In this study, we developed 11 microsatellite markers specific to A. crataegi using NGS to investigate the genetic relationships of A. crataegi populations from South Korea to 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. These results collectively suggest that northern populations, in particular, Mongolian populations can be considered as the most genetically compatible one as donee population, when the reintroduction program is launched. †These authors contributed equally to this paper.

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.

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.

The Japanese oak silkmoth, Antheraea yamamai Guérin-Méneville 1861 (Lepidoptera: Saturniidae), is one of the important natural resources possessing industrial value for silk fiber production. In this study, ten microsatellite markers and two mitochondrial DNA (mtDNA) gene sequences (COI and ND4) were used to investigate the genetic variation and geographic structure of A. yamamai populations in South Korea. Two mtDNA gene sequences revealed very low total genetic variation and resultant low geographic variation, validating to use further variable molecular markers. Population-based FIS, FST, RST, and global Mantel test consistently support that A. yamamai populations are overall well interconnected with a relatively high gene flow. Nevertheless, STRUCTURE analysis using microsatellite data and mtDNA sequences coincidently indicate the presence of two genetic pools in many populations.

본 연구는 한우의 경제형질 관련 유전적 표지인자(DNA marker) 개발을 목적으로 한우 도체형질과의 기능적 후보유전자로부터 검출된 10개의 유전마커(Single Nucleotide Polymorphism; SNP)에 대해서, 환경효과가 다양하게 포함되어 있는 상용축을 대상으로 마커효과를 검정하였다. 그 결과, 한우 후대 검정우에서 통계적 유의성이 인정되었던 다수의 SNP 좌위중 IP3R1 유전자에서 검출된 DNA 마커만 상용축의 근내지방도와 통계적 유의차를 보였으며, 이는 각 좌위마다의 환경 효과와의 보다 더 통계분석이 요구되는 것이며, 향후 근내지방도와 연관된 다수의 마커와 함께 혼합모델을 통하여 개체의 표현형 예측등에 활용이 가능할 것으로 사료된다.

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

Three tortricid pests, Grapholta dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura) are known as internal apple feeders in Korea. For identify young larvae which occurring serious damage in fruits, the molecular maker was developed from their mitochondrial DNA (mtDNA) sequences. To develop of PCR-RFLP marker, ND4 locus was digested with Swa Ⅰ. ND4-Swa Ⅰ digests showed two bands (396, 292 bp), one band (700 bp), and three bands (408, 178, and 103 bp) of G. dimorpha, G. molesta, and C. sasakii, respectively. Species-specific diagnostic PCR primers were developed in the ND4 locus and gave species-specific PCR products. Finally, these markers were applied to diagnose larvae infesting apples and showed species-specific fruit damage patterns, in which most feeders of G. dimorpha, G. molesta, and C. sasakii showed major feedings in apex, surface, and core of apple fruits, respectively.

The silkworm (Bombyx mori), as an industrial insect, possesses a high economic value. Casual discrimination and accumulated genetic information of silkworm varieties are essential ground for the practical utilization and long-term conservation. In this study, nine available microsatellite loci were successfully genotyped from ~50 silkworm strains preserved in Korea. According to genotyping analysis, we obtained 3 ~ 16 alleles per locus, with an average of 7.4, the observed heterozygosity ranging from 0.04 to 0.98, and the polymorphic information content (PIC) ranging from 0.06 to 0.88, revealing that some loci are highly variable. Among 54 strains 13 strains were casually identified by the presence of 17 strain-specific apomorphic alleles. Furthermore, 30 among remaining strains contained strain-specific allele combinations that are also apomorphic to each strain, allowing us to discriminate each of these from other strains by genotyping of multiple loci. These results collectively suggest that the silkworm microsatellite DNA is actually and potentially important molecular marker for the discrimination of the silkworm strains that are preserved as hundreds in Korea, as more loci are genotyped.

