유전자 친자확인시험법은 동일 종 내에서도 개체를 식별 할 수 있는 강력한 분자진단체계이다. 반달가슴곰 우수리아 종(Ursus thibetanus ussuricus)은 한반도를 비롯한 중국, 러시아 등지에 분포하나, 우리나라에서는 지역적으로 절멸된 것으로 추정되고 있으며, 현재는 멸종위기야생생물Ⅰ급으 로 지정되어 있고, 북한, 러시아, 중국 등에서 재도입되어 종복원이 진행 중에 있다. 본 연구는 우리나라에 재도입된 반달가슴곰집단의 친자확인, 가계도 작성, 미확인개체 추적 등 방사개체의 관리에 요구되는 분자진단체계의 구축을 위하여 차세대염기서열분석법을 통해 결정한 반달가슴곰 전 장유전체 서열(whole genome sequence, WGS)에서 미세 부수체(microsatellite, MS) 마커 개발을 목적으로 하였다. 전장유전체 서열은 총 12개체의 혈액 DNA를 이용하여 결정하였고, 결정된 서열에서 총 1,730,507개의 단순반복서열(simple tandem repeat, STR)의 정보를 발굴하였다. 이 중 반복단위(repeat unit)의 길이가 2-6 nt로 구성된 STR은 총 5,954개였다. WGS 상에서 검출된 STR의 관찰이형접합율(observed heterozygosity, Ho)을 고려하여 총 97종을 1차 선정하였다. 부-모-자 유전자형의 멘델유전(mendelian inheritance)을 만족하는 48종의 마커를 반달가슴곰 전체 집단에 적용하여 시험하였고, 중합효소연쇄반응(polymerase chain reaction, PCR)을 통해 유전자형을 결정하였다. 유전 자형의 다양성지수와다형정보량(polymorphic information content, PIC), 동일개체출현율을 고려하여 최종 15종의 MS 마커 세트를 구성하였다. 분석결과, 전체 집단에서 출현한 평균 대립유전자의 수는 6.267개였으며, 기대이형접합율(expected heterozygosity)는 평균 0.7242을 나타내었다. 평균 PIC는 0.6718, 동일개체 출현율은 1.867×10-14 수준으로 높았으나, 부권부정율은 0.00337로 다소 낮은 수준을 보였다. 재도입된 1세대집단 전체에서는 평균 대립유전자 수 5.867개, 평균 기대이형접합율 0.7217, 평균 PIC 0.6573을 나타내었고, 후손 전체에서는 평균 대립유전자 수 5.933개, 평균 기대이형접합율 0.7140, 평균 PIC 0.6554를 나타내었다. 하지만, 후손집단을 2세대와 3세대로 구분했을 때, 2세대는 평균 대립유전자 수 5.867, 평균 기대이형접합율 0.722, 평균 PIC 0.657, 3세대는 평균 대립유전자 수 4.467, 평균 기대이형접합율 0.694, 평균 PIC 0.601을 나타내어 세대가 진행될수록 대립유전자의 수와 기대이형접합율, PIC 모두 감소하는 경향을 나타내었다. 최종 선정된 15개의 MS 마커체계를 이용하여 반달가슴곰 집단에 대한 미확인 개체 추적, 친자확인 등을 시험하였다. 최근 포획된 개체들 중에서 개체명이 확인되지 않았던 4개체는 자연출생 2세대, 3세대, 전파발신기가 탈락된 방사 1세대로 확인 되었다. 또한 동일성검사 결과 올해 5월 교통사고를 당한 반달가슴곰은 KM-53이며, 올해 출생한 새끼 반달가슴곰들에 대한 검사를 통해 2개체가 인공수정에 의해 출생한 것으로 확인되었다. 이상의 결과들은 본 연구에서 확립한 MS 마커의 조합이 자연방사와 자연출생 개체들로 구성된 반달 가슴곰 집단의 개체관리를 위한 분자마커체계로 유용하게 이용될 수 있음을 보여주고 있다. 또한 본 연구를 통해서 확립된 MS 마커들은 현재 지리산과 수도산에 서식하고 있는 반달가슴곰 집단의 개체관리, 미확인 개체 추적, 친자확인 등 유전자분석에 활용될 수 있을 것으로 기대되며, 아울러 향후 유전적 다양성 증진, 집단간 교류개체 선정 등에도 필요한 유전 정보를 제공할 수 있을 것이다. 결론적으로 반달가슴곰 전장유전체 서열을 토대로 마련된 분자진단체계는 향후 반달가슴곰의 보호와 생태복원을 위한 종복원에 기여할 것으로 기대된다.

