Spodoptera litura Fabricius, 1775 (Lepidoptera: Noctuidae) is a serious crop pest with a long-distance migratory flight. To date, the DNA barcode region has been widely used in genetic diversity analysis studies of Spodoptera litura. However, the DNA barcode region showed maximum variation rate of S. litura, which from 18 regions in South Korea, was 0.608% (nine haplotypes) in previous study. In this study, four mitochondrial genes (ND4, ND4L, ND1, 16s rRNA) have higher intra-specific variation rates than the DNA barcode region. Among the four genes, The variation rate of the 16s rRNA region was confirmed to be a minimum of 0.203% (2bp) and a maximum of 1.824% (18bp). Finally, the 16s rRNA region with the highest PCR amplification efficiency and highest variation rate was selected as a high-efficiency molecular marker.

Mycoplasma hyorhinis (M. hyorhinis) is considered an etiological agent of arthritis in suckling pigs. Recently, some M. hyorhinis strains were shown to produce pneumonia that is indistinguishable from the mycoplasmosis caused by M. hyopneumoniae. In this study, we developed a sensitive and specific PCR assay to detect M. hyorhinis and applied the developed PCR assay for detection of Mycoplasma infection in clinical piglets infected with M. hyorhinis. We developed a new PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, designed from the Mycoplasma 16S-23S rRNA internal transcribed spacer (ITS) region. The primers and probe for the assay were designed from regions in the Mycoplasma 16S-23S rRNA ITS unique to M. hyorhinis. The developed PCR assay was very specific and sensitive for the detection of M. hyorhinis. The assay could detect the equivalent of 1 pg of target template DNA, which indicates that the assay was very sensitive. In addition, M. hyorhinis PCR assay detected only M. hyorhinis and not any other Mycoplasma or bacterial spp. of other genera. The new developed PCR assay effectively detected M. hyorhinis infection in pigs. We suggest that this PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, could be useful and effective for monitoring M. hyorhinis infection in pigs.

목 적: 콘택트렌즈 케이스에서 분리한 균주들을 동정하고, Serratia marcescens 균주들에 대해 항생 제 감수성 검사를 시행하였다. 방 법: 총 65개의 콘택트렌즈 케이스를 수거하여 분리한 균주들을 16s rRNA gene 염기서열을 분석하여 동정하였고, Serratia marcescens 균주들을 Nalidixic acid(30㎍), Ciprofloxacin(5㎍), Ofloxacin(5㎍), Levofloxacin(5㎍) 항생제를 이용하여 디스크 확산법으로 항생제 감수성 검사를 실시하였다. 결 과: 30개의 콘택트렌즈 케이스에서 세균이 검출되었으며, 균주로는 Achromobacter sp. 8균주, Achromobacter xylosoxidans 3균주, Achromobacter spanius 1균주, Pandoraea pnomenusa 1균주, Serratia marcescens 17균주가 동정되었다. Serratia marcescens 균주에 대한 항생제 감수성 검사에서는 Nalidixic acid, Ciprofloxacin, Ofloxacin, Levofloxacin 모두 감수성으로 나타났다. 결 론: 콘택트렌즈 케이스에서 분리한 균주들을 16S rRNA gene 염기서열을 분석하여 동정한 결과 모두 30 균주가 동정되었으며, 콘택트렌즈 케이스에서 많이 분리 동정된 Serratia marcescens 균주에 대한 Quinolone 계 항생제 감수성 검사에서 모두 감수성으로 나타났다. 그러나 항생제의 지속적인 사용으로 내성 균주들이 나타날 것으로 예측되며 Quinolone 항생제의 내성 발생률에 대한 체계적인 연구와 관리가 필요한 것으로 사료된다.

