Jung-Heon Han

Construction of an integrated pepper map using RFLP, SSR, CAPS, AFLP, WRKY, rRAMP, and BAC end sequences

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.

Three AFLP markers tightly linked to the genic male sterility ms 3 gene in chili pepper (Capsicum annuum L.) and conversion to a CAPS marker

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 ms3 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 ms3 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.

Functional Domain Marker (FDM): an In Silico Demonstration in Solanaceae Using Simple Sequence Repeats (SSRs)

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.

QTL Analysis of Fruit Length Using rRAMP, WRKY, and AFLP Markers 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.

Isolation and Characterization of Pepper mottle virus Infecting Tomato in Korea

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

Symptom and Resistance of Cultivated and Wild Capsicum Accessions to Tomato Spotted Wilt Virus

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

Isolation of an Rx homolog from C. annuum and the evolution of Rx genes in the Solanaceae family

The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this study, we isolated an RGA (CapRGC) from Capsicum annuum “CM334” using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural–function analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.

Physical and Chemical Properties of a Winter-Time Yellow Sand Event in Northeast Asia

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.This YS event had a bi-modal temporal structure with the daily average concentrations of 210∼349 µg m−3. The long-lasted second one followed the first arriving short and strong dust pulse. The dominant ion components were SO42−, NO3−, Ca2+ and Na+ with the concentration of 11.3, 7.6, 6.1 and 4.2 µg m−3, respectively during the passage of YS, compared to the corresponding concentrations of 4.1, 4.6, 0.4, and 1.2 µg m−3 after the passage of YS (AYS) over Korea. The mode diameter of these compounds of YS was around 4 µm, compared to 0.4∼0.9 µm of AYS. Concentrations of SO42− and NO3− were found to be highly correlated with that of Ca2+ in the coarse mode during the YS event, whereas they were well correlated with NH4+ during the non-YS period.

Specific Primer Sets for RT-PCR Detection of Major RNA Viruses of Tomato Plants in Korea

©The Korean Society of Plant Pathology ccThis is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Article Open Access Res. Plant Dis. 23(2): 193-201 (2017) https://doi.org/10.5423/RPD.2017.23.2.193 식물병연구

Simple and Rapid Detection for Rice stripe virus Using RT-PCR and Porous Ceramic Cubes

A rapid and simple RT-PCR diagnosis method for detection of Rice stripe virus (RSV), one of major virus infecting rice, was developed using porous ceramic cubes in this study. The porous ceramic cube can rapidly absorb biological molecules such as small-sized proteins and nucleic acid fragments into its pores. We examined whether this ability of porous ceramic cubes could be applied for isolating viral nucleic acids or particles from the RSVinfected plant tissues. In this study, we found that the porous ceramic cube was capable of absorbing a detection level of viruses from the rice tissues infected with RSV and established RT-PCR-based RNA diagnosis method using porous ceramic cubes.