Sung-Chur Sim

Development of genomic SSR markers and genetic diversity analysis in cultivated radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is a major vegetable cultivated worldwide. As a member of the family Brassicaceae, this species has diverse morphological characteristics in root. Radish cultivars have been classified based on morphological traits, including root shape and color. Despite its economic importance in Asia, genomic research in radish is less well developed relative to Brassica rapa, a close relative of radish. In this study, we developed genomic simple sequence repeat (SSR) markers using a SSR-enriched library and investigated genetic diversity in a collection of 144 radish cultivars. A total of 237 primer pairs for SSRs were designed and 184 (77.6%) primer pairs produced PCR amplicons. Of these, we selected 27 SSR markers (14.7%) based on polymorphism in a subset of 11 cultivars and then used to assess genetic relationships in the germplasm panel. For these markers, the number of alleles per marker ranged from 2 to 18 with an average of 7.77 alleles and the polymorphism information content (PIC) values ranged from 0.491 to 0.906. The estimates of pairwise Fst revealed significant genetic differentiation between the five market classes of 135 radish cultivars (74 big, 15 small, 29 young, 12 Altari, and 5 processing). Clustering analysis using NTSYS-pc and STRUCTURE software also found that the 74 big radishes were divided into 5–7 clusters. In addition, all 27 SSR markers were able to differentiate 64 big radish cultivars based on the UPGMA dendrogram and each of the 23 markers independently identified 1 to 17 big radish cultivars. These results suggest that cultivated radishes have different patterns of genetic variation and breeding practices should be a driving force for the genetic differentiation between and within market classes. The SSR markers developed in this study will be a useful resource for genetic study and protection of plant breeder’s intellectual property right through cultivar identification.

Assessing the genetic variation in cultivated tomatoes ( Solanum lycopersicum L.) using genome-wide single nucleotide polymorphisms

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop worldwide. Recently, a high-density single nucleotide polymorphism (SNP) array was developed based on genome-wide SNPs in tomato. In this study, we genotyped a collection of 48 Korean elite tomato varieties (26 fresh market and 22 cultivated cherry) using 7,720 SNPs of this array. Out of 6,652 polymorphic SNPs (86.1%) in the entire collection, there were 6,589 SNPs with < 10% missing data. The number of polymorphic SNPs in the fresh market and cultivated cherry subpopulations were 4,733 (61.3%) and 6,087 (78.8%), respectively. To examine the genetic variation between sub-populations, the SNP genotypes of the Korean tomato germplasm were analyzed along with the previously reported data on SNPs of the 277 Solanaceae Agricultural Coordinated Project (SolCAP) varieties (109 fresh market, 27 cultivated cherry, and 141 processing). Principal component analysis, pairwise Fst, and Nei’s standard genetic distance revealed genetic differentiation between these five sub-populations. Moreover, we validated another division within the Korean cherry varieties using the unweighted pair group mean algorithm (UPGMA). The genetic diversity of each sub-population was estimated based on allelic richness and expected heterozygosity. The fresh market and cultivated cherry sub-populations in the Korean tomato germplasm showed similar levels of genetic diversity as the corresponding SolCAP sub-populations. Visualization of the polymorphic information revealed genomic regions that differed between the two sub-populations in the Korean tomato germplasm. These results suggest that diversifying selection for market niches and environmental adaptation has led to allelic variation in cultivated tomatoes in Korea.

DNA profiling of commercial pumpkin cultivars using simple sequence repeat polymorphisms