벼의 약 및 현미 배양효율과 관련된 DNA marker를 이용하여 인디카형 벼 품종인 'IR 36'의 조직배양 효율을 개선하기 위하여 실험한 결과를 요약하면 다음과 같다. 벼품종 간에 약 및 현미배양 효율을 비교한 결과 자포니카 > 통일형 > 인디카 형의 순으로 나타났다. 그러나 MGRI집단의 약배양에서 식물체분화율이 높은 계통으로 선발된 'MGRI 079'와 'MGRI 036'의 약배양 효율은 각각 19.8%, 19.9%로 가장 높게 나타났다. 'MGRI 079'에 'IR 36'이 여교배되어 양성된 90 계통에 대한 marker검정을 실시하여 positive band를 나타내는 34계통을 선발할 수 있었다. 선발된 34계통 중 10 계통의 약배양에서 캘러스 형성률은 'IR 36' 보다 현저히 높았다. 선발된 10 계통의 현미배양에서도 캘러스형성 능력과 식물체재분화율이 'IR 36' 보다 높게 나타났다. 계통 중에서 식물체분화능력이 높은 계통으로 선발된 -28은 간장이 'IR 36'보다 큰 편이었으나 출수기와 미립특성은 'IR 36'과 비슷하였다.

The hemipteran whitefly Bemisia tabaci (Gennadius) is one of the most destructive pests damaging more than 600 agricultural crop species worldwide. The B and Q biotypes are most widely spread in Korea but they are not distinguishable based on morphological characters. In order to search for protein markers that can be employed for rapid and accurate diagnosis of biotypes, two-dimensional PAGE (2DE) in conjunction with mass spectroscopic analysis were conducted. Eleven biotype-specific spots were repeatedly identified during three repetitions of 2DE and analyzed by Q-TOF. One of the B type-specific protein spots was identified as carboxylesterase 2 (Coe2). The transcript level of coe2 was determined to be 6 times higher in B type than in Q type by quantitative real-time PCR. In addition, comparison of genomic DNA sequence of coe2 between B and Q types identified a biotype-specific intron, from which specific primer sets were designed. One-step PCR using these biotype-specific primers successfully distinguished the two biotypes in a high accuracy. Availability of the biotype-specific protein and DNA markers will greatly improve the detection of B. tabaci biotype in the field.

Neoseiulus womersleyi (Schicha) (Acari: Phytoseiidae) is one of the most important predators of spider mites in Japan. Various characteristics have been studied in this species. However, because there is a lack of genetic markers, genetic diversity within and among populations has not been well elucidated. Microsatellites, short stretches of tandem-repeated 1- to 5- nucleotide sequences, are ubiquitously present in eukaryotic genomes and are highly polymorphic. Their high polymorphism makes them suitable markers for studying intra-specific variation. In this study, we developed microsatellite markers in N. womersleyi, and then examined genetic diversity in their populations. Microsatellite enriched genomic DNA library was contracted and sequenced from a single female adult. Of the 40 plasmids sequenced, 31 plasmids showed microsatellite sequences and 24 plasmids were unique. Finally, we could design primers on three loci. When tested their diversity on one wild and two laboratory populations, five to 18 alleles were detected. The wild population showed highest genetic diversity, and this divergence decreased in rearing populations. To investigate the effects of different rearing conditions, genetic diversity in two rearing populations, which were different in population size, were compared with those in the original wild populations. The allelic richness and gene diversity were not significantly different between wild and large-size populations, while the values were significantly decreased in small-size populations. Thus, 40 to 60 females per generation was sufficient to conserve the genetic diversity in N. womersleyi populations during laboratory rearing. In conclusion, the microsatellite markers developed were useful to evaluate genetic diversity in wild and laboratory populations of N. womersleyi.

Two fruit moths of the oriental fruit moth, Grapholita molesta (Busck), and the peach fruit moth, Carposina sasakii (Matsumura), infest apples in Korea by internally feeding behavior. C. sasakii is a quarantine insect pest from some other countries importing Korean apples. G. molesta is not a quarantine insect pest, but can be incorrectly identified as C. sasakii especially when it is found inside apple fruits at its larval stages because it is not easy to identify the two species by morphological characters alone. This incomplete identification results in massive economical loss by fruits needlessly destroyed or turned away at border inspection stations of the importing nations. This difficulty can be overcome by molecular DNA markers. Several polymorphic regions of mitochondrial DNA of both species were sequenced and used for developing specific restriction sites and polymerase chain reaction (PCR) primers. Based on these sequences, three diagnostic PCR-restriction fragment length polymorphism (RFLP) sites were detected and validated for their practical uses. Also, species-specific PCR primers were devised to develop diagnostic PCR method for identifying the internal feeders.