Promalactis established by Meyrick in 1908, is one of the highest species richness genera of the family Oecophoridae. Over 240 described species are mainly distributed from Oriental and Palaearctic regions. They are usually a tiny-sized and exhibit subtle morphological differences which make problems on morphological-based identification. In this study, we performed DNA barcoding study for the Promalactis by using mitochondrial cytochrome c oxidase subunit I (COI). From analyzing 155 COI sequences, we observed the usefulness of the COI in species identification with intraspecific genetic variation (range 0.0-2.1%) and interspecific genetic divergence (range 2.8-15.4%).

Incursions of red imported fire ant (RIFA), Solenopsis invicta into Korea have been increasing. After a first interception of a colony of S. invicta on Gamman pier, Pusan port while intensive surveillance by Animal & Plant Quarantine Agency (APQA) in September 2017, three more RIFA colonies have been found in sea port piers of Pyeongtek, Incheon and Pusan cities. The social forms and mitochondrial DNA haplotypes of the intercepted RIFA colonies were analysed by allelic discrimination assay (peptide nucleic acid probe based RT-PCR) of Gp-9 gene and mt-DNA fragment of 768 bp, which is part of the Cytochrome oxidaseⅠ gene. The colony on Gamman pier, intercepted in Sep. 2017 was previously reported as a haplotype 5 (H5) of mitochondrial DNA and a social form of polygyne. The colony on Hutchison pier of Pusan port, intercepted in June 2018 were confirmed as a H22 haplotype and a monogyne. Those different social forms show different origins of each colonies. Those on piers of Pyeongtek and Incheon ports, also found in 2018 were confirmed samely as H22 and monogyne. However, it could be putatively assumed that those two colonies were differently introduced via different container cargoes, considering those colonies were found in container yards of distantly located different sea ports. More genetic variation analyses using diverse sets of molecular markers such as microsatellites, genome-wide single nucleotide polymorphisms, etc. in nuclear gens are being proceeded for more exact introduction routes (origins).

We conducted quarantine insect species diversity monitoring using DNA barcoding with 517 lepidopteran samples that were obtained from quarantine inspections of foreign vessels entering Korea. The DNA barcode of each sample was treated as a molecular operational taxonomic unit (MOTU). For species delimitation and species identification of the analyzed samples, we applied a 2% cutoff rule and then identified with using BLAST of NCBI GenBank and BOLD System ver. 3.0. Consequently, 517 analyzed samples were delimited as 214 putative species across 20 families. Of these 214 putative species 145 (368 samples) were considered taxonomically identified if the closest BLAST match was no more than 2% different. Therefore the number of samples that were identified to the species level was relatively low, at approximately 71%. 115 of the 145 species were known in Korea. Of the 30 species that were not known in Korea, three, i.e., Noctua pronuba (Noctuidae), Orthosia hibisci (Noctuidae), and Pieris brassicae (Pieridae), were checked as ‘Regulated pests’ in Korea. We suggest that the three regulated pest species could be prevented from being introduced to Korea if monitoring of the vessels that pass the navigation route that contains these three species is performed consistently. Therefore, we suggest that the monitoring of quarantined insect pest enables the prevention of the introduction of alien species.

The black-veined white, Aporia crataegi (Lepidoptera: Pieridae), which is distributed mainly in Eastern Asia is presumed to be extinct in South Korea, only with some numbers of dried specimens left, whereas the species is found casually in circumferential countries. One of the common conservation practices for such species is to launch introduction program, but prior population genetic analysis between donor and donee populations might be essential for long-term conservation. In this study, we developed 11 microsatellite markers specific to A. crataegi using Illumina paired-end sequencing to investigate the genetic relationships of A. crataegi populations from South Korea and circumferential Asian countries (China, Russia, Mongolia, and Japan). Further, two mitochondrial DNA (mtDNA) gene segments (COI and CytB) were sequenced from the samples. The population- and individual-based Principal Coordinates and STRUCTURE analyses collectively suggested that the South Korean population of A. crataegi is most differentiated from the Japanese population, whereas it was closer to Mongolian and Chinese populations. The STRUCTURE analysis based on two concatenated mtDNA gene sequences also supported different genetic composition of Japanese population from the remaining populations including that of South Korea and rather similar genetic composition between the populations of South Korea and Mongolia. These results collectively suggest that northern populations, in particular, Mongolian populations can be considered as the most genetically compatible one as doner population, when reintroduction program is launched.

