A karyotype analysis of five wild rose species (Rosa helenae, R. mulliganii, R. multiflora, R. rubus, and R. solieana) was performed using bicolor fluorescence in situ hybridization (FISH). All five species were found to be diploid (2n = 2x = 14). The chromosome length in the metaphase stage ranged from 1.29 to 2.05 μm in R. helenae, 3.32 to 6.82 μm in R. mulliganii, 1.58 to 2.24 μm in R. multiflora, 2.05 to 3.46 μm in R. rubus, and 1.62 to 2.46 μm in R. solieana. The chromosomes were either metacentric or submetacentric, with no subtelocentric or telocentric chromosomes being observed. Each p air of 5 S and 4 5S rDNA sites was detected at the proximal region of the long arm and the terminal region of the short arm of chromosome #7, respectively.

The genetic relationships among five genera, seven species of Theaceae were examined through a random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS). In RAPD analysis, five of 15 arbitrary primers showed polymorphic bands, which were able to classify different genera and species of Theaceae. The genetic variations of Theaceae were from 0.031 to 0.484. In ITS analysis, the ITS sequences were analyzed using BLAST and showed high identities with sequences of Theaceae, seven species published in NCBI GenBank database, which ranged from 98 to 100%. Sequence alignment of seven species showed 34.9% identities for ITS 1 region and 43.7% for ITS 2 region. Pairwise sequence divergences among seven species ranged from 0 to 0.330%. In phylogenetic tree, they were divided into three groups. In conclusion, the molecular data generated in the present investigation will help to understand the genetic relationships of Theaceae and also might be useful for further studies in intra-species, inter-species, and molecular evolution researches.

This work was performed to confirm the molecular discrimination through the nrITS1 sequences among 3 taxa of Scirpus L. sensu lato (s.l.) species. S. planiculmis represented only 2 base sequence variations with S. maritimus in spite that they showed different morphological features. The nucleotide sequences of the ITS1 region from S. planiculmis were shown to have 99.1% homology with S. maritimus and 60.4% homology with S. triqueter. Although the morphology of S. planiculmis is similar with S. triqueter, molecular basis of the size and sequences on ITS1 region were shown to have distinctive differences. For divergency investigation on same sites and metapopulation, sequencing was conducted on ITS1 region with partial 5.8S and 18S regions. All plants of each species collected at the same site had identical band size pattern and sequences. Intraspecific molecular divergency was not identified in spite that these species live in different wetland sites. The ITS1 sequences described here provided a powerful genetic tool for phylogenetic studies which was difficult by morphological identification as high rate of morphological plasticity

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

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.

Halacarid mites inhabit mainly in the bottom of the sea and their body lengths are approximately 0.5 mm. Few biological information about halacarids have been obtained. The phylogenetic study of halacarid mites has not been progressed and the phylogenetic relationship within the family Halacaridae is still unknown. Particularly there is no molecular study to infer phylogenetic relationship within the family. In the annual meeting of Acarological Society of Japan 2007, we developed the method for molecular analysis of 28S nuclear ribosomal gene in haracarid mites. Subsequently, we have further progressed the technique of the molecular analysis to infer the phylogenetic relationship of haracarids. The aim of the present study is to elucidate the phylogenetic position of the subfamily Rhombognathinae on the bases of molecular data. Halacarid specimens which were obtained from seaweeds and sandy segment at Japanese seacoasts, consist of eight genera in five subfamilies: Halacarinae (Halacarus sp., Agauopsis spp., Thalassarachna spp.), Halixodinae (Badyagaue sp., Agaue sp.), Copidgnathinae (Copidognathus spp.), Actacarinae (Actacarus spp.) and Rhombognathinae (Rhombognathus spp.). The template DNA was extracted and 28S nuclear ribosomal gene of the DNA was amplified by PCR reactions. And we determined approximately 400 base pair length of the gene. To construct phylogenetic trees by neighbor-joining method we calculated genetic distances of each operational taxonomic unit considering 1) transversions and 2) transitions and transversions based on Tamura - Nei model by using MEGA ver. 4 (Tamura et al. 2007). As a result, we got two trees both showing two main clusters. One of them includes only one subfamily Rhombognathinae and another consist of Halacarinae, Copidgnathnae, Actacarinae and Halixodinae. Rhombognathinae was greatly differentiated from the other subfamilies. Thus, Rhombognathinae possibly is a distinct taxon phylogenetically and this result supports the monophyletic nature obtained previously by morphological analysis.

