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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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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.

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Vance Baird

Prunus, which includes peach/nectarine, almond, apricot, cherry, and plum, is a large and economically important genus in the family Rosaceae. The size and long generation time of these tree crops have hampered improvement through classical breeding and long-term selection programs. With the advent of DNA-based molecular diagnostics, an exciting era in germplasm improvement has dawned. Efforts are underway, notably in the United States (e.g., California, Michigan, North Carolina, and South Carolina) and the European Community (e.g., England, France, Italy, and Spain), to apply the tools of molecular mapping and marker-assisted selection to this important genus. The objective of these projects is to develop molecular linkage maps of sufficient marker density to tag phenotypic trait loci of agronomic importance. These include traits controlled by single genes (e.g., flower color, compatibility, flesh color, pest resistance), as well as more-complex, quantitative traits (e.g., cold hardiness, tree architecture, sugar content). An immediate outcome of these mapping efforts has been the development of DNA “fingerprints,” allowing for the discrimination of cultivars—both scion and rootstock. The maps will be used by breeders and molecular biologists to monitor gene introgression from wild species into elite lines, for marker-assisted selection of desired trait combinations, and for map-based cloning of specific genes. The molecular markers used in these mapping projects include RFLPs, RAPDs, and microsatellites. Each has their appropriate applications and advantages depending upon the resources at hand and the project's specific goals.

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R.N. Trigiano, M.C. Scott, and G. Caetano-Anollés

The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars `Dark Charm', `Salmon Charm', `Coral Charm' and `Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of `Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing ≈37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars.

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Lianghong Chen and Mack Nelson

Randomly amplified polymorphic DNA (RAPD) technique is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. It has been widely used for genetic mapping, plant and animal breeding programs, and DNA fingerprinting. However, there is no single set of RAPD-PCR conditions that can be applied to all situations. In order to adjust reaction component concentrations within suggested ranges for efficient amplification during the use of RAPD in detection of genetic variation of genus Camellia, crucial factors, such as concentrations of MgCl2 and DNA, annealing temperature (37 to 44 °C), and the use of an AmpliTaq® DNA polymerase and Stoffel fragment were examined. Five camellia cultivars, `Winter's Beauty', `Pink Icicle', `Polar Ice', `Winter's Hope', and `Snow Flurry', were under investigation. Clear and reproducible amplification products were produced with 3.0 μM MgCl2 and 30 ng template DNA/25 μL reaction mixer at annealing temperature 37 °C and 40 °C, compared with MgCl2 at 1.5, 2.0, and 2.5 μM. When annealing temperature increased, the RAPD-PCR stringency was increased, as expected. Stoffel fragment was found to provide highly reproducible results.

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D. Struss, M. Boritzki, R. Karle, and A.F. Iezzoni

Two rootstocks from the Giessen (GiSelA) series of dwarfing cherry (Prunus sp.) rootstocks, GiSelA (GI) 5 (syn. 148/2) and GI 6 (syn. 148/1), are becoming commercially important and five other Giessen cherry rootstocks are being evaluated for horticultural traits. Since GI 5 and GI 6 are morphologically similar, a DNA fingerprinting project was undertaken to identify molecular markers that could be used by the nursery industry to differentiate these two rootstocks. The project was extended to include six additional Giessen rootstocks of varying pedigrees. Fourteen DNA primer pairs were tested for their ability to differentiate among the eight rootstocks. None of the primer pairs could differentiate all eight rootstock selections; however, three primer pairs could differentiate all but two selections. Two primer pairs, PMS 15 and PceGA59, were identified as the most suitable for high throughput screening of GI 5 and GI 6 due to the simplicity and the size of the base pair differences among the polymorphic fragments. These results demonstrate the utility of molecular markers to differentiate the Giessen cherry rootstocks.

