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Anthracnose is caused by Colletotrichum species and is one of the most virulent fungal diseases affecting chili pepper (Capsicum) yield globally. However, the noble genes conferring resistance to Colletotrichum species remain largely elusive. In this study, we identified CbAR9 as the causal locus underlying the large effect quantitative trait locus CcR9 from the anthracnose-resistant chili pepper variety PBC80. CbAR9 encodes a nucleotide-binding and leucine-rich repeat (NLR) protein related to defense-associated NLRs in several other plant species. CbAR9 transcript levels were induced dramatically after Colletotrichum capsici infection. To explore the biological function, we generated transgenic Nicotiana benthamiana lines overexpressing CbAR9, which showed enhanced resistance to C. capsici relative to wild-type plants. Transcript levels of pathogenesis-related (PR) genes increased markedly in CbAR9-overexpressing N. benthamiana plants. Moreover, resistance to anthracnose and transcript levels of PR1 and PR2 were markedly reduced in CbAR9-silenced chili pepper fruits after C. capsici infection. Our results revealed that CbAR9 contributes to innate immunity against C. capsici.

Chili pepper (Capsicumannuum) is an important fruit and spice used globally, but its yield is seriously threatened by anthracnose. Capsicum baccatum is particularly valuable as it carries advantageous disease resistance genes. However, most of the genes remain to be identified. In this study, we identified the C. baccatum-specific gene CbCN, which encodes a truncated nucleotide-binding and leucine-rich repeat protein in the anthracnose resistant chili pepper variety PBC80. The transcription of CbCN was greater in PBC80 than it was in the susceptible variety An-S after Colletotrichum acutatum inoculation. In order to investigate the biological function of CbCN, we generated transgenic tobacco lines constitutively expressing CbCN. Notably, CbCN-overexpressing transgenic plants exhibited enhanced resistance to C. acutatum compared to wild-type plants. Moreover, the expression of pathogenesis-related (PR) genes was remarkably increased in a CbCN-overexpressing tobacco plants. In order to confirm these results in chili pepper, we silenced the CbCN gene using the virus-induced gene silencing system. The anthracnose resistance and expressions of PR1, PR2, and NPR1 were significantly reduced in CbCN-silenced chili peppers after C. acutatum inoculations. These results indicate that CbCN enhances the innate immunity against anthracnose caused by C. acutatum by regulating defense response genes.

High-throughput molecular markers with high genotyping accuracy will be helpful for genetic analysis, mapping of interesting genes, and rice breeding program. To develop high-throughput and cost-effective molecular markers for Korean japonica rice varieties, which are closely-related genetically, we designed kompetitive allele-specific polymerase chain reaction (KASP) assays from the sequence data of 13 Korean japonica rice varieties. Of the 504 new KASP assays, 371 (73.6%) showed polymorphisms among the tested varieties. In addition to the 400 previously developed KASP markers, this resulted in 771 KASP markers being applicable for Korean japonica rice varieties. These KASP markers were used to map the quantitative trait loci (QTLs) for rice bakanae disease (BD) resistance. From the results of QTL mapping and determination of the mortality rate of BD in two F2:F3 populations, a major QTL, qFfR1-1, and a novel QTL, qFfR6, were revealed on chromosome 1 in the Junam/Nampyeong F2:F3 population and on chromosome 6 in the Saenuri/Nampyeong F2:F3 population, respectively. Further, the insertion/deletion markers in the qFfR1-1 region were developed to select BD-resistant japonica rice varieties. The 771 developed KASP markers will accelerate the molecular breeding in Korean japonica rice varieties, and the detected QTLs will be helpful in identifying candidate genes for BD resistance.

Tomato belongs to the Solanaceae family of plants. It is a diploid plant with 12 chromosomes. Previous studies have reported that its genome size is 950 MB with 35,000 protein-coding genes. Micro-Tom Tomato is a miniature dwarf determinate tomato cultivar. It has a small-sized genome, a short lifecycle, and a short seed-setting under fluorescent light. These features are similar to those of Arabidopsis. Consequently, Micro-Tom Tomato is considered as a model cultivar of tomato (Solanum lycopersicum) suitable for research. We sequenced its transcriptomes to identify tissue-specific gene candidate profiles in different plant tissues (petals, sepals, pistils, and stamens) at developmental stages.

