• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.41-41
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• 2015

Oomycetes belong to the kingdom Straminipila, a remarkably diverse group which includes brown algae and planktonic diatoms, although they have previously been classified under the kingdom Fungi. These organisms have evolved both saprophytic and pathogenic lifestyles, and more than 60% of the known species are pathogens on plants, the majority of which are classified into the order Peronosporales (includes downy mildews, Phytophthora, and Pythium). Recent phylogenetic investigations based on DNA sequences have revealed that the diversity of oomycetes has been largely underestimated. Although morphology is the most valuable criterion for their identification and diversity, morphological species identification is time-consuming and in some groups very difficult, especially for non-taxonomists. DNA barcoding is a fast and reliable tool for identification of species, enabling us to unravel the diversity and distribution of oomycetes. Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The mitochondrial cox2 gene has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. To determine which out of cox1 or cox2 is best suited as universal oomycete barcode, we compared these two genes in terms of (1) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (2) in terms of sequence polymorphism, intra- and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding type material. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. Including the two barcoding markers, ITS rDNA and cox2 mtDNA, the multi-locus phylogenetic analyses were performed to resolve two complex clades, Bremia lactucae (lettuce downy mildew) and Peronospora effuse (spinach downy mildew) at the species level and to infer evolutionary relationships within them. The approaches discriminated all currently accepted species and revealed several previously unrecognized lineages, which are specific to a host genus or species. The sequence polymorphisms were useful to develop a real-time quantitative PCR (qPCR) assay for detection of airborne inoculum of B. lactucae and P. effusa. Specificity tests revealed that the qPCR assay is specific for detection of each species. This assay is sensitive, enabling detection of very low levels of inoculum that may be present in the field. Early detection of the pathogen, coupled with knowledge of other factors that favor downy mildew outbreaks, may enable disease forecasting for judicious timing of fungicide applications.

• Korean Journal of Agricultural Science
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• v.39 no.1
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• pp.23-28
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• 2012

In order to cloning of PPO gene as a melanin formation related genes involved in hardening and pigmentation of insect integument or wing, we cloned cDNA and analyzed the sequence of PPO gene of H. axyridis. PPO2 primer were designed based on the sequences of PPO genes of Tribolium castaneum and Drosophila melanogaster, and then plasmid DNA were cloned from PCR products obtained from different two color patterns. When the plasmid DNA band pattern were digested by restriction enzymes, BamH1, Xba1, and EcoR1, we found same size band pattern. However, this sequence was not homologous to sequence of T. castaneum PPO gene. Using the primer designed based on the sequence of D. melanogaster, 209 bp PCR product was observed.

• Journal of Photoscience
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• v.7 no.2
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• pp.73-78
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• 2000

To analyze molecular traits determining pigmentation between Pharbitis nill violet and white, Amplified Fragment Length Polymorphism(AFLP) and Differential Display Reverse Transcription(DDRT) experiments were carried out with either genomic DNAs or total RNAs isolated from both plants. Results of AFLP experiment in combination of 8 EcoRⅠ primers with 6 MseⅠ primers showed 41 violet-and 60 white-specific DNA bands. In the subsequent experiment, 22 violet-and 22 white-specific DNA fragments were amplified by PCR with DNAs eluted. The sizes of the fragments range from 200 to 600bp. DDRT using total RNA produced 19 violet-and 17 white-specific cDNA fragments, ranging from 200 to 600bp. The fragments obtained by both AFLP and DDRT had been cloned into pGEM T-easy vector, amplified and subjected to the nucleotide sequence analyses. As a result of Blast sequence analysis, most of them sequenced up to date showed no similarity to any Known gene, while few has similarity to known animal or plant genes. An AFLP clone V6, for example, has a strong sequence similarity to the human transcription factor LZIP-alpha mRNA and a DDRT clone W19 to Solanum tuberosum 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA.

• BMB Reports
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• v.29 no.2
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• pp.146-150
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• 1996

A 2.1 kb cDNA clone for rat transketolase was isolated from rat liver ${\lambda}gt11$ cDNA library and its sequence was determined. The predicted rat transketolase (655 amino acids with $M_r$ 71,186) is highly similar (92%) to that of the human enzyme except that it contains an extra 32 amino acids at its N-terminus. Although it is less similar (<27%) to transketolases from non-mammalian species, the functional motifs such as the catalytic sites and thiamine binding domain are well conserved in the rat enzyme. Southern blot analysis of genomic DNA verified that transketolase appears to be derived from a single gene. Immunoblot and Northern blot analyses suggested that hepatic transketolase was activated pretranslationally by a 2.1-fold while little change was observed in brain enzyme, indicating a tissue-specific pretranslational activation during postnatal development.

