• The Plant Pathology Journal
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• v.35 no.2
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• pp.164-171
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• 2019

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with realtime detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of $1{\times}10^4$ to $1{\times}10^{12}$ ASSVd RNA $copies/{\mu}l$ with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from $1{\times}10^7$ to $1{\times}10^{12}$ copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2000.11a
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• pp.59-75
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• 2000

This study was carried out to develop a DNA marker for identifying between Korean cattle (Hanwoo) and other breeds. First experiment was performed to isolate Hanwoo specific DNA marker at sequence characterized amplified regions (SCARs). Five breeds of cattle including Hanwoo, Holstein, Hereford, Angus and Charolais were represented with the from 8 to 20 individuals. Fourteen primers of 300 arbitrary primers of 10 nucleotides showed reproducible polymorphism across the breeds. An amplified band of 0.9 kb in the primer MG-3 showed the specificity to Holstein breed. And MG-6 and MG-12 detected the Hereford and Hanwoo specific markers at the size of 2.0 kb and 1.0 kb, respectively. A 1.0 kb band of MG-12 was cloned and sequenced. A SCAR primer was designed based on the obtained sequences. It was possible to identify the Hanwoo from Holstein breed. Second experiment was carried out to observe the genotype frequencies of MC1R in 1,044 samples of imported beef and eight different cattle breeds including Hanwoo, Holstein, Angus, Brown-Swiss, Charolais, Limousin, Simmental and Hereford. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no.Y19103). A size of 350 bp was amplified by polymerase chain reaction(PCR), digested with two different restriction enzyme, BsrFI and MspA II, and electrophoresed in 2.5% Metaphore agarose gel for determination of genotypes. Genotype frequencies of Hanwoo were 0.10 in E+e and 0.90 in ee. Allele ED was shown in all of Holstein and Angus breeds tested which have black coat color phenotypes. We suggested that SCAR marker and the bovine MC1R gene could be used as a DNA marker for distinguishing beef between Hanwoo and Holstein.

• The Korean Journal of Mycology
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• v.39 no.1
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• pp.22-30
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• 2011

Weonhyeong is one of important commercial strains. It has good characteristics of bundle formation, grey colored pilei and high productivity. We previously reported grouping of 70 strains of Pleurotus ostreatus in which one group contained 35 strains including Weonhyeong. Four strains in that group showed same profiles implicating no variety distinction for mushroom cultivation. Now we developed a specific marker for identification of Weonhyeong. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. SCAR marker 'S-OPO5' produced only one band specific to 2183, 2240, 2595 and 2725 strains showing similar banding patterns to Weonhyeong in RAPD-PCR results. The sequence of 'S-OPO5' marker was unknown when compared with the data in the Genbank using BLASTN. BLASTX results indicated that the marker showed significant alignment with the protein sequences in Tricholoma bakamatsutake reverse transcriptase. The results indicate that this new SCAR marker ('S-OPO5') will be valuable to distinguish the Weonhyeong similar strains from Pleurotus spp.

• Korean Journal of Medicinal Crop Science
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• v.15 no.1
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• pp.1-5
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• 2007

To develop a convenient method for discriminating between violet flowered lines and white flowered lines in Chinese bellflower, RAPD analysis was carried out and SCAR markers were generated. Eighteen specific RAPD bands were obtained from 6 OPERON primer sets. Two out of eighteen RAPD bands were cloned into pGEM-T-Easy vectors and then subjected to the nucleotide sequence analysis. PgR1 and PgR2 DNA fragment, each specific for violet and white flowered lines, consist of 887 bp and 863 bp sequences, respectively. Two SCAR markers were developed from RAPD clones: SPgR1 (355 bp) from PgR1 and SPgR2 (493 bp) from PgR2. One (SPgR2) of these two markers was useful to differentiate between violet flowered lines and white flowered lines in Chinese bellflower.

• Horticultural Science & Technology
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• v.30 no.2
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• pp.171-177
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• 2012

A total of 94 tomato accessions were evaluated for the resistance to $Tomato$ $spotted$ $wilt$ $virus$ (TSWV) using a Sw5-2 SCAR marker and bioassay. PCR products of the marker were approximately 574 bp, 500 bp, and 462 bp, among which the longest was linked to TSWV resistance allele of Sw5-b. This allele was only found in three accessions (09-438, 10-318, and 10-321) in which some individuals showed apparent recovery or stem necrosis symptom to a tomato isolate of TSWV-pb1. Thirty-five individuals (one per each accession) which were non-infected by ELISA were selected for further observation. Among these, 26 individuals that did not show any symptom at 5 months after inoculation were confirmed for viral infection by RT-PCR. TSWV-specific PCR amplicon was weakly detected in all 26 individuals including 'Eureta', a commercial F1 possessing the resistance allele of Sw5-b. The resistant genes in the selected individuals may play an important role for reducing the viral concentration in tissues of inoculated tomato plants and seems to be quantitatively controlled by several factors including Sw5-b gene.

