멸구과(Delphacidae) 8종의 internal transcribed spacer 2 (ITS2) DNA 염기서열로 종간 차이 추정값을 비교하고 분자계통수를 추론하였다. ITS2 DNA 염기서열 길이는 종(species)마다 550 bp (흰등멸구)에서 699 bp (겨풀멸구)까지 차이를 보였다. 같은 Nilaparvata 속의 겨풀멸구와 벼멸구붙이 사이 의 염기서열 차이 추정값(d ± S.E.)은 0.001 ± 0.001로 가장 낮았으며, 사슴멸구와 일본멸구 사이는 0.579 ± 0.021로 가장 높았다. 벼멸구와 다른 멸구류들 과의 종간 염기서열 차이 추정값은 0.056 ± 0.008 (겨풀멸구)에서부터 0.548 ± 0.021 (일본멸구)로 구분되었다. 반면, Neighbor-joining 방법으로 추론된 분자계통수에서는 겨풀멸구와 벼멸구붙이를 제외하고 나머지 멸구류들은 독립된 다른 그룹으로 분지되었다. 벼멸구의 ITS2 염기서열을 참고하여 벼멸구 특이 고리매개등온증폭(loop-mediated isothermal amplification, LAMP) 프라이머 4 세트(BPH-38, BPH-38-1, BPH-207 및 BPH-92)를 제작하였다. 이들 각각을 벼멸구, 흰등멸구 및 애멸구의 게놈 DNA와 65°C에서 60분간 반응시켰을 때, 벼멸구 시료에서만 증폭 산물들이 관찰되었다. BPH-92 LAMP 프라이머 세트로 65°C에서 벼멸구 DNA의 양(0.1 ng, 1 ng, 10 ng, 100 ng)과 반응시간(20분, 30분, 40분, 60분)을 달리하여 형광반응을 관찰하였을 때, 20분과 30분 반응에서는 100 ng 까지에서도 발광여부 구별이 어려웠다. 그러나 40분 반응에서는 10 ng 이상에서, 60분 반응에서는 0.1 ng 이상에서 발광여 부가 명확히 구별되었다.

The Hawthorn spider mite Amphitetranychus viennensis is an important pest of various fruit trees as well as a variety of ornamental plants. Its presence has been reported in China, Japan, Korea, and middle east and European countries including Grorgia, Russia, Turkey, Ukraine. Thus, it is a quarantine species in American countries and Australia. Intra-specific variation of A. viennensis was investigated using 555 bp of ITS2 ribosomal DNA and 453 bp of the cytochrome oxidase I (COI) gene. Its variation of ITS2 and COI among China, Japan and Korea were 1.7-2.2% and 0.2-0.7%, respectively. In addition, we collected samples from 14 location in Korea. Intra-specific diversity of ITS2 and COI within Korean populations were ranged into 0.5-0.9% and 0.2-0.5%, respectively. Thus, A. viennensis is not much genetically diverse in different populations.

옥수수 포장에서 발생하는 Trichogramma속 조명나방 알기생벌의 종 분포를 조사하기 위해 채집된 알기생벌로부터 핵내 ITS2 DNA 전체 염기서열 정보를 해독하였다. 그리고 종 구별을 위한 참고정보로 NCBI GenBank에 등록된 Trichogramma속 60종의 ITS2 전체 염기서열을 확보하여 비교하였다. 국내 채집 알기생벌은 ITS2 DNA 길이와 3′ 말단 염기서열 패턴에 따라 3개 그룹(K-1, -2, -3)으로 구분되었다. 국내 채집 그룹 내 염기서열 차이 추정값(Evolutionary distance, d)은 0.005 이하로 그룹 간 비교 시 d 값(≥0.080)보다 낮았다. 그룹 및 GenBank 등록 종 간 비교 시 K-1은 T. ostriniae, K-2는 T. dendrolimi, K-3은 T. confusum과 d 값이 각각 0.016, 0.001, 0.002로 가장 작았다. 추론된 분자계통수에서 K-1은T. ostriniae, K-2는 T. dendrolimi와 각각 분지되었으나 K-3는 T. confusum, T. chilonis, T. bilingensis와 함께 분지되었다. NCBI BLAST 결과에서도 K-1은 T. ostriniae와 K-2는 T. dendrolimi와 99% identity를 보였다. 그러나 K-3의 홍천 채집 기생벌들은 T. confusum, T. chilonis와 99-100% identity를 보였지만, 고창 채집 기생벌은 T. bilingensis, T. confusum, T. chilonis와 98% identity를 보였다. 이상의 분석 결과 본 연구에서 채집된 알기생벌 K-1과 K-2는 각각 T. ostriniae와 T. dendrolimi, 단일한 종으로 추정되었으나 K-3는 ITS2 정보만으로 종을 추정하기 어려웠다.