Major tomato viruses in Korea are Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), and Tomato mosaic virus (ToMV). RT-PCR conditions for the viruses were examined, especially in primer set and RT-PCR mixture. Total 46 primer sets from the unique sequence of the viruses were tested for nonspecific background products in a RT-PCR mixture without template. Among them 16 primer sets were applied to healthy tomato RNA, resulting the compatibility between RT-PCR mixture and primer set influenced RT-PCR to reduce nonspecific background products. Based on the combinations among cDNA synthesis parameters and RT-PCR mixtures, two reaction mixtures were finally selected for ToCV detection. The condition allowed to determine more specific primer sets; C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), and C065 (ToMV). These primer sets are expected to be of use to specific detection of the major viruses in tomato plants.

Cucumber mosaic virus (CMV), a member of the Cucumovirus genus in the family Bromoviridae, is a frequently occurring virus in pepper worldwide. A broad host range, which includes many weed species and a large number of insects, means it is difficult to control CMV in the field. CMV resistance has been reported in Capsicum spp. In Korea, the C. annuum ‘Bukang’ cultivar is resistant to the CMVFNY and CMVKorean strains; however, a new strain (CMVP1) is able to infect the resistant cultivars. Therefore, it is necessary to pursue CMVP1 resistance in pepper breeding. In this study, we surveyed the quantitative trait loci (QTL) for resistance to CMVP1 using a single-seed-descent (SSD) F3 segregating population derived from a cross between a resistant line, ‘A1’, and a susceptible line, ‘2602.’ A total of 174 plants were evaluated for resistance to CMVP1 at 30 days after mechanical inoculation, with phenotypes confirmed by reverse transcription PCR. The number of resistant and susceptible plants was 88 and 86, respectively. Of these, 96 individuals including 48 resistant lines and 48 susceptible lines were used for a genotyping-by-sequencing (GBS) analysis. Approximately 19,000 single-nucleotide polymorphism (SNP) genotypes were obtained. Using the SNP data, a pepper genetic linkage map consisting of 906 SNP markers was constructed. The map showed 12 linkage groups with a total linkage distance of 1,272.9 cM. QTL analysis using a composite interval mapping (CIM) method revealed that two QTLs, cmvP1-5.1 and cmvP1-10.1, located on chromosomes 5 and 10, respectively, had R 2 (coefficient of determination) values of 17.81% and 22.78%. This information will be helpful for developing SNP markers linked to CMVP1-resistant QTLs and for developing new CMVP1-resistant pepper cultivars.

Capsaicinoids are pungent compounds used for industrial and medical purposes including food, medicine and cosmetics. The Indian local variety ‘Bhut Jolokia’ (Capsicum chinense Jacq.) is one of the world's hottest chilli peppers. It produces more than one million Scoville heat units (SHUs) in total capsaicinoids. In this study, our goal was to identify quantitative trait loci (QTLs) responsible for the high content of capsaicin and dihydrocapsaicin in ‘Bhut Jolokia’. Capsicum annuum ‘NB1’, a Korean pepper inbred line containing 14 000 SHUs, was used as a maternal line. An F2 population derived by crossing between ‘NB1’ and ‘Bhut Jolokia’ was generated to map QTLs for capsaicinoids content. A total of 234 markers, including 201 HRM, 21 SSR, 2 CAPS and 10 gene-based markers of the capsaicinoid synthesis pathway, were mapped. The final map covered a total distance of 1175.2 cM and contained 12 linkage groups corresponding to the basic chromosome number of chilli pepper. Capsaicin and dihydrocapsaicin content were analysed in 175 F2 pepper fruits using the HPLC method. The maximum total capsaicinoids content was 1389 mg per 100g DW (dry weight), and the minimum content was 11 mg per 100g DW. Two QTLs (qcap3.1 and qcap6.1) for capsaicin content were identified on LG3 and LG6, and two QTLs (qhdc2.1 and qdhc2.2) for dihydrocapsaicin content were located on LG2. We did not detect QTLs for total capsaicinoids content. The QTL positions for capsaicin content were different from those for dihydrocapsaicin content. These results indicate that the complexity of selecting for more pungent chilli peppers must be considered in a chilli pepper breeding programme. The QTL-linked markers identified here will be helpful to develop more pungent pepper varieties from ‘Bhut Jolokia’, a very hot pepper.

Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.

