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Paul A. Wiersma, Deniz Erogul, and Shawkat Ali

DNA fingerprinting of highly related individuals. Although SSR markers can be used to discriminate sweet cherry cultivars as previously suggested, finding markers that differentiate closely related cultivars, such as the Lapins and ‘Sweetheart, groups

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A. Vainstein and H. Ben-Meir

Mini- and microsatellite probes were hybridized to DNA of 24 rose (Rosa×hybrida) genotypes. The resultant DNA fingerprints were shown to be genotype-specific, thereby enabling cultivar identification at the DNA level. Restriction enzyme Dra I yielded the most informative band patterns. Full-sib family analysis of DNA fingerprints revealed 32 parental-specific bands out of the 128 observed in the parents. These bands were revealed cumulatively by phage (M13), human (33.6), and oligonucleotide (GACA)4 probes. Only one pair of these loci was found to be allelic, and no linked pairs were detected in the progeny analyzed. The probability of two offspring from this cross having identical DNA fingerprints was calculated to be 2 × 10-8.

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R.N. Trigiano, B.H. Ownley, A.N. Trigiano, J. Coley, K.D. Gwinn, and J.K. Moulton

tools in plant biotechnology, which includes genomics and proteomics, are gel electrophoresis, polymerase chain reaction (PCR), and DNA fingerprinting. To incorporate these techniques into middle and high school and college curricula, content instruction

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Victor Luk and John Carlson

DNA fingerprinting is a potentially powerful molecular genetic technique that can be used to distinguish subtle differences in genome structure among closely related genotypes, such as many horticultural varieties. A DNA fingerprinting project is currently in progress at the Univ. of British Columbia (UBC) Biotechnology Laboratory to produce a set of DNA markers and an easy, reliable, and legally recognized analysis protocol that will enable the UBC Botanical Garden Plant Introduction Scheme (PISBG) to unambiguously identify any of their released varieties, even in dormant or juvenile form, wherever it is being propagated or sold. High-quality genomic DNA was isolated from the leaf samples of six PISBG species (Anagallis monellii, Artemesia stelleriana, Clematis, Genista pilosa, Microbiota decussata, and Penstemon fruticosa) using a modified CTAB DNA isolation protocol, and further purified by cesium chloride/ethidium bromide gradient. Samples of these genomic DNA preparations (10 ng) were then amplified by a 45-cycle polymerase chain reaction (PCR) protocol using 1.5-μm 10-nucleotide primers of arbitrary nucleotide sequence that amplify a variety of sites distributed across the genome. Following the amplification, PCR products [random amplified polymorphic DNA (RAPD) markers] were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. More than 70% of the 51 primers tested so far generated distinctive banding patterns (2–11 bands) with DNA samples from each species. Subtle changes in the genome or differences between genotypes can be detected by screening a series of such primers against DNA samples from the genotypes in question. Once a RAPD primer has been identified that consistently generates a different banding pattern between genotypes, it can be used as an identification tool for discriminating between those genotypes at any time in the future.

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Naomi R. Smith and Robert N. Trigiano

Flowering dogwood (Cornus florida L.) is an important tree of forests and urban landscapes in the eastern United States. Currently, there are over 100 cultivars of flowering dogwood commercially available. An identification process based on genotype would be of use to researchers, breeders, and nurserymen, as many cultivars are similar phenotypically. Molecular markers offer a promising way of definitively identifying flowering dogwood cultivars. Amplified fragment length polymorphism (AFLP) is a technique that can be used to generate DNA fingerprints. DNA was isolated from leaves of 17 common cultivars of dogwood and AFLP fingerprints were generated by a Beckman Coulter CEQ™ 8000. Fingerprints were converted to binary data and verified manually. Two drafts of a cultivar identification key were generated based on the corrected, verified binary data and cultivar-specific peaks. Six primer combinations were used to construct all keys and were tested with seven unknown dogwood cultivar samples. Six unknown samples were correctly identified using the keys. Only one unknown, `Cherokee Brave', was unidentifiable with any key. In all cases, some intracultivar variation was observed. A similarity index was calculated and visualized with a tree of genetic relatedness using NTSYSpc. Intracultivar variation was observed in the similarity index as well. This database for cultivar-specific molecular markers will serve as a starting point to which other cultivars can be added and also can be used in breeding applications, patent application and other projects, such as mapping the C. florida genome.

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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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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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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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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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U. Lavi, J. Hillel, A. Vainstein, E. Lahav, and D. Sharon

Application of four DNA fingerprint probes to avocado (Persea americana Mill.) resulted in identification of various cultivars, characterization of the three avocado races, and a genetic analysis of family structure. Genomic DNA from 14 cultivars was probed with four DNA fingerprint probes. Three of the probes gave well-resolved bands. The individual-specific patterns obtained for each cultivar validate the use of this technique for definitive cultivar characterization, with the probability of obtaining a similar pattern for two different cultivars being 2 × 10-9. DNA mixes representing either Mexican, Guatemalan, or West-Indian avocado races were hybridized with the DNA fingerprint probes, and a band pattern characteristic for each race was obtained. Progeny of a cross between the cultivars Ettinger and Pinkerton were analyzed. Their DNA fingerprints revealed one pair of linked bands and another band allelic to one of them. The application of these observations to identification, evolutionary studies, and breeding is discussed.