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Dror Sharon, Jossi Hillel, Samir Mhameed, Perry B. Cregan, Emanuel Lahav, and Uri Lavi

The detection of association between DNA markers and traits of interest in an outbred population is complicated and requires highly polymorphic markers. A genetic linkage map of avocado (Persea americana Mill.) recently generated consists of simple sequence repeat (SSR) markers as well as DNA fingerprint (DFP) and randomly amplified polymorphic DNA (RAPD) markers. These markers were used to detect putative quantitative trait loci (QTLs) of eight avocado fruit traits. Two statistical methods were used: one-way analysis of variance and interval mapping. Six traits were found to be associated with at least one of the 90 DNA markers. Based on the two statistical approaches, a putative QTL associated with the presence of fibers in the flesh, was found to be located on linkage group 3. This putative QTL was found to be associated with the SSR marker AVA04 having a high significant value (P = 4.4 × 10-8). The haplotype analysis of linkage group 3 showed a putative dominant interaction between the alleles of this locus.

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James A. Schrader and William R. Graves*

Long regarded as a genus of two species, Dirca L. was expanded to include a third North American shrub discovered in 1994 as one population in the Sierra Madre Oriental of Tamaulipas in northeastern Mexico. The designation of Dirca mexicana Nesom & Mayfield as a third species in the genus was based in part on geographical separation from Dirca palustris L. and Dirca occidentalis Gray, which occur farther north in eastern North America and in a small region of California, respectively. Morphologically, D. mexicana was regarded as more similar to D. occidentalis than to D. palustris. Our objectives were to obtain fruits of all species, germinate seeds, and compare the three species genetically through analyses of seedling DNA. Drupes of D. mexicana, D. palustris (from populations in Iowa), and D. occidentalis were collected as they abscised naturally from plants in native habitats in mid-May, late May to early June, and mid-June, respectively. Embryo extraction, gibberellin, and cold stratification were used to promote germination, and DNA was extracted from leaves of seedlings by using the fully automated Autogen Autogenprep 740 DNA extraction system. Genomic DNA templates were used to compare sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of the nuclear ribosomal DNA repeat and to examine polymorphisms in inter-simple sequence repeats (ISSRs). These analyses reinforce the present morphologically based classification of the three Dirca species by confirming species-level divergence at the molecular level. ITS sequences and ISSR banding patterns also enabled us to reconstruct the phylogenetic relationship among the three extant species of Dirca.

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Xiaomeng Li, Rangjin Xie, Zhenhua Lu, and Zhiqin Zhou

of cultivated citrus were mainly based on morphological, biochemical, and isozyme data ( Barrett and Rhodes, 1976 ; Fang, 1993 ; Scora, 1988 ; Torres et al., 1978 ). Recently, DNA markers such as restriction fragment length polymorphisms (RFLPs

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W. Vance Baird, Agnes S. Estager, and John K. Wells

Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera [Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)], two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60±0.03 pg (or 5.8×108 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (× = 8).

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C.J. Simon and N.F. Weeden

The ribosomal genes of the two crab apple (Malus) genotypes White Angel' and `Robusta 5' were characterized to determine the extent of between- and within-genotype heterogeneity. Initial investigations with a cloned sequence of soybean rDNA failed to detect some Malus intergenic spacer region fragments. An alternative probing method that used electrophoretically purified Malus rDNA was developed. Double-digests of total genomic DNA with combinations of 13 restriction endonucleases identified the positions of 35 restriction sites. Restriction site polymorphism was observed both between and within the crab apple genotypes. Ribosomal DNA from White Angel' was cloned in phage and plasmid vectors and mapped with 11 enzymes. The region of the spacer causing length heterogeneity was identified. These clones should be useful as genetic markers and for examining population dynamics and systematic of Malus and closely related taxa.

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Channapatna S. Prakash, Guohao He, and Robert L. Jarret

Highly polymorphic DNA markers were identified in sweetpotato (Ipomoea batatas) using PCR amplification and arbitrary primers. More than 100 accessions representing US cultivars and their progenitors, and germplasm lines from around the world were analyzed. Sweetpotato germplasm exhibited high genetic variability and individual-specific profiles were obtained for all accessions. US cultivars formed a tight cluster in the principal coordinate analysis suggesting a narrow genetic base. The genetic relationship data of US cultivars and their progenitors based on DNA polymorphisms was in agreement with their known pedigree. The putative paternal parents of certain cultivars selected through open pollination were identified based on shared polymorphisms. The PCR-based markers are valuable in the characterization of sweetpotato germplasm and in ensuring a broad genetic base for future cultivars.

