• Title/Summary/Keyword: PCR polymorphic

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Identification of Rice Variety Using Simple Sequence Repeat (SSR) Marker (Simple sequence repeat (SSR) marker를 이용한 벼 품종 식별)

  • Kwon, Yong-Sham;Park, Eun-Kyung;Park, Chan-Ung;Bae, Kyung-Mi;Yi, Seung-In;Cho, Il-Ho
    • Journal of Life Science
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    • v.16 no.6
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    • pp.1001-1005
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    • 2006
  • The objective of this study was carried out to evaluate the suitability of simple sequence repeat (SSR) markers for genetic diversity assessment and identification of rice varieties. The 23 primers selected from 50 SSR primers showed polymorphisms in the 21 rice varieties. The 2 to 9 SSR alleles were detected for each locus with an average of 3.00 alleles per locus. The polymorphism information content (PIC) ranged form 0.091 to 0.839. Based on band patterns, UPGMA cluster analysis was conducted. These varieties were separate into 4 groups corresponding to rice ecotype and pedigree information and genetic distance of cluster ranging from 0.59 to 0.92. The 4 SSR primer sets (RM206, RM225, RM418, RM478) selected form 23 polymorphic primers were differentiated all the rice variety from each other by markers genotypes. These markers may be used wide range of practical application in variety identification of rice.

Intergeneric Transfer of Isolated Nuclei from Lentinus edodes into Protoplasts of Pleurotus florida (사철느타리버섯 원형질체내에 표고 핵의 전이)

  • Yoo, Young-Bok;Shin, Pyung-Gyun
    • The Korean Journal of Mycology
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    • v.24 no.2 s.77
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    • pp.111-126
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    • 1996
  • Transfer of the isolated nuclei from Lentinus edodes into protoplasts of Pleurotus florida was induced with polyethlene glycol (PEG) and $CaCl_2$. The intergeneric transfer products were classified into nuclear hybrid, heterokaryon or synkaryon, and reconstituted cell. These progenies except nuclear hybrids formed mature fruiting bodies on sawdust rice bran medium. Formation of fruit bodies was influenced by several factors such as light, temperature, nutrition and physic state of the culture media. Most of fruiting body characters were similar to those of P. florida in synkaryon and L. edodes in reconstituted cell, respectively. All these basidiocarps had clamp connections though initial heterokaryon colonies were lacking. Isozyme patterns of intergeneric progenies were quite different from those of parents. DNA polymorphisms of transfer products were also compared by random amplified polymorphic DNAs (RAPD) analysis based on polymerase chain reaction. The RAPD patterns were different from those of donor and recipient. DNA fingerprints ranged in size from 0.25 to 4.0 Kb. On the basis of RAPD, the transfer products were classified into five groups. Two synkaryon were analysed with distribution of progenies and segregation of genetic markers by random spore analyses. The genetic markers were segregated into wild type and riboflavine requiring auxotrophs.

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Morphological and Genetic Characteristics of Colletotrichum gloeosporioides Isolated from Newly Emerging Static-Symptom Anthracnose in Apple