지렁이과 (family Megascolecidae) 왕지렁이속 (genus Amynthas) 그룹을 rDNA ITS 유전자를 이용하여 계통 유연관계를 알아보고자 하였다. 2과 10속에서 26종의 DNA 염기서열을 이용하여 종간 계통적 유연관계를 MP (Maximum Parsimony), NJ (Neighbor Joining), QP (Quartet Puzzling)로 계통도를 작성하였다. 염기서열이 확보된 Megascolecidae (지렁이과)의 국내외

국내에서 재배하여 생산되고 있는 상황버섯의 일종인 PMO-P4균주에 대한 ITS 영역의 염기서열 분석을 실시하였으며 목질 진흙버섯으로 잘 알려져 있는 P. linteus와 함께 RFLP분석을 통하여 상호 비교한 결과 PMO-P4균주는 P. baumii로 판명되었다. 이 결과를 토대로 이미 보고 되어 있는 Phellinus속 균주들과의 종간 ITS 영역의 상동성을 비교한 결과 48.6%-72.2%였으며 본 연구에서 비교한 종들 가운데서는 P. linteus와 상동성이 가장 높았으며 P. gilvus와 상동성이 가장 낮았다.

곤충자원의 대량사육을 위한 병 발생 예방과 해충의 효과적인 방제를 위하여 흰점박이꽃무지 이병충으로부터 곤충병원 사상균을 분리하였다. 전자현미경 관찰 결과 분리균주 KMA-1은 Metharizium속의 전형적인 쇠사슬형의 분생자를 paliside-like masse에 형성하였다. 따라서, 정확한 동정을 위하여 28S rRNA와 ITS염기 서열을 바탕으로 제작한 특이 프라이머쌍을 사용하여 PCR 반응을 수행하였다. 각각의 프라이머쌍을 사용한 PCR반응으로부터 특이 밴드가 검출되었으며 이 증폭 산물들의 염기 서열을 결정, 비교하였다. 분리 균주 KMA-1의 PCR산물인 28S rRNA와 ITS DNA염기서열을 GenBank데이터베이스에 등록된 염기서열 정보와의 상동성을 검색한 결과, 모두 Metarhizium anisopliae와 가장 높은 서열 상동성을 보였다. 이상의 결과로서 본 실험에서 분리 명명된 KMA-1는 M. anisopliae로 동정되었다.

The 12 cultivars of the Jeju native Citrus are considered to have originated from China. However, the origin of the cultivar ‘Byungkyool’ (Citrus platymamma Hort. ex Tanaka) is not clearly known. We performed PCR analysis by using three primer sets designed from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) to analyze the phylogenetic relationship between the traditional citrus cultivars and the Byungkyool cultivar. Sequence length of the nrDNA ITS1 region of JNCPCRI (Jeju Native Citrus platymamma Citrus Research Institute) cultivar was 247 bp, 8the ITS2 region was 228 bp and the total ITS region (ITS1-5.8S-ITS2) was 638 bp. Analysis of the genetic relationship based on the sequence analysis at the ITS region of the JNCPCRI cultivar revealed that the ITS1 region of the cultivar was genetically the same as that of the Byungkyool (JQ990189) cultivar, and the ITS2 region was genetically similar to the Binkyool (JQ990180), Hongkyool (JQ990178), Dangyooja (JQ990179), and Pyunkyool (JQ990181) cultivars. Moreover, the total ITS region in the 5.8S rDNA region was genetically similar to the Hongkyool (JQ990178) cultivar. In addition, the total ITS region of the JNCPCRI cultivar was the most closely related to the Cheongkyool (JQ990183) cultivar and has been reported to originate from the Binkyool (JQ990180) and Pyunkyool (JQ990181) cultivars. Although the JNCPCRI cultivar was morphologically the same as the Byungkyool (JQ990189) cultivar, the ITS region showed genetic heterogeneity. Taken together, we conclude that the genetic variation in the ITS region of JNCPCRI cultivar suggests that it was propagated through fertilization with the surrounding citrus cultivars.