ITS2(Internal Transcribed Sequence 2) DNA 영역은 핵내 5.8S와 28S 리보솜 RNA 사이에 존재하는데 종간 변이가 크고 적은 양으로도 PCR 증폭이 잘 되기 때문에 곰팡이나 벌목 분류에 자주 사용되고 있다. 본 연구에서는 공중포충망과 유아등에 포획된 벼 멸구류들로부터 간편하게 DNA를 추출하고, 형태적 구별이 어려운 유사 벼 멸구류들을 구별하는데 ITS2 DNA 영역을 활용할 수 있는지 검토하였다. 그 결과 벼 멸구류의 ITS2영역은 전체길이가 551∼700bp였으며 그중 벼멸구, 애멸구, 흰등멸구가 각각 694bp, 601bp, 551bp로 종간 DNA 염기서열 길이 차이가 있었다. 또한 DNA 염기서열을 배열하여 비교 후, 유전적 거리 추정방법과 추론된 분자계통도를 통해 종간 차이를 구별할 수 있었다.

옥수수 포장에서 발생하는 Trichogramma 속(genus) 조명나방 알기생벌의 종 (species) 분포를 조사하기 위해 홍천 외 7개 지역에서 6 ~ 8월에 수집된 알기생벌로 부터 핵내 리보솜 RNA 유전자의 ITS2(internal transcribed spacer 2) DNA 염기서 열 정보를 213 개 해독하였다. 종 구별을 위한 참고정보로서 GenBank 데이터베이 스에 등록되어 있고 종이 명확하게 확인된 Trichogramma 속 ITS2 염기서열 정보 를 58 종 총 404 개 확보하여 MEGA 5를 이용하여 국내에서 조사된 결과와 비교 분 석하였다. ITS2 DNA 염기서열 정보를 이용한 phylogenetic tree 결과들을 통해서 GenBank에 등록되어 있는 같은 종들이 여러 가지(branch)의 그룹에 흩어져 있어 일부 종들을 제외하고 명확한 구별이 어려웠다. 국내의 결과는 3 개의 가지(branch) 그룹으로 나눠졌으며 조명나방 알기생벌로 Trichogramma dendrolimi Matsumura 를 포함하여 최소한 3 개의 종이 있을 것으로 추정되었다. 앞으로 조명나방 알기생 천적을 이용한 생물적 방제 방법을 개발을 위해서는 명확한 종 동정이 되어야하며 그러기 위해서 수컷의 생식기에 관한 형태적 분석들이 필요할 것으로 보인다.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

서해안 지역 논에서 벼 줄기속을 가해하고 있는 벼밤나방 유사 유충과 논 주변에 설치한 벼밤나방 성페로몬 트랩에 포획된 벼밤나방 유사 성충으로부터 게놈 DNA 를 추출하고 미토콘드리아 시토크롬옥시다제I(mtCOI) 부분영역을 비교하였다. mtCOI 염기서열로부터 추론된 아미노산 서열의 경우 한 위치에서 국내 채집 나방 모두가 GenBank 데이터베이스에 등록되어 있는 중국산 벼밤나방 일부와 다른 아 미노산(Gly vs. Ser) 변이가 관찰되었다. 그리고 동일 지역 채집 나방에서도 일부 추론된 아미노산에서 변이가 있었다. 이러한 결과가 벼밤나방 종내변이 때문인지 다른 종이 섞여서 나타난 종간변이 때문인지는 앞으로 명확한 규명이 필요할 것으 로 보인다. 한편 종동정을 위한 참고자료로서 핵내 리보솜 RNA 유전자의 ITS2(rRNA internal transcribed spacer 2) 전체영역을 해독하고 비교하였다. 최근 우리나라에 발생하여 피해를 주고 있는 벼 줄기속 가해 나방의 종류를 밝히고 분포 지역과 생리·생태, 약제저항성 등에 관한 연구가 필요할 것으로 보인다.

