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.
M.C. Scott, G. Caetano-Anollés and R.N. Trigiano
Xiaomeng Li, Rangjin Xie, Zhenhua Lu and Zhiqin Zhou
of cultivated citrus were mainly based on morphological, biochemical, and isozyme data ( Barrett and Rhodes, 1976 ; Fang, 1993 ; Scora, 1988 ; Torres et al., 1978 ). Recently, DNA markers such as restriction fragment length polymorphisms (RFLPs
James A. Schrader and William R. Graves*
Long regarded as a genus of two species, Dirca L. was expanded to include a third North American shrub discovered in 1994 as one population in the Sierra Madre Oriental of Tamaulipas in northeastern Mexico. The designation of Dirca mexicana Nesom & Mayfield as a third species in the genus was based in part on geographical separation from Dirca palustris L. and Dirca occidentalis Gray, which occur farther north in eastern North America and in a small region of California, respectively. Morphologically, D. mexicana was regarded as more similar to D. occidentalis than to D. palustris. Our objectives were to obtain fruits of all species, germinate seeds, and compare the three species genetically through analyses of seedling DNA. Drupes of D. mexicana, D. palustris (from populations in Iowa), and D. occidentalis were collected as they abscised naturally from plants in native habitats in mid-May, late May to early June, and mid-June, respectively. Embryo extraction, gibberellin, and cold stratification were used to promote germination, and DNA was extracted from leaves of seedlings by using the fully automated Autogen Autogenprep 740 DNA extraction system. Genomic DNA templates were used to compare sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of the nuclear ribosomal DNA repeat and to examine polymorphisms in inter-simple sequence repeats (ISSRs). These analyses reinforce the present morphologically based classification of the three Dirca species by confirming species-level divergence at the molecular level. ITS sequences and ISSR banding patterns also enabled us to reconstruct the phylogenetic relationship among the three extant species of Dirca.
W. Vance Baird, Agnes S. Estager and John K. Wells
Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera [Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)], two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60±0.03 pg (or 5.8×108 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (× = 8).
Channapatna S. Prakash, Guohao He and Robert L. Jarret
Highly polymorphic DNA markers were identified in sweetpotato (Ipomoea batatas) using PCR amplification and arbitrary primers. More than 100 accessions representing US cultivars and their progenitors, and germplasm lines from around the world were analyzed. Sweetpotato germplasm exhibited high genetic variability and individual-specific profiles were obtained for all accessions. US cultivars formed a tight cluster in the principal coordinate analysis suggesting a narrow genetic base. The genetic relationship data of US cultivars and their progenitors based on DNA polymorphisms was in agreement with their known pedigree. The putative paternal parents of certain cultivars selected through open pollination were identified based on shared polymorphisms. The PCR-based markers are valuable in the characterization of sweetpotato germplasm and in ensuring a broad genetic base for future cultivars.
C.J. Simon and N.F. Weeden
The ribosomal genes of the two crab apple (Malus) genotypes White Angel' and `Robusta 5' were characterized to determine the extent of between- and within-genotype heterogeneity. Initial investigations with a cloned sequence of soybean rDNA failed to detect some Malus intergenic spacer region fragments. An alternative probing method that used electrophoretically purified Malus rDNA was developed. Double-digests of total genomic DNA with combinations of 13 restriction endonucleases identified the positions of 35 restriction sites. Restriction site polymorphism was observed both between and within the crab apple genotypes. Ribosomal DNA from White Angel' was cloned in phage and plasmid vectors and mapped with 11 enzymes. The region of the spacer causing length heterogeneity was identified. These clones should be useful as genetic markers and for examining population dynamics and systematic of Malus and closely related taxa.
C.L. Boehm, I.K. Lee, G. Jung, H.C. Harrison, J. Nienhuis, M. Sass and Moore Hall
Random amplified polymorphic DNA (RAPD) may have utility as genetic markers facilitating selection in ginseng crop improvement. This experiment determined chemical buffer and root tissue-type combinations that yield repeatable bands. The results allow further experiments using RAPD markers for estimating the genetic distance between ginseng landraces, selection for crop improvement, and extensive fingerprinting for use in determining the origin of tissue samples. This experiment determined mean band yields for all combinations of dry, fresh, and powdered root with cetyltrimethylammonium bromide, potassium/sodium ethyl xanthogenate, and urea buffers. The buffers were applied in replication to the tissue-types with other extraction protocol factors constant. Replications were amplified four times with four different primers using constant PCR and agarose gel electrophoretic protocols. Distinct bands were counted in each replication, and the summation of the replication repeats considered an observation. Least squares means for several response variables were analyzed. The most significant difference found was between buffers. The buffers ctab and urea were productive, and the pex was not. Significant difference was found when buffers were crossed with tissue. The applications of urea to fresh root, ctab to dry root, urea to dry root, and ctab to powdered root were productive. Based on these results we conclude 1) urea and ctab are productive when applied to all tissue-types, 2) dry root, which is easily collected and stored, yields sufficient DNA for analysis, and 3) powdered root, often the form of commercial products that might be tested for genetic origin, will yield sufficient DNA for analysis.
Carlos A. Urrea, Phillip N. Miklas, James S. Beaver and Ronald H. Riley
Molina, José Vélez, and Maximo Halpay for assisting with greenhouse disease screening; and Nada Abbas for assisting with randomly amplified polymorphic DNA analyses. The cost of publishing this paper was defrayed in part by the payment of page charges
Frank A. Buffone, Don R. La Bonte and Christopher A. Clark
DNA isolated from Fusarium lateritium Nees: Fr.-infected `Jewel' sweetpotato [Ipomoea batatas (L.) Lam.] plants was compared to F. lateritium-free `Jewel' plants for differences in random amplified polymorphic DNA (RAPD) marker products. Differences in RAPD marker products were detected. Amplified DNA isolations from F. lateritium-infected `Jewel' plants generated additional, unique DNA fragments not found in amplified DNA isolations of F. lateritium-free `Jewel' plants. These unique amplified DNA fragments were consistent with those obtained from amplified DNA isolations of the F. lateritium isolate, 91-27-2, used for inoculation. We found that F. lateritium DNA successfully competes with sweetpotato DNA in the polymerase chain reaction for priming sites in a 3: 1 ratio of sweetpotato DNA to F. lateritium DNA. Our results indicate the importance of avoiding plant material infested with pathogens to avoid spurious marker bands.
Patricia M. Sweeney and T. Karl Danneberger
As the number of perennial ryegrass (Lolium perenne L.) cultivars increases, the development of reliable identification methods becomes more important. Randomly amplified polymorphic DNA (RAPD) markers show promise in cultivar identification. Since perennial ryegrass cultivars are composites of genotypes rather than a single genotype, finding markers that distinguish cultivars is difficult. The ideal cultivar identification procedure would use seed tissue as a DNA source and evaluate a single sample as representative of a cultivar. The objective of this research was to determine whether RAPD markers could be used to consistently distinguish bulk seed samples of perennial ryegrass cultivars. Two extraction protocols were evaluated. A quick, simple extraction resulted in the amplification of few consistent RAPD markers. The more labor-intensive extraction with hexadecyltrimethyl ammonium bromide (CTAB), however, produced more reliable RAPD markers. Eight of 11 cultivars were distinguished by using RAPD markers produced using bulk seed samples together with four of 30 primers that were screened. These results show the potential of RAPD markers to provide the turfgrass industry, breeders, and certification agencies additional options to ensure the genetic integrity of perennial ryegrass seed lots and cultivars.