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.

Occurrence spontaneous mushrooms in eastern Jeju Muryeongarioreum, Sillyecheon-Iseungak, Kyoraegotjawal, Geomunoreum were examined in 2016. The collected mushrooms were classified by 15 order 56 family 149 genera 317 species. Among the collected mushrooms, 87 species of mushrooms were successfully incubated in mushroom complete medium in which DNA of 25 species were completely extracted and amplified using universal primers ITS1 (5’-CTTG GTCA TTTA GAGG AAGT AA-3’) and ITS4 (5’-TCCT CCGC TTAT TGAT ATGC-3’). Four species of them were differently identified between the classification by morphological observation and by analysis of DNA sequences i.e. by morphological identification Ganoderma applanatum, Armillaria gallica, Macrolepiota procera and Trametes gibbosa were identified as G. gibbosum, A. cepistipes, M. detersa and T. elegans by analysis of DNA sequences, respectively. Based on these results both identification ways might be necessary for correct classification of mushrooms. On the other hand, among the collected 317 mushrooms, 48 species could be identified to genus but not to species by the morphological observations. However, by the analysis of DNA sequences, 15 species were identified to species in which 6 species were unregistered mushrooms in Korea. Therefore, the analysis of DNA sequence may be useful to identify the mushrooms which are difficult to recognize by morphological observation.

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.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), was originally observed in a few Asian countries, but is now found even in North America and Europe. Genetic information on geographic variation and relationship may broaden our understanding of origin and migration. As a first step, in this study, a portion of mitochondrial COI gene was sequenced to understand genetic relationship and diversity in Korea. Sequencing of 104 individuals provided 57 haplotypes, with the maximum sequence divergence of 1.5%, suggesting high haplotype diversity and moderate sequence divergence. Comparison to GenBankregistered D. suzukii haplotypes (possibly from Spain, Portugal, USA) has shown 100% sequence identity to most of the haplotypes found in this study, but two USA sequences were found to be independent haplotypes, with the sequence divergence ranging from 0.5% ~ 1.4% from our samples in the 553-bp comparison. Phylogenetically, no separable group was found, but, population genetically, the only Chinese population, Sandong, was significantly differentiated (p < 0.05) from all Korean populations, without sharing any haplotype. Among 28 pairwise comparisons of Korean populations only two comparisons showed a significant genetic differentiation, indicating that no population in Korea is completely isolated. Geographically, one haplotype (SWDBA06) was relatively widespread (five among nine localities) and a few haplotypes were found in more than one locality, but most haplotypes were restricted in a locality as a single individual. Overall, high rate of per generation female migration (Nm = 0.75 ~ infinite) and low level of geographic separation (FST=0~0.40) among localities were characteristic. Current data is limited mainly to Korean localities, thus, an expanded study may provide further scrutinized analysis for the fly.

본 연구는 국내 유통 방풍(S. divaricata, P. japonicum, G. littoralis) 한약재의 원산지 판별을 위해 수행 되었다. 이를 위 해, 형태적 특징비교 뿐만 아니라 엽록체(nrDNA-ITS2) 및 핵 DNA 바코드 유전자(cpDNA-matK, psbA-trnH, rpoB2와 rpoC1)를 이용한 염기서열 분석을 수행하였다. 방풍류 3종의 형태적 특징을 비교한 결과, 잎 모양과 거치의 형태에서 가장 큰 차이를 보인 반면, 건조약재의 경우 육안으로 구별하기에 어려움이 있었다. DNA 수준에서의 차이를 비교하기 위해 DNA 바코드 후보 유전자들을 이용한 방풍류 3종의 식물자원 에 대한 염기서열 분석을 수행한 후 판별 마커 개발 가능성이 있는 ITS2와 matK, psbA-trnH 세 primer를 선발하였다. 이 를 국내 유통 한약재에 적용한 경우 방풍은 S. divaricata와, 식방풍은 P. japonicum과, 해방풍은 G. littoralis와 동일한 것 으로 확인되었다. 중국산 방풍은 S. divaricata와 동일종으로 바르게 표기되어 유통되나, 국산 방풍은 P. japonicum과 동일 종으로 식방풍(갯기름나물)의 뿌리를 건조시켜 방풍으로 혼용 유통됨을 확인하였다. ITS2 구간의 염기서열 분석 결과, 식방 풍의 경우 두 그룹으로 나눠져 동일 종 내에 유전적 변이가 있음이 확인되었다. 따라서 본 연구결과 방풍류 3종 판별을 위 해 nrDNA-ITS2와 cpDNA-matK, psbA-trnH의 사용이 유용 할 것이며, 차후 방풍류 한약재의 판별을 위한 마커 개발 연 구의 기초 자료로서 활용될 수 있을 것으로 사료된다.

