Gray leaf spot caused by Stemphylium spp., is a major disease of tomatoes, and it threatens its cultivation worldwide, especially in warm and humid areas. This study was conducted on 223 tomato germplasm conserved at the National Agrobiodiversity Center to select the resources resistant to the gray leaf spot pathogen strain previously isolated in Korea, using a bioassay and genotypic analysis of the resistance gene (Sm). Two weeks after inoculation with Stemphylium lycopersici, the disease index (rated on a scale of 0-4) of gray leaf spot was assessed in detached tomato leaves. The results showed that 22 resources were resistant, with a disease index of 0-1. Additionally, 65 genetic resources were found to be moderately resistant, with a disease index between 1.0 and 2.0. Subsequently, Hybridization Probe Melting (HPM) analysis of the 22 resistant genetic resources confirmed the genotype of the gray leaf spot resistance gene (Sm). Among them, 20 genetic resources showed a homozygous resistant genotype. The resources selected in this research may contribute to the breeding of new tomato varieties resistant to gray leaf spot and may serve as a basis for further genotypic analysis studies.

고추역병균(Phytophthora capsici)은 고추 생육 전반에 걸쳐 병을 발생시켜 농가 소득에 큰 손실을 일으키고 있다. 고추 역병균 저항성은 양적 형질 유전자좌(Quantitative Trait Loci, QTL)에 의해 조절되며 주동 유전자는 고추의 5번 염색체에 존재한다고 보고 되었지만, 후보 유전자의 선발 및 저항성 유전자 규명 연구는 아직 초기 단계이다. 특히, 고추는 형질전환이 어려운 작물로써 병원균과의 상호작용 연구를 통한 저항성 유전자 동정에 제한이 많다. 반면 고추와 같은 가지과 작물인 담배(Nicotiana benthamiana)는 병원균 상호작용 모델로 알려져 형질전환을 통해 저항성 유전자 규명에 활용된다. 본 연구에서는 고추 역병 저항성 기작 규명을 위한 기초 연구로써, 식물 저항성 유사 유전자(Resistance Gene Analog, RGA)를 선발하고, 이들 유전자들에 대한 담배 형질전환 기법 최적화 연구를 수행하였다. 고추 5번 염색체에 존재하는 고추 역병 저항성 분자표지들을 분석하여 RGA 후보 유전자인 CaNBARC105, CaNBARC112 유전자를 동정하였다. 이들 유전자들에 대해 Agrobacterium tumefaciens를 매개체로 하여 고추 RGA가 삽입된 담배 형질전환체를 개발하였다. 형질전환 여부는 유전자 특이적인 서열을 이용한 genomic PCR과 RT-PCR 검증을 통해 이들 형질전환 된 담배들의 생육 및 발달에 영향이 없다는 것을 확인하였다. 본 연구는 향후 고추 병 저항성 후보 유전자들이 삽입된 담배 형질전환체는 고추 역병 저항성 유전자 규명 및 기작 연구에 기반이 될 것이다.

The detection of the genome-based antibiotic resistance gene is an essential analysis process for the purpose of verifying the safety of probiotic strains, including lactic acid bacteria. In this study, 4 analysis platforms (AMRFinderPlus, staramr, rgi, ABRicate) were used for cross-comparison of 782 genomes corresponding to 19 kinds of probiotic species notified as functional foods. As a result of the analysis, the relatively fewest number of antibiotic resistance genes were detected in strains belonging to the order Lactobacillales, and antibiotic resistance genes were detected in 322 genomes used in the case of 2 types of Enterococcus genus. In addition, the presence and type of antibiotic resistance gene detection showed a lot of difference even for the same genome sequence depending on the database and analysis algorithm used by the analysis platform. These results can be confused in evaluating the potential for transmission of antibiotic resistance genes inherent in specific lactic acid bacteria and predicting potential risks that may occur in the future. Accordingly, it is judged that the antibiotic resistance gene-related analysis criteria need to be established more clearly and specifically in the safety evaluation of probiotic bacteria.