Pumpkin (Cucurbita spp.) is a major vegetable crop grown worldwide. Three species, C. pepo, C. moschata, and C. maxima, are economically important cultivated pumpkins. To develop a core set of markers for DNA profiling and cultivar identification, we used a total of 300 SSRs consisting of 158 CMTp and 142 CMTm that were previously identified in C. pepo and C. moshata, respectively. Polymorphisms in these primers were tested using a subset of 22 cultivars selected from a collection of 160 commercial cultivars. A total of 12 CMTp and 28 CMTm markers were selected based on polymorphism and number of alleles, and these 40 markers were used to genotype all 160 cultivars. Of these, 29 markers (5 CMTp and 24 CMTm) accurately detected a total of 215 alleles with an average of 7.41 alleles per marker in our collection of pumpkin cultivars. Their PIC values ranged from 0.327 to 0.894 with an average of 0.674. Analysis of genetic similarity using the 29 SSR markers revealed that the 160 cultivars were divided into five major clusters representing C. maxima×C. moshata hybrids (cluster I), C. moshata (cluster II), C. maxima (cluster III), C. pepo (cluster IV), and C. ficifolia (cluster V). In clusters I-IV, the cultivars were further separated into 2-3 sub-clusters. In addition, we found that 29 SSR markers were able to differentiate all 160 cultivars. Results from our study will facilitate genetic study and protection of breeders’ intellectual property rights in pumpkins.

Genetic variation of flower characteristics in a population derived from a cross between the chrysanthemum cultivars ‘Falcao’ and ‘Frill Green’

Chrysanthemum is a major floriculture species and a breeding goal is to develop cultivars with unique flower forms and colors. This study was conducted to investigate the genetic basis of six flower traits including petal color, flower shape, ray floret length, the level of ray floret twisting, the shape of the ray floret tips, and the attitude of the basal part of the ray florets. A pseudo-testcross population derived from a cross between the ‘Falcao’ and ‘Frill Green’ cultivars was used. The female parent ‘Falcao’ is a popular pompon type that has an orange-pink color and lacks twisted ray florets. The male parent ‘Frill Green’ is a single type with green twisted ray florets. For color, six different colors were observed in 130 progeny: white, yellow, green, orange, orange-pink, and pink and 52 (40%) and 52 (40%) progeny showed green and yellow colors, respectively. The progeny was grouped into 4 classes based on flower form. The semi-double class was the most frequent (70%) in the population, suggesting an incomplete dominance for the double and single flower. Among four ray floret traits, the shape of the ray floret tips seems to be controlled by a single gene, while the level of twisting of the ray floret showed a significant deviation from the expected 3:1 or 1:1 ratio (p < 0.05). The progeny were divided into three classes in terms of the attitude of the basal part of the ray florets (ascending, descending, and horizontal). Four and three progeny for green pompon or green double flowers were selected, respectively. Two of the four green pompon selections have twisted ray florets. These results will help breeders for developing new chrysanthemum cultivars with unique flower forms and colors.

Comparative genome analysis between Agrostis stolonifera and members of the Pooideae subfamily, including Brachypodium distachyon.

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.

Development of SCAR Marker for the Detection of QTL for Resistance to Dollar Spot in Creeping Bentgrass (Agrostis stolonifera L.)

Dollar spot (caused by Sclerotinia homoeocarpa F. T. Bennett) is the most economically important fungal turf disease on golf courses in North America. This disease is mainly controlled by using multiple fungicides. However, the causal fungus has developed resistance to seveal classes of fungicides and host resistance is an alternative control strategy. A creeping bentgrass (Agrostis stolonifera L.) cultivar improved with dollar spot resistance would greatly reduce the costs and environmental impacts of fungicide applications. Our previous QTL analysis in the creeping bentgrass ‘549 × 372’ population detected one QTL with a large effect for dollar spot resistance and several QTLs with smaller effects. One RAPD primer, AW10, amplified an 650-bp band in the parent ‘372’, was named 3.AW10.650. This RAPD marker was located on linkage group 7.1 and was significantly (p < 0.0001) associated with the QTL for dollar spot resistance based on 697 progeny of the ‘549 × 372’ population. In this study, we converted the RAPD marker 3.AW10.650 into a sequence characterized amplified region (SCAR) marker, named CreepDSp.650. The SCAR marker showed a significant association (p = 0.006) with dollar spot resistance in an additional segregating population developed at the University of Illinois. We report here the first PCR-based SCAR marker associated with a major-effect QTL for dollar spot resistance in creeping bentgrass. This SCAR marker will facilitate evaluating relatively large numbers of breeding progeny for marker-assisted selection (MAS) in bentgrasses.