Lysiphlebus orientalis is previously recorded in 2010, which is a more recently recorded than Lysiphlebia japonica. Host range of Lysiphlebus orientalis range is narrow, while that of Lysiphlebia japonica is very broad. Although two species, Lysiphlebus orientalis and Lysiphlebia japonica, are belonged to different genus, respectively, they are morphologically similar each other, which make us confused. Therefore, we have to identify these two cryptic species using COI DNA barcode. We used the ‘NCBI-BLAST (National center for biotechnology information-Basic Local Alignment Search Tool)’ to perform COI DNA barcode identification, and introduce preliminary results in this presentation.

The yellow-legged hornet, Vespa velutina nigrithorax (Hymenoptera: Vespidae), invaded into South Korea in 2003. Currently, V. velutina is distributed throughout South Korean regions,. A genetic trait of invasive species provides important data for environmental risk assessment. In this study, we analysed the genetic variation of the V. velutina among the populations of South Korea and other foreign countries using partial sequences of mitochondrial COI, CytB and 16S rRNA. We further developed intergenic spacers (IGS2 and IGS3) to detect genetic variation among Korean populations. Each single haplotype was recovered from the domestic V. velutina from each coding gene sequence. Korean individuals shared the haplotype with that of Japan and China. Phylogenetic analysis (excluding 16S rRNA) using available sequences and ours showed the presence of two groups: the Indonesia and Malaysia group and another group (Korea, Japan, China, France, Vietnam and Thailand). The newly developed IGS2 and IGS3 markers resulted in each seven and four haplotypes, providing better resolution than coding gene sequences. Analysis of Molecular Variance using the combined sequences of IGS2 and IGS3 showed that the majority of variance was allocated to each population and only minority of variance is allocated to among-populations. This result may indicate that the V. v. nigrithorax invaded in Korea shows a single panmictic population and this possibly suggest that Korean population may have originated from small founder individuals.

The pea aphid, Acyrthosiphon pisum, requires the nutritional endosymbiont, Buchnera, for the production of essential amino acids. However, it is unclear if host plant diet that varies in essential amino acids influences aphid regulation of its nutritional symbioses. We hypothesized that aphid genes respond to host plant diet when aphids feed on their specialized (alfalfa) compared to universal host plant diet (fava), which vary in essential amino acid concentrations. Using RNA-Seq and whole genome bisulfite sequencing, we compared the gene expression profiles and DNA methylation distributions of specialized aphid cells that harbor Buchnera (bacteriocytes) when aphids feed on their specialized compared to their universal host plant diets. Our results show that bacteriocyte transcription and methylation patterns differ between host plant diets. When aphids feed on their specialized host plant, they significantly up-regulate and/or hypo-methylate key aphid genes in bacteriocytes related to the amino acid metabolism, including glutamine synthetase (GS) and the glutamine transporter ApGLNT1. Moreover, regardless of which host plant aphids feed on, we observed significant up-regulation and differential methylation of the key genes in the amino acid metabolism and the glycine/serine metabolism in aphid bacteriocytes. We suggest that the regulatory response of key symbiosis genes in bacteriocytes allows aphids to feed on a specialized host plant diet with suboptimal nitrogen concentrations.

Pear psyllids belong to the most serious pests of pear. They damage pear trees by excessive removal of phloem sap, by soiling the fruits with honeydew which, in turn, provides a substrate for sooty mold, and by transmission of Candidatus Phytoplasma, the causal agents of the pear decline disease. The morphological similarity, the presence of seasonal dimorphism that affect adult colour, size and wing characters, and uncritical use of species names, led much confusion in the taxonomy of pear psyllid species. As a result, pear psyllids have been frequently misidentified. Here we analysed DNA barcodes of eleven pear psyllid species from eastern Asia, Europe and Iran using four mitochondrial gene fragments. The efficiency of identification was notably high and considerable barcoding gaps were observed in all markers. Our results confirm the synonymies of the seasonal forms. Previous misidentifications are also corrected. There is no evidence for the presence of European pear psyllid species in East Asia.

