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A. Adato, D. Sharon, U. Lavi, J. Hillel, and S. Gazit

DNA fingerprint information was used for identification of mango (Mangifera indica L.) cultivars for genetic relatedness analysis of20 mango cultivars and for genetic analysis of a family structure. Genomic DNA was extracted from young leaves, digested with Hind III or Dra I, and hybridized with 10 different DNA probes. Jeffreys' minisatellite probe 33.6 was the most useful, resulting in well-resolved bands representing highly polymorphic loci. Specific patterns were obtained for each cultivar. The probability of obtaining a similar pattern for two different cultivars was 9.4 × 10-6. Based on DNA fingerprint information, genetic distances between 20 mango cultivars were evaluated and an evolutionary tree was established. Analysis of DNA fingerprint band patterns of 12 progeny resulting from a cross between `Tommy Atkins' and `Keitt' mango revealed neither linked nor allelic bands. Application of the reported results for identification, genetic analyses, and mango breeding is discussed.

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Terri Woods Starman and Shane Abbitt

The objective was to distinguish between cultivars and evaluate genetic relatedness of poinsettia (Euphorbia pulcherrima) using two methods of DNA fingerprinting—DNA Amplification Fingerprinting (DAF) and Arbitrary Signatures from Amplification Profiles (ASAP). Eleven red poinsettia cultivars were studied, including `Celebrate 2', `Darlyne', `Freedom Red', `Lilo', `Nutcracker Red', `Peterstar Red', `Petoy', `Red Sails', `Supjibi', `V-14 Glory', and `V-17 Angelika'. Amplification was with 10 octamer primers. Gels were visually scored for presence or absence of bands. The 10 primers generated 336 bands. The average number of bands (≈1000 bp) per primer was 34 ranging from 19 to 43. Thirty-one percent of bands were polymorphic and distinguished between each cultivar. The number of unique profiles varied from two to nine. Genetic relationships were evaluated by SAHN cluster analysis based on the distance estimator of Jaccard using the NTSYS-pc program (Numerical taxonomy and multivariate analysis system, version 1.8). The resulting dendrogram closely agreed with known pedigree data. ASAP analysis was used to further assess cultivar identification of two cultivars that were genetically and morphologically similar. Markers were found that separated `Nutcracker Red' and `Peterstar Red'. ASAP analysis separated cultivars within the Freedom series that DAF failed to distinguish. Two cultivars in the Freedom series, `Jingle Bells' and `Marble', were characterized from other cultivars in the series with ASAP.

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D.G. Ranamukhaarachchi, R.J. Henny, C.L. Guy, and Q.B. Li

Randomly amplified polymorphic DNA (RAPD) markers were utilized to determine the genetic relationships of nine morphologically similar pot plant cultivars of Anthurium sp. by developing DNA fingerprints (DFP). Of 25 arbitrary primers screened, nine generated DFPs that were used in computing the genetic distance (d) and similarity coefficient (C) values. All cultivars tested exhibited a high degree of genetic similarity. `Lady Ann' and `Lady Beth' possessed the closest relationship with d and C values of 0.06 and 0.98, respectively. The next closest genetic relationship was between `Red Hot' and `Southern Blush' (d = 0.33, C = 0.89). These two cultivars exhibited a more distant relationship to the other seven cultivars as indicated by higher `d' values. However, this study showed that the nine Anthurium cultivars examined were genetically closely related. These cultivars share specific DNA bands with three possible parental species (A. andraeanum Linden ex Andre, A. antioquens L., and A. amnicola Dressler) included in this study, which may indicate similarities in their pedigree. This study shows that RAPDs can be a useful tool to distinguish Anthurium pot plant cultivars as well as identify their genetic relationships.

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James J. Polashock and Nicholi Vorsa

DNA fingerprinting has been useful for genotypic classification of American cranberry (Vaccinium macrocarpon Ait.). Polymerase chain reaction (PCR) based methodologies including randomly amplified polymorphic DNA (RAPD) markers are relatively easy to use, and inexpensive as compared to other methods. However, RAPD markers have some limitations including seamless interlaboratory transferability and susceptibility to certain types of error. An alternative method, sequence characterized amplified regions (SCARs), was developed for cranberry germplasm analysis. Nine primer sets were designed from RAPD-identified polymorphic markers for use in two multiplex PCR reactions. These primer sets generated 38 markers across a cranberry germplasm collection. Estimates of genetic relatedness deduced from employment of the RAPD and SCAR methods were compared among 27 randomly chosen cranberry germplasm accessions. Although both methods produced comparable results above 0.90 coefficient of similarity, branches below this level exhibited variation in clustering. SCAR and RAPD markers can be employed for identifying closely related genotypes. However, the inferences of more distant genetic relationships are less certain. SCAR marker reactions provided more polymorphic markers on a per reaction basis than RAPD marker reactions and as such more readily separated closely related progeny. When SCAR primers were fluorescent dye-labeled for computerized detection and data collection, reduced marker intensity relative to unlabeled reactions was one problem encountered.

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Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin, and Mark K. Ehlenfeldt

Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.

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Mark Hubbard, John Kelly, Albert Abbott, and Robert Ballard

To protect plant patents, rose breeders would benefit from a reliable and sensitive method for differentiating cultivars at the genetic level. Rcombinant DNA technologies are being employed to characterize individual DNA structure of numerous rose cultivars. Restriction fragment length polymorphisms (RFLPs) are being studied to develop a characteristic pattern, or fingerprint for each cultivar. DNA from various cultivars is restriction enzyme digested and the fragments separated by agarose gel electrophoresis. The gel is Southern blotted and hybridized with probes from the rose DNA library to yield RFLPs. RFLPs are being located and will eventually result in a characteristic fingerprint for each cultivar.

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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.

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John H. Culpepper, Luis A. Sayavedra-Soto, Brant J. Bassam, and Peter M. Gresshoff

Several horticulturally important members of the genus Cornus were characterized at the DNA level to identify genotypes. Random genomic DNA fragments from Cornus florida L. `Barton' were cloned into pBR322 and λ Gem-11 and used to search for restriction fragment length polymorphisms (RFLPs) among C. sericea L., C. kousa Hance., and four cultivars of C. florida: `Barton', `Cherokee Princess', `Cloud 9', and `Mary Ellen'. Total DNA from these genotypes was restricted with several endonucleases (of which BamHI, EcoRI, and HindIII were used to search for RFLPs), vacuum-blotted onto nylon membranes, and probed with the C. florida `Barton' DNA clones. RFLPs were common among the Cornus species sericea, kousa, and florida, suggesting considerable DNA sequence divergence at the species level. RFLPs were less common among the cultivars of C. florida. These cultivars were selected from a narrow geographical area in North America from nursery-grown trees and exhibit much less DNA sequence divergence.

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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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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.