본 연구는 국내 유통 감초약재의 원산지 판별을 위해 수행되었다. 이를 위해 약재로 쓰이는 만주감초(Glycyrrhiza uralensis) 및 유럽감초(G. glabra)와 약재로 사용되지 않는 국내 자생종 개감초(G. pallidiflora) 식물의 형태적 차이 및 엽록체와 핵 DNA 구간의 염기서열의 차이를 구명하였다. 감초식물 3종의 형태적 특징을 비교한 결과, 잎과 뿌리에서 차이를 보였다. 특히 잎의 형태에 있어서 중국감초와 유럽감초는난형 혹은 타원형인 반면, 개감초는 엽장/엽폭비가 큰 피침형이었다. 또한 건조된 뿌리로 시중되는 감초 한약재의 경우, 중국산과 한국산은 백색인 반면 우즈베키스탄산은 황백색이었다.감초식물 3종을 대상으로 DNA 수준에서의 차이를 비교한 결과 rpoB2, rpoC1에서는 G. uralensis와 G. glabra가 구분되지않았으며, psbA-trnH의 경우 단 한 구간의 SNP에서 차이를 보였다. 반면 ITS2에 의해 증폭된 450bp의 염기서열을 비교한 결과, G. glabra는 98, 99, 100번째 위치에서, G. pallidiflora는 137, 161, 164, 203, 296위치에서 감초 종간판별을 위한 특이적 SNP가 확인되었다. 또한, phylogenetic tree 분석을 통해, 약재로 쓰이는 G. uralensis과 G. glabra는 약재로 쓰이지 않는 G. pallidiflora에 비하여 유전적 거리가 가까움을 확인하였다. 본 표준시료에서 얻은 결과를 유통 한약재 원산지 판별에 적용한 결과, 중국산과 한국산의 한약재는 G. uralensis와 염기서열이 일치하였고, 우즈베키스탄으로부터 수입된 한약재는 G. glabra와 염기서열이 일치됨을 확인하였다. 이에 감초의 생체 및 한약재의 원산지 판별을 위해 ITS2를 이용한 분자수준에서의 판별이 유용하다고 판단된다.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) of the species collected from seven Korean localities. A total of 17 haplotypes (CACOI01 ~ CACOI17), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 16 sequence types (ITS2CA01 ~ ITS2CA16), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in COI gene sequence. Phylogenetically, the COI gene provided two haplotype groups with a high nodal support (≥ 87%), whereas ITS2 provided one sequence type group with a high nodal support (≥ 92%). The result of COI gene may suggest the presence of historical biogeographic barriers that bolster genetic subdivision in the species. Different grouping pattern between COI gene and ITS2 sequences were interpreted in terms of recent dispersal, reflected in the ITS2 sequence. Finally, finding of unique haplotypes and sequence types only from Beakryeng-Islet population was interpreted as an intact remnant of ancient polymorphism. As more samples are analyzed using further hyper-variable marker, further fruitful inference on the geographic contour of the species might be available.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from seven Korean localities. A total of 18 haplotypes (BARCA01 ~ BARCA18), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 17 sequence types (ITS2CA01 ~ ITS2CA17), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in mitochondrial gene sequence. Phylogenetically, the mitochondrial DNA has shown several haplotypes formed independent groups with substantially high node support (≥ 90%), whereas no such grouping was evidenced for ITS2, indicating different behaviors of the two molecules. Such difference may reflect a diverse dynamics of the species such as biogeographic history, mating behaviors, and also possibly different mode of inheritance of the two molecules, but requires further scrutinized examination of the dataset. In terms of population genetic perspective, overall no population subdivision was detected from both molecules, except for locality 7 (Eocheong islet) from mitochondrial DNA. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), known also as the seven-spot ladybird, is natural enemy for aphids and has a broad ecological range, living almost anywhere there are aphids for it to eat. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from nine Korean localities. A total of 21 haplotypes (CSCOI01 ~ CSCOI21), with the maximum sequence divergence of 4.56% (30 bp) were obtained from COI gene sequence (from 78 individuals), whereas 65 sequence types (CSITS201 ~ CSITS265), with the maximum sequence divergence of 2.06% (11 positions) were obtained from ITS2 (from 79 individuals), indicating substantially larger sequence divergence in mitochondrial gene sequence. Both COI gene and ITS2 shows the distribution pattern that only a few haplotypes or sequence types are widely distributed, whereas majority of them are highly restricted in one geographic location, even represented as a single individual. Unlikely the ITS2 sequence types the mitochondrial COI haplotypes evidenced the presence of two main phylogenetic groups, reciprocally monophyletic to each other. Geographically, these two groups are spread in all localities surveyed. Considering both COI gene and ITS2 sequence together, current our data may suggest the presence of ancestral polymorphism, rather than on-going speciation, but more scrutinized analysis will be performed soon. Due partially by the presence of both COI groups in all surveyed localities, the genetic diversity estimates of all localities are similar from the perspective of COI gene, but ITS data showed extremely lower genetic diversity of one islet locality, Anmyeon-do (locality 2; 0.002530 vs. 0.008054 ~ 0.012060). Analysis of gene flew estimates between localities indicates that most populations are highly interconnected to each other. However, one islet locality, Anmyeon-do (locality) has shown statistically significant distance from the remaining localities on the basis of only ITS2 data (FST = 0.19 ~ 0.34), requiring scrutinized phylogeographic inference on this population with expanded sampling. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

