• The Plant Pathology Journal
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• 제39권1호
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• pp.149-157
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• 2023

Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrI and 16SrXXXII groups) in Jeju Island (Republic of Korea).

• The Plant Pathology Journal
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• 제40권2호
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• pp.205-217
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• 2024

Brown rot disease, caused by Monilinia spp., poses a significant threat to pome and stone fruit crops globally, resulting in substantial economic losses during pre- and post-harvest stages. Monilinia fructigena, M. laxa, and M. fructicola are identified as the key agents responsible for brown rot disease. In this study, we employed the amplified fragment length polymorphism (AFLP) method to assess the genetic diversity of 86 strains of Monilinia spp. isolated from major stone fruit cultivation regions in South Korea. Specifically, strains were collected from Chungcheong, Gangwon, Gyeonggi, Gyeongsang, and Jeolla provinces (-do). A comparative analysis of strain characteristics, such as isolation locations, host plants, and responses to chemical fungicides, was conducted. AFLP phylogenetic classification using 20 primer pairs revealed the presence of three distinct groups, with strains from Jeolla province consistently forming a separate group at a high frequency. Furthermore, M. fructicola was divided into three groups by the AFLP pattern. Principal coordinate analysis and PERMANOVA were applied to compare strain information, such as origin, host, and fungicide sensitivity, revealing significant partition patterns for AFLP according to geographic origin and host plants. This study represents the utilization of AFLP methodology to investigate the genetic variability among M. fructicola isolates, highlighting the importance of continuous monitoring and management of variations in the brown rot pathogen.

• 한국자원식물학회지
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• 제21권1호
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• pp.41-46
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• 2008

차나무의 분자학적 유연관계 및 유전적 다양성을 알아보기 위하여 한국에서 자생하는 차나무 집단 21개 지역 차나무에 대하여 DFR 유전자 부위를 이용하여 PCR-RFLP분석을 하였다. DFR 4+5 primer 쌍을 이용하여 증폭결과 DFR유전자의 크기는 약 1.4kb에서 PCR산물을 획득하였다. PCR산물에 제한효소 Hpa II 및 Mse I를 이용하여 RFLP분석 결과 차나무 집단간 또는 집단내 차나무간의 유전적 다양성을 보였다. Hpa II 제한효소를 이용한 RFLP 밴드패턴은 3가지 형태로 구분되었으며, 같은 차나무 집단내에서도 유전적 다양성이 나타났다. Mse I 제한효소를 이용한 밴드패턴은 6가지의 형태로 다양성을 보였으며, 웅포집단 차나무의 경우 집단 내 다양성이 2가지 형태로 나타나 같은 집단내에서의 유전적 변이는 적은 것으로 보인다. 본 연구에서 사용한 2가지의 제한효소의 결과 차나무의 집단간 또는 같은 집단내의 차나무간에서 유전적 다양성을 확인할 수 있었다.

• The Plant Pathology Journal
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• 제25권4호
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• pp.322-327
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• 2009

A total of 23 isolates of Botrytis cinerea causing the grey mold were collected from infected ginseng in several fields of Korea. The sensitivity to carbendazim and the mixture of carbendazim plus diethofencarb was determined through a mycelial inhibition test on PDA amended with or without fungicides. B. cinerea isolates were classified as 3 phenotypes, which were the first phenotype resistant to both of carbendazim and the mixture ($Car^RMix^R$), the second one resistant to carbendazim and sensitive to the mixture ($Car^RMix^S$), and the last one sensitive to both of them ($Car^RMix^S$). Carbendazim resistance correlated with a single mutation $\beta$-tubulin gene of B. cinerea amplified with primer pair tubkjhL and tubkjhR causing a change of glutamate to alanine at amino acid position 198. Furthermore, the substitution of valine for glutamate led the resistance to carbendazim and the mixture at the same position of amino acid. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction endonuclease, Tsp451 and BstUI allowed differentiation of the PCR fragment of $\beta$-tubulin gene of $Car^SMix^S$ isolates from that of $Car^RMix^R$ and $Car^RMix^S$ isolates. This method will aid in a fast detection of resistance of carbendazim and the mixture of carbendazim plus diethofencarb in B. cinerea in ginseng field.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.615-621
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• 2007

AMP-activated protein kinase alpha 2 (PRKAA2) plays a key role in regulation of fatty acid and cholesterol metabolism. This study investigated the porcine PRKAA2 gene as a positional candidate for intramuscular fat and backfat thickness traits in pig chromosome 6. A partial fragment of the porcine PRKAA2 gene, amplified by PCR, contained a putative intron 3 including a part of exon 3 and 4, comparable with that of human PRKAA2 gene. Within the fragment, several single nucleotide polymorphisms were identified using multiple sequence alignments. Of these, TaqI restriction enzyme polymorphism was used for genotyping various pig breeds including Korean reference family. Using linkage and physical mapping, the porcine PRKAA2 gene was mapped in the region between microsatellite markers SW1881 and SW1680 on chromosome 6. Allele frequencies were quite different among pig breeds. The full length cDNA of the porcine PRKAA2 (2,145 bp) obtained by RACE containing 1,656 bp open reading frame of deduced 552 amino acids, had sequence identities with PRKAA2 of human (98.2%), rat (97.8%), and mouse (97.5%). These results suggested that the porcine PRKAA2 is a positional candidate gene for fat deposition trait at near telomeric region of the long arm of SSC 6.