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D.W. Lickfeldt, N.E. Hofmann, J.D. Jones, A.M. Hamblin, and T.B. Voigt

An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

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G. S. Varadarajan and C. S. Prakash

The sweet potato (Ipomoea batatas) and its relatives (the batatas complex) appear to have evolved in the New World and radiated over several geographic centers in the tropics and subtropics. Traditional studies on taxonomy, cytogenetics, and reproductive biology of the batatas complex have enabled us to investigate certain evolutionary aspects. We conclude that this complex is a monophytetic, “polyploid pillar”, evolved by chromosome doubling (euploidy) and interspecific hybridization. We apply molecular genetic techniques to detect variation [restriction fragment length polymorphism (RFLP) and DNA fingerprinting analyses] to reexamine some of the evolutionary issues that could not be satisfactorily addressed by the conventional approaches, e. g., phylogenetic history of the batatas group, the diploid ancestors of the polyploid members, homology/diversity of genome(s) within the entire group. We find DNA variation in the hypervariable or multiple copy regions of the genome in Ipomoea species. In addition, we are investigating polymorphism in unique/low copy regions using a battery of DNA sequences from homologous as well as heterologous sources. The success of this study will hopefully shed a new light on the subject of evolutionary biology and may also have potential applications in the sweet potato breeding.

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Kenneth L. Deahl, Richard W. Jones, Frances M. Perez, David S. Shaw, and Louise R. Cooke

The oomycete, Phytophthora infestans, is a devastating pathogen of potato worldwide. Several genotypes of P. infestans are able to infect other cultivated and weed species of the family Solanaceae and cause symptoms similar to late blight. Changes in P. infestans populations have stimulated investigations to determine if potato strains from new immigrant populations infect nonpotato hosts more often than those from the older population. Expansion of the effective host range may be one of the mechanisms involved in pathogenic changes in natural populations of P. infestans and to determine its significance, it is necessary to establish if the pathogen strains on nonpotato hosts represent distinct genotypes/populations or are freely exchanging with those on potato. This article reports characterization of P. infestans isolates from four solanaceous hosts (black nightshade, hairy nightshade, petunia, and tomato) growing within and around fields of blighted potatoes in four U.S. locations and one U.K. location and their comparison with isolates collected from adjacent infected potatoes. Isolates were characterized for mitochondrial DNA haplotype, mating type, metalaxyl resistance, allozymes of glucose-6-phosphate isomerase and peptidase, and DNA fingerprint with the RG57 probe. Analysis showed close similarity of the petunia, hairy and black nightshade isolates to potato isolates. However, tomatoes from New Jersey and Pennsylvania, respectively, were infected by two distinct and previously unreported pathogen genotoypes, which had quite different fingerprints from P. infestans isolates recovered from nearby infected potatoes. Potato growers should be aware that both weed and cultivated solanaceous species can be infected with P. infestans and may serve as clandestine reservoirs of inoculum. Because some of these plants do not show conspicuous symptoms, they may escape detection and fail to be either removed or treated and so may play a major role in the introduction and spread of pathogens to new locations.

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S.D. Reid, M. Ali-Ahmad, and H.G. Hughes

The use of random amplified polymorphic DNA (RAPD) markers has been shown to be a potentially useful technique for identifying buffalograss breeding lines. Analysis of RAPD markers has also revealed considerable variation within, as well as among, each of four natural buffalograss populations surveyed. Identification of genetic markers for quantitative traits, such as physiological components of tolerance to salt stress, can provide important information for plant improvement programs. The objectives of this study were to develop DNA fingerprints for buffalograss clones selected from an in vitro seedling screening program for survival at high NaCI (200–250 mM) levels, identify markers for future analysis, and assess the variability among the lines. DNA was extracted from leaves of 10 salt-selected and 15 non-selected buffalograss clones. Fifty-two 10-mer primers were screened for ability to produce bands with DNA from four clones as visualized on ethidium-stained agarose gels. Bands were most reproducible with a genomic template DNA concentration of 1 ngμl–1 reaction volume. Primers selected for ability to produce a moderate number of clear bands were used to produce RAPD profiles of the 25 clones. Abundant polymorphism to distinguish among clones was found. Four primers produced a total of 45 polymorphic markers. The primer 5′-CGGAGAGCCC-3′ produced 11 readily scored markers, allowing identification in 94.67% of pair-wise comparisons. As a group, RAPD profiles of salt-selected clones are more variable than non-selected clones from the same population; however, no unique pattern of markers generated by primers screened to date differentiates all salt-selected clones from the non-selected group.