The purpose of this study was to identify fast and effectively the Nosema disease of bumble bees from Gangwon province in Korea. Bumble bees are crucial pollinators of various crops and microsporidia are the critical infections of these hosts. The origin of the bumble bees is probably being known from the Britain. Also, the following species of bumble bees have been used around the world: Bombus terrestris, Bombus lucorum, Bombus occidentalis, Bombus ignites and Bombus impatiens. When bumble bees are infected with N. bombi, their abdomens can become distended, paralyzed and infected workers and they often become sluggish and die early. We have identified the morphology of the microsporidium by light and electron microscopy and have found that the morphology of the microsporidium is rounded spore morph, with fairly small spores as described before in many other articles. For the specific and sensitive diagnosis of the microsporidian parasite N. bombi in bumble bees, we have developed the improved method of polymerase chain reaction (PCR) conditions for expeditious diagnosis. Two pairs of primers were tested on N. bombi and the related microsporidia Nosema apis and Nosema sp., both of which infect bumble bees and honey bees and further we have verified and analyzed DNA sequence data of N. bombi in bumble bees by using the BLAST server at the National Center for Biotechnology Information.

Worldwide studies on Apis cerana variation for biogeography and genetic diversity depended largely on a 86~93 bp-long mitochondrial non-coding region (internal spacer region) located between tRNALeu and COII (named as NC2), possibly due to higher variability among available markers. In order to incorporate the A. cerana occurring in South Korea into world extensive data, we also sequenced the NC2 from 118 A. cerana samples collected over nine Korean localities and 66 A. cerana samples over seven Asian localities, such as China, Vietnam, and Thailand. These data were combined with preexisting world data to scrutinize genetic relationships of A. cerana in South Korea to outside distributional range. Sequencing of 184 samples provided a total of ten haplotypes: five from Korea, six from China, one from Vietnam, and two from Thailand. Among them eight were new, whereas two were previously reported ones. Phylogenetic analysis of A. cerana NC2 haplotypes so far found including ours has confirmed the presence of four major groups of A. cerana (Asian mainland group, Sundaland group, Palawan group, and Luzon-Mindahnao group) and all haplotypes found in this study also were included in the Asian mainland group. In order to find further variable regions that can be used as sequence-based marker several mitochondrial non-coding regions and nuclear intron regions are in the middle of testing.

In DNA barcoding, the DNA degradation of old museum specimens has been limited full-length (658bp) sequencing. The challenges associated with the retrieval and authentication of degraded DNA extracts from fossil and old museum specimens were principally limited to analyze the relatively short sequences (<300 bp). Furthermore, almost protocols in other to analyzed the degraded DNA contained the cloning process after PCR causing the time-consuming and the rising costs. To overcome these problematic circumstances, we tried a modified method to analyze full-length of DNA barcoding region in 30~60 year-old butterfly specimens (225 samples in 28 species), using direct sequencing after PCR with species-specific overlapping primer sets per each species. As a result, all of 28 species have been successfully analyzed, although 178 samples (79%) are completely generated barcoding sequences ranged from 640 to 658 bp and 47 samples (21%) are partially sequenced ranged from 100 to 500 bp. Thus, the result showed that the direct PCR sequencing using the overlapping primer sets per species appears to have great potential efficiency for analysis of degraded DNA without incorrect sequences.

