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A. Levi, C.E. Thomas, T. Trebitsh, A. Salman, J. King, J. Karalius, M. Newman, O.U.K. Reddy, Y. Xu, and X. Zhang

Seventy-one amplified fragment length polymorphism (AFLP), 93 sequence related amplified polymorphism (SRAP), and 14 simple sequence repeat (SSR) markers were used to extend an initial genetic linkage map for watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai]. The initial map was based on 151 randomly amplified polymorphic DNA (RAPD) and 30 and inter-simple sequence repeat (ISSR) markers. A testcross population previously used for mapping of RAPD and ISSR markers was used in this study: {plant accession Griffin 14113 [C. lanatus var. citroide (L.H. Bailey) Mansf.] × the watermelon cultivar New Hampshire Midget (C. lanatus var. lanatus)} × PI 386015 [C. colocynthis (L.) Schrad.]. The linkage map contains 360 DNA markers distributed on 19 linkage groups, and covers a genetic distance of 1976 cM with an average distance of 5.8 cM between two markers. A genomic DNA clone representing 1-amino-cyclopropane-1-carboxylic acid (ACC-) synthase gene, involved in ethylene biosynthesis, was also mapped. As in previous mapping studies for watermelon, a large number of AFLP and SRAP markers were skewed away from the 1:1 segregation ratio, and had to be excluded from the final mapping analysis. The stringent mapping criteria (JoinMap 3.0 mapping program) produced linkage groups with marker order consistent with those reported in previous mapping study for watermelon.

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Timothy A. Rinehart, Brian E. Scheffler, and Sandra M. Reed

Using 14 codominant microsatellite markers that amplify loci across 14 different Hydrangea L. species, we analyzed gene diversity and genetic similarity within Hydrangea. Samples also included Dichroa Lour., Platycrater Sieb. and Zucc., and Schizophragma Sieb. and Zucc. genera to establish their relatedness to Hydrangea species since previous work suggests they may be closely related. Our results support the close affiliation between Macrophyllae E.M. McClint. and Petalanthe (Maxim.) Rehder subsections and their separation from the other Hydrangea species. Most of the Hydrangea species analyzed cluster within their designated sections and subsections; however, genetic distance between species within each subsection varied considerably. Our data suggest that morphological analyses which labeled H. serrata (Thunb.) Ser. as a subspecies of H. macrophylla (Thunb. Ex J.A. Murr.) Ser. are probably more accurate than recent genome size data suggesting H. macrophylla ssp. macrophylla (Thunb.) Ser. and H. macrophylla ssp. serrata (Thunb.) Makino are separate species. Gene diversity estimates indicate that 64.7% of the total diversity is due to differences between species and 49.7% of the overall variation is due to differences between subsections. Low diversity suggests a lack of gene flow between species and subsections and underscores the difficulty in making wide hybrids. Since only 35.3% of the genetic variation is common to all species, unique alleles were used to develop a molecular key for unambiguous species identification and interspecific hybrid verification. Genetic similarity estimates for all 85 samples suggests a roadmap for introgressing horticulturally important traits from different Hydrangea species.

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James D. McCreight, Jack E. Staub, Anabel López-Sesé, and Sang-Min Chung

Genetic variation among 378 melon (Cucumis melo L.) germplasm accessions collected in India in 1992 and 26 accessions in China in 1994 was evaluated with 19 isozyme loci. `Top Mark' and `Green Flesh Honeydew', which represented two distinct C. melo ssp. melo L. groups, Cantalupensis and Inodorus, respectively, were used as reference cultivars. Genetic distances among accessions were calculated, and an initial cluster analysis using these distances resulted in 148 groups of varying size, ranging from two to 47 accessions. One accession from each of the 148 groups was chosen at random and used in a second cluster analysis that identified 11 accession groups. Group 1 was unique and consisted of only two C. melo ssp. agrestis (Naudin) Pangalo accessions. Two large branches were detected at cluster node 2. One branch was comprised of three groups of 3, 12, and 34 accessions, while the other branch contained seven groups of 2, 3, 14, 16, and 47 accessions, and the reference cultivars. Of the 148 accessions, 132 were from 41 sites in Rajasthan and Madhya Pradesh, India, which were distributed unequally across the 11 groups. The 14 Chinese accessions originating from seven provinces were also dispersed unequally in the four major cluster groups. `Top Mark' and `Green Flesh Honeydew' were genetically distinct and uniquely clustered in the same group. These results indicate that additional collections of melon germplasm should be made in eastern and southern India.

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Gerald S. Dangl, Keith Woeste, Mallikarjuna K. Aradhya, Anne Koehmstedt, Chuck Simon, Daniel Potter, Charles A. Leslie, and Gale McGranahan

One hundred and forty-seven primer pairs originally designed to amplify microsatellites, also known as simple sequence repeats (SSR), in black walnut (Juglans nigra L.) were screened for utility in persian walnut (J. regia L.). Based on scorability and number of informative polymorphisms, the best 14 loci were selected to analyze a diverse group of 47 persian walnut accessions and one J. hindsii (Jepson) Jepson ex R.E. Sm × J. regia hybrid (Paradox) rootstock. Among the 48 accessions, there were 44 unique multi-locus profiles; the accessions with identical profiles appeared to be synonyms. The pairwise genetic distance based on proportion of shared alleles was calculated for all accessions and a UPGMA (unweighted pair group method with arithmetic mean) dendrogram constructed. The results agree well with what is known about the pedigree and/or origins of the genotypes. The SSR markers distinguished pairs of closely related cultivars and should be able to uniquely characterize all walnut cultivars with the exception of budsports. They provide a more powerful and reliable system for the molecular characterization of walnut germplasm than those previously tested. These markers have numerous applications for the walnut industry, including cultivar identification, verification of pedigrees for cultivar and rootstock breeding programs, paternity analysis, and understanding the genetic diversity of germplasm collections.