  • Jeon, Yongho;Cheon, Wonsu
    • 한국균학회소식:학술대회논문집
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    • 2014.10a
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    • pp.34-34
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    • 2014
  • Filamentous fungi of the genus Colletotrichum (teleomorph, Glomerella) are considered major plant pathogens worldwide. Cereals, legumes, vegetables, and fruit trees may be seriously affected by this pathogen (1). Colletotrichum species cause typical disease symptoms known as anthracnoses, characterized by sunken necrotic tissue, where orange conidial masses are produced. Anthracnose appears in both developing and mature plant tissues (2). We investigated disease occurrence in apple orchards from 2013 to 2014 in northern Gyeongbuk province, Korea. Typical anthracnose with advanced symptoms was observed in all apple orchards studied. Of late, static fruit spot symptoms are being observed in apple orchards. A small lesion, which does not expand further and remains static until the harvesting season, is observed at the beginning of fruit growth period. In our study, static symptoms, together with the typical symptoms, were observed on apples. The isolated fungus was tested for pathogenicity on cv. 'Fuji apple' (fully ripe fruits, unripe fruits, and cross-section of fruits) by inoculating the fruits with a conidial suspension ($10^5$ conidia/ml). In apple inoculated with typical anthracnose fungus, the anthracnose symptoms progressed, and dark lesions with salmon-colored masses of conidia were observed on fruit, which were also soft and sunken. However, in apple inoculated with fungi causing static symptoms, the size of the spots did not increase. Interestingly, the shape and size of the conidia and the shape of the appressoria of both types of fungi were found to be similar. The conidia of the two types of fungi were straight and cylindrical, with an obtuse apex. The culture and morphological characteristics of the conidia were similar to those of C. gloeosporioides (5). The conidia of C. gloeosporioides germinate and form appressoria in response to chemical signals such as host surface wax and the fruitripening hormone ethylene (3). In this study, the spores started to germinate 4 h after incubation with an ethephon suspension. Then, the germ tubes began to swell, and subsequently, differentiation into appressoria with dark thick walls was completed by 8 h. In advanced symptoms, fungal spores of virtually all the appressoria formed primary hyphae within 16 h. However, in the static-symptom fungus spores, no primary hyphae formed by 16 h. The two types of isolates exhibited different growth rates on medium containing apple pectin, Na polypectate, or glucose as the sole carbon. Static-symptom fungi had a >10% reduction in growth (apple pectin, 14.9%; Na polypectate, 27.7%; glucose, 10.4%). The fungal isolates were also genetically characterized by sequencing. ITS regions of rDNA, chitin synthase 1 (CHS1), actin (ACT), and ${\beta}$-tubulin (${\beta}t$) were amplified from isolates using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, ACT-512F and ACT-783R, and T1 and ${\beta}t2$ (5), respectively. The resulting sequences showed 100% identity with sequences of C. gloeosporioides at KC493156, and the sequence of the ${\beta}$t gene showed 100% identity with C. gloeosporioides at JX009557.1. Therefore, sequence data from the four loci studied proves that the isolated pathogen is C. gloeosporioides. We also performed random amplified polymorphic DNA-PCR, which showed clearly differentiated subgroups of C. gloeosporioides genotypes. The clustering of these groups was highly related to the symptom types of the individual strains.

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Amplified fragment length polymorphism fingerprinting analysis of Staphylococcus aureus isolated from bovine mastitis milk (소 유방염 유래 Staphylococcus aureus의 AFLP 지문분석)

  • Kim, Yeon-soo;Kim, Sang-kyun;Hwang, Eui-kyung
    • Korean Journal of Veterinary Research
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    • v.41 no.2
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    • pp.157-165
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    • 2001
  • Amplified fragment length polymorphism(AFLP) technique is based on the polymorphism detection through selective PCR amplification of restriction fragments from digested genomic DNA and thus includes the procedures of the total DNA digestion by endonucleases, ligation of adapters to the ends of the fragments, and following the selective amplification of the restricted DNA fragments. This study were aimed to : (1) determine the genetic variability of S aureus strains, (2) estimate genetic diversity within and among these strains, (3) compare phylogenetic relationships among these strains as genetic markers using AFLP techniques. Genomic DNA was digested with a particular combination of three restriction enzymes with specific recognition sites and the DNA fragments were ligated to restriction specific adapters and amplified using the selective primer combinations. In the S aureus strain, the number of scorable AFLP bands detected per each primer combination varied from 29 to 102, with an average of 61.59 using 27 primer combinations. A total of 1,663 markers were generated, 904 bands of which were polymorphic, showing a 33.48% level of polymorphism with these primer combinations. Among the primer combinations, E02/T02, E02/T03, E04/H02, E02/T01 and E04/H03 primer combinations showed a high level of polymorphism with 0.78, 0.76, 0.74, 0.71 and 0.70, respectively. But T03/H01, E01/T02 and E01/T03 primer combinations showed a low level of polymorphism with 0.38, 0.37 and 0.15, respectively, Therefore, the former primer combinations will be the most effective for AFLP analysis of S aureus. In SA1 sub-types the level of polymorphism of S aureus KCTC 1927 was similar to that of S aureus CU 01(0.825) and higher than those of other strains such as S aureus CU 02 (0.715), S aureus KCTC 2199(0.625), S aureus KCTC 1916(0.607) and S aureus KCTC 1621 (0.553). In SA2 sub-types the level of polymorphism of S aureus CU 07 was similar to that of S aureus CU 08(0.935) and higher than those of both S aureus CU 04(0.883) and S aureus CU 05(0.883) and lower than those of S aureus CU 03(0.583). In SA3 subtypes the level of polymorphism of S aureus CU 11 was similar to that of S aureus CU 12(0.913) and lower than that of S aureus CU 15(0.623). The results proved that AFLP marker analysis of S aureus strain could be used to study the epidemiology of mastitis and in addition, common genotype in geographic region could be useful for the development of an effective vaccine or DNA marker for easy diagnosis of mastitis caused by S aureus infection.

