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.
Ruwanthi C. Wettashinghe and Ellen B. Peffley
Peggy Ozias-Akins and Robert L. Jarret
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).
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.
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.
Stacie L. Aragon, Keng-Chang Chuang and Adelheid R. Kuehnle
Isolation of high quality nucleic acids from aroids can be difficult due to the presence of carbohydrates, phenolics, and other compounds that bind to and/or co-precipitate with the DNA or RNA. Methods previously used for marine algae, mango, and papaya were modified and successfully used for the simultaneous isolation of high quality genomic DNA and RNA from Anthurium, Colocasia, and Spathiphyllum leaves. Genomic DNA yields averaged 477 μg·g-1 fresh weight for Anthurium and 322 and 177 μg·g-1 fresh weight, respectively, for Colocasia and Spathiphyllum. Total RNA yields averaged 129 μg·g-1 fresh weight for Anthurium and 61 and 50 μg·g-1 fresh weight, respectively, for Colocasia Spathiphyllum. This method may be useful in co-isolating high quality nucleic acids from additional aroids and other plants.
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.
Dror Sharon, Jossi Hillel, Samir Mhameed, Perry B. Cregan, Emanuel Lahav and Uri Lavi
The detection of association between DNA markers and traits of interest in an outbred population is complicated and requires highly polymorphic markers. A genetic linkage map of avocado (Persea americana Mill.) recently generated consists of simple sequence repeat (SSR) markers as well as DNA fingerprint (DFP) and randomly amplified polymorphic DNA (RAPD) markers. These markers were used to detect putative quantitative trait loci (QTLs) of eight avocado fruit traits. Two statistical methods were used: one-way analysis of variance and interval mapping. Six traits were found to be associated with at least one of the 90 DNA markers. Based on the two statistical approaches, a putative QTL associated with the presence of fibers in the flesh, was found to be located on linkage group 3. This putative QTL was found to be associated with the SSR marker AVA04 having a high significant value (P = 4.4 × 10-8). The haplotype analysis of linkage group 3 showed a putative dominant interaction between the alleles of this locus.
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.