Background : Plants belonging to 5 species of the genus Eleutherococcus are currently distributed in the Korean peninsula. The traditional medicine ‘Ogapi’, derived from Eleutherococcus sessiliflorus and other related species, and ‘Gasiogapi’, derived from Eleutherococcus senticosus, are frequently mixed up and marketed. Therefore, accurated identification of their origins in urgently required. Methods and Results : Candidate genes from nuclear ribosomal DNA (nrDNA) and chloroplast DNA (cpDNA) of Eleutherococcus plants were analyzed. Whereas the nrDNA-internal transcribed spacer (ITS) regions were useful in elucidating the phylogenetic relationships among the plants, the cpDNA regions were not as effective. Therefore, a combined analysis with nrDNA-ITS was performed. Various combinations of nrDNA and matK were effective for discriminating among the plants. However, the matK and rpoC1 combination was ineffective for discriminating among some species. Based on these results, it was found that OG1, OG4, OG5, OG7, GS1, GS2, and GS3 were derived from E. sessiliflorus. In particular, it was confirmed that GS1, GS2, and GS3 were not derived from E. senticosus. However, more samples need to be analyzed because identification of the origins of OG2, OG3, OG6 and GS4 was not possible. Conclusion : The ITS2, ITS5a, and matK combination was the most effective in identifying the phylogenetic relationship among Eleutherococcus plants and traditional medicines based on Eleutherococcus.

Totally, 26 collections, 17 from Korea and 9 from China, were investigated for their sequences of 5S rDNA, especially the non-transcribed spacers (NTSs). Sequences of 5S rDNA were isolated by PCR using the primers, 5s-rRNA1 and 5s-rRNA2. Genomic DNA PCR produced single amplification of 300, 330, or 350 base pair fragments. Sequence analysis revealed that all inserts contained the part of 5S rDNA gene sequence and the full length of the NTS region. Three different sizes of the fragments were confirmed due to different size of NTS and their length were 300bp, 330bp and 350bp, respectively. Among 17 Korean foxtail millets tested, 14 collections showed single 300bp amplification. Longest fragment amplification, 350bp, was obtained only from the foxtail millet from China origin, even though 2 of them include 300bp fragment. CLUSTALW multiple alignments of 26 foxtail millets clearly revealed 4 areas with certain degree of sequence heterogeneity (region I, II, III, IV). Among 4 boxed areas, foxtail millet genotypes from China have distinct insertion especially in region III. Five of them have extra insertion of sequence and their additional sequences were either 45 or 48 base pair. Three Korean foxtail millets have 32 bp insertion. Other 8 Korean collections have short insert sequences (6 to 8 bp), 3 with 8 bp and 5 with 6 bp. In addition to insert, deletion sequences were also confirmed as major deletion was observed in region II of Chinese collection. The size of deletion was 7 bp long. According to phylogenic tree constructed using MEGA4 program, clear grouping was not revealed. To obtain more convincing results various collections from many countries should be obtained and analyzed to distinguish different germplasm from different origin.

Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.

We performed phylogenetic analyses of a total of 21 acessions covering 5 species in the Korean Trigonotis and one outgroup species using nuclear ribosomal ITS and chloroplast rbcL, matK, ndhF sequences. Outgroup were chosen from the closely related genus Lithospermum zollingeri. Both parsimony and Bayesian Inference methods were used to reconstruct the evolutionary history of the group. The evidence collected indicated that phylogenetic relationships among Korean Trigonotis species are unresolved based on nuclear marker (ITS), as the same as based on separated chloroplast sequences. While the phylogenetic relationships of Korean Trigonotis species almost clearly were resolved in combined chloroplast sequences. Thus, the members of Trigonotis coreana can be distinguished to the members of Trigonotis peduncularis in combined cpDNA sequences and Trigonotis nakaii was treated as a synonymed to Trigonotis radicans var. sericea. In addition, the MP and BI analysis showed Trigonotis icumae as sister of the remained Korean Trigonotis species based on combined molecular markers (BI: PP = 1).

한국, 중국, 러시아에서 자생하고 있는 가시오갈피에 대한 유전적 유연관계를 파악하기 위해 ITS (internal transcribed spacer)의 염기서열을 분석하였다. Universal primer를 사용하여 PCR로 증폭시킨 후 염기서열을 결정한 결과 ITS의 길이는 162 bp의 5.8S rRNA 유전자를 포함하여 한국산과 중국산은 608 bp 그리고 러시아산은 611 bp로 나타났다. ITS영역의 G+C 함량은 한국산과 중국산이 60.20%이고 러시아산이 60.06%였다. 한국산과 중국산의 ITS영역은 100% 동일하였으며, 러시아산은 한국산과 비교했을 때 3 bp의 염기삽입과 2 bp의 염기치환이 존재하여 99.2%의 상동성을 나타내었다. 따라서 한국산 가시오갈피는 러시아산보다 중국산과 유전적 유연관계가 더 가까운 것으로 추정된다. 또한 동일 속내에서는 오갈피나무보다는 음나무와 ITS 염기서열이 유사한 것으로 나타났다.