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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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John McCallum, Susan Thomson, Meeghan Pither-Joyce, Fernand Kenel, Andrew Clarke, and Michael J. Havey

Bulb onion (Allium cepa L.) is a globally significant crop, but the structure of genetic variation within and among populations is poorly understood. We broadly surveyed genetic variation in a cultivated onion germplasm using simple sequence repeat (SSR) markers and sequenced regions flanking expressed sequence tag (EST)-SSRs to develop single-nucleotide polymorphism (SNP) markers. Samples from 89 inbred and open-pollinated (OP) bulb onion populations of wide geographical adaptation and four related Allium L. accessions were genotyped with 56 EST-SSR and four genomic SSR markers. Multivariate analysis of genetic distances among populations resolved long-day, short-day, and Indian populations. EST-SSR markers frequently revealed two major alleles at high frequency in OP populations. The median proportion of single-locus polymorphic loci was 0.70 in OP and landrace populations compared with 0.43 in inbred lines. Resequencing of 24 marker amplicons revealed additional SNPs in 17 (68%) and five SNP assays were developed from these, suggesting that resequencing of EST markers can readily provide SNP markers for purity testing of inbreds and other applications in Allium genetics.

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Metka Sisko, Branka Javornik, Aleksander Siftar, and Anton Ivancic

A sample of 94 pear (Pyrus L.) genotypes, traditionally present in the Slovenian landscape, was analyzed by amplified fragment length polymorphism (AFLP) and SSR (SSR), focusing on the assessment of genetic relationships. The analyzed samples involved germplasm of Pyrus communis L., P. nivalis Jacq., and P. pyraster L. The AFLP technique, using five EcoRI and MseI primer combinations, revealed molecular polymorphism at a level of 65.95%, representing 93 polymorphic bands among the total of 141 scored. With SSR analysis, 64 polymorphic alleles were found at seven microsatellite loci, with an average of 9.14 alleles per locus. Genetic distances among the individuals being studied were calculated using the Dice coefficient of similarity, and a dendrogram supported by AFLP and SSR data was constructed using the neighbor-joining method. The clustering method grouped the analyzed genotypes into three main clusters. The first cluster included the P. communis germplasm. The genotypes resembling P. nivalis were grouped in the second largest cluster, which could be divided in to four subclusters. The germplasm of P. pyraster, in this cluster, was found to be much less distinct than we had assumed. The most typical cultivar group in the third cluster was ‘Vinska Mostnica’. The study indicated that P. nivalis germplasm is frequently present in Slovenia, but not as a pure species.

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Jan G. Tivang, Neal DeVos, James Nienhuis, and Paul Skroch

Individual heads (capitula) from five discrete artichoke, Cyara scolymus L., populations were evaluated using RAPD markers. One vegetatively-propagated cultivar; Green Globe; two seed-propagated cultivars, Imperial Star and Big Heart XR-1; and two breeding populations were examined. Twenty-seven RAPD primers were scored yielding 2 to 16 polymorphic bands resulting in a total of 178 bands. Our objective was to determine if RAPD markers could be used to distinguish between and within populations. The genetic relationships among populations as well as among individuals within each population were estimated using the ratio of discordant to total bands scored. Data reduction (MDS) provided a plot indicating five clusters corresponding to the five populations. Confirmation of the presence of five discrete clusters was obtained by analysis of variance of the marker frequencies. The genetic diversity index (GDI) was calculated for each populations as the pooled variance of band frequency for each population. The GDI values were highly correlated to the mean genetic distance within each population. The homogeneity of variance for the GDI values associated with each population were compared using the Siegel-Tukey test for homogeneity of spread.

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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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Hongwen Huang and Desmond R. Layne

The pawpaw is the largest tree fruit native to the United States and the only temperate member of the tropical Custard Apple family (Annonaceae). In 1995, Kentucky State Univ. was established as the USDA-ARS-National Clonal Germplasm Repository for Asimina spp. Seedling trees from 400 pawpaw accessions representing 70 distinct geographic regions from 17 states are currently being grown at our research farm. In a preliminary study, 18 pawpaw cultivars were assayed in 30 enzyme systems using an isoelectric focusing polyacrylamide slab gel system of pH 4-9. Twelve enzymes produced high resolution without tissue specificity and were further used for evaluation of allozyme diversity of geographic populations. Degree of genetic diversity within populations and differentiation between populations as evaluated by the expected heterozygosity (He), the proportion of polymorphic loci (P), the average number of alleles per locus (A), chi-squared analysis of allele frequency heterogeneity, Nei's standard genetic distance (D), and identity (I) will be discussed. Dendrograms were generated by cluster analysis using the unweighted pair group method to demonstrate the relationships of geographic populations in the 17 states evaluated. The strategy for germplasm conservation and cultivar development through breeding will also be discussed.