본 연구에서는 오징어류에 해당하는 대왕오징어, 오징어, 문어, 한치 및 이를 이용한 가공식품에 대해서 분자생물 학적 기법을 활용한 시험법을 검토하였다. 시료 중 원료 성분 확인을 위하여 오징어류 4종에 대해 최적의 종 특이 프라이머를 디자인하였으며, 시료로부터 직접 genomic DNA를 추출하여 PCR을 실시하였다. PCR 수행과정에서 반응을 저해하는 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR을 실시한 결과, Single PCR의 경우 대왕오징어(552 bp), 오징어(463 bp), 문어(247 bp), 한치(354 bp)에 해당하는 종 특이적인 증폭산물을 확인하였으며, Multiplex PCR 의 경우 서로 다른 종 사이의 교차반응없이 동시다발적으로 증폭이 일어남을 확인할 수 있었다. 또한 이들 4종에 대해 PCR 민감도를 조사한 결과, 모두 약 0.1 ng/μl의 농도까지 검출이 가능함을 확인하였으며 multiplex PCR의 경우 약 0.25 ng/μl의 농도까지 검출이 가능함을 확인하였다. 이를 이용하여 오징어류가 함유된 수산물 가공식품 8건에 대해 적용성을 검토한 결과, 모든 시료에서 유효한 결과를 확인할 수 있었다. 따라서 본 연구에서 제작된 오징어류 4종에 대한 종 특이적 프라이머는 생물 상태뿐만 아니라 수산물 가공식품에 대해서도 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다.

Nuclear ribosomal DNA (rDNA) was analyzed to identify inter-specific genetic relationships among 8 Cymbidium species (Cymbidium insigne, C. ensifolium, C. marginatum, C. faberi, C. gyokuchin, C. kanran, C. forrestii, and C. goeringii). Nuclear rDNA including 2 internal transcribed spacer (ITS) regions and 5.8S, was amplified using polymerase chain reaction and sequenced. The sequences were compared via pair-wise multiple alignment to determine the genetic relationships among the studied species. The lengths of the ITS1, ITS2, and 5.8S regions were 235 bp, from 255 bp to 257 bp, and from 153 bp to 165 bp, respectively. Sequence similarities in the ITS region ranged from 78.7% between C. gyokuchin and C. kanran to 96.8% between C. ensifolium and C. kanran. A phylogenetic tree was constructed from nuclear rDNA nucleotide sequence data of the 8 cymbidiums and 1 outgroup species to estimate genetic relationships. The tree revealed that cymbidiums could be classified by their ecological traits, such as their temperature preference or inflorescence pattern. The phylogenetic data is applicable for identification, classification, and breeding of cymbidiums.

Previously, several levels of phylogenetic relationships in an insect order Odonata have been estimated using morphological and molecular markers. For the molecular phylogeny rRNA sequences were mainly, but other markers were not frequently employed. In this study, we sequenced both two mitochondrial genes (COI and 16S rRNA) and nuclear genes (28S rRNA and elongation factor-1α), composed of ~4,002 bp from 71 species of Odonata, occurring mostly in South Korea. These concatenated sequences were utilized to test the previous phylogenetic hypotheses of Odonata via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms, along with the data partition option available in BI method. Each families and superfamilies represented by multiple taxa consistently supported monophylies with the highest nodal supports in both Anisoptera and Zygoptera. A close relationship of Anisozygoptera to Anisoptera represented by a single species was obvious. On the other hand, familial relationships within each suborder of Anisoptera and Zygoptera have shown two compelling topologies. The topology obtained by BI method with partitioning of the four genes showed an unresolved relationship among Gomphidae, Aeshnidae, and the suborder Anisozygoptera in Anisoptera clade, presenting the relationships ((((Libellulidae + Corduliidae) + Macromiidae) + (Gomphidae + Aeshnidae + Anisozygoptera)) + (((Coenagrionidae + Platycnemdidae) + Calopterygidae) + Lestidae)). Another topology obtained by both BI and ML methods without partitioning, on the other hand, placed Anisozygoptera the basal lineage of Anisoptera, but Lestidae in Zygoptera was placed as the sister to Anisoptera + Anisozygoptera, presenting the relationships (((((((Libellulidae + Corduliidae) + Macromiidae) + Aeshnidae) + Gomphidae) + Anisozygoptera) + Lestidae) + ((Coenagrionidae + Platycnemdidae) + Calopterygidae)). Topological test to find out better supported tree turned out a slight higher support for the former topology, but the monophyly of Zygoptera with the inclusion of Lestidae was supported only poorly (BPP = 0.68) in the former topology.