The Scarites aterrimus (Coleoptera: Carabidae), is one of the carabid beetles dwelling exclusively on coastal sandy dunes. Recent habitat deterioration has greatly concerned population declines in several species dwelling on the coastal sandy dunes. Asa first step to establish long-term conservation strategy, we investigated the nation-wide magnitude and nature of genetic diversity of the species. As a first step, we sequenced a portion of mitochondrial COI gene, corresponding to “DNA Barcode” region (658 bp) from a total of 24 S. aterrimus individuals collected over nine sandy dunes belonging to four Korean provinces. The sequence analysis evidenced moderate to low magnitude of sequence diversity compared with other insect species distributed in Korean peninsula (0.152% to 0.912%). The presence of closely related haplotypes and relatively high gene flow estimate collectively suggest that there had been no historical barriers that bolster genetic subdivision. Population decline was postulated on the basis of several missing haplotypes that are well found in the species with a large population size. This interpretation is consistent with field observation of small population size in the coastal sandy dune habitats. The highest genetic diversity estimates were found in the coastal sand dune population of Seogwipo, Jeju Island, justifying a prior attention to the population, in order to sustain overall genetic diversity of the species. Further scrutinized study might be required for further robust conclusion.

The rice leaf roller, Cnaphalocroci smedinalis Guenée (Lepidoptera: Pyralidae) is a leaf-feeding pest of rice world-widely distributed. For better understanding of the pest insect, geographic sequence variation of the species were performed using the mitochondrial A+T-rich region, with the samples collected from seven Korean and six Chinese localities. A total of 94 haplotypes obtained from 187 individuals showed the length variation, ranging from 339 bp to 348 bp. The maximum divergence of 4.57% appears to evidence a substantial sequence variation, indicating the applicability of this molecular marker to the study of geographic variation. Overall, a high per generation migration ratio (Nm = 3.67742 ~ infinite), a low level of genetic fixation (FST = 0 ~ 0.11969), and no discernable isolated population were noted in the most C. medinalis populations. AMOVA analysis to find out allocation of genetic variability of C. medinalis populations has shown allocation of majority of variation to the within-population, rather than among-populations and between-region, suggesting that the C. medinalis populations in both China and Korea are largely well connected. This result is consistent with current knowledge of the dispersal ability of the species. The structure analysis of the A+T-rich region has shown that the typical structural elements found in other lepidopteran insects also is well preserved in the C. medinalis A+T-rich region (i.e., a poly-T stretch and a microsatellite-like A/T repeat).

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.

In order to understand the nature of population genetic structure of the notorious pest insect, Spodoptera litura (Lepidoptera: Noctuidae), a total of 159 individuals of the tobacco cutworm collected from six Korean and five Chinese localities were cloned and sequenced their mitochondrial A+T-rich region. A total of 90 haplotypes ranged 324 bp ~ 372 bp in length and 0.30% ~ 4.85% in sequence divergence. Compared to the preliminary mitochondrial COI gene sequence data that provided nearly identical sequences in all samples, the A+T-rich region provided substantially high sequence variation, validating the applicability of this molecule to the study of intraspecific variation. Overall, a very high per generation migration ratio (Nm = 5.91 ~ infinite), a very low level of genetic fixation (FST = 0 ~ 0.077796), and no discernable isolated population were noted to exist among populations of S. litura, although some significant genetic differentiations were found between several pairs of populations. These results collectively suggest that the S. litura populations in both Korea and China are very well interrelated to each other. The structural analysis revealed that the S. litura A+T-rich region harbored two stretches of the [TA(A)]n sequence, the poly-A stretch, and a stem-and-loop structure that are well found in other lepidopteran A+T-rich region. Furthermore, the region harbors the 13 ~ 19-bp long T stretch and 5-bp long motif that have been suggested to function as a possible recognition site for the initiation of replication of the minor strand of mtDNA also were found.