In this study, we aimed to develop a simple analysis method for measuring the carotenoids content of pepper powder. A 96-wells polystyrene microplate and an ELISA reader were used for analysis. Although ELISA reader with 450 nm filter was applicable to measure carotenoid contents, the surface of microplates were degenerated by acetone used for carotenoids extraction. However, ten-folded dilute of the color extract with methanol did not affect the surface of polystyrene microplate and components of the color extract could be successfully measured by a ELISA reader, showing a high corelation with ASTA-20.1 method. In addition, this method uses 10 fold less acetone than ASTA-20.1 method resulting less acetone waste. The microplate method using ELISA reader has potential power for analyzing a large number of samples which may be very useful to the practical breeding program for high-colored peppers.

The resistance to Tobamovirus in Capsicum spp. has been known to be controlled by five different alleles (, , , , and ) of L locus on the telomere of long arm of pepper chromosome 11. To develop a set of molecular markers differentiating all the alleles of L locus, we used five pepper differential hosts including Capsicum annuum Early California Wonder (ECW, ), C. annuum Tisana (), C. annuum Criollo de Morelos 334 (CM334, ), Capsicum chinense PI 159236 (), and Capsicum chacoense PI 260429 (). Developing a series of CAPS or SCAR markers specifically linked to the alleles was allowed by the sequence comparison of PCR amplicons of the -linked markers (189D23M, A339, and 253A1R) and BAC sequences (FJ597539 and FJ597541) in the pepper differentials. Genotypes deduced by these markers in 48 out of 53 hybrids of commercial pepper varieties were consistent with their phenotypes by bioassay using Tobamovirus pathotypes (, , and ). Consequently, these markers can be useful to differentiate L alleles and for breeding Tobamovirus resistance in pepper with marker-assisted selection.

A total of 94 tomato accessions were evaluated for the resistance to tomato spotted wilt virus (TSWV) using a Sw5-2 SCAR marker and bioassay. PCR products of the marker were approximately 574 bp, 500 bp, and 462 bp, among which the longest was linked to TSWV resistance allele of Sw5-b. This allele was only found in three accessions (09-438, 10-318, and 10-321) in which some individuals showed apparent recovery or stem necrosis symptom to a tomato isolate of TSWV-pb1. Thirty-five individuals (one per each accession) which were non-infected by ELISA were selected for further observation. Among these, 26 individuals that did not show any symptom at 5 months after inoculation were confirmed for viral infection by RT-PCR. TSWV-specific PCR amplicon was weakly detected in all 26 individuals including ‘Eureta’, a commercial F 1 possessing the resistance allele of Sw5-b. The resistant genes in the selected individuals may play an important role for reducing the viral concentration in tissues of inoculated tomato plants and seems to be quantitatively controlled by several factors including Sw5-b gene.

Phytophthora root rot has been causing a serious yield loss in pepper production. Since 2004, the year in which commercial cultivars resistant to the disease were firstly commercialized, it has been necessary to introduce the resistance into domestic pepper cultivars for dried red pepper. Therefore, developing molecular markers linked to the resistance is required for an accurate selection of resistant plants and increasing breeding efficiency. Until now, several markers associated with the major dominant gene resistant to Phytophthora root rot have been reported but they have some serious limitations for their usage. In this study, we aimed to develop molecular markers linked to the major dominant gene that can be used for almost of all genetic resources resistant to Phytophthora root rot. Two segregating populations derived from a 'Subicho' 'CM334' combination and a commercial cultivar 'Dokyacheongcheong' were used to develop molecular markers associated with the resistance. After screening 1,024 AFLP primer combinations with bulked segregant analysis, three AFLP (AFLP1, AFLP2, and AFLP3) markers were identified and converted into three CAPS markers (M1-CAPS, M2-CAPS, and M3-CAPS), respectively. Among them, M3-CAPS marker was further studied in ten resistants, fourteen susceptibles, five hybrids and 53 commercial cultivars. As a result, M3-CAPS marker was more fitted to identify Phytophthora resistance than previously reported P5-SNAP and Phyto5.2-SCAR markers. The result indicated that the M3-CAPS marker will be useful for resistance breeding to Phytophthora root rot in chili pepper.