본 연구에서는 보육시설 실내공기에서 분리된 식중독 균 주의 독소 유전자 분포와 항생제 내성을 분석하여 보육시설 실내공기에 의한 식중독 발생을 사전 예방하고 식중독 발생 시 적절한 치료를 위한 기초자료를 제공하고자 하였다. 어린이집 실내공기에서 분리된 Staphylococcus aureus 16주, Bacillus cereus 37주를 실험대상으로 하였다. S. aureus와 B. cereus 독소 유전자는 PCR 방법으로 검출하였다. 항생제 감수성 실험은 Clinical and Laboratory Standard Institute의 디스크 확산법에 따라 실험하였다. S. aureus 16 균주 중 11 균주(68.6%)에서 seg와 sei 독소 유전자가 검출되었다. B. cereus 37 균주 모두에서 nheA와 nheB 독소 유전자가 검출되었다. B. cereus 독소 유전자 패턴은 총 12개로 나타났으며 nheA-nheB-nheC 독소 유전 자가 가장 중요한 패턴으로 나타났다. S. aureus 16 균주의 항생제 감수성실험 결과 ampicillin과 penicillin 항생제 에 93.8%, 87.5% 내성을 나타내었으나 methicillin resistance Staphylococcus aureus와 vacomycin resistance Staphylococcus aureus는 검출되지 않았다. B. cereus 37 균주의 항생제 감수성 실험 결과 ampicillin과 penicillin 항생제에 100% 내성을 나타냈었다. 이러한 결과를 종합하여 볼 때 보육시 설 실내공기에 오염된 S. aureus와 B. cereus에 의한 식중독을 발생을 예방하기 위하여 주기적인 환기와 공기 질 관리가 필요한 것으로 판단되었다.

The two-spotted spider mite, Tetranychus urticae, is a worldwide agricultural pest that invades a wide range of host crops and rapidly develops resistance to pesticides. T. urticae can be resistant to one acaricide or exhibit multiple resistances or cross resistance to various other acaricides. Acequinocyl inhibits respiration in mitochondria at the ubiquinol oxidation site (Q0) of Complex III of the electron transfer chain. Pyridaben is a METI acaricide that inhibits mitochondrial electron transport at complex I. In this study, we investigated the cross resistance to seven acaricides in acequinocyl- and pyridaben-resistant strain. Furthermore, the frequencies of the I256V and N321S mutations in mitochondrial cytochrome b (cytb) of pyridaben-resistant and fieldcollected strain was analyzed.

Control of the vector mosquitoes is critical for implementation of patient’s decrease. Vector mosquito control has been mainly carried out using pesticides in Republic of Korea (ROK), which has developed the resistance against selected insecticides. Voltage-gated sodium channel (VGSC) and Acethylcholine esterase (AchE) are the target of several classes of insecticides. Genetic mutations of VGSC and AchE have been documented to confer resistances to pyrethroids and organophosphates in mosquitoes, respectively. In our studies, the insecticidal resistable level of An. sinensis from four malaria risk areas near Demilitarized Zone (DMZ) was measured. The partial sequence of An. sinensis voltage gated sodium channel (VGSC) IIS6 domain for Kdr gene and ace1 gene for AchE was sequenced. An. sinensis from two areas demonstrated significantly higher resistance to pyrethroids and mutation rates of Kdr gene were comparatively higher. In all tested areas An. sinensis showed high insecticidal resistance to organophosphates and > 70% of mutation rates of ace1 gene ware higher. Understanding the mechanisms of insecticide resistance among vector mosquitoes populations is very important to efficient and effective vector control.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pest species devastating many horticultural, ornamental crops and fruit trees. Difficulty in managing this mite is largely attributed to its ability to develop resistance to many acaricides. Development of 3,700 folds resistance to etoxazole was found in the population of T. urticae collected from rose greenhouses in Buyeo, Chungnam Province in August 2000. This population has been selected for eleven years with etoxazole (over 500 times), and increased over 5,000,000 fold in resistance as compared with susceptible strain (S). Etoxazole-resistant strain was shown to be maternally inherited. The objective of this study was to determine whether resistance of T. urticae to etoxazole was linked with point mutations in the mitochondrial gene. DNA sequencing of cytochrome c oxidase subunit I (COX1), COX2, COX3, cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), ND2, ND3, ND4, ND5, and ND6 were analyzed by comparing two isogenic etoxazole-susceptible (EtoS) and etoxazole-resistant (EtoR) strains. As a result, all genes revealed no point mutations between the two strains.