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Development and Characterization of Chloroplast Simple Sequence Repeat markers in Pinus koraiensis (잣나무 엽록체 Simple Sequence Repeat 표지자 개발 및 특성 분석)

  • Lee, Jei-Wan;Baek, Seung-Hoon;Hong, Kyung-Nak;Hong, Yong-Pyo;Lee, Seok-Woo;Ahn, Ji-Young
    • Journal of Korean Society of Forest Science
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    • v.104 no.4
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    • pp.549-557
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    • 2015
  • Novel cpSSR primers were developed based on the sequence information of the Pinus koraiensis chloroplast genome. A total of 30 cpSSR loci were detected in the chloroplast genome, and a total of 30 primer sets flanking those loci were designed. All primer sets were successfully amplified for chloroplast DNA in P. koraiensis. The cross-species transferability of the 30 primer sets was considerably high in P. pumila (100%) and P. paviflora (97%) belonging to the same Subgenus (Strobus) of P. koraiensis. Meanwhile, the transferability was relatively low (73%) in P. densiflora and P. sylvestris belonging to Subgenus Pinus. A total of 13 cpSSR loci out of the 30 loci were polymorphic in the Mt. Jumbong population of P. koraiensis. The mean of haploid diversity(H) was 0.512. The number of haplotypes(N) and the haplotype diversity($H_e$) were 25 and 0.992, respectively. Of the 25 haplotypes, 22 were unique in the analyzed population. The unique haplotypes differentiated 22 individuals (79%) from the total of 28 individuals. In conclusion, the novel cpSSR primers developed in this study would be applicable to other Pinus species, especially the subgenus Strobus, and provide a high level of polymorphism for the study of genetic variation of P. koraiensis.

Classification of Microsatellite Alterations Detected in Endoscopic Biopsy Specimens of Gastric Cancers (단순반복염기서열의 변이 형태에 따른 위암 내시경 조직의 유전자형 분류)

  • Choi Young Deok;Choi Sang Wook;Jeon Eun Jeong;Jeong Jeong Jo;Min Ki Ouk;Lee Kang Hoon;Lee Sung;Rhyu Mun Gan
    • Journal of Gastric Cancer
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    • v.4 no.2
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    • pp.109-120
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    • 2004
  • Purpose: Individual gastric cancers demonstrate complicated genetic alterations. The PCR-based analysis of polymorphic microsatellite sequences on cancer-related chromosomes has been used to detect chromosomal loss and microsatellite instability. For the purpose of preoperative usage, we analyzed the correspondance rate of the microsatellite genotype between endoscopic biopsy and surgical specimens. Materials and Methods: Seventy-three pairs of biopsy and surgical specimens were examined for loss of heterozygosity and microsatellite instability by using 40 microsatellite markers on eight chromosomes. Microsatellite alterations in tumor DNAs were classified into a high-risk group (baselinelevel loss of heterozygosity: 1 chromosomal loss in diffuse type and high-level loss of heterozygosity: 4 or more chromosomal losses) and a low-risk group (microsatellite instability and low-level loss of heterozygosity: 2 or 3 chromosomal losses in diffuse type or $1\∼3$ chromosomal losses in intestinal type) based on the extent of chromosomal loss and microsatellite instability. Results: The chromosomal losses of the biopsy and the surgical specimens were found to be different in 21 of the 73 cases, 19 cases of which were categorized into a genotype group of similar extent. In 100 surgical specimens, the high-risk genotype group showed a high incidence of nodal involvement (19 of 23 cases: $\leq$5 cm; 23 of 24 cases: >5 cm) irrespective of tumor size while the incidence of nodal involvement for the low-risk genotype group depended on tumor size (5 of 26 cases: $\leq$5 cm; 18 of 27 cases: >5 cm). Extraserosal invasion was more frequent in large-sized tumor in both the high-risk genotype group ($\leq$5 cm: 12 of 23 cases; >5 cm: 23 of 24 cases) and the low-risk genotype group ($\leq$5 cm: 7 of 26 cases; >5 cm: 16 of 27 cases). The preoperative prediction of tumor invasion and nodal involvement based on tumor size and genotype corresponded closely to the pathologic tumor stage (ROC area >0.7). Conclusion: An endoscopic biopsy specimen of gastric cancer can be used to make a preoperative genetic diagnosis that accurately reflect the genotype of the corresponding surgical specimen.

