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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.

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Hilde Nybom

Hybridization of minisatellite DNA with an M13 probe yields DNA fingerprints that usually are highly cultivar-specific. However, 15 different sports of `Red Delicious' apples (Malus × domestics Borkh.) exhibited almost identical fingerprints. The mutations determining the morphological differences between the sports could not be detected by the minisatellite probe. These hypervariable DNA sequences appear rather stable in apples, making them ideal for differentiating between cultivars derived through genetic recombination but probably not very useful for differentiating between vegetative sports.

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Stacey M. Sakakibara and John E. Carlson

Random amplified polymorphic DNA (RAPD) markers were evaluated for use in DNA fingerprinting of commercial Rhododendron cultivars. DNA was isolated from Rhododendron leaves and subjected to PCR amplification with single primers, 10 nucleotides in length, and of arbitrary sequence. Amplification products were visualized by agarose gel electrophoresis and ethidium bromide staining. Fingerprints were readily identifiable for a number of cultivars, and a high level of polymorphism was observed among clones of 10 rhododendron varieties. The technique was consistently reproducible in different trials using the thermocycler, between different thermocyclers, and using different DNA isolation from the same plant. This method will be applied to large-scale fingerprinting of Rhododendron cultivars and for distinguishing material propagated in tissue culture.

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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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James Polashock and Nicholi Vorsa

We have used RAPDs (Randomly Amplified Polymorphic DNAs) to successfully fingerprint cranberry. Although this method is simple and inexpensive, disadvantages include limited reproducibility in other labs and it is not easily computer-analyzed. RAPDs can also be labor-intensive because multiple primers are required to adequately fingerprint a single sample. As an alternative, we have utilized a method called SCARs (Sequence Characterized Amplified Regions). Clear polymorphic RAPD markers were cloned and sequenced. Primers were designed to amplify each polymorphic band and contained the original 10-mer RAPD primer sequence and 10 to 12 additional “clone-specific” bases. Primer sets were tested on eight common cranberry cultivars to determine if the desired polymorphic marker was amplified. The success rate of developing ëgoodí primer sets was ≈25%. The most common problem was loss of polymorphism, suggesting that selectivity was contained within the original 10-mer RAPD primer. The amplification of many similarly sized markers, suggesting the primer set amplified a repeat region, was another problem. Useful primer sets were multiplexed in PCR reactions to establish a “fingerprint.” The SCARs system we developed to fingerprint cranberry is powerful enough to distinguish individual clones in both crosses and selfed progeny. To further simply the system, computer automation for detection and analysis using fluorescently labeled primers is underway. One problem we are addressing is reduced product in the labeled multiplex reactions. Reduced product yield is presumably because the dye molecule (Cy5) is very large and may reduce primer binding and/or polymerization efficiency. This problem has been somewhat alleviated using a patented form of Taq DNA polymerase.

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Teresa A. Cerny and Terri W. Starman

Seed of five species of petunia and 10 cultivars of Petunia xhybrida were obtained from several sources and plants were fingerprinted using DNA amplification fingerprinting (DAF). Within some species, variable fingerprints were generated between individual plants from the same seed source and/or different sources. Consistencies were found among DAF profiles by bulking the leaf tissue from 10 different plants, but not five plants. Each of 10 octamer primers used during the study revealed polymorphic loci between the species and cultivars. Among the 201 bands produced, 146 (73%) loci were polymorphic and these could be used to distinguish between each of the species and cultivars. Scoring for presence and absence of the amplified bands was used to generate a phylogenetic tree and to calculate the pairwise distances between each of the taxa using parsimony (PAUP) analysis. The tree generated using DAF molecular markers separated P. axillaris from P. parodii (two white-flowered species), and distinguished between the violet-flowered species, P, inflata, P. integrifolia, and P. violacea.

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Claudio Cantini, Amy F. Iezzoni, Warren F. Lamboy, Manuela Boritzki, and Darush Struss

The U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) tetraploid cherry (Prunus L. sp.) collection at Geneva, N.Y., contains ≈75 accessions of sour cherry (P. cerasus L.), ground cherry (P. fruticosa Pall.), and their hybrids. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Simple sequence repeats (SSRs) are currently the markers of choice for germplasm fingerprinting because they characteristically display high levels of polymorphism. Recently SSR primer pairs from sweet cherry (P. avium L.), sour cherry, and peach [(P. persica L. Batsch (Peach Group)] have been reported. Ten SSR primer pairs were tested on 59 tetraploid cherry accessions to determine if they could differentiate among the accessions. Scorable SSR fragments were produced with all primer-accession combinations. The cherry accessions exhibited high levels of polymorphism with 4 to 16 different putative alleles amplified per primer pair. Most of the putative alleles were rare with frequencies <0.05. Heterozygosity values ranged from 0.679 to 1.00, while gene diversity values ranged from 0.655 to 0.906. The primer pairs differentiated all but two of the 59 cherry accessions. Based upon the ability of the SSR data to differentiate the cherry accessions and the high level of gene diversity, we propose that all the tetraploid cherry accessions in the USDA/ARS collection be fingerprinted to provide a mechanism to verify the identity of the individual accessions. The fingerprinting data are available on the World Wide Web (http://www.ars-grin.gov/gen/cherry.html) so that other curators and scientists working with cherry can verify identities and novel types in their collections and contribute to a global database.

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Paul Skroch and Jim Nienhuis

The genetic variation in a population of one hundred Snap Bean varieties, including processing and garden types, was studied using RAPD markers. All one hundred genotypes were distinguished by unique combinations of banding patterns. These unique “fingerprints” were tested for repeatability. Certain bands were very reliable and can be used for varietal identification. The RAPD marker data was also used to estimate genetic relationships among a subset of the one hundred lines. The results of the analysis agreed with known pedigree information. These markers will allow more precise monitering and control of germplasm by those who are involved with the breeding and production of superior seed.

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Masao Yoshida, T. Shimada, and M. Yanaguchi

Twenty-eight Prunus species were examined in order to survey their genetic diversity. Genomic DNA was extracted from 36 varieties and used for the template DNA of PCR. DNA fingerprints were generated by random primers or semi-random primers, some primers consensus to the repeated units as telomers, and three sets of sequence-tagged primers specific to domains of chloroplast DNA (psbA, rbcL-ORF106, atpB-rbcL). PCR products generated from these three domains were digested by 12 restriction enzymes. RFLPs were detected among varieties and subjected to the UPGMA. Thirty-six varieties were classified approximately into two groups: “Plum group” and “Cherry group.” It was inferred that these two groups were divided in old time. P. tomentosa, P. japonica, P. glandurosa, and P. besseyi, which are classified into the cherries, showed the same fingerprint patterns from chloroplast DNA of the plum group; plums and cherries have a large genetic diversity. It was supposed that the diversity of plums depended on nuclear DNA, besides the diversity of cherries on both nuclear and chloroplast DNA.

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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.