This study was carried out to investigate the antimicrobial resistance pattern and distribution of resistance gene determinants in fecal E. coli from chicken. Antimicrobial susceptibility test was performed on a total of 109 fecal E. coli isolates from chicken, collected in Gyeonggi, Chungbuk, Jeonnam and Jeonbuk province from March to November 2003, by the disk agar diffusion method. Eighteen commonly used antimicrobial agents approved in Korea as veterinary medicine were tested: ampicillin (AM), amoxicillin/clavulanic acid (AMC), cephalothin (CF), cefozolin (CZ), cefoxitin (FOX), cefotaxime (CTX), cefepime (FEP), imipenem (IPM), streptomycin (S), gentamicin (GM), amikacin (AN), ciprofloxacin (CIP), enrofloxacin (ENO), norfloxacin (NOR), trimethoprim/sulfamethoxazole (SXT), erythromycin (E), chloramphenicol (C) and tetracycline (TE). Higher resistance rates (≥50%) were observed against 9 antimicrobial agents including AM, CF, S, CIP, ENO, NOR, SXT, E and TE. Resistance was most frequent for TE in 105 E. coli isolates (96.3%). Twenty-two isolates (20.2%) of the isolates showed multiple antimicrobial resistance to 8, and 19 isolates (17.4%) showed to 7 antimicrobial agents. The distribution of the resistance gene determinants for S and TE was assessed by PCR in resistant isolates. Thirty isolates possessed the strA, strB, and aadA gene, 25 isolates possessed the strA and strB gene among the 66 streptomycin-resistant isolates. Fifty one isolates possessed only the tetA gene, 22 isolates possessed the tetA and tetB gene, 11 isolates possessed only the tetB gene among the tetracycline-resistant isolates.

벼멸구에 대한 벼품종의 저항성요인으로 주목되고 있는 선호성, 란 및 약충기간, 부화 및 우화률, 성비, 성충의 수명 및 산란력 등을 조사하였다. 식이 및 산란선호성은 동률벼에서 생태형 모두 높은 선호성을 보였으며, 청청벼와 밀양63호에서는 각각 생태형-2와 3에서 약간 높은 선호성을 보였으나, 동률벼에 비해서는 현저히 낮은 선호성이었다. 난 및 약충기간과 부화율, 우화률 및 생장 지수는 생태형-1의 경 우 타 품종에 比해 密陽 23號에 서 짧고, 높았으며 , 또態型-2와 3 은 各各 좁 팍벼 와 密陽 63號에 서 密陽 23號에서와 같이 짧고, 높게 나타났다. 성충의 수명과 산란수도 생태형-1의 경우 타 품종에 비해 밀양 23호에서 길고, 많았으며, 생태형-2와 3은 각각 청청벼와 밀양 6 63호에서 밀양 23호에서와 같이 수명이 길고 산란수도 많았다.

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse pepper production. In contrast to most epiphytic powdery mildew species, L. taurica is an endophytic fungus which colonizes in the mesophyll tissues of the leaf. In the genus Capsicum, several studies have been conducted to identify resistance sources to L. taurica. In previous studies, five quantitative trait loci (QTLs) for powdery mildew resistance have been identified. An F2 population derived from self-pollination of the commercial cultivar Capsicum annuum ‘PM Singang’ was used for genetic analysis of powdery mildew resistance. Resistance of the F2 plants was tested under the natural environmental conditions. Sporulation intensity on infected leaves was used as a disease scale to assign resistance levels to plants, where 0-5% is Resistant, 6-15% Moderate resistant and 16-100% Susceptible. A total of 83 F2 plants were evaluated for resistance. The results showed that 59 plants were resistant, 10 susceptible and 14 moderately resistant. If we consider MR as S, segregation ratio fitted to a single dominant resistance gene model. In the future study, closely linked molecular marker will be developed and tested to locate this gene. The developed marker will be used to identify the powdery mildew resistance gene.

Leaf mold disease in tomato (Solanum lycopersicum) is caused by Cladosporium fulvum, a fungal leaf pathogen. One of effective ways to control leaf mold is to breed disease-resistant tomato cultivars. Cf-4 and Cf-9 resistance (R) genes encode proteins that carry a leucine rich repeat domain and are located in plasma membrane. They trigger hypersensitive response following recognition of corresponding Avr4 and Avr9 proteins of C. fulvum, respectively. Cf-4 and Cf-9 genes are originated from wild tomato species S. habrochaites and S. pimpinellifolium and have been introgressed into commercial tomato cultivars. These two highly homologous orthologs exist as a cluster with four highly homologous paralogs. Due to this reason, development of genetic markers to distinguish these two functional R genes from their orthologs and paralogs is difficult. In this study, we tried to develop single-nucleotide polymorphism (SNP) markers to select tomato cultivars carrying resistant Cf-9 genotype. The genomic sequences of resistant Cf-4 and Cf-9 alleles, susceptible cf-9 alleles, and their paralogs were obtained from the GenBank database, and two functional SNPs causing non-synonymous substitution were found among them. Based on two SNPs, the Cf-9_2-SNP-F/R primer set for high resolution melting (HRM) analysis was developed. HRM analysis with this primer set could successfully distinguish tomato cultivars carrying resistant Cf-9 allele among 30 commercial tomato cultivars, which were characterized with the gene-based marker. These indicate that the SNP marker developed in this study is useful to trace Cf-9 genotype efficiently in marker-assisted selection in tomato.

Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threaten tomato (Solanum lycopersicum) worldwide. So far, eight TSWV resistance genes, Sw1a, Sw1b, sw2, sw3, sw4, Sw-5b, Sw-6, and Sw-7 have been identified and Sw-5b has been incorporated into tomato for prevention of TSWV. The objectives of this research are first to discover single nucleotide polymorphisms (SNPs) in Sw-5 alleles and then to develop SNP markers to distinguish resistant genotypes against TSWV for marker-assisted breeding in tomato. First, DNA sequences of Sw-5b alleles from both resistant and susceptible cultivars amplified using known Sw-5 gene-based marker was analyzed. The single functional SNP (G→A) was detected as non-synonymous substitution because this SNP causes change of arginine (Arg599) to glutamine (Gln599). Next, the primer pair for high resolution melting analysis (HRM) was designed around this SNP. To determine accuracy of this SNP marker to distinguish resistant Sw-5b genotypes against TSWV, genotypes of 32 commercial tomato cultivars were checked. The newly developed SNP marker could select six cultivars carrying resistant Sw-5b genotype, which was 100% correlated with genotypes based on the gene-based marker. These results indicate that the SNP maker developed in this study could be useful for better tracking resistance to TSWV in tomato breeding.

Root-knot nematode, Meloidogyne incognita is a virulent pest of solanaceaous crops worldwide. The M. incognita resistance gene Me7 derived from Capsicum annuum CM334, is located on chromosome 9. In the present study, an F2 population derived from a cross between ECW03R and CM334 was used to locate the Me7 gene. An F2 population was inoculated using approximately 1,000 second-stage juveniles per individual plant. Phenotype screening was done 45 days after inoculation by using gall index system. The phenotype study of 503 F2 individual showed 391 resistant and 112 susceptible plants. The 3:1 phenotypic ratio confirmed that resistance phenotype is controlled by a single dominant gene. Previously reported two markers were tested to reveal the linkage of markers to phenotype. Two markers, CAPS_F4R4 and SCAR_PM6a were located at 4.3 and 2.7 cM from the resistance gene, respectively. Additional SNP markers were developed using CM334 reference genome information to narrow down the position of the gene, but no closer markers could be developed due to errors of DNA sequence assembly. The closest marker was positioned on telomere of the chromosome 9 long arm, where tens of other NB-LRR genes are clustered. NB-LRR genes are being used as candidates to identify the Me7 gene.

For efficient introgression of the downy mildew resistance gene from a resistant cultivar into domestic breeding lines, molecular markers used for marker-assisted backcrossing (MAB) were developed in onion (Allium cepa L.). The resistance gene (Pd) was originally introgressed from a wild species, A. roylei, by interspecific hybridization, and the resistant gene was known to be positioned at the end of chromosome 3. Therefore, cDNA sequences of loci located at the ends of chromosome 3 of two linkage maps were obtained from a transcriptome database. Primer pairs were designed on exon sequences of eight loci. Among them, the PCR products of the i25255 locus showed length polymorphism between A. roylei and onions, and both large and small-sized PCR products were observed in the resistant cultivar. Sequence analysis showed that a 67-bp indel existed in the intron sequences. Based on this indel polymorphism, a simple PCR marker, designated DMR1, was developed. Analysis of diverse onion accessions showed that no accessions contained the A. roylei-specific marker genotype except for the resistant cultivar. These results indicated that the DMR1 marker was successfully tagging the A. roylei fragment harboring the downy mildew resistance gene, and the resistant cultivar was heterozygous for the resistance gene. After further analysis of multiple loci positioned at chromosome 3, a range of the A. roylei fragment introgressed in the resistant cultivar was determined in two linkage maps. On the basis of the range of the A. roylei fragment, three molecular markers used for recombinant selection in MAB were also developed.