임의증폭 다형 DNA(RAPD)와 미토콘드리아 12S, 16S rRNA 유전자의 제한단편 DNA 표식자에 의해 한국에서 발생하는 담배가루이 개체군들의 biotype을 판별하였다. 진천의 장미 온실과 서울 내곡동의 포인세티아 온실에서 발생한 담배가루이는 일본, 이스라엘, 호주의 B biotype 과 동일한 DNA 단편들을 보유하였다. 여러 지역의 노지 콩 (Glycine max), 고구마 (Ipomea batatas), 들깨 (Perilla frutescens)에서 채집된 담배가루이 개체군들은 일본 시코쿠의 인동덩굴(Lonicera japonica)에서 채집된 담배가루이와 같은 DNA로 표식되었다. 이들 non-B biotype은 한국, 일본 등 극동아시아 지역에 고유한 계통으로 보인다. 최근 한국에서 발견된 담배가루이 Bemisia tabaci(Gennadius)의 주사전자현미경 관찰에 의한 형태적 특정을 온실 원예작물의 주요해충인 온실가루이 Trialeurodes vaporariorum (Westwood)와 비교하여 기재하였다.

Six field bean (GI-vcine soza S and Z ) plants were examined for their genetic polymorphisms and intraspecific variations using randomly amplified polymorphic DNA(RAPD) markers. In RAPD analysis of 5 random primers (Rp-1, Rp2, Rp-3, Rp-4, Rp-5), 30 of tot

Eleven Italian ryegrass cultivars were examined for their genetic polymorphisms and phylogenetic relationships using randomly amplified polymorphic DNA (RAPD) markers. In RAPD analysis of 34 random primers, 96 of total 162 bands obtained from 16 primers w

Background : Kalopanacis Cortex (海桐皮) is listed in「The Korea Pharmacopoeia (KP)」as the original plant of Kalopanax septemlobus (Thunb.) Koidz. However, the dried cortex of Erythrina eariegata L (刺桐) is an adulterant, one of the most indiscriminately used herbal medicines because of its similar morphologic. Due to the morphological similarities of the dried cortex of this plant to those of K. septemlobus which is used as a substitute herbal for E. eariegata, distinguish these two species is extremely difficult. Meanwhile, K. septemlobus is a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, it is conducted to develop molecular markers to distinguishing these K. septemlobus with E. eariegata by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of K. septemlobus, E. eariegata was analyzed from four barcode regions of chloroplast DNA (psbA-trnH, rbcL, matK, atpH-atpF) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcode region. The present study aimed to analyze the percent of variable sites were provided the highest ITS2 (2.3%) followed by rbcL (8.2%), in oder to develop a species-specific primer that can distinguish K. septemlobus form E. eariegata. Conclusion : The INDEL markers were developed based on the divergence of each sequence, and it is possible now to identify the two species of K. septemlobus with just a single performance of PCR. This will not only prevent misused of the plant, but also to maintain the quality of the herbal medicine as well as to verify and guarantee safety for public health.