사슴풍뎅이는 꽃무지아과 중에서 수컷이 유일하게 특이한 뿔을 갖고 있을 뿐 아니라 긴 앞다리를 가진 모습으로 주목을 받아왔다. 이 종은 한반도에서 처음 신종으로 발표된 이래 베트남 북부, 티벳동부, 중국 서부와 중북부에 이르기까지 아종의 분화 없이 단일 종으로 다루어왔다. 최근까지 사슴풍뎅이속(Dicronocephalus)은 동양구에서 한반도와 극동러시아 지역에 걸쳐 8종 7아종이 분포하며, 종 또는 아종 사이에 형태적으로 매우 유사하여 이들 종간에 유연관계는 아직까지 알려지지 않았다. 따라서 국내 집단은 지리적으로 먼 중국서부 등의 집단과 단일 종을 형성하는 것인지 또는 타 지역 분포 종과의 유연관계는 어떠한 지 곤충자원의 보전과 관리 측면에서 검토할 필요성이 있었다. 이에 따라 1차적으로 국내 사슴풍뎅이 집단을 포함하여 5종 5아종을 대조 분류군으로 삼아 mtCOI과 16S rRNA마커를 이용한 분자분류를 시도하였다. 그 결과, 한국산 사슴풍뎅이 집단은 중국산 사슴풍뎅이 집단과 유전적 차이가 없는 것으로 확인되었다. 다만, 형태분류학자의 주장과 달리 대만산 D. yui와 sister group을 형성하는 것이 확인되었다. 따라서 향후 추가적인 샘플의 확보를 통해 사슴풍뎅이 종의 분화를 밝히고, 사슴풍뎅이속의 지리적 분포와 더불어 새로운 종의 발굴 가능성을 타진하는 연구를 계속하고자 한다.

After pea aphid Acyrthosiphon pisum genome project, hologenome concept was applied to pea aphid and symbioant such as Buchnera aphidicola. Here we screened symbiotic microorganism in four lab strains (two genetically different insecticide susceptible strains, host plant: cucumber and two different host adopted imiacloprid resistance strains, host plants: cucumber and potato) and four field populations (Jeju, Goryeong, Gimjae and Muju, host plant: potato) of cotton aphid based on GS-FLX pyrosequencing which were conducted with universal primer amplified partial fragments of 16S rRNA from total DNA which was extracted from each strain and population. B. aphidicola occupied over 90% of all identified prokaryotic microorganisms which all tested samples. It’s interesting that the ratio of B. aphidicola occupied over 99% in all of the tested lab strains. However, specific enterobacteriaceae occupied six to seven percents of all field populations which closely related endosymbiont of Glycaspis brimblecombei. That means B. aphidicola occupied only 91~92% of all identified prokaryotic microorganisms. Futhermore, other actinobacteridae and bacillaceae also were detected in field populations. The results obtained for these ratios suggested that there has some interaction between symbioant and environment NOT in imidacloprid resistance.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial in several perspective. The present study sequenced a total of ~ 3,500 bp from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) in 80 nymphalid species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae), along with those of six lycaenid species as outgroups. Phylogenetic analyses via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), with high nodal support for monophyletic subfamilies and tribes. This result is largely consistent with a previous study performed with a substantially large sequence information and morphological characters, except for the position of Libytheinae that has previously been placed as the sister to all reminder of Nymphalidae.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial. The present study sequenced approximately 1,099 bp from cytochrome oxidase subunit I (COI), 1,336 ~ 1,551 bp from 16S ribosomal RNA (16S rRNA), and 1,066 bp from elongation factor-1 alpha (EF-1α) in 80 species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae) of Nymphalidae, along with those of six lycaenid species as outgroups. The average base compositions for the three genes (COI, 16S rRNA, and EF-1α) are as follows: A (30.6%, 38.8%, and 25.8%), G (14.7, 5.2%, and 23.6%), T (39.8%, 45.2%, and 23.4%), and C (14.9%, 10.8%, and 27.3%). This result shows the A/T bias in the mitochondrial genes, but not for the nuclear EF-1α. Between the two mitochondrial genes, the 16S rRNA gene evidenced a significantly higher A/T content than was detected in the COI gene. These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms. Both analyses concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), along with highly supported monophyletics of tribes within subfamilies. This result is largely consistent with a previous study performed with a large sequence information and morphological characters, except for the position of Libytheinae, which was suggested to be the basal lineage of Nymphalidae.