• 대한소아치과학회지
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• 제25권2호
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• pp.292-303
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• 1998

The arbitrary primer polymerase chain reaction(AP-PCR) and Southern blot restriction fragment length polymorphism(RFLP) were used to genotype the cariogenic pathogen S. mutans in children. Following the morphologic chracteristics of colony on selective medium for S. mutans, total genomic DNA from 155 strains was extracted by conventional methods. Among 155 strains, 143 strains (92.3%) were confirmed S. mutans by PCR with dexA gene and 114 strains were used in this study. Three random sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 2% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI and BamHI, separated on a 0.7 % agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterised 1.6 kilobases (kb) DNA fragment cloned from gtf B gene of S. mutans. The probe was labeled with isotope[$^{32}P-{\alpha}CTP$], and hybridized fragments were detected with intensifying screen. AP-PCR produced 4-8 DNA bands in the 0.25-10 kb regions and distinguished 9, 10 or 12 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 2 hybridization patterns consisting of 1 DNA fragments 450, 500 bp. These results indicate that AP-PCR is more discriminative method for genotyping of S. mutans.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.323-330
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• 2009

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84-58) and M. chelonae (477-84-80-58), and M. kansasii (141-136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.

• BMB Reports
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• 제34권2호
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• pp.114-117
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• 2001

Cervi Parvum Cornu is widely used as a hemopoietic, tonifying, growth-promoting, cardiotonic, and immuno-modulating agent in Korea. In order to develop the quality control method of Cervi Parvum Cornu by the identification of the biological source or origin, the molecular approach was applied using PCR (polymerase chain reaction) and PCR-RFLF (PCR-restriction fragment length polymorphism) analysis. In the PCR analysis of the mitochondrial 12S rRNA gene and cytochrome b gene regions, no distinctive DNA bands from Cervidae (deer) antlers and Rangifer (reindeer) antlers were observed. However, when the amplified products in the mitochondrial cytochrome b gene region were subjected to restriction digestion with TaqI, Cervidae antlers showed an undigested state of 380 by band, differently from two bands of 230 by and 1S0 by from Rangifer antlers. Based on this finding, the base sequences of amplified PCR products in the range of mitochondria) cytochrome b gene from Cervidae antlers and Rangifer antlers were determined and subjected to restriction analysis by various endonucleases. The results showed that antlers from Rangifer species could be simply discriminated with other antlers from 8 Cervidae species (Chinese deer, Russian deer, Hong Kong deer, New Zealand deer, Kazakhstan deer, elk, red deer and Sika deer) by PCR-RFLP analysis using AtuI, HaeIII, HpaII or Sau3AI(MboI) as well as TaqI in the range of the mitochondrial cytochrome b gene.

• The Plant Pathology Journal
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• 제25권3호
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• pp.247-255
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• 2009

Root-knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria, and M. javanica are the most economically notorious nematode pests, causing serious damage to a variety of crops throughout the world. In this study, DNA sequence analyses were performed on the D3 expansion segment of the 28S gene in the ribosomal DNA in an effort to characterize genetic variations in the three Meloidogyne species obtained from Korea and four species from the United States. Further, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) PCR and RAPD (Randomly Amplified Polymorphic DNA) were also utilized to develop methods for the accurate and rapid species identification of the root-knot nematode species. In the sequence analysis of the D3 expansion segment, only a few nucleotide sequence variations were detected among M. incognita, M. arenaria, and M, javanica, but not M. hapla. As a result of our haplotype analysis, haplotype 5 was shown to be common in M. arenaria, M. incognita, M. javanica, but not in the facultatively parthenogenetic species, M. hapla. PCR-RFLP analysis involving the amplification of the mitochondrial COII and large ribosomal RNA (lrRNA) regions yielded one distinct amplicon for M. hapla at 500 bp, thereby enabling us to distinguish M. hapla from M. incognita, M. arenaria, and M. javanica reproduced via obligate mitotic parthenogenesis. SCAR markers were used to successfully identify the four tested root-knot nematode species. Furthermore, newly attempted RAPD primers for some available root-knot nematodes also provided some species-specific amplification patterns that could also be used to distinguish among root-knot nematode species for quarantine purposes.

• 한국작물학회지
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• 제49권1호
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• pp.69-72
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• 2004

Somaclonal variation, defined as phenotypic and genetic variations among regenerated plants from a parental plant, could be caused by changes in chromosome structure, single gene mutation, cytoplasm genetic mutation, insertion of transposable elements, and DNA methylation during plant regeneration. The objective of this study was to evaluate DNA variations among somaclonal variants from the cotyledonary node culture in soybean. A total of 61 soybean somaclones including seven $\textrm{R}_1$ lines and seven $\textrm{R}_2$ lines from Iksannamulkong as well as 27 $\textrm{R}_1$ lines and 20 $\textrm{R}_2$ lines from Jinju 1 were regenerated by organogenesis from the soybean cotyledonary node culture system. Field evaluation revealed no phenotypic difference in major agronomic traits between somaclonal variants and their wild types. AFLP and SSR analyses were performed to detect variations at the DNA level among somaclonal variants of two varieties. Based on AFLP analysis using 36 primer sets, 17 of 892 bands were polymorphic between Iksannamulkong and its somaclonal variants and 11 of 887 bands were polymorphic between Jinju 1 and its somaclonal variants, indicating the presence of DNA sequence change during plant regeneration. Using 36 SSR markers, two polymorphic SSR markers were detected between Iksannamulkong and its somaclonal variants. Sequence comparison amplified with the primers flanking Satt545 showed four additional stretches of ATT repeat in the variant. This suggests that variation at the DNA level between somaclonal variants and their wild types could provide basis for inducing mutation via plant regeneration and broadening crop genetic diversity.