Spider mites are one of major pests in cultivations of various ornamental plants and also important in plant quarantine service. Due to the very small body size and high similarity within the Genus the identification of species is difficult even at the microscopic observation. To identify 5 major species (Tetranychus cinnabarinus, T. urticae, T. phaselus, T. kanzawai and T. truncatus) in the Genus Tetranychus at the molecular level, we designed 4 species-specific primer sets using nucleotide sequences of the internal transcribed spacer (ITS2) region in the nuclear ribosomal repeat unit. At the PCR diagnosis of extracted genomic DNAs of 5 species using each primer set, specific primers of both T. phaselus and T. truncates were species-specific to their own species samples. However, specific primer set of T. urticae detected T. cinnabarinus as well as T. urticae. Specific primer set of T. kanzawai detected T. truncates as well as T. kanzawai, even though detection intensities were lower in non-target species.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species

In an effort to gain a better understanding of the nature of the population genetic structure of the pest insect, Spodoptera litura (Lepidoptera: Noctuidae), tobacco cutworms were collected from six Korean and five Chinese localities and their mitochondrial A+T-rich region and nuclear internal transcribed spacer 2 (ITS2) regions were cloned and sequenced. A total of 106 A+T-rich region haplotypes and 92 ITS2 sequence types were obtained from 158 individuals. Compared to the preliminary mitochondrial COI gene sequence data that provided all identical sequence types in all samples, the A+T-rich region and nuclear ITS2 are highly variable. Overall, a low level of genetic fixation in the A+T-rich region (FST = 0 ~ 0.02965) and nuclear ITS2 (FST = 0 ~ 0.34491), and no discernable isolated population was noted to exist among most S. litura populations. The presence of a majority of within-population variations, rather than variations among populations or between China and Korea, indicates that the S. litura populations are composed of heterogeneous individuals. The fixation index at hierarchical level of among regions shows no significance, suggesting that the S. litura populations in both Korea and China are profoundly interrelated with one another. This finding is in accord with the current knowledge that S. litura has sufficient flight capacity for dispersal.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability.

The bumblebees, Bombus species are valuable natural resources being utilized for greenhouse pollination. Low level of genetic variation of Bombus species has been reported previously. In this study, we sequenced complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA from 100 individuals of B. ardens collected from seven localities in Korean peninsula. The ITS2 sequence of B. ardens is longest, ranging in size from 1,940 bp-1,954 bp among known in insects, which ranges approximately from 241 bp-1,728 bp. The ITS2 sequences have ~51% of C+C content and contain each two 27 bp repeats, 20 bp repeats, 33 bp repeats, and 34 bp repeats at the beginning. Such repeats were not found in other insects. Uncorrected pairwise distance among 96 sequences that were obtained from 100 individuals revealed a maximum sequence divergence of 1.03%. Genetic diversity (π) of each population ranged from 0.007801 to 0.009627, and the lowest diversity was obtained from islet population of Ulleungdo, indicating possibly small, isolation of the population. Significant level of genetic distance was only found when Ulleungdo population was compared to two other mainland populations. Except for this, overall, a very high rate of per generation migration ratio (Nm=7.1-infinite) and a very low level of genetic fixation (FST=0-0.06546) were detected between pairs of localities. Analysis of hierarchical relationships among localities consistently revealed no statistically significant structure among populations. Taken these together, the B. ardens populations on the Korean peninsula are panmictic this is consistent with our understanding of the dispersal capability.

DNA barcoding is the use of short DNA sequences of the genome for large scale species identification. The Consortium for the Barcode of Life (CBOL) plant-working group recommended the 2-locus combination as the standard plant barcode. The evolutions of the chloroplast regions combine with nuclear gens are sufficiently rapid to allow discrimination between closely related species. We evaluated the efficacy of the proposed plant barcoding loci matK along with ITS2 for barcoding Vigna species. To assess the discrimination ability of barcoding loci to resolve Vigna species, we sampled 52 of the taxonomically best known groups in the genus. Topologies of the phylogenetic trees based on ITS2 and matK analyses were similar but a few accessions were placed into distant phylogenetic groups. Neither ITS2 nor matK analyses were able to discriminate some closely related Vigna species alone. Thus, we used concatenated data to increase the resolving power of ITS2 and used matK as an additional tool for phylogenetic analysis in Vigna because characterization of the nucleotide sequences of matK region was easier to recover and more cost-effective than those of the ITS region.