• Asian Journal of Turfgrass Science
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• v.23 no.2
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• pp.307-316
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• 2009

Creeping bentgrass (Agrostis palustrics Huds.) is cool season turfgrasse that is used for putting green in golf course. Creeping bentgrass cultivars are difficult to distinguish with the same species because of similar morphological characters and low level of genetic diversity. The SCAR markers using the specific DNA can be useful for differentiating between creeping bentgrass cultivars. Five RAPD primers were used for specific band detection among creeping bentgrass cultivars, penncross, penn A-4, crenshaw, L-93, CY-2, T-1. The pairs of SCAR primers for six cultivers were designed by the specific sequences of the bands that amplified by RAPD. Three of the six SCAR primers could not make the use as SCAR primers because the specific false bands were detected in all cultivars. The remaining pairs of SCAR primer, CY850F/R, T700F/R, L2900F/R, amplified the specific band at expected size for three cultivars, CY-2, T-1, L-93, respectively. The CY850F/R primer amplified a band of 850bp in CY-2 cultivar, the T700F/R primer amplified a band of 700bp in T-1 cultivar, and the L2900F/R primer amplified a band of 2.9kb in L-93 cultivar. In this study we developed the SCAR markers to identify and distinguish the inerseeded creeping bentgrass cultivars in a golf course green.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.724-730
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• 2006

A sequence characterized amplified region (SCAR) marker, which was tightly linked with the A1 mating type locus in Phytophthora infestans, was developed. During the random amplified polymorphic DNA-based phylogenic studies of 33 isolates of P infestans collected from year 2002 to 2004, we found an A1 mating type-specific DNA fragment. This 573-bp DNA fragment was generated only in the genomic DNA of the A1 mating types, when OPC-5 primer was used. Based on the specific DNA sequence, we designed the primer sets for generating the A1 mating type-specific 569-bp DNA fragment. When 33 genomic DNAs of P. infestans were subjected to PCR amplification using different primer combinations, the A1 mating type-specific DNA was amplified, when LB-1F and LB-2R primers were used. The specific 569-bp DNA fragment was generated only from all 18 A1 strains, but not from 15 A2 mating type strains. These results corresponded to the mating type discriminating bioassay of 33 isolates of P. infestans. Therefore, the primer combination of LB-1F/LB2R was chosen as a SCAR marker. Overall, this study indicates that the SCAR marker could be developed into a useful tool for mating type determination of P. infestans.

• Korean Journal of Breeding Science
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• v.42 no.2
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• pp.139-143
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• 2010

Late blight caused by Phytophthora infestans is historically a serious epidemic disease in potato and tomato cultivations. Accession L3708 (Solanum pimpinellifolium), a new source for late blight resistance was identified in AVRDC, and carries the resistance gene, Ph-3, incompatible to P. infestans race 3. The AFLP markers linked to Ph-3 were previously developed from the L3708 accession (Chunwongse et al. 2002). To facilitate tomato breeding with the Ph-3 gene, an attempt was made to convert AFLP markers to sequence-characterized amplified region (SCAR) markers. Among 6 AFLP markers, only one AFLP marker, L87, was successfully converted to SCAR marker. The resistance-specific 230 bp AFLP fragment was cloned and sequenced, and the PCR primer amplifying a 123 bp fragment was designed. This SCAR marker could discriminate resistant and susceptible individuals with high stringency. The developed SCAR marker could be used for the marker assisted-selection in tomato breeding programs.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.36-45
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• 2006

Twenty isolates of Didymella bryoniae were isolated from infected cucurbit plants in various growing areas of southern Korea in 2001 and 2002. Random Amplified Polymorphic DNA (RAPD) group [RG] I of D. bryoniae was more virulent than RG IV to watermelon. Virulence of the RG I isolate was strong to moderate to cucumber, whereas that of the RG IV varied from strong, moderate to weak. Two hundred seventy-three amplified fragments were produced with 40 primers, and were analyzed by a cluster analysis using UPGMA method with an arithmetic average program of NTSYSPC. At the distance level of 0.7, two major genomic DNA RAPD groups were differentiated among 20 isolates. The RG I included 7 isolates from watermelon and one isolate from melon, whereas the RG IV included 12 isolates from squash, cucumber, watermelon and melon. Amplification of internal transcribed spacer (ITS) region and small subunit rRNA region from the 20 isolates yielded respectively a single fragment. Restriction pattern with 12 restriction enzymes was identical for all isolates tested, suggesting that variation in the ITS and small subunit within the D. bryoniae were low. Amplification of the genomic DNAs of the tested isolates with the sequence characterized amplified regions (SCAR) primer RG IF-RG IR specific for RG I group resulted in a single band of 650bp fragment for 8 isolates out of the 20 isolates. Therefore, these 8 isolates could be assigned into RG I. The same experiments done with RG IIF-RG IIR resulted in no amplified PCR product for the 20 isolates tested. An about 1.4 kb-fragment amplified from the RG IV isolates was specifically hybridized with PCR fragments amplified from genomic DNAs of the RG IV isolates only, suggesting that this PCR product could be used for discriminating the RG IV isolates from the RG I isolates as well other fungal species.

• The Plant Pathology Journal
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• v.17 no.5
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• pp.300-304
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• 2001

Apple is the most economically important fruit in Korea. The suspected viroid disease of dapple apple was found in apple fruits cultivated in Kyungpook province. Symptoms begin in mid-July as small circular spots, which stand out against the background color on the young fruit. Dappling of the fruit becomes more intense and easier to detect as the fruit approaches maturity; the affected spots remain yellowish as the fruit matures. no leaf or bark syndromes have been associated with this disease. The infected fruits are downgraded considerably during quality grading. The low molecular weight RNA containing viroid RNA molecules were extracted from the peels of the apples with dapple symptoms. The RNA molecules were extracted from the apples using Qiagen column chromatography. The purified RNAs were used for the synthesis of cDNA with RT-PCR. The PCR products were then ligated into a pGEM-T Easy vector, cloned and sequenced. The sequence of the viroid RNA molecule shows 331 nucleotides with one base difference ("G" insertion between the position of 133 and 134) compared with that of the Apple scar skin viroid (ASSVd) reported by Hashimoto and Koganezawa in Japan. This is the first report on the occurrence of the ASSVd in apple trees cultivated in Korea, as well as the identification of a new Korean strain of the ASSVd.the ASSVd.