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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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Amnon Levi, Lisa J. Rowland, and John S. Hartung

A procedure for identifying reproducible RAPD markers from woody plant DNA is presented. The procedure relies on using a PCR buffer that contains 1% Triton-X-100 and 0.1 % gelatin [previously described for successful polymerase chain reaction (PCR) amplification of 16S/23S rRNA intergenic spacer regions from eubacteria], and amplification conditions of 50 cycles: 30 sec at 94C, 70 sec at 48C, and 120 sec at 72C. The combination of this buffer and these conditions amplified consistent fragments in higher amounts, as compared to other standard PCR buffers and conditions generally used for RAPD analysis. This procedure resulted in reliable RAPD patterns for all organisms tested. Chemical name used: α-[4-(1,1,3,3,-tetramethylbutyl)phenyl]-cohydroxypoly(oxy-l,2-ethanediyl) (Triton-X-l00).

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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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A.M. Torres, T. Millán, and J.I. Cubero

Five rose (Rosa spp.) cultivars were analyzed using random amplified polymorphic DNA (RAPD) markers. Using eight primers, all cultivars were distinguished by comparing differences in DNA banding patterns. The RAPD technique fingerprints rose cultivars rapidly and inexpensively for identification and patent protection purposes.

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Rodomiro Ortiz, D.E. Costich, T.P. Meagher, and N. Vorsa

DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.

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W. Patrick Wechter, Ralph A. Dean, and Claude E. Thomas

PI161375 DNA for this project. This work was supported by USDA—ARS and Clemson Univ. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked

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Sandy Lin, Hsiao-Ching Lee, Wen-Huei Chen, Chi-Chang Chen, Yen-Yu Kao, Yan-Ming Fu, Yao-Huang Chen, and Tsai-Yun Lin

Nuclear DNA contents were estimated by flow cytometry in 18 Phalaenopsis Blume species and Doritis pulcherrima Lindl. DNA amounts differed 6.07-fold, from 2.74 pg/diploid nuclear DNA content (2C) in P. sanderiana Rchb.f. to 16.61 pg/2C in P. parishii Rchb.f. Nuclear DNA contents of P. aphrodite Rchb.f. clones, W01-38 (2n = 2x = 38), W01-41 (2n = 3x = 57), and W01-22 (2n = 4x = 76), displayed a linear relationship with their chromosome numbers, indicating the accuracy of flow cytometry. Our results also suggest that the 2C-values of the Phalaenopsis sp. correlate with their chromosome sizes. The comparative analyses of DNA contents may provide information to molecular geneticists and systematists for genome analysis in Phalaenopsis. Endoreduplication was found in various tissues of P. equestris at different levels. The highest degree of endoreduplication in P. equestris was detected in leaves.

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Stacie L. Aragon, Keng-Chang Chuang, and Adelheid R. Kuehnle

Isolation of high quality nucleic acids from aroids can be difficult due to the presence of carbohydrates, phenolics, and other compounds that bind to and/or co-precipitate with the DNA or RNA. Methods previously used for marine algae, mango, and papaya were modified and successfully used for the simultaneous isolation of high quality genomic DNA and RNA from Anthurium, Colocasia, and Spathiphyllum leaves. Genomic DNA yields averaged 477 μg·g-1 fresh weight for Anthurium and 322 and 177 μg·g-1 fresh weight, respectively, for Colocasia and Spathiphyllum. Total RNA yields averaged 129 μg·g-1 fresh weight for Anthurium and 61 and 50 μg·g-1 fresh weight, respectively, for Colocasia Spathiphyllum. This method may be useful in co-isolating high quality nucleic acids from additional aroids and other plants.

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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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Patricia Sweeney, Robert Golembiewski, and Karl Danneberger

Random amplified polymorphic DNA (RAPD) markers from leaf tissue extractions are effective for discrimination of turfgrass varieties. The usefulness of RAPD markers for turfgrass variety identification can be enhanced by use of seed rather than leaf tissue for DNA extraction. To determine whether DNA extracted from turfgrass seed was suitable for amplification, DNA was extracted from bulk samples and individual seeds of bermudagrass [Cynodon dactylon (L.) Pers.], chewings fescue (Festuca rubra var. commutata Gaud.), Poa annua L., Poa supina Schrad., creeping bentgrass [Agrostis stolonifera L. var. palustrus (Huds.) Farw.], Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.). All samples were successfully amplified using an arbitrary primer. Amplification intensity varied among species. With an almost infinite number of arbitrary primers available, it is likely that suitable primers can be found to amplify DNA from most turfgrass species. Amplification of turfgrass seed DNA, whether bulk or individual seed, is possible and should prove more useful than amplification of leaf tissue DNA for discrimination of turfgrass varieties.