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 complete nucleotide sequences of the mitochondrial genome (mitogenome) from the white-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Cetoniidae) was determined. The 20,319-bp long circular genome is the longest among the completely sequenced arthropods. This extraordinary length of the genome stemmed from 5,654-bp long A+T-rich region composed of twenty-eight 117-bp tandem repeats, seven 82-bp tandem repeats, and each two 19-bp and 38-bp tandem repeats. The P. brevitarsis contains a typical gene complement, order, and arrangement identical to most common type found in insects. The P. brevitarsis COI gene does not have typical ATN codon. Thus, we also designated it as AAC (asparagine), which is found in the start context of all sequenced Polyphaga within Coleoptera. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNASer (AGN), DHU arm of which could not form stable stem-loop structure. The 5bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand also was found betweent RNASer (UCN) and ND1, as have been detected in all sequenced coleopteran insects.

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.

The bumblebee, Bombus ardens, is a valuable natural resource, and is most notably utilized for greenhouse pollination. In order to gain a greater understanding of the population genetic structure and the genetic diversity of this species, we sequenced a portion of the mitochondrial COI gene corresponding to the “DNA Barcode” region (658 bp) from 160 individuals collected over 15 Korean localities. Uncorrected pairwise distances among the eight haplotypes suggested low intraspecific genetic diversity, with a maximum sequence divergence of 0.3%. Such a low level of intraspecific genetic diversity was further reflected in local populations, particularly to islet populations, such as those of Youngheungdo, Jakyakdo, and Ulleugdo, which had zero genetic diversity. Geographically, one haplotype (BARBA01) was widespread and dominant, with a frequency of 90.6% (145 among 160 individuals). Other haplotypes were restricted to one to three localities and had low frequency. Overall, a very high rate of per generation female migration ratio (Nm= 4.6 to infinite) and a very low level of genetic fixation (FST= 0 to 0.099) were detected between pairs of localities, suggesting that the B. ardens population sonthe Korean peninsula are panmictic, which is consistent with our understanding of their dispersal capability.

We analyzed a portion of mitochondrial COI gene sequences (658 bp) to investigate the genetic diversity and geographic variation of the swallowtail butterfly, Papilioxuthus L., and the cabbage butterfly, Pieris rapae (Lepidoptera: Papilionidae). P. xuthus showed a moderate level of sequence divergence (0.91% at maximum) in 15 haplotypes, whereas P. rapae showed a moderate to high level of sequence divergence (1.67% at maximum) in 30 haplotypes, compared with other relevant studies. Analyses of population genetic structure showed that most populations are not genetically differentiated in both species. The distribution pattern of both species appears to be consistent with category IV of the phylogeographic pattern sensu Avise (Avise et al. 1987): a phylogenetic continuity, an absence of regional isolation of mtDNA clones, and extensive distribution of close clones. The observed pattern of genetic diversity and geographic variation of the two butterfly species seems to reflect the abundant habitats, abundant host plants, and flying abilities in connection with the lack of historical biogeographic barriers.

The 15,338-bp long complete mitochondrial genome (mitogenome) of the Japanese oak silkmoth, Antheraeayamamai (Lepidoptera: Saturniidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNAMet to a position 5’-upstream of tRNAIle. No typical start codon of the A. yamamai COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for A. yamamai COI gene. Three of the 13 protein-coding genes (PCGs) harbor the incomplete termination codon, T or TA. All tRNAs formed stable stem-and-loop structures, with the exception of tRNASer(AGN), the DHU arm of which formed a simple loop as has been observed in many other metazoan mt tRNASer(AGN). The 334-bp long A+T-rich region is noteworthy in that it harbors tRNA-likestructures, as has also been seen in the A+T-rich regions of other insect mitogenomes. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As has been previously suggested, Bombycidae (Bombyxmori and B.mandarina) and Saturniidae (A.yamamai and Caligula boisduvalii) formed a reciprocal monophyletic group.