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Hot Pepper Genome : Basic Genetic Tools for Molecular Breeding
Kang,Byoung Cheorl,Nahm,Seok Hyeon,Huh,Jin Hoe,Lee,Je Min,Lee,Moon Hwan,김병동 한국생명과학회 2000 한국생명과학회 학술발표회 Vol.29 No.-
Pepper fruits are consumed as food additives for their unique color, pungency, and aroma in many regions of the world, particularly in Asia and South and Central America. Five species of Capsicum peppers, including C. annuum, C. chinense, C. baccatum, C. frutescens and C. pubescens, are cultivated in different parts of the world. Among them C. annuum is most widely grown in both Asia and worldwide. It includes most of the Mexican chile peppers, most of the hot peppers of Africa and Asia, and various cultivars of sweet peppers grown in temperate regions of Europe and North America. During the last decade, the construction of molecular linkage map has become an essential tool for plant molecular genetics and breeding research. Despite the pepper genome research is being conducted by only a small number of research groups worldwide, development of a linkage map in Capsicum has been greatly aided by use of tomato-derived RFLP probes, We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206 - 60.3 cM) and 5 small (32.6 - 10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones and these markers were evenly distributed across the genome. By using 30 primer combinations, 444 AFLP markers were generated in the F2 population. The majority of the AFLP markers clustered in each linkage group, although PstI/Msel markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. We have developed SSR markers and these markers were informative for pepper genome research. Genes for biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper.
Molecular Mapping of The Tsw Locus for Resistance to Tomato spotted wilt virus (TSWV) in Capsicum
Byoung-Cheorl Kang,Ngoc Huy Hoang,Hee-Bum Yang,Bong Nam Chung 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07
Tsw, a single dominant resistant gene against Tomato spotted wilt virus (TSWV), has been mapped on chromosome 10 in Capsicum. Previously found molecular markers linked to the Tsw gene are not transferable for all pepper breeding materials. To develop segregating populations for the Tsw, commercial F1 cultivar C. annuum ‘Telmo’ was self-pollinated. An F2 population was obtained from the self-pollination of F1 plants deriving from a cross between C. annuum ‘Special’ and C. chinense ‘PI152225’. Twelve additional molecular markers linked to the Tsw gene were developed using tomato and pepper genome sequence database. A tomato scaffold sequence of 7841 kb in size covering the corresponding region of the Tsw locus was identified based on the sequence of Tsw-linked marker. Analyzing the tomato scaffold sequence, two sequences of pepper scaffold and contig at down and up site of the Tsw locus, respectively, were located. Three SNP markers linked to the Tsw locus (HRM1, HRM2, and HRM3) were developed using the pepper scaffold sequence of 419 kb in size. All three markers showed 2 recombinants (1.0 cM) out of 198 individuals of F2 ‘Telmo’ population. When analyzing these SNP markers in an F2 population deriving from C. annuum ‘Special’ and C. chinense ‘PI152225’, we detected 5 recombinants (0.76 cM) out of 659 individuals. HRM4, a SNP marker linked to the Tsw gene, was developed with a 99 kb pepper contig sequence. It showed 7 recombinants (3.5 cM) out of 198 individuals of F2 ‘Telmo’ population. We found 5 recombinants (0.76 cM) out of 659 individuals when HRM4 was analyzed in F2 population derived from C. annuum ‘Special’ and C. chinense ‘PI152225’. To narrow down the molecular markers linked to the Tsw locus, four SNP markers, HRM5, HRM6, HRM7, and HRM8, were developed with the pepper scaffold sequence. All of them showed 5 recombinants (0.76 cM) out of 659 individuals of F2 ‘SP’ population. Four other SNP markers, HRM9, HRM10, HRM11, and HRM12, were developed using the pepper contig sequence. HRM9 showed 5 recombinants (0.76 cM), HRM10 showed 4 recombinants (0.61 cM), and HRM11 and HRM12 showed 3 recombinants (0.46 cM) out of 659 individuals of F2 ‘SP’ population. The SNP markers developed in this study will be useful for fine mapping of the Tsw gene and for developing cultivars which carry TSWV-resistance gene.
Genome-based fine mapping of the Tomato spotted wilt virus resistance gene, Tsw, in Capsicum
Byoung-Cheorl Kang,Ngoc Huy Hoang,Hee-Bum Yang,Won-Hee Kang,Bong Nam Chung 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07
Tsw, a single dominant resistant gene against Tomato spotted wilt virus (TSWV), has been mapped on chromosome 10 in Capsicum chinense species. Previously reported molecular markers linked to the Tsw gene are not transferable for all pepper breeding materials. To develop additional markers and do genome-based fine mapping of the Tsw gene, approaches of mapping comparison, pooled transcriptome analysis, and genome walking were applied. Eleven additional SNP molecular markers tightly linked to the Tsw gene were developed using tomato and pepper whole genome sequencing databases. Among them, four SNP markers, SNP7715-1, SNP68-1, SNP17918-1, and SNP1072-1, showed no recombination in two segregating populations of F2 ‘Telmo’ (210 individuals) and ‘SP’ (843 individuals). Three scaffold sequences from the C. annuum BAC database and two BAC clones from the BAC library of C. annuum ‘CM334’ covering the Tsw gene were identified by transcriptome analysis and genome walking. A pepper scaffold sequence covering three pepper scaffold sequences was identified from a final version of the C. annuum BAC database. The Tsw gene was delimited within 149 kb by alignment analysis of two BAC clone sequences and the pepper scaffold sequence. A total of 22 predicted genes were resided in the target region between SNP7715-1 and SNP1072-1 co-segregating markers. Among them, five predicted genes showing annotations of CC/TIR-NBS-LRR resistance proteins, mRNA-6, mRNA-7, mRNA-11, mRNA-12, and mRNA-13, were identified. The transcriptome analysis and gene expression study showed that the mRNA-13 was expressed in ‘PI152225’ but was absent in ‘Special’, demonstrating the mRNA-13 could be a strong candidate gene for the Tsw gene. This result will be favorable for cloning the Tsw gene and developing cultivars which carry the TSWV-resistance gene.
Genome-assisted marker development for disease resistance in pepper
Byoung-Cheorl Kang 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
The last decade has witnessed tremendous progress in genome sequencing and explosion of genome sequence information. This remarkable advancement in genomics provides unprecedented opportunities for crop improvement. Pepper (Capsicum spp.) is an important vegetable crop worldwide. Pepper production is constantly challenged by various pathogens and developing cultivars harnessing multiple disease resistance genes are ever increasing. Molecular markers linked to disease resistance genes will expedite the gene pyramiding. Here, I introduce genome-assisted development of molecular markers linked to resistance genes, in pepper. Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of Capsicum annuum against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F2 population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 x 103 /mL). Resistance phenotype segregation ratios for the populations were close to 3:1 and 1:1 (resistant:susceptible), respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 were used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to Phytophthora resistance. Markers linked to these genes were developed and validated by testing 100 F1 commercial cultivars. Among the markers, Phyto5NBS1 showed about 90% accuracy in predicting resistance phenotypes to a low-virulence Phytophthora isolate. These results suggest that Phyto5NBS1 is a reliable marker for Phytophthora resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5