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Identification and Characterization of Polymorphic Microsatellite DNA Markers Using Next-generation Sequencing in Parapristipoma trilineatum (차세대 염기서열 분석법을 사용한 벤자리(Parapristipoma trilineatum)의 microsatellite 마커의 개발 및 유전학적 특성 분석)

  • Chun Mae Dong;Mi-Nan Lee;Jae Koo Noh;Jin Woo Park;Young-Ok Kim;Eun-Mi Kim
    • Journal of Life Science
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    • v.33 no.8
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    • pp.623-631
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    • 2023
  • This study was conducted to develop microsatellite markers in Parapristipoma trilineatum using next-generation sequencing. A total of 402,244,934 reads were generated on the Illumina Hiseq X Ten System, yielding 60,738,985,034 bp of sequences. The de novo assembly resulted in 1,320,995 contigs. A total of 952,326 contigs (0.016%) including 151 microsatellite loci were derived from the 1,320,995 contigs longer than 640 bp. A total of 34 primer sets were designed from the 151 microsatellite loci. As a result, 15 microsatellite loci were chosen and used for assuming population genetic parameters in the wild and farmed populations. The mean number of effective alleles was 12, ranging from 6 to 25. The observed heterozygosity (HO) and the expected heterozygosity (HE) ranged between 0.530 and 0.873, with an average of 0.750, and from 0.647 to 0.895, with an average of 0.793, respectively. According to these results, the developed set of 15 microsatellite markers is expected to be useful for the analysis of genetic characteristics in the population of P. trilineatum in Korea. There are requirements now for further genetic information, fishery resource management, breeding guidelines, support with the selection of breeds and studies on the effects of release, all of which will improve species conservation, and through future research, we aim to offer genetic foundational data with that goal.

Association Analysis of MUC5AC Promoter Polymorphism with Asthma (MUC5AC 프로모터의 유전자 다형성과 천식과의 연관성)

  • Han, Seon-Sook;Sung, Ji Hyun;Lee, Mi-Eun;Lee, Seung-Joon;Lee, Sung Joon;Kim, Woo Jin
    • Tuberculosis and Respiratory Diseases
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    • v.63 no.3
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    • pp.235-241
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    • 2007
  • Background: Airway mucus hypersecretion plays an important role in the pathogenesis of asthma, and is associated with the induction of MUC5AC expression in airway secretion. The MUC5AC gene is highly polymorphic; however, there are few studies about the association between the polymorphisms of the MUC5AC gene and asthma susceptibility or asthma phenotypes. We have investigated the association of MUC5AC promoter polymorphisms with the risk of asthma and asthma phenotypes. Methods: We determined the genotypes of the MUC5AC promoter (-1274G>A) in 78 asthma patients and in 78 age, sex-matched control individuals in the Korean population. Genomic DNAs from blood were analyzed by PCR and RFLP (restriction fragment length polymorphism) determination. We examined $FEV_1$, total eosinophil count, serum IgE level, $PC_{20}$ and the presence of atopy (by a skin test) in asthma patients. Results: The mean age of the patients was $47.7{\pm}16.1$ years and 38.5% were men, and the mean $FEV_1$ was $84.4{\pm}22.3%$ of predicted in the asthma patients. The -1274G>A polymorphism of the MUC5AC promoter in asthma patients was not significantly different as compared with normal individuals (GG 57.7%, AG 34.6% and AA 7.7% in asthma patients vs. GG 56.4%, AG 38.5% and AA 5.1% in control subject, p = 0.752, Cod). Several clinical parameters in asthma patients such as $FEV_1$, total eosinophil count, serum IgE level, $PC_{20}$ and the presence of atopy, were not associated with the -1274G>A polymorphism of the MUC5AC promoter. Conclusion: The -1274G>A single nucleotide polymorphism (SNP) frequency of the MUC5AC promoter was not associated with asthma in a Korean population.

