Ki-Taek Kim

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.

SNP Marker Integration and QTL Analysis of 12 Agronomic and Morphological Traits in F8 RILs of Pepper (Capsicum annuum L.)

Red pepper, Capsicum annuum L., has been attracting geneticists’ and breeders’ attention as one of the important agronomic crops. This study was to integrate 41 SNP markers newly developed from comparative transcriptomes into a previous linkage map, and map 12 agronomic and morphological traits into the integrated map. A total of 39 markers found precise position and were assigned to 13 linkage groups (LGs) as well as the unassigned LGe, leading to total 458 molecular markers present in this genetic map. Linkage mapping was supported by the physical mapping to tomato and potato genomes using BLAST retrieving, revealing at least two-thirds of the markers mapped to the corresponding LGs. A sum of 23 quantitative trait loci from 11 traits was detected using the composite interval mapping algorithm. A consistent interval between a035_1 and a170_1 on LG5 was detected as a main-effect locus among the resistance QTLs to Phytophthora capsici at high-, intermediate- and low-level tests, and interactions between the QTLs for high-level resistance test were found. Considering the epistatic effect, those QTLs could explain up to 98.25% of the phenotype variations of resistance. Moreover, 17 QTLs for another eight traits were found to locate on LG3, 4, and 12 mostly with varying phenotypic contribution. Furthermore, the locus for corolla color was mapped to LG10 as a marker. The integrated map and the QTLs identified would be helpful for current genetics research and crop breeding, especially in the Solanaceae family.

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.

Rapid and practical molecular marker development for rind traits in watermelon

A three-locus model for rind phenotypes in watermelon (Citrullus lanatus) was previously proposed based on genetic analysis. These three loci, S (foreground stripe pattern), D (depth of rind color), and Dgo (background rind color), segregate in a Mendelian manner. Whole genome sequencing of watermelon offers a new strategy for marker development in these rind phenotype-related loci. A genotype analysis using subsets of 188, 273, 287 and 113 probes was performed for the ‘0901’, ‘10909’, ‘109905’ and ‘90509’ rind trait-segregating F2 populations, respectively. A total of 26, 34, 30 and 15 linkage groups with 175, 254, 269 and 79 probes were constructed for the ‘0901’, ‘10909’, ‘109905’ and ‘90509’ populations, respectively. The genetic order of the probes was mostly collinear with the physical order on the reference genome, except for some probes on chromosomes 1, 3 and 11. The three rind-related loci, S, D, and Dgo were anchored near chr6_25767 on chromosome 6, chr8_26061 on chromosome 8 and chr4_150/chr4_249 on chromosome 4, respectively. The three loci are located on different chromosomes, and the three-locus model was therefore verified through molecular genetic analysis. We suggest a rapid and practical marker development strategy that can be used not only for rind traits but also for other agriculturally important traits in watermelon and applied for conventional breeding.

Development of gene-based markers for the Bs2 bacterial spot resistance gene for marker-assisted selection in pepper (Capsicum spp.)

The Bs2 gene of pepper (Capsicum spp.) confers resistance to the most commonly detected races of Xanthomonas campestris pv vesicatora (Xcv). Using information from a published database, we developed gene-based markers for selection of Bs2 in pepper. High polymorphism was detected in exon 3, intron 2 and 3’-UTR of the Bs2 gene. The three regions were sequenced and numerous SNPs and In/Dels were identified between resistant and susceptible inbred lines. Seven SNPs were selected to develop SNP markers using the tetra-primer amplification refractory mutation system-PCR methodology (Tetra-primer ARMS-PCR). One SNP (G/C) was used to develop a molecular marker that can perfectly identify the presence of the bacterial spot resistance. The gene-based markers were further validated to test association with resistance in 80 lines and cultivars. The markers successfully identified a bell pepper commercial cultivar 62-2 that harbors the Bs2 gene. The successful development of the markers in this study provides a useful tool for MAS to screen the Bs2 gene in pepper.