Foxglove aphid, Aulacorthum solani (Kaltenbach), is a Hemipteran insect that infected a wide variety of plants worldwide and caused serious yield losses in crops. The foxglove aphid resistance gene, Raso2 was previously mapped from PI 366121 (Glycine soja Sieb. and Zucc.) to a 26cM marker interval on soybean chromosome 7. The development of additional genetic markers, which are mapped closer to Raso2 were required to accurately position the gene to improve the effectiveness of marker assisted selection. The objective of this study was to narrow down the putative QTL region, which is responsible to foxglove aphid resistance in PI366121 using recently developed high-density 180K Axiom SoyaSNP genotyping array. One hundred and forty one F8-derived F12 recombinant inbred lines developed from a cross of susceptible Williams 82 and resistant PI 366121, were used to generate a fine map of Raso2 interval. The phenotyping of antibiosis and antixenosis was done through choice and no-choice assays with total plant damage (TPD) and primary infestation leaf damage (PLD). The composite interval mapping analysis showed that the physical interval between two flanking makers, which was corresponding to Raso2, was narrowed down to 500kb on the Williams 82 genome assembly (Glyma2.0), instead of 4Mb in the previous report using Goldengate assay. In the Raso2 interval, there are about 60 candidate genes, including 4 of NBS-containing putative R genes. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars.

The depletion of stratospheric ozone has resulted in increased amount of ultraviolet-B radiation (UV-B: 280-320 nm) reaching the Earth’s surface and could cause significant biological effect in plants. In this study, putative quantitative trait loci (QTL), which is responsible to UV-B resistance in soybean, was identified using recently developed high-density 180K Axiom SoyaSNP genotyping array. A population of 115 recombinant inbred lines (RILs) derived from a cross between susceptible Keunolkong and resistant Iksan 10 was analyzed. A total 8,970 polymorphic SNP markers were used to construct linkage map. The both parents and RILs were grown with supplemental UV-B radiation in a greenhouse condition. Three categories of UV-B induced morphological damage, degree of leaf chlorosis, leaf shape change, and total plant damage were evaluated. Using composite interval mapping analysis, one major QTL associated with all of the phenotypic traits was detected on 7.7cM of soybean chromosome 7 with 22 of LOD score accounting for about 60% of phenotypic variance. Also, the allele from Iksan 10 were responsible for the UV-B resistance. Thus, the UV-B resistance QTL on chromosome 7 from Iksan 10 was designated to qUVBR1, corresponding to 30kb on the Williams 82 genome assembly (Glyma2.0) including 7 candidate genes. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars. In addition, these results provided useful information not only for marker-assisted selection for UV-B resistance soybean, but also for the future identification of putative candidate genes, responsible for UV-B resistance in soybean.

Blackleg disease caused by Leptosphaeria maculans, is the most devastating disease of Brassica germplam worldwide that causes million tonnes of crop losses per year throughout the world. To date, a total of 12 race-specific resistance genes of Brassica napus to L. maculans have been reported but linkage mapping analysis reveals that all of those loci are located in A genome i.e., in B. rapa chromosomes. B. oleracea has high ancestral synteny with B. rapa through their evolution. We believe that presence of qualitative resistance is possible in B. oleracea germplasm. The present study was therefore planned to find out any race-specific qualitative resistance gene present in C genome of B. oleracea. A total of 16 microsatellite markers were used which are linked to seven different Rlm and Lep genes of B. napus to screen 32 inbred lines of cabbage. Primers were designed based on homology assessment in corresponding nucleotide sequence available in Bolbase (a B. oleracea genome database, http://www.ocri-genomics.org/bolbase/index.html), located in B. oleracea scaffolds/chromosomes. Out of 16 SSR markers, 13 were found polymorphic which indicates possible existence of resistant genes in cabbage lines. The inbred lines are then assessed against two L. maculans stains with known avirulent genes. Some inbred lines were hypersensitive against gene-specific virulent strains of L. maculans that confirmed existence of Rlm1, Rlm2, Rlm4, LepR3 and LepR4 in the cabbage lines. In this way we were able to select out resistant and susceptible lines against each resistant gene. The gene-specific polymorphic SSR marker regions were cloned and sequenced and candidate SNPs were identified for confirmation of their functionality.