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Patrick J. Conner and Bruce W. Wood

Genetic variation among pecan [Carya illinoinensis (Wangenh.) C. Koch] cultivars was studied using randomly amplified polymorphic DNA (RAPD) markers. Using a combination of primers, a unique fingerprint is presented for each of the pecan genotypes studied. The genetic relatedness between 43 cultivars was estimated using 100 RAPD markers. Genetic distances, based on the similarity coefficient of Nei & Li, varied from 0.91 to 0.46, with an average value of 0.66 among all cultivars. The phenetic dendrogram developed from cluster analysis showed relatively weak grouping association. However, cultivars with known pedigrees usually grouped with at least one of the parents and genetic similarity estimates appear to agree with known genetic relationships.

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R.G. Fjellstrom, D.E. Parfitt, and G.H. McGranahan

RFLP markers were used to investigate genetic diversity among California walnut (Juglans regia) cultivars and germplasm collected worldwide. Sixteen of 21 RFLP markers were polymorphic in the 48 walnut accessions tested. RFLP markers were useful for identifying walnut cultivars. All genotypes were heterozygous at ≈20% of the loci for both California and worldwide germplasm. California walnut germplasm contained 60% of the worldwide allelic diversity. Cluster analysis of genetic distance between accessions and principal component analysis of allelic genotypes showed two major groups of walnut domestication. California germplasm was associated with germplasm from France, central Europe, and Iran and had less genotypic similarity with germplasm from Nepal, China, Korea, and Japan.

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Mario Crespo, James Nienhuis, Jan Tivang, and Paul Skroch

Knowledge of relative genetic distance among genotypes is useful in a breeding program because it permits organization of germplasm resources. Genetic distance (GD) was estimated among 113 faba bean, Vicia faba L. genotypes, which included three botanical varieties from different geographical areas around the world. The genotypes included 87 accessions from Bolivia, 14 accessions from the Middle East and North Africa, five accessions from Australia, and seven commercial varieties from Europe. Twenty-three RAPD primers were scored yielding four to 13 polymorphic bands resulting in a total of 165 bands. Our objective was to determine genetic relationships among accessions and cultivars as measured by RAPD markers. The genetic relationships were estimated using the ratio of discordant to total bands scored. A multidimensional scaling (MDS) plot indicated four clusters corresponding to: i) European commercial cultivars; ii) the Middle East, North Africa, and Australian accessions; iii) the Bolivian highland landraces; and iv) the Bolivian collection maintained in a valley environment. A permutation test confirmed the four clusters (P < 0.01). Sampling variance results indicated that a CV of 10% could be obtained with as few as 148 bands between groups. Selection and drift appears the main cause of divergence of two populations in the Bolivian faba bean collection. The results of this study indicated that RAPDs are a powerful tool for evaluation of germplasm conservation methods in faba bean.

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Antonio Figueira, Jules Janick, and Peter Goldsbrough

RAPD markers were used to examine genetic similarity in cacao. DNA from 30 cacao cultivars amplified using 15 arbitrary oligonucleotide primers, produced a total of 112 fragments, of which 88% were polymorphic. A phenogram was developed which illustrates the genetic relationships among the cacao cultivars representing the four major geographic groups of cacao (Criollo, Trinitario, Forastero Lower Amazonian, and Forastero Upper Amazonian). The phenogram indicated a general separation of the four groups into three clusters. Criollos and Trinitarios (supposedly hybrids between Forastero and Criollos types) appeared in a single cluster. Lower Amazonian cultivars (mainly selections made in Bahia, Brazil) appeared in a separate cluster. The third cluster consisted of the Upper Amazonian cultivars, which were originally collected from the region believed to be the center of origin of this crop. This cluster displayed the furthest genetic distance from the others. Crosses between Upper Amazon germplasm and local selections have shown heterosis in clonal crosses, which has been exploited in all genetic improvement programs for cacao. We propose that genetic distances based on RAPD markers can be potentially used as a criterion to select parents capable of producing superior hybrids and populations. Genetic relationships can also be useful to define germplasm collections and conservation strategies. Studies are underway to compare phenograms derived from RAPD markers and ribosomal RNA gene polymorphisms.

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A. Levi, C. E. Thomas, J. Thies, A. Simmons, Y. Xu, X. Zhang, O.U.K. Reddy, A. Davis, S. King, and T. Wehner

Genetic linkage map is being constructed for watermelon based on a testcross population and an F2 population. About 51.0% and 31.8% of the markers in the testcross and F2 populations are skewed form the expected segregation ratios. AFLP markers appeared to be clustered on linkage regions, while ISSR and RAPD markers are randomly dispersed on the genome. AFLP markers also have greater genetic distances as compared with ISSR and RAPD markers, resulting in significant increase of map distance. An initial genetic map (based on the testcross population) that contains 27 ISSR and 141 RAPD markers has a total linkage distance of 1,166.2 cM. The addition of 2 ISSR, 8 RAPD and 77 AFLP markers increased the genetic distance of the map to 2,509.9 cM. Similar results with AFLP markers were also shown in mapping experiments with an F2S7 recombinant inbred line (RIL) population that was recently constructed for watermelon. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and the F2 population. Experiments with SSR, and EST markers are being conducted to saturate the linkage map of watermelon genome.