• Korean Journal of Microbiology
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• v.37 no.2
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• pp.137-144
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• 2001

In order to investigate the diversity of bacterial community in the mud flat of Sunchon Bay, Chunnam province, diversity of amplified 16S rDNA was examined. Total DNA was extracted from sediment soils and 16S rDNAs were amplified using PCR primers based on the universally conserved sequences in bacteria. Clonal libraries were constructed and 111 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII. Clones were clustered based on restriction patterns using computer program, GelCompar II. One hundred different RFLP types were detected from 111 clones. The 20 clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the bacterial diversity in the clone libraries. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA databases and GenBank. All sequences showed between 77 and 96.8% similarity to the known 16s rRNA sequence from cultured organisms. The 20 clones sequenced fell into seven major lineages of the domain Bacteria: alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga) and Cyanobacteria (chloroplast). Among the clones, the Proteobacteria were dominant.

• Journal of Animal Science and Technology
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• v.46 no.6
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• pp.937-946
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• 2004

Duroc is widely used to improve the meat quality and productivity. To elucidate the phylogenetic relation and the sequence specificity for the maternal property, the complete sequence of mitochondrial genome was determined and the population diversity of Duroc was investigated in this study. The length of mtDNA tested is 16,584-bp. There are several insertion/deletion mutations in the control region and coding regions for tRNA and rRNA, respectively, but not in peptide-coding regions. Four peptide-coding genes(COⅡ, COⅢ, ND3 and ND4) showed incomplete termination codon sequences such as T--, and two(ND2 and ND4L) did alternative initiation codons(AIC), respectively. Especially, the initiation codon sequences of ND2 gene were polymorphic in this population. Polymorphisms were detected in 11-bp duplication motif within control region as well as ND2 and CYTB. Variation patterns observed from the tests on three mtDNA regions were linked completely and then two haplotypes obtained from combining the data dividing this population. Duroc mtDNA is observed at the European pig cluster in the phylogenetic tree, however, the results from the population analyses supported previous opinions. This study suggests that the breed Duroc was mainly originated from the European pig lineage, and Asian lineage was also used to form the pig breed Duroc as maternal progenitors.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.212-220
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• 2008

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.45-51
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• 2003

Genetic diversity of local populations among geologically isolated groups of Rana amurensis was refined by sequence comparison of the mitochondrial (mt) 165 rDNA genes. Each 401 base pairs of DNA sequences, which was determined from four local populations of Rana amurensis, two local populations of R. nigromacutata, and three species of the genus Rana were used in this analysis. Despite morphological similarity of Rana amurensis, Korean populations were well distinguished from the other groups on the basis of 105 rDNA gene difference. Further analyses for additional local populations belonging to R. amurensis will be necessary to clarify the taxonomic status.

• Journal of Plant Biotechnology
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• v.42 no.2
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• pp.93-103
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• 2015

The isolation, cloning, and characterization of an R2R3-MYB transcription factor gene (MtMYB70) from the model legume Medicago truncatula is reported. MtMYB70 consists of a 768-bp coding sequence corresponding to 255 amino acids. Sequence alignment revealed that MtMYB70 cDNA contains conserved R2R3-type MYB domains with highly divergent C terminal regions. MtMYB70 was found to have relatively low sequence homology with known R2R3-MYB genes. Phylogenetic analysis placed the R2R3-MYB domain of MtMYB70 closest to PtMYB1, a known activator of lignin biosynthesis. Overexpression of MtMYB70 under the control of the 35S promoter in transgenic poplar did not cause a significant difference in total lignin content relative to the control, but glucan content was significantly increased in transgenic poplar. Therefore, MtMYB70 might have regulatory role in the biosynthesis of cell wall structural carbohydrates.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.215-228
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• 1999

To choose one or more appropriate molecular markers or gene regions for resolving a particular systematic question among the organisms at a certain categorical level is still a very difficult process. The primary goal of this review, therefore, is to provide a theoretical information in choosing one or more molecular markers or gene regions by illustrating general properties and phylogenetic utilities of nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) that have been most commonly used for phylogenetic researches. The highly conserved molecular markers and/or gene regions are useful for investigating phylogenetic relationships at higher categorical levels (deep branches of evolutionary history). On the other hand, the hypervariable molecular markers and/or gene regions are useful for elucidating phylogenetic relationships at lower categorical levels (recently diverged branches). In summary, different selective forces have led to the evolution of various molecular markers or gene regions with varying degrees of sequence conservation. Thus, appropriate molecular markers or gene regions should be chosen with even greater caution to deduce true phylogenetic relationships over a broad taxonomic spectrum.