국내 판매되고 있는 사슴벌레종[넓적사슴벌레(Dorcus titanus), 참넓적사슴벌레(Serrognathus consentaneus), 남방넓적사슴벌레(D. titanus jejuensis, 가칭), 애사슴벌레(D. rectus) 및 왕사슴벌레(D. hopei)]과 부산 지역에서 채집한 넓적사슴벌레 12개체와 경남 의령군에서 채집한 1개체의 미토콘드리아 DNA 중 cytochrome oxidase I(COI) 를 이용하여 계통을 비교분석하였다. 아울러 NCBI에 기등록된 북한, 일본, 대만, 필리핀, 인도네시아 및 태국의 넓적사슴벌레의 COI 서열을 이용하여 추가 분석하였다. 먼저 판매되고 있는 사슴벌레종은 넓적사슴벌레, 남방넓적사슴벌레 및 참넓적사슴벌레의 group I과 애사슴벌레 및 왕사슴벌레의 group II로 구분되었다. 부산지역 및 의령에서 채집한 넓적사슴벌레의 경우 COI 및 COII로 분석 결과 북한에서 기보고 된 3개체를 포함해 유전적 변이에 있어 큰 차이가 나타내지 않았다. 또한 한반도와 일본 일부지역에서 높은 상동성을 확인하였는데 그 외 아시아 지역에 서식하는 넓적사슴벌레와는 다른 그룹을 형성함을 확인하였다. 이러한 결과들은 국내 서식 사슴벌레 종의 유전적 분석과 함께 외래 유입종을 판별하는 근거자료로 활용될 수 있을 것이다.

DNA bacoding is a popular DNA diagnostic technique especially for specific and generic level identification during various quarantine activities. BOLD Systems, an internet accessible DNA barcode data portal, currently includes over 12,000 DNA barcodes for the fruit fly family Tephritidae, of which 8,940 barcodes are open to public. These data represent 655 tephritid species, majority of which are pest species. These figures are rapidly increasing as the tephritid barcoding research is increasing every year. Therefore, BOLD has established as the most important DNA barcode source for tephritid identification. It is, therefore, also important to understand the limitations and problems of the DNA barcoding analysis. We here discussed the following potential problems and their possible solutions: (1) misidentification of species listed in BOLD; (2) sibling species with identical or near-identical barcodes; (3) NUMT (nuclear mitochondrial DNA) or pseudogene; and (4) introgression by hybridization between closely related species.

목적 : Coagulase-negative Staphylococci 분리 균주에 대한 quinolone 계열 및 그람양성균에 효과적인 vancomycin과 erythromycin 항생제 감수성 정도를 알아보고 DNA gyrase 유전자를 분석하고자 한다. 방법 : 세균성 각막염이 의심되는 사람의 각막에서 검출한 Coagulase-negative Staphylococci 6 균주를 대상으로 ofloxacin, ciprofloxacin, levofloxacin, vancomycin, erythromycin 항생제 감수성을 실시한 후 각 균주들의 genomic DNA를 추출하여 PCR 증폭한 후 gyraseA 유전자의 DNA를 분석하였다. 결과 : 항생제 감수성 검사에서 S. epidermidis (SE-2, SE-3), S. hominis (SH-1) 그리고 S. saprophyticus (SS-1) 균주에서는 ofloxacin, ciprofloxacin, levofloxacin, vancomycin 감수성, erythromycin 내성으로 나타났으며, 나머지 균주에서는 모두 감수성으로 나타났다. gyraseA 유전자 분석에서는 S. epidermidis (SE-1) 균주에서 Met75→Thr, Met93→Val 으로 아미노산 이중 mutation이 관찰되었으며, S. warneri (SW-1)와 S. saprophyticus (SS-1) 균주에서는 다수의 silent mutation이 관찰되었다. 결론 : Coagulase-negative Staphylococci 분리균주에서 erythromycin 항생제의 내성이 있다는 것을 확인 할 수 있었으며, gyraseA 유전자 분석결과 아미노산 이중 mutation 및 silent mutation이 관찰되었다. 따라서 지금은 내성이 아니지만 silent mutation이 언제든지 내성을 일으킬 수 있을 것으로 생각된다.

