The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei. Ploidy level and DNA content were significantly correlated within the genus, but more highly so within species that contained multiple cytotypes. DNA content of cultivated Z. batatas (L.) Lam. (4.8 to 5.3 pg/2C nucleus) and feral tetraploid I. batatas (3.0 to 3.5 pg/2C nucleus) was estimated from the known DNA content of chicken erythrocytes (2.33 pg), which were used as an internal standard. Tetraploid forms of Z. cordato-triloba Dennstedt also were identified. Ploidy analysis using flow cytometry is rapid and suitable for large-scale experiments such as studying the genetic structure of populations of Z. batatas and related species. Chemical name used: 4′,6-diamidino-2-phenylindole (DAPI).
Peggy Ozias-Akins and Robert L. Jarret
Ruwanthi C. Wettasinghe and Ellen B. Peffley
Random amplified polymorphic DNA (RAPD) have potential as genetic markers that may facilitate selection in plant improvement. To obtain clear, reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared. DNA extraction followed modified Tai and Tanksley (PMBR), Dellaporta et al. (PMBR), and Guilllemant et al. (PMBR) protocols, and a plant tissue DNA isolation kit from Gentra Systems was used. The modified Guillemant protocol was selected because of ease of extraction and cost effectiveness. Genotypes studied were TG1015Y (Allium cepa). Polymerases compared were Taq and Taq Stoffel fragment. Results are based on separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.
Ruwanthi C. Wettashinghe and Ellen B. Peffley
Random amplified polymorphic DNA (RAPD) are genetic markers that facilitate selection in plant breeding. To obtain clear reproducible, and repeatable RAPD bands, four DNA extraction protocols and two Taq polymerases were compared using thirteen TG1015Y (Allium cepa) genotypes. Protocols for DNA extraction followed those of a modified Tai and Tanksley, 1989 (PMBR); a modified Dellaporta et al., 1983 (PMBR); a modified Guillemunt et al., 1992 (PMBR); and extracted with a plant tissue DNA isolation kit from Gentra System (Minneapolis). The modified Guillemunt protocol was selected due to ease of extraction and cost effectiveness. Polymerases compared were Taq and Taq Stoffel fragment. Results are based on three separate amplifications and electrophoretic assays. PCR amplifications of Stoffel fragment produced more scorable and reproducible RAPD bands compared to bands produced using Taq polymerase.
Sriyani Rajapakse, Mark Hubbard, Albert Abbott, John Kelly, and Robert Ballard
Restricted Fragment Length Polymorphisms (RFLPs) were investigated in closely and distantly related rose cultivars as means of identifying those cultivars for the purpose of patent protection. A random genomic DNA library was constructed using the cultivar `Confection' and the Escherichia Coli strain JM83 plasmid vector pUC8. Clones with interspersed repeat sequences were then identified by hybridizing restriction digested cloned DNA fragments with nick translated genomic DNA of the rose cultivar `Confection'. Hybridization positive clones were screened for polymorphism by Southern hybridization on restriction digested genomic DNA of various rose cultivars. About 75% of the interspersed repeat copy probes screened revealed polymorphisms. We have identified probes that give fingerprint patterns for rose cultivars. From this information, a dichotomous key which differentiates the rose cultivars examined was prepared. Current research involves screening more probes and rose cultivars for polymorphisms, and examining single copy probes for potential use in RFLP genetic linkage map construction in roses.
Donglin Zhang, Michael A. Dirr, and Robert A. Price
The correct identification of horticultural taxa becomes more and more important for intellectual property protection and economic reasons. Traditionally, morphological characteristics have been used to differentiate among the horticultural taxa. However, the morphological characteristics may vary with plant age, cultural conditions, and climate. Modern technologies, such as DNA markers, are now employed in the identification of horticultural taxa. Currently, technologies of DNA sequencing (gene sequences) and DNA fingerprinting (RAPD, RFLP, SSR, and AFLP) are available for distinguishing among horticultural taxa. The literature and our personal experience indicate that the application of each technique depends on the taxon and ultimate goal for the research. DNA sequencing of a variety of nuclear or chloroplast encoded genes or intergenic spacers (rbcL, ndhF, matK, ITS) can be applied to distinguish different species. All DNA fingerprinting technologies can be used to classify infraspecies taxa. AFLP (the most modern technique) is the better and more-reliable to identify taxa subordinate to the species, while RAPDs can be employed in clonal or individual identification. Techniques of RFLP and SSR lie between AFLP and RAPD in their effectiveness to delineate taxa. Mechanics, laboratory procedures, and inherent difficulties of each technique will be briefly discussed. Application of the above technologies to the classification of Cephalo taxus will be discussed in concert with the morphological and horticultural characteristics. Future classification and identification of horticultural taxa should combine DNA technology and standard morphological markers.
