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Ivan Simko and Jinguo Hu

The association mapping technique is a useful tool for detecting markers linked to the genes underlying the variation of a trait among elite cultivars. To avoid false-positive results due to unrecognized population structure in the analyzed set of individuals, the subpopulations need to be identified. Fifty-four lettuce (Lactuca sativa L.) cultivars representing five horticultural types important in North America, together with six accessions from two wild species (Lactuca saligna L. and Lactuca serriola L.), were assayed for polymorphism with target region amplified polymorphism (TRAP) marker loci. The model-based clustering approach recognized three main subpopulations in cultivated lettuce that are well separated from wild species. Although the clustering based on molecular markers was generally in good agreement with horticultural types, some cultivars were classified differently or showed mixed origin. The effect of population structure on association mapping was tested on four traits with strong or weak correlation to the lettuce horticultural type and monogenic or polygenic mode of inheritance. Traits that were strongly correlated with lettuce types displayed many false-positive results when population structure was ignored, but the spurious associations disappeared when estimates of population structure or relative kinship (both based on molecular markers) were included in the statistical model. Using of horticultural types as covariate was not sufficient to control for spurious associations in the monogenic trait with strong correlation to lettuce types. The best approach to avoid spurious associations in lettuce association studies is to assess relatedness of accessions with molecular markers and to include this information into the statistical model.

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Jinguo Hu, Beiquan Mou and Brady A. Vick

Target region amplified polymorphism (TRAP) markers were used to evaluate genetic variability among 48 accessions of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Thirty-eight accessions collected and preserved by the USDA National Plant Germplasm System (NPGS) and 10 commercial hybrids were used in the current study. For assessing genetic diversity within accessions, DNA samples were prepared from nine to 12 individual seedlings from six germplasm accessions and two hybrids. Relatively high levels of polymorphism was found within accessions based on 61 polymorphic TRAP markers generated with two fixed primers derived from the Arabidopsis-type telomere repeat sequence and two arbitrary primers. For evaluating inter-accession variability, DNA was extracted from a bulk of six to 10 seedlings of each accession. Of the 1092 fragments amplified by 14 primer combinations, 96 (8.8%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice, Nei) was 57.5% with a range from 23.2 to 85.3%. A dendrogram was constructed based on the similarity matrix. It was found that the genetic relationships were not highly correlated with the geographic locations in which the accessions were collected. However, seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities have effect on the genetic variability. This study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability of spinach germplasm.

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Zhanao Deng, Fahrettin Goktepe, Brent K. Harbaugh and Jinguo Hu

Caladium (Caladium ×hortulanum Birdsey) is an important aroid widely used in the ornamental plant industry. Concerns have been raised about possible loss of genetic diversity due to a drastic decline in the number of cultivars in the last century. This study assessed genetic diversity and relationships among caladium cultivars and species accessions. Forty-five major cultivars and 14 species accessions were analyzed based on 297 DNA fragments produced by the target-region amplification polymorphism marker system. A low level of diversity (44.4% polymorphism) was exhibited in cultivars, while a high level of diversity (96.8% polymorphism) was present among seven accessions of Caladium bicolor (Aiton) Vent., Caladium marmoratum Mathieu, Caladium picturatum C. Koch, and Caladium schomburgkii Schott. A small percentage (7.6%) of DNA fragments was present in cultivars but absent in the seven species accessions, while a high percentage (32.2%) of DNA fragments was present in the seven species accessions but absent in cultivars. Cultivars shared a higher level of similarity at the molecular level with an average Jaccard coefficient at 0.802, formed a large group in cluster analysis, and concentrated in the scatter plot from a principal-coordinate analysis. Two accessions of C. bicolor and C. schomburgkii were very similar to cultivars with Jaccard similarity coefficients from 0.531 to 0.771, while the rest of the species accessions had small similarity coefficients with cultivars (0.060 to 0.386). Caladium steudnirifolium Engler and Caladium lindenii (André) Madison were very dissimilar to C. bicolor, C. marmoratum, C. picturatum, and C. schomburgkii, with Jaccard similarity coefficients from 0.149 to 0.237 (C. steudnirifolium) and from 0.060 to 0.118 (C. lindenii). There is a limited amount of molecular diversity in caladium cultivars, but the great repertoire of unique genes in species accessions could be used to enhance the diversity in future cultivars and reduce potential genetic vulnerability.

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Rose Palumbo, Wai-Foong Hong, Jinguo Hu, Charles Krause, David Tay and Guo-Liang Wang

The Ornamental Plant Germplasm Center (OPGC) maintains a collection of herbaceous ornamental plants in order to protect future breeders from a loss of genetic diversity. The current Pelargonium collection includes ≈870 accessions. Our preliminary studies showed that TRAP (Target Region Amplified Polymorphism) has promise for analyzing the variation in our collection, and so we have expanded the study to analyze the entire Pelargonium collection. We have used the same primers for this screening of the Pelargonium collection as were used on sunflowers, and TRAP results run on a sequencing gel showed 90–150 bands that segregate the population into groups of similar accessions. In order to facilitate analysis of OPGC's large population, we have converted the method to a high throughput technique that efficiently analyzed the entire population. We used a 96-well DNA extraction kit from Qiagen that produced high quality DNA in spite of the high phenol levels in some Pelargonium species. Also, the use of labeled primers allowed analysis of the gels to be aided by a computer. These results produce a categorization of the collection that, combined with morphology and taxonomy, will form the basis for future studies that will use target genes specific to Pelargonium.