A pepper genetic map was constructed from F2 mapping population of 93 individuals from a cross between Capsicum annuum ‘F68’ and C. chinense ‘Habanero’. Surrey was made for the map distribution and polymorphism level of these marker groups; reverse random amplification microsatellite polymorphism (rRAMP), WRKY and amplified fragment length polymorphisms (AFLP). A total of 912 molecular markers [356 rRAMP, 190 WRKY, 305 AFLP, and 61 simple sequence repeats (SSR)] were developed in this study. The rRAMP and WRKY markers were more evenly scattered in the pepper genome than the AFLP and SSR markers, and filled the gaps not populated by the other markers. The interspecific pepper map contained 28 linkage groups with 625 linked markers and covered 3377.2 cM with an average interval of 5.9 cM. On the basis of the map, the fruit length quantitative trait locus (QTL) was analyzed and these QTL regions were detected near rRAMP and WRKY markers on the chromosome 3, 5, 11, and LG3. These marker system, map information, and detected QTLs could be one of basic information for pepper research.

Agricultural Microbiology Division, National Academy of Agricultural Science, Suwon 441-707, Korea(Received on November 22, 2010; Accepted on March 7, 2011)One hundred Capsicum accessions were screened for symptomatic response and resistance to Tomato spottedwilt virus-pb1 (TSWV-pb1). Symptom and its severity rating were checked by visual observation at 9, 12, 14,and 45 days after inoculation, respectively. Enzyme-linked immune-sorbent assay was performed all testedindividuals on non-inoculated upper leaves after the third rating to indentify viral infection. Leaf curling waspredominant in almost susceptible individuals of each accession. Stem necrosis was most frequent in wildspecies while yellowing in commercial hybrids and Korean land race cultivars. Ring spot, a typical symptomof TSWV, was rarely detected in some of a few accessions. Different levels of resistance to TSWV-pb1 wereobserved among the tested accessions. High level of resistance was detected in 4 commercial cultivars of Kpc-35, -36, -57, and -62, and 8 wild species of PBI-11, C00105, PBC076, PBC280, PBC426, PBC495, PBC537, andPI201238 through seedling test by mechanical inoculation. Keywords : Chili pepper, Resistance, Tomato, Tomato spotted wilt virus 토마토반점위조바이러스(Tomato spotted wilt virus;TSWV)는 Bunyaviridae과 Tospovirus속으로 분류되는 구형 바이러스로 꽃노랑총채벌레(Frankliniella occidentalis)에 의해 주로 전반된다(Whitfield 등, 2005). 1919년에 호주의 토마토에서 처음 발생한 이후 TSWV는 하와이, 브라질, 유럽의 여러 국가, 남아프리카공화국 등에서 보고되었으며(조 등, 2005), 국내에서는 2003년에 충남 예산신안면 파프리카 재배 농가에서 최초로 발생하였다(Choi등, 2010). 이후 강원도 지역을 포함한 총 23개 지역에서발생하였고, 고추와 토마토뿐만 아니라 국화, 콩 등에도감염하여 지속적으로 관찰해야 할 주요 바이러스로 인식되고 있다(Choi 등, 2010). 우리나라에 발생하는 TSWV는 식물 종류별, 발생 지역별 병원성 분화가 일어나는 것으로 확인되어 각 작물 별 저항성 유전자원 확보가 매우중요한 것으로 보고되었다(조 등, 2009). 한편, 국내에서는 TSWV 발생현황과 바이러스에 대한특성 연구가 주로 보고 되었으나 외국에서는 TSWV 저항성 연구가 활발하게 수행되었다. Capsicum chinensePI159236과 PI152225에서는 단일 우성 유전자인 Tsw 이보고되었으며, 이와 연관 분자 표지도 개발되어 실제 상업 육종에 이용되고 있다(Boiteux와 Avila, 1994; Boiteux,1995). 비록 Tsw 유전자가 도입된 상용 파프리카가 판매되고 있지만, 1992년부터 브라질(Boiteux와 Nagata, 1992),루이지아나(Hobbs 등, 1994), 이탈리아(Roggero 등, 2002),스페인(Margaria 등, 2004), 호주(Sharman와 Persley, 2006)에서는 Tsw 저항성을 극복하는 새로운 TSWV 계통이 보

Genic male sterility (GMS) has long been used as a tool for hybrid seed production in chili pepper (Capsicum annuum L.). We developed DNA markers linked to the GMS ms 3 gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 512 primer combinations. Three AFLP markers (Eagg/Mccc276, Eagc/Mctt178, and Ecag/Mtgc204) were identified as tightly linked to the ms 3 locus. Among them, we converted the AFLP marker Ecag/Mtgc204 to the cleavage amplified polymorphic sequence (CAPS) marker, named GMS3-CAPS, based on sequencing analysis of internal and flanking regions for the markers between male-fertile and sterile plants. This marker will be useful for pepper breeding using the GMS system.