Reproductive Ecology and Genetic Variations in the Sea Urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus in the Cheju Coast (제주연안에 서식하는 보라성게 Anthocidaris crassispina와 말똥성게 Hemicentrotus pulcherrimus의 지역별 번식생태학적 특성과 유전적 변이의 비교)

  • 이정재;김범규;강상균;정상철;이기완;최광식
    • Journal of Aquaculture
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    • v.13 no.2
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    • pp.129-135
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    • 2000
  • Genetic variabilities of local populations of Anthocidaris crassispina and Hemicentrotus pulcherrimus were analysed by random amplified polymorphic DNA (RAPD) technique with phenotypic variabilities in timing of gonadal maturation, reproductive output and size of individuals in Cheju. H, pulcherrimus, collected from 4 locations during March 1997, indicated that Daepo individuals were significantly smaller than that at Hamdok, Wimi site A and Wimi site B (ANOVA, p>0.001). Gonodal-somatic index (GSI) of the Wimi site B population was significantly higher than that of three other locations (ANOVA, P>0.0001). An ANOVA test conducted on test size of A. crassispina, harvested from six different locations of Cheju during June 1997, indicated that the size of individuals from Pophwan was significantly smaller (P>0.0001) than that from five other locations. GSIs of urchin m Wimi and Hanlim were significantly higher than that from Pohwan and Oedo (ANOVA, p>0.0001). Genetic similarity, calculated from k13 primer analysis of total DNA, among the six different populations of A. crassispina varied from 0.67 to 0.92, and the values for H. pulcherrimus from 0.60 to 0.73; thus there was no genetic variation among different populations of the same species. Therefore, the populations are genetically homologous and the observed phenotypic variabilities were possibly associated with water temperature and food.

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Construction of a Genetic Linkage Map in Radish(Raphanus sativus L.) Using RAPD Markers (RAPD 마커를 이용한 무의 유전자지도 작성)

  • Ahn, Choon-Hee;Choi, Su-Ryun;Lim, Yong-Pyo;Chung, Hae-Joon;Yae, Byeong-Woo;Yoon, Wha-Mo
    • Journal of Plant Biotechnology
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    • v.29 no.3
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    • pp.151-159
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    • 2002
  • Genetic map and molecular marker have a great importance in improving and facilitating crop breeding program as well as in genome analysis and map-based cloning of genes representing desirable characters. This study aimed at developing RAPD markers and constructing a genetic linkage map using 82 BC$_1$F$_1$individuals originated from the cross between '835' and B$_2$in radish (Raphanus sativus L.). One of the parents for genetic linkage map construction, '835'(P$_1$) of egg type is susceptible to Fusarium wilt and have medium resistance to virus infection and the other parent, B$_2$(P$_2$) of round type, is susceptible to Fusarium wilt and virus, Screening of 394 RAPD primers in BC$_1$F$_1$) population resulted in selecting 128 polymorphic markers which displayed 1:1 segregation pattern. Two markers failed to display 1:1 segregation and showed the segregation ratio skewed to maternal genotype. Selected markers were categorized into 14 linkage group based on LOD score represented by MAPMAKER/EXP program. Five groups composed of single marker among them were excluded from the linkage map, and consequently, the remaining groups are well matched with the number of radish chromosome (n=9). The linkage map constructed with 128 markers covers 1,688.3 cM and the average distance between markers was 13.8 cM. For developing STS marker, we determined the partial nucleotide sequence of OPE10 marker at both ends and designed a oligonucleotide primer pair based on this sequence. STS PCR using the primer pair displayed a single, clear band of which segregation is perfectly matched with that of OPE10 marker. This implies that RAPD markers could readily convert into clear and reliable STS markers.