 3000 lux, anaerobe conditions for 7 days cultivated in basal medium. For pure isolation from 13 samples, we used agar-shake tube method (0.4 % agar) and separated 5 strains through 13-repetition test. EGH-24 and EGH-30 was identified as the same strain through the RAPD(Random Amplified Polymorphic DNA)-PCR of strain EGH-9, EGH-13, EGH-23, EGH-24, EGH-30. Four isolates cultivated in synthesis wastewater for wastewater biodegradation test. EGH-24 was selected with efficient wastwater treating strain. Based on the results obtained from morphology, nutrient requirements, major bacteriochlorophyll content, 16S-rDNA phylogenetic analysis, EGH-24 strain may be identified as a new strain of the genus Rhodobacter and named Rhodobacter sp. EGH-24.

In vitro Propagation of Junos Orange (Citrus junos Sieb.) through Nucellar Polyembroid Cultures

  • Park, Woo-Jin;Kang, Young-Min;Min, Ji-Yun;Park, Dong-Jin;Kim, Yong-Duck;Karigar, C.S.;Choi, Myung-Suk
    • Korean Journal of Medicinal Crop Science
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    • v.12 no.5
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    • pp.384-390
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    • 2004
  • An in vtro nucellar polyembryo propagation method was established with mature seed of the Citrus junos Sieb. 7-8 nucellar polyembryos per seed were induced on MS basal medium without plant growth regulators. The polyembryos developed to complete plantlets on teatment with IBA. These shoots grew further in MS medium without plant growth regulators. Rooting of shoots occurred on MS medium supplemented with IBA. These plantlets were successfully transplanted to small plastic pot containing soil mixture. Somatic embryos were induced from nucellar polyembryo and maturation occurred spontaneously from proliferating cultures on MS medium without growth regulators. Random Amplified Polymorphic DNA (RAPD) marker analysis of in vitro and in vivo grown junos orange showed identical polymorphism indicative of their genetic stability. The RAPD polymorphism produced revealed same banding pattern in each regenerant. Hence, propagaton of junos orange by nucellalr polyembryos was efficient and produced in genetically stable plants under in vitro conditions.

Molecular Biological Species Identification of Imported Groupers(Epinephelus moara ♀×E. lanceolatus ♂) (수입산 바리과(Family Serranidae) 잡종 어류(Epinephelus moara ♀×E. lanceolatus ♂)의 분자생물학적 판별)

  • Kim, Yong Hwi;Park, Jong Yeon;Kim, Jae Hoon;Bang, In-Chul
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.53 no.4
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    • pp.566-571
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    • 2020
  • To classify a presumed hybrid of imported grouper species acquired from the National Fishery Products Quality Management Service, maternal and paternal lines were identified based on partial sequencing of mitochondrial cytochrome c oxidase 1 (co1) and nuclear recombination activation gene 1 (rag1) genes. The matrilineal species was identified as Epinephleus moara by a partial (760 bp) co1 sequence. Ambiguous sequences with base pairs belonging to E. moara or E. lanceolatus were found in a total of 15 different base pairs in the partial 1,159 bp of the rag1 gene, and the patrilineal species was found to be E. lanceolatus. Therefore, all of the groupers examined in the study were identified to be hybrids of E. moara and E. lanceolatus. In addition, a fast and convenient method using random amplification of polymorphic DNA (RAPD) was established for hybrid discrimination. Hybrids between E. moara ♀ and E. lanceolatus ♂ were identified through specific bands of 387 bp and 433 bp in PRIMER 6.

PCR-mediated Fingerprinting to Identify Dang-Gui(당귀) (당귀류 한약재의 유전자 감별 연구)

  • 최호영;정유헌;고지완
    • The Journal of Korean Medicine
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    • v.20 no.4
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    • pp.11-15
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    • 2000
  • Radix Angelicae Gigantis is sweet and pungent in flavor, warm in property. Its effects are tonifying the blood, promoting blood circulation, relieving pain and moistening the bowels. Its indications are blood deficiency syndrome characterized by sallow complexion, dizziness, irregular menstruation, amenorrhea, pains due to blood stasis, and rheumatic arthralgia. Using genes of A. gigas, A. acutiloba, and A. sinensis, the origin of which is identified, as criteria, we analysed many kinds of Angelica with RAPD and RFLP on ITS region, in order to compare and discriminate genes extracted from crude drugs ‘Dang-gui’, that are produced in Korea on the one hand and imported on the other hand. We reached the following conclusion. 1. We could extract DNA from both original plant and dried plant. 2. Especially Uniprimer #1, Uniprimer #2, Uniprimer #4 and Uniprimer #9 were useful. 3. Among the restriction enzymes Sma I, Msp I, Hae III, and Hinf I, used in this experiment, four restriction enzymes except Hinf I could be used properly in discriminating all samples used as A. gigas. We think that this result can be used as a method of discriminating crude drug of Angelica L. related drugs, and used in controlling quality and circulation.

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Identification and Characteristics of a Purple, Non-Sulfur Bacterium Rhodobacter sp. EGH-24 from Korea Coast

  • 김기한;차미선;이나은;이정은;이상준;박재림
    • Proceedings of the Korean Environmental Sciences Society Conference
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    • 2003.11b
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    • pp.233-235
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    • 2003
  • 한국 서해안과 남해안 47개소의 해수와 mud 시료로부터 광합성세균으로 유추되는 13개의 균주를 분리하였고, agar-shake tube method와 RAPD-PCR을 이용하여 4개 서로 다른 균주를 순수분리 하였다. 4개의 균주 중 폐수분해능이 가장 뛰어난 균을 선정하여, 형태학적, 배양적, 생화학적 특성 및 16S rRNA sequencing에 의한 동정결과 purple, sulfur bacteria 쪽에 가까웠으나 형태학적, 배양학적, 생화학적 특성이 purple, non-sulfur bacteria의 Rhodobacter 속에 가장 근접하여, Rhodobacter sp. EGH-24로 명명하였다.

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