Jae Bok Yoon

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.

Identification of QTLs for resistance to anthracnose to two Colletotrichum species in pepper

Pepper (Capsicum spp.) anthracnose caused by Colletotrichum spp. is a serious disease damaging pepper production in Asian monsoon regions. For QTL mapping analyses of anthracnose resistance, an introgression BC1F2 population was made by interspecific crosses between Capsicum annuum ‘SP26’ (susceptible recurrent parent) and Capsicum baccatum ‘PBC81’ (resistant donor). Both green and red fruits were inoculated with C. acutatum ‘KSCa-1’ and C. capsici ‘ThSCc-1’ isolates and the disease reactions were evaluated by disease incidence, true lesion diameter, and overall lesion diameter. On the whole, distribution of anthracnose resistance was skewed toward the resistant parent. It might indicate that one or two major QTLs are present. The introgression map consisting of 13 linkage groups with a total of 218 markers (197 AFLP and 21 SSR), covering a total length of 325 cM was constructed. Composite interval mapping analysis revealed four QTLs for resistance to ‘KSCa-1’ and three QTLs for ‘ThSCc-1’ isolate, respectively. Interestingly, the major QTLs (CaR12.2 and CcR9) for resistance to C. acutatum and C. capsici, respectively, were differently positioned but there were close links between the minor QTL CcR12.2 for C. capsici and major QTL CaR12.2 as well as the minor QTL CaR9 for C. acutatum and major QTL CcR9. These results will be helpful for marker-assisted selection and pyramiding two different anthracnose-resistant genes in commercial pepper breeding.

A CAPS marker associated with the partial restoration of cytoplasmic male sterility in chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS), an economically important trait for hybrid seed production in many crops, is a maternally inherited trait in which a plant fails to produce functional anthers, pollen grains, or male gametes. It has long been reported that the restoration of CMS in chili pepper is controlled by a major nuclear gene termed restorer-of-fertility (Rf), along with several modifiers and some environmental factors. In this study, we identified the partial restoration (pr) locus related to the fertility restoration of CMS, demonstrated the inheritance of the trait, and developed a CAPS marker closely linked to the locus. The partially restored plant had normal anthers that produced a mix of normal and aborted pollen grains that stuck tightly to the anther wall, even after dehiscence. This trait was expressed only when the pepper plant had the sterile (S) cytoplasm and homozygous recessive pr alleles. A total of 768 AFLP primer combinations were screened, and bulked segregant analysis (BSA) was performed by preparing two pools of eight Pr/Pr (fully fertile) and eight pr/pr (partially fertile) plants, respectively, selected from the 87 individuals of the F2 segregating population. Of the eight Pr-linked AFLP markers that were identified, E-AGC/M-GCA122 and E-TCT/M-CCG116 were the closest to the locus, estimated at about 1.8 cM in genetic distance. E-AGC/M-GCA122 was converted into a CAPS marker, PR-CAPS, based on the sequences of the internal and flanking regions of the AFLP fragment. This PR-CAPS marker could be useful in selecting fully fertile lines (Pr/Pr) and eliminating partially fertile (pr/pr) and potential (Pr/pr) lines in segregant populations during the development of new inbred restorer lines.

Development of STS markers linked to the major QTLs for resistance to the pepper anthracnose caused by Colletotrichum acutatum and C. capsici

Anthracnose (Colletotrichum spp.) causes significant yield losses in chili pepper (Capsicum annuum L.), and several loci conferring resistance to this disease have been identified. Seven and one amplified fragment length polymorphic (AFLP) markers were additionally located on LG 12 (CaR12.2, major locus resistant to Colletotrichum acutatum) and LG 9 (CcR9, major locus resistant to C. capsici), respectively, through AFLP analysis combined with extreme bulked segregant analysis (BSA). Among these, two AFLP markers, EtagMcgt04 (CaR12.2) and EtacMccg13 (CcR9), were converted into sequence tagged site (STS) markers (CaR12.2M1-CAPS and CcR9M1-SCAR, respectively), via sequencing analysis of internal and flanking regions of each AFLP marker. The selection efficiencies were 72% for CaR12.2M1-CAPS and 82.5% for CcR9M1-SCAR. These simple PCR-based markers will be useful for breeding cultivars with enhanced resistance to anthracnose, for pyramiding resistances to both C. acutatum and C. capsici, and for further characterization of the locus, including isolation of genes responsible for resistance.

QTL mapping of resistance to the Cucumber mosaic virus P1 strain in pepper using a genotyping-by-sequencing analysis

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 R2 (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.

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