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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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A. Fabbri, J.I. Hormaza, and V.S. Polito

Seventeen olive (Olea europaea L.) cultivars, including oil and table olive cultivars originating from throughout the Mediterranean area, were screened using random amplified polymorphic DNA (RAPD) markers. The results indicate that a high degree of polymorphism is evident in the olive germplasm reexamined. Forty random decamer primers were screened; seventeen of these produced 47 reproducible amplification fragments useful as polymorphic markers. Each of the 17 cultivars can be discriminated with a few primers. Results were analyzed for similarity among the cultivars and a cluster analysis was performed. These analyses revealed two main groups: one comprising primarily small-fruited cultivars grown mainly for oil production, and the other characterized by having large fruit. There was no apparent clustering of olive cultivars according to their geographic origins.

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Salih Kafkas, Hakan Ozkan, and Mehmet Sutyemez

Turkey has more than 4 million walnut trees (Juglans regia L.), most of which are derived from seedlings, and are nongrafted trees. This characteristic leads to a huge opportunity to select superior walnut genotypes from natural populations for cultivation and for breeding programs. Several selection studies have been performed in the last decades and few genotypes were selected. The goal of this study was to characterize and determine genetic relationships among 21 walnut genotypes with potential in walnut production using amplified fragment length polymorphism (AFLP) and selective amplification of microsatellite polymorphic loci (SAMPL) techniques. Eight primer combinations (six for AFLP and two for SAMPL) were applied to 21 walnut genotypes and a total of 230 bands of which 50.4% of them were polymorphic were obtained. The SAMPL technique was more effective than AFLP in the separation of very closely related genotypes. Genotypes of the pairs `Maras-18' with `Maras-46', `KSU-5' with `Sutyemez-1', `Maras-12' with `Sutyemez-2,' `Kaman-3' with `Kaman-4', and `KSU-11' with `Maras-10' were the most closely related.

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Alan T. Bakalinsky, Hong Xu, Diane J. Wilson, and S. Arulsekar

A total of eight random amplified polymorphic DNA (RAPD) markers were generated in a screen of 77 primers of 10-base length and were detected reproducibly among nine different grape (Vitis) rootstocks. Occasional failed amplifications could not be explained rationally nor easily corrected by systematic replacement of individual reaction components. In an effort to improve their reliability, the RAPD markers were cloned, their termini sequenced, and new sequence-specific primer pairs were synthesized based on addition of 10 to 14 bases to the 3' termini of the original 10-mers. Six pairs of the new primers were evaluated at their optimal and higher-than optimal annealing temperatures. One primer pair amplified a product the same size as the original RAPD marker in all rootstocks, resulting in loss of polymorphism. Post-amplification digestion with 7 different restriction endonucleases failed to reveal restriction site differences. Three primer pairs amplified an unexpected length variant in some accessions. Two other pairs of primers amplified a number of unexpected bands. Better approaches for exploiting the sequence differences that account for the RAPD phenomenon will be discussed.

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M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano

The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assessed using DAF. Thirteen cultivars of chrysanthemum included in the study were members of the following series: Charm (five), Davis (four), and Pomona (four). The genetic variability within and between series were evaluated using 11 arbitrary octamer primers. A few polymorphic loci were evident that uniquely identified closely related cultivars within a series. In contrast, there were many polymorphisms between members of different series. Genetic distances between cultivars within and between series were calculated using marker comparison and UPGMA (cluster analysis). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series also were bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique possibly could be used for patent protection and phylogenetic studies and may be useful in breeding for chrysanthemums.

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B. Sosinski and D.S. Douches

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

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M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano

The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assess using DAF. Twenty-three cultivars of chrysanthemum included in the study were members of the following series: Anne (3), Blush (3), Boaldi (4), Charm (5), Davis (4), and Pomona (4). The genetic variability within and between series were evaluated using 11 arbitrary octamer primers. A few polymorphic loci were evident that uniquely identified closely related cultivars within a series. In contrast, many polymorphisms were observed between members of different series. Genetic distances between cultivars within and between series were evaluated using marker comparison and analyzed with PAUP (phylogenic analysis using parsimony) and UPGMA (unweighted pair group cluster analysis using arithmetic means). The average distance between series was 10-fold greater than between cultivars within a series. Furthermore, series with similar flower morphology, pompon or daisy-like, were more closely related than those with different phenotypes. DNA from all cultivars belonging to a series were also bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique can possibly be used for patent protection and phylogenetic studies, and may be useful in breeding chrysanthemums.

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Dongyan Hu*, Zuoshuang Zhang, Donglin Zhang, and Qixiang Zhang

Ornamental peach (Prunus persica (L.) Batsch) is native to China. The ornamental value of peach is gaining popularity for its use in urban landscape and everyday gardens. However, the genetic relationship among ornamental peach cultivars is not clear, which limits the further studies of its molecular systematic. A sample of 51 cultivars of ornamental peach, originated from P. persica and Prunus davidiana, had been studied by using AFLPs. All samples were collected from China, Japan, and the US. A total of 275 useful markers between 75 to 500 base pairs were generated from 6 EcoRI/MseI AFLP primer combinations. Among them, 93% of bands were polymorphic markers. Total markers for each cultivar ranged form 90 to 140, and the average number of markers for each cultivar was 120. Two distinguished clad generated from PAUP-UPGMA tree. P. davidiana, as a species, was apparently an out-group to P. persica, which implied that P. davidiana was far away genetically from ornamental peach (P. persica). Within P. persica clad, 15 out of 17 upright ornamental peach cultivars in this study were grouped to one clad, which indicated cultivars that with upright growth habit had close genetic relationship. Five dwarf cultivars were grouped to one clad, with 81% bootstrap supported. The genetic relationships between these five dwarfs were much closer than any other cultivars, and showed that they probably derived from the similar gene pool. The results demonstrated that AFLP are powerful markers for revealing genetic relationships in ornamental peach. The genetic relationships among ornamental cultivars established in this study could help future ornamental peach germplasm identification, conservation, and new cultivars development.

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Fenny Dane and Hongwen Huang

The genetic diversity within and between four geographic populations of the Ozark chinkapin was evaluated and partitioned in order to gain an understanding of the overall genetic diversity and structure of this species, which will be instrumental for its preservation and germplasm enhancement. Nuts of chinkapin trees along the natural range of the species in the Sylamore Ranger District of the Ozark National Forest in Arkansas were collected and evaluated with isozyme and RAPD markers scattered across the genome. Allozyme differences were detected among the geographic populations. Allele frequencies will be determined and subjected to genetic diversity statistics. A conservation plan will be recommended.

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Dror Sharon, Avital Adato, Samir Mhameed, Uri Lavi, Jossi Hillel, Maria Gomolka, Conny Epplen, and Jorg Thomas Epplen

Plant genomes contain polymorphic repetitive sequences that can be used as DNA markers. Minisatellites (16 to 64 bp per repeat) and simple-sequence repeats (2 to 6 bp per repeat) are the most polymorphic markers found in plant and animal genomes. In this study, the hybridizations between genomic DNA and variable number of tandem repeat probes were examined in Arabidopsis thaliana L. (Heynn), onion (Allium cepa L.), tomato (Lycopersicon esculentum L.), wheat (Triticum aestivum L.), avocado (Persea americana Mill.), litchi (Chinensis Sonn.), mango (Mangifera indica L.), and Carica species. Some of the probes detected polymorphic sequences in all the species, but others were useful only for one or two species. None of the probes gave clear band patterns in either onion or wheat. The in-gel hybridization method was similar to Southern blot hybridization using the simple-sequence repeat probes.