본 연구에서는 한국 연안에 서식하는 대표적인 15종 패 류의 핵DNA 함량을 조사하였다. 복족류에서 DNA 함량 (pg DNA nucleus-1)은 3.3±0.08 (Haliotis discus hannai)과 2.4±0.18 (Batillus cornutus)이었다. 이매패류에서 DNA 함 량 (pg DNA nucleus-1)은 2.0±0.15 (Scapharca broughtonii), 3.0±0.12 (Mytilus galloprovincialis), 2.9±0.05 (Meretrix lusoria), 2.2±0.03 (M. lamarkii), 2.6±0.05 (Fulvia mutica), 1.8±0.18 (Tegillarca granosa), 3.3±0.01 (Solen corneus), 2.2±0.04 (Barnea manilensis), 2.5±0.32 (Crassostrea gigas), 3.9±0.24 (Atrina pectinate), 3.5±0.15 (Patinopecten yessoensis), 1.9±0.16 (Amygdala philippinarum) 및 2.3±0.14 (Pseudocardium sachalinensis)이었다. 본 연구 결과는 본 연구에 사용된 패류의 genomic 진화과정을 더욱 잘 이해하는 새로운 정보를 제공한다.

한국산 나문재속 식물에 대한 계통학적 유연관계를 밝히고, 분자계통학적 연구를 통해 나문재속 종간 유연관계를 확인할 수 있는 분자마커를 찾아내기 위해 연구를 수행하였다. 핵 리보솜 DNA ITS와 엽록체 DNA matK, psbA-trnH 그리고 trnL-trnF를 분자마커로 사용하였다. ITS 영역은 칠면초와 해홍나물 그리고 해홍나물과 방석나물을 구분하지 못하였다. psbA-trnH와 trnL-trnF 영역의 염기서열은 칠면초와 방석나물을 구분하지 못하였다. 그러나 4종의 분자마커 영역을 조합하여 분석한 결과 나문재속 식물 5종이 각각 독립적인 계통을 형성하는 것을 확인하였다. 따라서 나문재속 계통관계 분석을 위해서 여러 개의 분자마커 조합이 유용할 것으로 판단된다. 나문재속 내 분류군 간의 계통관계를 명확히 밝히기 위해 차후에 좀 더 많은 생태학적, 형태학적 자료를 조사해야 할 것으로 보인다.

The soft scales (Hemiptera: Coccidae) are plant-sucking insects, some of which are considered as serious agricultural pests. Here, we performed phylogenetic analysis of Coccidae, based on molecular fragments 1813bp containing mitochondrial DNA (COI), nuclear ribosomal RNA genes (18S and 28S), and elongation factor 1α (EF1-α). Data sets of 64 taxa were analyzed using maximum likelihood (ML) and Bayesian inference (BI). Our molecular analyses represent: 1) Monophyletic clade of Ceroplastinae is nested within the major clade of Coccinae; 2) Four tribes of Coccinae are paraphyletic, especially Megapulvinaria and Paralecanium not clustered within the major clade of Coccinae; 3) Didesmococcus is separated from the clade of Eulecaniinae; 4) Filippiinae and Cardiococcinae are paraphyletic with respect to some of Coccinae and Eulecaniinae.

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

느타리버섯류(Pleurotus spp.) 우량 품종개발에 많이 이용되는 교잡육종법 중에서 단핵-단핵간(mono-mono) 교잡에 관한 특성을 구명하기 위하여 느타리 6계통 및 사철느타리 1계통으로 단핵-단핵간 7조합 85개 교잡주를 얻어 교잡율, 핵 DNA 패턴 양상, 자실체의 갓 색깔과 수량성을 분석한 결과는 다음과 같다. 단핵-단핵간 교잡율은 50~93.75%로 나타났으며, 단핵간 85 교잡주의 핵 DNA 양상을 분석한 결과 양친주의 핵을 공유하고 있어 DNA 패턴은 양친의 중간이지만 유전유사도는 어느 한쪽 친주와 조금 더 가까운 양상을 나타냈다. 계통간교잡주 모두 양친의 핵이 공존하는 DNA 패턴을 나타내었지만 양친 중 한쪽 친과 유연관계가 가까운 것으로 나타났다. 사철느타리와 느타리간 교잡주는 유사도가 사철느타리에 가까웠고, 느타리간의 교잡주도 한쪽 모균주에 가까운 유연관계로 나타났다. 단핵-단핵간 교잡에서 자실체 갓 색은 사철느타리와 느타리간 교잡주는 대부분 양친주의 중간정도의 색을 나타냈으나 양친주 중 어느 한 쪽 친주에 좀 더 가까운 갓 색을 띄는 경향을 나타냈다. 자실체 수량성은 느타리간의 교잡주는 양친과 유사한 것이 82 %, 양친보다 높은 것이 0%, 양친보다 낮은 것이 18%였다. 본 연구는 느타리 계통간 교잡주의 핵 DNA 양상과 자실체 특성을 구명하였다. 단핵-단핵간 교잡법의 장점을 충분히 활용하여 앞으로 육종방법으로서 느타리버섯류의 우량 품종을 개발하는데 유용하게 이용될 수 있을 것으로 기대된다.