A simple sequence repeat–functional domain marker (SSR-FDM) relies on development of molecular markers for putative functional domains using simple sequence repeats and in silico annotated information of those sequences using biological databases. A total of 148,921 tomato ESTs and 115,598 pepper ESTs were analyzed, resulting in the identification of 439 tomato SSR-FDMs and 489 pepper SSR-FDMs. Among them, 54 pepper SSR-FDMs were tested on pepper. Several genomic databases were used for the in silico annotation of the SSR-FDM sequences that revealed a wide range of candidate genes. This study demonstrates that SSR-FDMs provide information regarding transcribed genetic markers and putative function as a genomic resource database for Solanaceae. This system could be applied to the development of a functional marker database for any crop species.

Map-based cloning to find genes of interest, markerassisted selection (MAS), and marker-assisted breeding (MAB) all require good genetic maps with high reproducible markers. For map construction as well as chromosome assignment, development of single copy PCR-based markers and map integration process are necessary. In this study, the 132 markers (57 STS from BAC-end sequences, 13 STS from RFLP, and 62 SSR) were newly developed as single copy type PCR-based markers. They were used together with 1830 markers previously developed in our lab to construct an integrated map with the Joinmap 3.0 program. This integrated map contained 169 SSR, 354 RFLP, 23 STS from BAC-end sequences, 6 STS from RFLP, 152 AFLP, 51 WRKY, and 99 rRAMP markers on 12 chromosomes. The integrated map contained four genetic maps of two interspecific (Capsicum annuum 'TF68' and C. chinense 'Habanero') and two intraspecific (C. annuum 'CM334' and C. annuum 'Chilsungcho') populations of peppers. This constructed integrated map consisted of 805 markers (map distance of 1858 cM) in interspecific populations and 745 markers (map distance of 1892 cM) in intraspecific populations. The used pepper STS were first developed from end sequences of BAC clones from Capsicum annuum 'CM334'. This integrated map will provide useful information for construction of future pepper genetic maps and for assignment of linkage groups to pepper chromosomes.

A peculiar virus-like disease of tomato showing yellow mosaic and necrotic spots on leaves and necrosis on veins, petioles and stems was observed at the Tomato Experimental Station (TES), Buyeo, Chungcheongnamdo, Korea. The disease incidence at TES fields ranged from 21 to 35% infecting different tomato cultivars. For this reason, to identify the virus infecting tomato and to characterize the virus based on biology, serology, cytology and at molecular level. Here, leaf samples were randomly collected from different infected tomato cultivars at TES fields and greenhouses and tested by ELISA using Pepper mottle virus (PePMoV) and Tomato mosaic virus (ToMV) antisera. Infected saps were mechanically inoculated in different host plants to test for pathogenicity, symptomatology and host ranges. Infected tissues and ultrathin sections were examined by electron microscopy. Finally, putative coat protein and 3`-untranslated region (CP/3`-UTR) fragment was amplified and cloned for sequence determination and analyzed its genetic relationship to existing PepMoV and PVY sequences at the Genbank. Results showed 69% of the samples were positive with PepMoV, 13% with ToMV and 19 % were doubly infected with PepMoV and ToMV. Symptoms greatly varied from different host plants inoculated with tomato leaf sap infected with PepMoV alone and discussed in detailed in this paper. Electron microscopy from infected tissues showed filamentous particles of 720-750nm in length, a typical morphology and size of PepMoV. In addition, cylindrical inclusion bodies, pinwheels, scrolls and laminates with masses of fibrillar inclusions were also found in ultrathin sections. Alignment of the sequences of the CP/3`-UTR revealed >96% sequence identity with PepMoV and only

Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation. To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation. In this procedure, positive transformants could be clearly differentiated from the nontransformed plants. Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV). In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants. These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.

The long range transport of mineral dust such as Yellow Sand (YS) is not restricted to the springtime periods in Northeast Asia. A YS phenomenon was observed during 25∼27 January 1999, which was a remarkably distinctive episode in the occurrence time and intensity that had ever been observed in the wintertime in Korea.