The phylogenetic relationships among true butterfly families (superfamily Papilionoidea) have been a matter of substantial controversy, and that debate has led to several competing hypotheses. Two of the most compelling of those hypotheses involve the relationships of (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae) and (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae). In this study, approximately 3,500 nucleotide sequences from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) were sequenced from 83 species belonging to four true butterfly families, along with those of eight outgroup species belonging to the skipper family (superfamily Hesperioidea). These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP) algorithms. All phylogenetic analyses among the four true butterfly families strongly indicated a sister relationship between the Nymphalidae and Lycaenidae on one hand, and relatively strongly indicated a sister relationship between the Pieridae and Papilionidae on another hand, thus supporting the hypothesis: (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae).

Microsporidia are obligate fungal intracellular parasites of all animal taxa. Among them the genus Nosema (Nosematidae) is known as the most common entomopathogen. Of these parasites, the ribosomal organization is one of the most pronounced molecular characteristics. One type is the normalarrangement of small subunit (SSU)-internal transcribed spacer (ITS)-large subunit (LSU) in the DNA sequence order. The other is the reverse arrangement of LSU-ITS-SSU. The latter is assigned to be the ‘true’ Nosema in the Nosema/Vairimorpha clade. However, we found that the SSU sequence of a strain of Nosema species having the normal arrangement of its rRNA sequence seemed to be more closely related to the ‘true’ Nosemagroup. Consequently we have further analyzed the complete sequence of rRNA. The results imply that there might be arecombination event in its rRNA evolution and/or the strain may form a novel group near the ‘true’ Nosema group. Interestingly both SSU and LSU of the ‘true’ Nosema and others may be under different selection pressure. We have also found that the size of ITS is distinct between the ‘true’ Nosema and other microsporidian species within the Nosema/Vairimorpha clade. This feature should be a useful diagnostic tool to distinguish the ‘true’ Nosema from others in the clade.

The subfamily Hoplolaiminae included economically important plant parasitic nematodes and consisted of more than 400 species, all having the diagnostic characters of a strongly annulated cuticle and a large stylet. Among the Hoplolaiminae genera, the genus Hoplolaimus species include species such as H. columbus, and H. galeatus that cause serious damage to crops and turf grass in the Southeastern United States. Traditional identification of species has been approached by interspecific variation of phenotypic traits that rely on morphological and morphometric characters. However, these taxonomic criteria are sometimes not practical because of their limited ability to discriminate species among closely related groups due to overlapping of important taxonomic characters. The exact species identification is needed to control target nematode and also quarantine. Therefore, genetic studies for development of molecular diagnostics, population biology, and disease management are required. In recent years, many molecular diagnostic methods have been used for the identification of plant parasitic nematodes. Advanced molecular techniques have been used that test traditional identification methods. In our studies, Hoplolaimus species showed that high genetic divergence in rDNA sequence is combined with low morphological diversity. Based on genetic information, we developed multiplex PCR for H. columbus, H. galeatus, and H. magnistylus and successfully amplified mixed populations. In molecular phylogeny, the subfamily Hoplolaiminae is an important out‐group of the Heteroderidae, a notorious plant parasite nematode group. Molecular phylogeny of the Hoplolaiminae will help us understand of pathways of pathogenesis. In our phylogenetic analysis using D2 and D3 expansion segments of 28S gene, the molecular data supported morphological based taxonomic schemes. To reconstruct more reliable phylogenetic analysis, correct assignment of each nucleotide within multiple sequence alignment is an important step. Sequence alignments based on secondary structure have been proposed as new alternative methods to obtain this goal. We predicted the secondary structure of D2 and D3 domain using computational predictions method such as the minimization energy method and comparative sequence analysis (co‐variation). Predicted secondary structure included 18 species with two outgroup species, Globodera rostochiensis, Rotylenchulus reniformis. Consensus secondary structure was obtained from closely related and distantly related species. Phylogenetically informative characters were distributed in the stem region (86.7%). These results support the effectiveness of stem and loop regions for phylogenetic analysis of the Hoplolaiminae.

본 연구는 위장관염을 일으키는 Vibrio fluvialis의 16S-23S rRNA intergenic spacer region을 분석하였다. ISR을 PCR 증폭 후 plasmid vector에 클로닝하여 염기서열을 분석하였다. 그 결과, ISR의 염기서열은 tRNA gene 조성과 크기에 따라 총 6개의 type으로 분류되었다. 각 type은 tRNA gene 조성과 수에 따라 ISR-A, ISR-E, ISR-El, ISR-lA, ISR-EKV,