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Zhanao Deng, Jinguo Hu, Fahrettin Goktepe, Brady A. Vick and Brent K. Harbaugh

Cultivated caladiums are valued for their bright colorful leaves and are widely used in containers and landscapes. More than 1500 named cultivars have been introduced during the past 150 years, yet currently only about 100 cultivars are in commercial propagation in Florida. Caladium tubers produced in Florida account for 95% of the world supplies. Loss of caladium germplasm or genetic diversity has been a concern to future improvement of this plant. In addition, the relationship among the available cultivars, particularly those of close resemblance, has been lacking. This study was conducted to assess the genetic variability and relationship in commercial cultivars and species accessions. Fifty-seven major cultivars and 15 caladium species accessions were analyzed using the target region amplification polymorphism marker technique. This marker system does not involve DNA restriction or adaptor linking, but shares the same high throughput and reliability with the amplified fragment length polymorphism system (AFLP). Eight primer combinations amplified 379 scorable DNA fragments among the caladium samples. A high level of polymorphism was detected among the species accessions as well as among cultivars. These markers allowed differentiation of all the cultivars tested, including those hardly distinguishable morphologically. Clustering analysis based on these DNA fingerprints separated the cultivars into five clusters and Caladium lindenii far from other caladium species. The availability of this information will be very valuable for identifying and maintaining the core germplasm resources and will aid in selecting breeding parents for further improvement.

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Wai-Foong Hong, Chang-Qing Bai, Michael Broe, Jinguo Hu, Charles Krause, David Tay* and Guo-Liang Wang

Pelargonium is one of the important flower crops in USA. It is a priority genus for conservation at the USDA Ornamental Plant Germplasm Center (OPGC). It belongs to Geraniaceae family and comprises of about 280 species. To understand the genetic variation of the Pelargonium collection at OPGC, the PCR-based TRAP (target region amplified polymorphism) marker system which was newly developed in sunflower was used in this study. Twelve sets of primers were used to fingerprint 46 accessions representing 21 commercial P. hortorum, 17 scented geraniums and 8 other unidentified Pelargonium taxa. About 150 DNA bands could be detected in each primer and accession combination. Cluster analysis showed that molecular data was highly correlated with the phenotypes. Cultivars with similar morphological traits were clustered together. These results demonstrated that the TRAP system is a useful technique for the characterization and classification of Pelargonium collections.

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Rose E. Palumbo, Wai-Foong Hong, Jinguo Hu, Charles Krause, James Locke, Richard Craig, David Tay and Guo-Liang Wang

Pelargonium is one of the priority genera collected by the Ornamental Plant Germplasm Center (OPGC). In order to protect future breeders from a loss of genetic diversity, the OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Our project was designed to analyze the current collection in order to facilitate the maintenance of a more-diverse core collection. We have expanded our TRAP (Target Region Amplified Polymorphism) analysis from 120 plants with one primer set to include 780 plants with four primer sets. Each primer set consists of a labeled arbitrary primer paired with a gene-specific primer, and two different fluorescent labels were used to allow multiplexed PCR reactions. We scored about 90 markers in each of the first two primer sets and about 60 markers in each of the second two. In comparisons between the phylogeny and the morphology and taxonomy of these plants, we show some matching clusters that may be explained by the breeding history of the plants.

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Rose Palumbo, Wai-Foong Hong, Guo-Liang Wang, Jinguo Hu, Richard Craig, James Locke, Charles Krause and David Tay

Pelargonium was a priority genera collected by the Ornamental Plant Germplasm Center (OPGC) until a recent reorganization. To preserve genetic diversity for future breeders, OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection at OPGC consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Target region amplification polymorphism (TRAP) has the advantage of producing a large number of markers through use of sequence information that is already available. Our first goal was to determine the feasibility of TRAP for the analysis of this large collection, so that in the future the most diverse genotypes may be retained. To achieve this goal, we first modified existing DNA extraction techniques to account for the high levels of phenolic compounds present in some Pelargonium species by combining several washes to remove the phenolics with the addition of high levels of antiphenolic compounds. Second, we evaluated the TRAP procedure using the DNA isolated from 46 accessions. For 44 accessions, one or two primer combinations generated enough fragments to discriminate each of the accessions, and similar clades were produced by cluster analysis of the polymorphic fragments amplified by different primer combinations. All the scorable fragments were polymorphic, for one primer combination there were 148 markers from one image and the other produced 160 markers on two images. These results demonstrate that TRAP is an effective method for molecular characterization of ornamental collections.