The primary objective of the present study is the characterization of the somatic hybrids of dikaryon-monokaryon (di-mono) crosses in mushroom breeding. We employed this technique for developing superior strain from Pleurotus ostreatus strains with 56 intraspecific hybrids of 14 combinations between six Pleurotus ostreatus strains and one Pleurotus florida strain. In this study, the results of analysis on hybridization rate, nuclear DNA patterns, and colors and morphology of fruit-bodies, are presented as follows. In di-mono crosses, somatic hybrids among Pleurotus strains showed 100% of crossability as seen in those among Pleurotus strains indicating that nuclei of a dikaryon migrated to a recipient. 89.3% of the somatic hybrids among Pleurotus strains were similar to the donor dikaryons, and 10.7% had combined DNA patterns of both parents. In the 14.3% di-mono cross between P. ostreatus and P. florida, the nuclear DNA patterns of the all hybrid strain showed the same or similar patterns compared to the donor dikaryons. 75.0% of the hybrid between P. ostreatus and P. ostreatus were similar to the donor dikaryons; 10.7% had combined DNA patterns of both parents. 82.2% of fruiting body morphology of the hybrids among Pleurotus strains were similar to the dikaryons, and 17.8% had combined DNA patterns of both parents. All hybrid strains between dikaryon P. florida and monokaryon P. ostreatus showed the fruiting body whose colors were similar to those of the dikaryon, while the hybrids between dikaryon P. ostreatus and monokaryon P. florida were all showed combined colors of both parents but are more similar to the dikaryon. Therefore, the fruiting body color of P. florida tends to be generally dominant. The present study was able to find out and suggest superior hybrid strains by identifying the nuclear DNA patterns of hybrids between Pleurotus strains as well as the characteristics of their fruiting bodies. This study expects that the advantages of the di-mono crossing are needs to be fully utilized in mushroom breeding and it is better to develop superior strains of Pleurotus strains.

느타리(Pleurotus ostreatus)의 이핵-단핵 계통간(di-mono)교잡주의 DNA 유전에 관한 특성을 구명하기 위하여 느타리 6계통 및 사철느타리 1계통으로 이핵-단핵 계통간 12조합 48교잡주를 얻어 교잡율, 교잡주의 핵 DNA패턴 양상과 유연관계도, 자실체의 형태, 갓 색깔을 분석하였다. 이핵-단핵 계통간 교잡에서 느타리와 느타리간, 느타리와 사철느타리간 교잡은 모두 교잡율 100%로 나타났다.이핵-단핵 계통간 교잡주는 공여체(donor) 이핵체의 핵이수용체(recipient) 단핵체로 전이되었다. 이핵-단핵 계통간교잡주의 DNA 패턴은 이핵체와 유사하거나 동일한 것이87.5%, 양친의 중간 패턴이 12.5%였다. 즉, 느타리 이핵주와 느타리 단핵 계통간 교잡주는 이핵체와 유사한DNA 패턴이 70.9%, 양친의 핵이 공존하는 중간 패턴이12.5%였으며, 사철느타리와 느타리간 이핵-단핵 계통간교잡주는 16.6%로 모두 사철느타리 핵 DNA 패턴과 유사하거나 동일하였다. 교잡주의 핵 DNA 패턴은 교잡조합에 따라 차이가 나타났는데 12교잡조합 중에서 4조합에서만 단핵주와 유사하거나 중간 형태를 나타내었고 나머지는 이핵주와 동일한 양상이었다. 교잡주의 자실체 형태는 이핵주 형태가79.2%, 양친의 중간형태 또는 단핵체 모군주의 형태가20.8%였다. 하지만 이핵체 형태라 하더라도 자실체 색깔은 다소 달랐다. 사철느타리 이핵-느타리 단핵주간 교잡주의 자실체 갓 색깔은 모두 이핵체 사철느타리와 유사하거나 동일하였다. 느타리 이핵-사철느타리 단핵 계통간교잡주는 양친의 중간 갓 색깔로 모두 나타났으며 다소이핵체와 유연관계가 가까운 색깔이었다. 따라서 사철느타리가 다소 우성으로 나타나는 경향이었다. 이러한 결과로 보아 교잡주는 3종류의 핵이 모두 공존하는 세포가 많을 것으로 생각되며, 이핵-단핵 계통간 교잡 방법은 우수한 계통을 육성하는 훌륭한 방법으로 이용될 수 있을 것으로 기대된다.