본 연구는 국내에서 생산되거나 해외에서 수입되어 국내에서 유통되는 수산물 중에서 두족류를 문어류, 낙지류, 오징어류, 주꾸미류, 꼴뚜기류의 5개 그룹으로 구분하여 분석하였다. 두족류 5개 그룹을 판별을 하기 위해 미토콘드리아에 존재하는 유전자를 분석하였고, 그 중에서 COI (mitochondrial cytochrome C oxidase subunit I), 16s rRNA (16s ribosomal RNA), 12s rRNA (12s ribosomal RNA) 내에서 상당히 유사한 DNA 서열 부분과 일부 서열 변화 부분이 확인되었다. 명확하게 두족류 5개 그룹 판별을 하기 위해 COI, 16s rRNA, 12s rRNA 유전자의 일부 서열 변화 부분에서 그룹 특이적 프라이머 세트를 디자인하였다. 국내·외에서 확보한 두족류 시료(참문어, 낙지, 살오징어, 아메리카 대왕오징어, 갑오징어, 주꾸미, 모래주꾸미, 하이야주꾸미, 참꼴뚜기, 창꼴뚜기, 한치꼴뚜기)의 genomic DNA을 추출하여 각 그룹의 특이적 프라이머를 이용하여 SYBR 기반의 real-time PCR 시스템에 의해 분석되었고, threshold cycle (Ct) value와 같은 real-time PCR 결과 분석에 의해 두족류 내 그룹 판별이 가능하였다(Table 3).

유전분석을 위해서, CTAB buffer를 이용하여 가시나무류 4종의 genomic DNA를 분리하였다. CTAB buffer를 이용하여 분리한 genomic DNA의 순도는 Edward buffer를 이용했을 때보다 더 높게 나타났다. 가시나무(Q. myrsinaefolia)로부터적정 순도 gDNA는 2% CTAB에 0, 1 또는 2% PVP가 첨가된 buffer를 이용했을 때 얻어졌다. 종가시나무(Q. glauca)로부터 1% CTAB buffer와 1% PVP를 첨가한 buffer를 이 용하여 얻은 gDNA 순도는 1.84±0.02(A260/280)였다. 참가시나무(Q. salicina)의 적정순도 gDNA는 2% CTAB와 1 또는 5% PVP가 첨가된 buffer를 이용하여 분리할 수 있었다. 2% CTAB와 2% PVP가 첨가된 buffer를 이용했을 때, 졸가시나무(Q. phillyraeoides)의 gDNA의 순도는 1.85±0.01(A260/280)였다. 18개 의 RAPD primer를 사용하여 PCR을 수행하였을 때, 가시나무에서는 15개 primer에 의해 53개의 다형질 DNA가 발견되었다. 종가시나무에서는 11개의 primer에 의해 40개의 다형질 DNA가 나타났다. 참가시나무의 경우 16개의 primer에 의해 50개의 증폭된 DNA밴드를 확인 할 수 있었다. 졸가시나무에서 14의 primer가 증폭반응을 일으켰고, 53개의 다형질 DNA가 나타났다. 이 결과들은 가시나무류의 유전분석에 이용할 수 있다.

A colony of Solenopsis invicta was first intercepted on Gamman pier, Pusan port in Korea at September, 2017 by Animal and Plant Quarantine Agency. The mitochondrial DNA (mt-DNA) of workers was analyzed and compared with vary libraries of mt-DNA haplotypes to elucidate the origin of the introduced colony with the trade pattern of the Gamman pier. The mt-DNA fragment of 768 bp, which is part of the Cytochrome oxidae I gene, was amplified and sequenced. The results showed that the mt-DNA was in the clade of haplotype 5, which is endemic in southern USA, China, Taiwan, and Australia. More than 60% of containers are imported from China into Gamman pier, it may be possible to assume that the colony was inadvertently invaded through containers from China.

Previously, a partial mitochondrial COI and CytB gene sequences have been used to infer genetic diversity and gene flow of the species. In this study, we additionally collected C. tripartitus from one previous and two new localities, sequenced the COI and CytB genes. Sequence divergence of current samples showed slightly lower values, but nearly equivalent to previous study. Haplotype diversity was still high and nucleotide diversity indicating that Seogwi-dong showed the highest estimates in both gene sequences. Gene flow among populations is high, but a significance difference was detected between Gulupdo and Anmado; and between Gulupdo and Seogwi-dong only in COI sequences (P < 0.05). Considering the high genetic diversity and gene flow in C. tripartitus populations, one of the major issues regarding conservation seems not to be the recovery of genetic diversity as has been suggested before.