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C.L. Boehm, I.K. Lee, G. Jung, H.C. Harrison, J. Nienhuis, M. Sass, and Moore Hall

Random amplified polymorphic DNA (RAPD) may have utility as genetic markers facilitating selection in ginseng crop improvement. This experiment determined chemical buffer and root tissue-type combinations that yield repeatable bands. The results allow further experiments using RAPD markers for estimating the genetic distance between ginseng landraces, selection for crop improvement, and extensive fingerprinting for use in determining the origin of tissue samples. This experiment determined mean band yields for all combinations of dry, fresh, and powdered root with cetyltrimethylammonium bromide, potassium/sodium ethyl xanthogenate, and urea buffers. The buffers were applied in replication to the tissue-types with other extraction protocol factors constant. Replications were amplified four times with four different primers using constant PCR and agarose gel electrophoretic protocols. Distinct bands were counted in each replication, and the summation of the replication repeats considered an observation. Least squares means for several response variables were analyzed. The most significant difference found was between buffers. The buffers ctab and urea were productive, and the pex was not. Significant difference was found when buffers were crossed with tissue. The applications of urea to fresh root, ctab to dry root, urea to dry root, and ctab to powdered root were productive. Based on these results we conclude 1) urea and ctab are productive when applied to all tissue-types, 2) dry root, which is easily collected and stored, yields sufficient DNA for analysis, and 3) powdered root, often the form of commercial products that might be tested for genetic origin, will yield sufficient DNA for analysis.

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Valentina Scariot, Aziz Akkak, and Roberto Botta

Six polymorphic sequence-tagged microsatellite sites (STMSs) were used to characterize 65 accessions of old garden roses [OGRs (Rosa L. spp.)] from seven botanical sections and 13 horticultural groups. Aims of the study were to define the genetic profiles of accessions and to provide information useful for the classification and pedigree reconstruction of OGRs. In roses, a precise botanical classification is difficult due to repeated hybridization carried out in breeding; OGRs are classified in horticultural groups on the basis of their original parentage or of their morphological traits. A total of 82 alleles were detected at six loci. The number of alleles per locus ranged from six to 21, with an average of 13.7 alleles per locus. A dendrogram was constructed by cluster analysis, displaying the relative genetic similarities between species' accessions, hybrids, and cultivars. Cluster analysis grouped the genotypes into seven major clusters that were substantially consistent with their classification into botanical sections and horticultural groups. Several hypotheses of apportionment of accessions to horticultural groups were evaluated on the basis of the relative position in the dendrogram of the analyzed individuals. Results demonstrated that DNA analyses can contribute to drawing the botanic classification of rose accessions, improving the genetic knowledge on the background of modern rose, and providing the basis for breeding programs.

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Carlos A. Urrea, Phillip N. Miklas, James S. Beaver, and Ronald H. Riley

Molina, José Vélez, and Maximo Halpay for assisting with greenhouse disease screening; and Nada Abbas for assisting with randomly amplified polymorphic DNA analyses. The cost of publishing this paper was defrayed in part by the payment of page charges

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Patricia M. Sweeney and T. Karl Danneberger

As the number of perennial ryegrass (Lolium perenne L.) cultivars increases, the development of reliable identification methods becomes more important. Randomly amplified polymorphic DNA (RAPD) markers show promise in cultivar identification. Since perennial ryegrass cultivars are composites of genotypes rather than a single genotype, finding markers that distinguish cultivars is difficult. The ideal cultivar identification procedure would use seed tissue as a DNA source and evaluate a single sample as representative of a cultivar. The objective of this research was to determine whether RAPD markers could be used to consistently distinguish bulk seed samples of perennial ryegrass cultivars. Two extraction protocols were evaluated. A quick, simple extraction resulted in the amplification of few consistent RAPD markers. The more labor-intensive extraction with hexadecyltrimethyl ammonium bromide (CTAB), however, produced more reliable RAPD markers. Eight of 11 cultivars were distinguished by using RAPD markers produced using bulk seed samples together with four of 30 primers that were screened. These results show the potential of RAPD markers to provide the turfgrass industry, breeders, and certification agencies additional options to ensure the genetic integrity of perennial ryegrass seed lots and cultivars.