종의장거리산포는집단의확산과집단간개체의흐름에중요한역할을한다. 본연구는독도에서발견된식물을형태학적특징과핵및엽록체DNA의분자마커를이용하여종을식별한결과, 참빗살나무(노박덩굴과)로확인되었다. 얕은토양층과험준한지형등입지적으로열악한대양섬인독도에서목본의분포는의미있는결과이며, 향후외부유입종의지속적인모니터링이요구된다.

Polyploidy analysis being one of the important mechanisms to study the genetic resources and plant diversity which plays significant role in crop evolution, breeding programs, and crop improvement was conducted in this study. The root tips of nine Cymbidium genotypes were used as materials to determine their chromosome number and flow cytometric analysis of chromosomal ploidies. The chromosome number (2n=40) has been observed in all the nine genotypes. Results showed that among nine cymbidium genotypes, six were tetraploid (2n=4x=80), two diploid (2n=2x=40), and one triploid (2n=3x=60). This study provides useful chromosome landmarks and evidence about genome evolution, heteromorphic chromosomes based heterozygosity, basic chromosome number and ploidy level in the genus Cymbidium. These results can be used as theoretical base line for crossbreeding in the genus Cymbidium.

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.

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

The reactive oxygen species (ROS) generated during the somatic cell transfer nuclear (SCNT) procedures may cause the mitochondrial dysfunction and DNA damage, which may result in restricts the reprogramming of SCNT embryos and play a key direct role in apoptosis. The present study was conducted to investigate the effect of antioxidant treatment during the SCNT procedures on the inhibition of mitochondria and DNA damages in bovine SCNT embryos. The reconstituted oocytes were treated with antioxidants of 25 μM β-mercaptoethanol (β-ME) or 50 μM vitamin C (Vit. C) during the SCNT procedures. In vitro fertilization (IVF) was performed for controls. Mitochondrial morphology and membrane potential (ΔΨ) were evaluated by staining the embryos with MitoTracker Red or JC-1. Apoptosis was analyzed by Caspase-3 activity assay and TUNEL assay, and DNA fragmentation was measured by comet assay at the zygote stage. Mitochondrial morphology of non-treated SCNT embryos was diffused within cytoplasm without forming clumps, while the IVF embryos and antioxidant treated SCNT embryos were formed clumps. The ΔΨ of β-ME (1.3±0.1, red/green) and Vit. C-treated (1.4±0.2, red/green) SCNT embryos were significantly higher (p<0.05) than that of non-treated SCNT embryos (0.9±0.1, red/ green), which similar to that of IVF embryos (1.3±0.1, red/green). Caspase-3 activity was not difference among the groups. TUNEL assay also revealed that little apoptosis was occurred in SCNT embryos as well as IVF embryos regardless of antioxidant treatment. Comet tail lengths of β-ME and Vit. C-treated SCNT embryos (337.8±23.5 μm and 318.7 ±27.0 μm, respectively) were shorter than that of non-treated SCNT embryos (397.4± 21.4 μm) and similar to IVF embryos (323.3±10.6 μm). These results suggest that antioxidant treatment during SCNT procedures can inhibit the mitochondrial and DNA damages of bovine SCNT embryos.

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

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.

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum‐starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or 150 μM roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine‐treated groups (27.6%) than that of the roscovitine‐treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine‐treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine‐treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine‐treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine‐treatment creates a more suitable condition for nuclear reprogramming after SCNT.