A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). Six T1 selections were free of deleterious traits, consistently high expressers of GFP, and produced fertile pollen.
Rose E. Palumbo and Richard E. Veilleux*
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.
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.
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.