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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.

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J.F. Hancock, P.A. Callow, and Douglas V. Shaw

Eight strawberry cultivars or advanced selections from the Univ. of California, Davis, breeding program were screened for polymorphisms using the polymerase chain reaction (PCR) and 43 random 10-base DNA primers. Over 60% of the primers screened resulted in replicable polymorphic banding patterns (amplification profiles), and a subset of ten primers that exhibited high levels of amplification profile polymorphism was used to identify each of the eight genotypes uniquely. There was also a significant product-moment correlation (r = 0.64, P < 0.01) between number of shared amplification profile phenotypes and pairwise coefficient of coancestry. This technology shows high promise as a means of verifying the identity of cultivars and developing a genetic map of the octoploid cultivated strawberry.

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Richard E. Harrison, James J. Luby, and Glenn R. Furnier

Restriction fragment-length polymorphisms (RFLPs) of chloroplast DNA (cpDNA) were used to study phylogenetic relationships among twenty-six Fragaria taxa and two closely related species, Potentilla fruticosa L. and Duchesnea indica (Andrews) Focke. Sixteen restriction enzymes and probes of the entire Nicotiana tabacum L. chloroplast genome revealed a very low level of variation among the Fragaria taxa, limiting phylogenetic resolution. However, Fragaria appears to be more closely related to Potentilla than Duchesnea. The diploid taxa, F. iinumae Makino, F. nilgerrensis Schlect. and F. vesca L. were the most divergent Fragaria taxa and F. iinumae appears to be the most ancestral taxon. Little variation was revealed within the economically important octoploid group of taxa, which gave rise to the cultivated strawberry, and no progenitor taxa to the octoploid group could be identified. The lack of variation in the chloroplast genome suggests that these Fragaria species may be of relatively recent evolutionary origin.

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Jon T. Lindstrom and Matthew C. Pelto

Itea virginica (Virginia Sweetspire) is a woody landscape shrub that has recently gained much popularity in the landscape. Several cultivars of Itea have been selected for fall leaf color and plant habit. Visual identification of some of these cultivars is difficult and confusion exists in the trade. RAPDs (randomly amplified polymorphic DNA) were used to identify Itea virginica cultivars. A single 10-base primer was sufficient to separate the cultivar Saturnalia from the cultivar Henry's Garnet. Two dwarf cultivars of I. virginica, `Merlot' and `Sprinch' (= Little Henry), were separable from `Henry's Garnet' using a single 10-base primer. A primer that distinguishes between these two dwarf cultivars has not yet been found. The technique of RAPDs appears sufficiently sensitive and repeatable to resolve questions of identity that may exist among several cultivars of Itea.

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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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Njung’e Vincent Michael, Pamela Moon, Yuqing Fu, and Geoffrey Meru

; Paris, 2016 ). Measurement of genetic diversity and elucidation of phylogenetic relationships among C. pepo genotypes has been accomplished using a variety of marker systems including allozymes ( Decker, 1985 ) and DNA markers [random amplified

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Roger J. Sauve, Suping Zhou, Yingchun Yu, and Wolfram George Schmid

A randomly amplified polymorphic DNA (RAPD) technique was used to identify and determine the phylogenetic relationships of 37 hosta accessions representing the major subgenera, sections and groups in the genus Hosta. Results of this study show that RAPD markers were able to differentiate not only the main groups, whose plants shared many genetic traits, but also cultivars within a species. Some accessions were identified by a single primer while others had high intercross linkage and required many markers for their separation. The phylogenetic clustering showed that H. plantaginea, the only night-blooming species, and H. ventricosa, the only known natural tetraploid, are unique and should be classified separately. The four species in the subgenus Bryocles, section Lamellatae H. venusta, H. minor, H. capitata, and H. nakaiana have very low genetic similarity since they do not share many amplified fragments. The other accessions were classified into four main clusters; cluster 1: H. venusta, H. tardiva, H. pycnophylla, H. tsushimensis `Ogon', H. montana, H. tibae, H. montana f. macrophylla, H. kikutii `Kikutii', H. longissima `Longifolia', H. rectifolia `Rectifolia', H. takahashii and H.`Undulata'; cluster 2: H. laevigata, H. sieboldiana, H. pycnophylla × H. longipes f. latifolia, H. longipes `Urajiro' and H. ibukiensis; cluster 3: H. capitata, H. kikutii `Polyneuron', H. nigrescens, H. kikutii `Yakusimensis', H. pachyscapa, H. kikutii `Caput-Avis', H. longipes f. latifolia, H. hypoleuca, H. okamotoi, H. densa and H. takiensis; and cluster 4: H. aequinoctiiantha, H. rupifraga, H. `Amanuma', H. minor and H. kikutii `Densa'.

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Arthur Q. Villordon and Don R. LaBonte

Clonal propagation assures the maintenance of genetic purity of a sweetpotato variety. The existence of foundation seed programs further contributes to the conservation of favorable genetic constitution in a commercial cultivar. However, the improvement of current maintenance procedures is necessary as shown by the occurrence of mutations and the decline of certain commercial varieties. Information on the nature and extent of changes in sweetpotato would therefore be useful in this regard.

`Jewel' clones obtained from eight state foundation seed programs were subjected to yield tests and a RAPD-based assay. Differences in nearly all yield grades were detected during the 1991, 1992, and 1993 seasons. The yield of U.S. No. 1 grade roots varied from 27% to 46%. The quality factors measured also varied: % alcohol insoluble solids varied by 13%, while sucrose ranged from 9.6% to 19%. Total DNA was extracted from each clone and assayed against 40 primers. All primers produced amplified fragments. A total of 110 reproducible bands was generated by 38 primers. Putative polymorphic markers were scored in 21 (18.58%) of these bands based on the presence or absence of amplified products. The results suggest an underlying cause for the variability observed in phenotypic traits within sweetpotato clones.

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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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Xiaofeng Yang and Carlos F. Quiros

To characterize the celery (Apium graveolens L. var. dulce, 2n = 2x = 22) genome, 126 celery cDNA clones and 340 random 10-mer primers were used to generate restriction fragment-length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers between two cultivated types. Different abundance classes of the genomic sequences represented by the cDNA clones and the RAPD markers were observed. Most of the cDNA clones were single-copy sequences, suggesting the true diploid nature of the celery genome. Nearly half of the 39 RAPD markers tested by Southern hybridization were multiple-copy sequences. Of the RAPD markers tested, 28% was single- and low-copy, and 26% was high-copy sequences. The polymorphism level of the cDNA clones was 23% when tested with four restriction enzymes (Eco RI, Eco RV, Hin dIII, and Hae III). A positive association was observed between RFLP level and the size of cDNA inserts or hybridized restriction fragments. Deletion, insertion, and base substitution were important in the formation of the RFLP markers. Eighty-two (23%) of the 340 primers tested yielded useful RAPD markers, but only 3.8% of the amplified products were polymorphic. Base substitution may be the most important mechanism for the RAPD markers in celery. The RAPD fragments revealed no RFLP markers when tested by Southern hybridization, implying that RAPD markers are an important complement to RFLP markers in genomic mapping in celery. Random methylation of cytosine was determined in 5S rDNA on Bam HI and Hin dIII cutting sites that produced ladder patterns characteristic of tandem repeats.