Sweetpotato, Ipomoea batatas is in the morning glory family, Convolvulaceae, genus Ipomoea, group Batatas. It has many wild Ipomoea relatives that serve as a reservoir of many needed pest and stress-resistance genes. A major barrier to introgression of useful genes is the ploidy gap—sweetpotato is a hexaploid and wild Ipomoeas are diploids and tetraploids. The wild species can be successfully crossed using 2n pollen or by first increasing ploidy by colchicine treatment. The ploidy of such hybrid offpsring can be determined by DNA flow cytometry. My objective was to develop a technique to determine DNA content in Ipomoea and values for DNA content for the major Ipomoea species using the EPIC flow cytometer with a UV detector. Nuclei were extracted and pretreated with cellulase and pectolyase before staining with propidium iodide (PI). A highly linear relationship was found between the DNA content determined by DNA flow cytometry and the ploidy of the closest sweetpotato relatives as determined by chromosome counts. These species were diploid I. trifida, tetraploid I. batatas, and hexaploid I. batatas. DNA content was most similar among other diploid Ipomoea species in the group Batatas and was significantly different in other Ipomoeas not in group Batatas.
A. Virginia Freire, John E. Preece, and David A. Lightfoot
Silver maple has great potential as a biomass feedstock. We compared three clones from each of seven provenances located on east to west and north to south transects across the natural range of silver maple and one red maple. DNA extracted by a modification of the CTAB technique (Murray and Thompson, 1980) was not suitable for RAPD analysis. Using this technique, polymorphism was either not reproducible or there was poor amplification for some clones. A new DNA extraction technique using PVPP, chloroform, and cesium chloride was tested (a modification of Yoon et al., 1991). this method yielded DNA that was more suitable for PCR amplification. Both RAPD and DAF (Caetano-Anolles and Gresshoff, 1994) methods were used for amplification. Polymorphism was detected among and within provenances. DAF was more efficient than RAPDs for determination of the genetic relationship among silver maple clones.
Jean-Guy Parent and Danièl Pagé
Characterization and identification of 13 red raspberry (Rubus idaeus L.) and two purple raspberry (R. × neglectus Peck) cultivars were obtained by nonradioactive genetic fingerprinting. DNA from leaves was digested with Hae III and Hin f I restriction enzymes and probed with alkaline phosphatase-labeled oligonucleotide. All tested cultivars could be identified by a unique band pattern. No differences were noted within cultivars when the reproducibility of the fingerprints was evaluated by analyzing the effects of age of the raspberry plantation, developmental stage during the growing season, or position of the sampled leaf on stem. These results suggest that simple nonradioactive DNA fingerprinting can be routinely used to identify raspberry cultivars.
M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano
DNA amplification fingerprinting (DAF) was used to study genetic relationships between closely related chrysanthemum cultivars (Dendranthema grandiflora Tzvelev.). Twenty-one cultivars were examined that belonged to the Anne, Blush, Boaldi, Charm, Davis, and Pomona series (families). The genetic variability of cultivars within and between series was evaluated using eleven arbitrary octamer primers. A few polymorphic characters uniquely identified closely related cultivars within each of the series. In contrast, many DNA polymorphisms were observed between members of the different series. Phenetic patterns were established by unweighted pair group cluster analysis using arithmetic means (UPGMA) and principal coordinate analysis (PCO). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series were also bulked to generate profiles containing unique amplified products for each series. Cluster analysis and PCO of bulked DNA clearly grouped Charm and Pomona together. However, series grouping did not correspond to morphology of inflorescence types. The results demonstrate the utility of the DAF technique in distinguishing clonal materials and its potential use for patent protection, phylogenetic studies, and for identifying useful markers in breeding applications.
Jinggui Fang, Chih Cheng Chao, Richard J. Henny, and Jianjun Chen
Plant tissue culture can induce a variety of genetic and epigenetic changes in regenerated plantlets, a phenomenon known as somaclonal variation. Such variation has been widely used in the ornamental foliage plant industry as a source for selection of new cultivars. In ornamental aroids alone, at least 63 somaclonal-derived cultivars have been released. In addition to morphological differences, many somaclonal aroid cultivars can be distinguished by amplified fragment length polymorphism (AFLP) analysis. However, a few cultivars have no detectable polymorphisms with their parents or close relatives by AFLP fingerprints. It is postulated that DNA methylation may be involved in the morphological changes of these cultivars. In this study, methylation-sensitive amplification polymorphism (MSAP) technique was used to study DNA methylation in selected somaclonal cultivars of Alocasia, Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium. Results showed that polymorphisms were detected in the somaclonal cultivars, suggesting that DNA methylation polymorphisms may associate with tissue culture-induced mutation in ornamental aroids. This is the first study of methylation variation in somaclonal variants of ornamental foliage plants. The results clearly demonstrate that the MSAP technique is highly efficient in detecting DNA methylation events in somaclonal-derived cultivars.