Background : In the KHP (the Korea Herbal Pharmacopoeia), the Cuscutae Semen (菟絲子) is defined as the seed of the Cuscuta chinensis Lamark (family: Convolvulaceae). Using authentic raw herbal materials is fundamental to herbal medicine quality and Cuscutae Semen is widely distributed in many asian countries. Due to having tiny bodies of seeds, it is extremely difficult to differentiate them from adulterants and closely related species by morphologic characteristics, leading to serious safety problems. For this reason, there was conducted to develop molecular markers to distinguishing these Cuscuta chinensis with Cuscuta japonica and Cuscuta pentagona by using conventional polymerase chain reaction (PCR). Method and Results : In this study we developed a clearly and efficient method to identify Cuscutae Semen on the market. These samples (C. chinensis, C. japonica and C. pentagona) were analyzed from two barcode regions of chloroplast DNA (rbcL, psbA-trnH) and nuclear ribosomal DNA (ITS2). Based on genetic distance, the precent of variable sites were provided the highest psbA-trnH value (38.7%), followed by ITS2 (23.4%), rbcL (9.9%), in order to develop a specific primer that can distinguish C. chinensis, C. japonica and C. pentagona. Conclusion : From the above results, DNA barcoding was proved to be a successful tool for authentication the three species of Cuscutae semen. The adoption of DNA barcoding as an authentication tool by food safety agencies can safeguard the interests of both consumers and traders.

Background : Curcumae Longae Rhizoma (薑黃) is listed in「The Korea Pharmacopoeia (K P)」as the original plant of Curcuma longa L (Zingiberaceae). Meanwhile, Zeodariae Rhizoma (莪朮) is listed in 「The Korea Pharmacopoeia (KP)」as the original plant of C. phaeocaulis, C. aromatica and C. Kwangsiensi (Zingiberaceae). Due to the morphological similarities of the dried roots of this plant to those of C. phaeocaulis, C. aromatica and C. Kwangsiensis which is used as a substitute herbal for C.longa, distinguish these four species is extremely difficult. Methods and Results : A total of 90 collected samples were used in this study, In order to clearly distinguish of Curcumae Longae Rhizoma and Zeodariae Rhizoma were analysis based on sequence of the chloroplast DNA (trnK, rbcL, trnL-F, atpB-rbcL) and nuclear ribosomal DNA (ITS2). The present study aimed to analyze the percent of variable sites were provided the highest trnK (2.3%), in oder to develop a species-specific primer that can distinguish C.longa form C. phaeocaulis, C. aromatica and C. Kwangsiensis. In addition, the complete chloroplast genome of C. longa were sequenced by a 454 sequencing platform, and the structure of the obtained chloroplast genome was also analyzed. the result used that INDEL (insertion/deletion) marker for distinguish C.longa form C. phaeocaulis, C. aromatica and C. Kwangsiensis. Conclusion : The INDEL markers were developed based on the divergence of each sequence, and it is possible now to identify the four species of Curcumae Longae Rhizoma with just a single performance of PCR. This will not only prevent misused of the plant, but also to maintain the quality of the herbal medicine as well as to verify and guarantee safety for public health.

Background : Chrysanthemi Indici Flos (甘菊) is listed in 「The Korea Herbal Pharmacopoeia (KHP)」as the original plant of Chrysanthemum indicum L. C. indicum was one of the most representative medicinal plants in Asteraceae, Dried flowers of this plant have been valid chemical composition such as flavonoids, phenylpropanoids, terpenoids, and polysaccharides, possessing broad spectrum antibacterial, antiviral, antihypertensive and anti-oxidation functions. Meanwhile, C. indicum was a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, there was conducted to develop molecular markers to distinguishing these C. indicum with C. morifolium, C. zawadskii var. latilobum and Aster spathulifolius by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of Chrysanthemi Indici Flos, these samples (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius) were analyzed from five barcoding regions of chloroplast DNA (rbcL, matK, rpoB, atpF-atpH) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcoding region. Based on genetic distance, the percent of variable sites were provided the highest ITS2 value (56.9%), followed by atpF-atpH (48.18%), matK (27.2%), psbK (8.2%), and rbcL (2.9%). Comparative analysis based on the complete genome sequence of the petL-petG region INDEL (insertion/deletion) that the gene annotations were registered to the GenBank (accession number: JN-867592.1, NC-020092.1, MF-034027.1, NF-279514.1). Conclusion : From the above results, we may suggest that the petL-petG region INDEL analysis were conducted for molecular authentication of four plants (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius). The findings of results indicated that petL-petG region might be established INDEL analysis systems and hence were proved to be an effective tools for molecular evaluation and comparison of “Chrysanthemi Indici Flos” with other plants.