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Peter Boches, Lisa J. Rowland, Kim Hummer, and Nahla V. Bassil

Microsatellite markers for blueberry (Vaccinium L.) were created from a preexisting blueberry expressed sequence tag (EST) library of 1305 sequences and a microsatellite-enriched genomic library of 136 clones.

Microsatellite primers for 65 EST-containing simple sequence repeats (SSRs) and 29 genomic SSR were initially tested for amplification and polymorphism on agarose gels. Potential usefulness of these SSRs for estimating species relationships in the genus was assessed through cross-species transference of 45 SSR loci and cluster analysis using genetic distance values from five highly polymorphic EST-SSR loci. Cross-species amplification for 45 SSR loci ranged from 17% to 100%, and was 83% on average in nine sections. Cluster analysis of 59 Vaccinium species based on genetic distance measures obtained from 5 EST-SSR loci supported the concept of V. elliotii Chapm. as a genetically distinct diploid highbush species and indicated that V. ashei Reade is of hybrid origin. Twenty EST-SSR and 10 genomic microsatellite loci were used to determine genetic diversity in 72 tetraploid V. corymbosum L. accessions consisting mostly of common cultivars. Unique fingerprints were obtained for all accessions analyzed. Genetic relationships, based on microsatellites, corresponded well with known pedigree information. Most modern cultivars clustered closely together, but southern highbush and northern highbush cultivars were sufficiently differentiated to form distinct clusters. Future use of microsatellites in Vaccinium will help resolve species relationships in the genus, estimate genetic diversity in the National Clonal Germplasm Repository (NCGR) collection, and confirm the identity of clonal germplasm accessions.

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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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Robert Griesbach* and Ronald Beck

The genetic distance for three Petunia species was determined based upon an intron in the chalcone synthase gene. The sequence of the intron was obtained for P. integriflolia ssp. integriflolia var. depauperata, P. integriflolia ssp. integriflolia Torres ecotype, P. altiplana and P. littoralis. These species are very closely related and believed by some taxonomists to be part of a large single species complex. In all the taxa, the intron contained multiple repeated and inverted sequences. The P. integriflolia ssp. integriflolia Torres ecotype intron differed from the P. integriflolia subsp. integriflolia var. depauperata intron in 3 of 930 nucleotides. While, the P. littoralis intron differed from the P. integrifolia subsp. integrifolia var. depauperata intron in 15 of 930 nucleotides. As compared to the P. integrifolia subsp. integriflolia var. depauperata intron, the intron in P. altiplana intron was longer (1125 bp), had a section of 338 nucleotides with a completely different sequence, and differed by 27 of 787 nucleotides in the common sequence.

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Angela K. Anderson and Chad E. Finn

Trailing blackberry cultivars, such as `Marion', can be traced to relatively few chance selections of Rubus ursinus Cham. & Schlecht. Wild R. ursinus offer a range of horticulturally desirable traits to breeders, from high fruit quality to improved cold hardiness. Cuttings from 460 plants, representing 20 populations in southern British Columbia, Washington, and Oregon, collected in 1993. Rooted clones were planted in 1994 in a replicated field trial to assess morphological variation. A greenhouse study was also undertaken, with 10 clones represented from each site, in two replications. Preliminary data from the greenhouse and field studies show variability in the following morphological characters: Glandular hairs; cane and prickle color; cane diameter; prickle density; internode length; leaf color, size, shape and density; and senescent leaf drop and color change. Floricane morphology will be assessed in 1995. Analysis of these data will determine relative genetic distances among the populations and enhance the understanding of the diversity available in R. ursinus.

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Winthrop B. Phippen, James R. McFerson, and Stephen Kresovich

Genetic variation and relationships in genetic resources collections can be assessed using molecular genetic markers. We examined the applicability of the RAPD assay for quick, cost-effective, and reliable use in improving collection management. Fourteen accessions of Brassica oleracea spp. capitata `Golden Acre' (cabbage) were screened using nine decamer oligonucleotide primers. We obtained 110 reproducible fragments, of which 80 were polymorphic, ranging in size from 370-1730 bp. Individual accessions were readily distinguished. A cluster analysis of genetic distances generated by bootstrapping reflected all known genetic relationships, except one. Bulking strategies were also investigated. RAPD markers can be applied to gene bank management to measure variation, identify accessions, and establish genetic similarity at the intra-specific level addressing the needs of both curators and users.