A relationship between pepper trichome and pepper mottle virus (PepMoV) resistance was examined. In an intraspecific F(2) mapping population from the cross between Capsicum annuum CM334 (trichome-bearing and PepMoV resistant) and Chilsungcho (glabrous and PepMoV susceptible), major QTLs for both traits were identified by composite interval mapping in linkage group (LG) 24 corresponding a telomere region on pepper chromosome 10. Ptel1 of putative trichome enhancing locus was a common major QTL for trichome density on the main stem and calyx. Ptel1 apart from HpmsE031 at a 1.03 cM interval was specifically associated to the trichome density on the main stem, whereas Ptel2 near m104 marker on LG2 was specific for the calyx trichome. Epistatic analysis indicated that Ptel1 engaged in controlling the trichome density by mutual interactions with the organ-specific QTLs. For PepMoV resistance, two QTLs (Pep1 and Pep2) were identified on the LG 24. Pep1 was located with Ptel1 in the R-gene cluster (RGC) for potyvirus resistance including Pvr4 with broad spectrum resistance to potyviruses. Pep1 flanking TG420 marker seemed to be the major factors determining correlation with PepMoV resistance. These results indicate that the level of trichome density on pepper main stem can be used as a morphological marker for Pvr4 in pepper breeding.

Trichomes are present on nearly all land plants and protect plants against insect herbivores, drought and UV radiation. The trichome-bearing phenotype is conferred by the dominant allele of the pepper trichome locus 1 (Ptl1) in Capsicum annuum, Mexican 'Criollo de Morelos-334' (CM334). A genetic analysis using simple sequence repeats from pepper cDNA identified the HpmsE031 marker as tightly linked to Ptl1 in 653 individuals of an F(2) population derived from a cross between CM334 and Chilsungcho varieties. A bacterial artificial chromosome (BAC) library from CM334 covering 12x of the genome was screened using the HpmsE031 SSR marker as a probe and three BAC clones were identified. The Ptl1 region was covered by one 80 kb BAC clone, TT1B7. Fluorescence in situ hybridization (FISH) confirmed that TT1B7 localized to pepper chromosome 10. One co-dominant marker, Tco, and one dominant marker, Tsca, were successfully developed from the TT1B7 BAC sequence. Tco mapped 0.33 cM up from Ptl1 and Tsca mapped 0.75 cM down from Ptl1. Analysis of the BAC sequence predicts the presence of 14 open reading frames including 60S ribosomal protein L21-like protein (Solanum demissum), protein kinase 2 (Nicotiana tabacum), hypothetical proteins, and unnamed protein products. These results will provide not only useful information for map-based cloning of Ptl1 in Capsicum but also the starting points for analysis of R-gene cluster inked with Ptl1.

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.

To develop molecular markers linked to the L4 locus conferring resistance to tobamovirus pathotypes in pepper plants, we performed AFLP with 512 primer combinations for susceptible (S pool) and resistant (R pool) DNA bulks against pathotype 1.2 of pepper mild mottle virus. Each bulk was made by pooling the DNA of five homozygous individuals from a T10 population, which was a near-isogenic BC4F2 generation for the L4 locus. A total of 19 primer pairs produced scorable bands in the R pool. Further screening with these primer pairs was done on DNA bulks from T102, a BC10F2 derived from T10 by back crossing. Three AFLP markers were finally selected and designated L4-a, L4-b and L4-c. L4-a and L4-c each underwent one recombination event, whereas no recombination for L4-b was seen in 20 individuals of each DNA bulk. Linkage analysis of these markers in 112 F2 T102 individuals showed that they were each within 2.5 cM of the L4 locus. L4-b was successfully converted into a simple 340-bp SCAR marker, designated L4SC340, which mapped 1.8 cM from the L4 locus in T102 and 0.9 cM in another BC10F2 population, T101. We believe that this newly characterized marker will improve selection of tobamovirus resistance in pepper plants by reducing breeding cost and time.

Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsungcho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to F2 genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.

The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO(3) under a 16-h photoperiod. After 3-4 weeks of culture, 21.9-80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2-3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO(3) were diploid.

Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation. To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation. In this procedure, positive transformants could be clearly differentiated from the nontransformed plants. Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV). In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants. These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.