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M. Mcharo, D. LaBonte, R.O.M. Mwanga, and A. Kriegner

Molecular markers linked to resistance to sweetpotato chlorotic stunt closterovirus [SPCSV (genus Crinivirus, family Closteroviridae)] and sweetpotato feathery mottle virus [SPFMV (genus Potyvirus, family Potyviridae)] were selected using quantitative trait loci (QTL) analysis, discriminant analysis and logistic regression. Eighty-seven F1 sweetpotato [Ipomoea batatas (L.) Lam.] genotypes from a cross of `Tanzania' and `Wagabolige' landraces were used to generate DNA marker profiles for this study. Forty-five of the clones were resistant to SPCSV while 37 were resistant to SPFMV. A combination of 232 amplified fragment length polymorphism (AFLP) markers and 37 random amplified polymorphic DNA (RAPD) markers obtained were analyzed to determine the most informative markers. All three statistical procedures revealed that AFLP marker e41m33.a contributed the greatest variation in SPCSV resistance and RAPD marker S13.1130 accounted for most of the variation in SPFMV resistance. The power of discriminant and logistic analyses is that you do not need a parent-progeny population. An evaluation of these two models indicated a classification and prediction accuracy rates of 96% with as few as four markers in a model. Both multivariate techniques identified one important discriminatory marker (e44m41.j) for SPCSV and two markers (e41m37.a and e44m36.d) for SPFMV that were not identified by QTL analysis.

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Ryutaro Tao, Tsuyoshi Habu, Hisayo Yamane, Akira Sugiura, and Kazuya Iwamoto

Self-compatible cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) have a horticultural advantage over self-incompatible ones because no pollinizer is required. Self-incompatibility is gametophytic, as in other Prunus species. We searched for molecular markers to identify self-compatible cultivars based on the information about S-ribonucleases (S-RNases) of other Prunus species. Total DNA isolated from five self-incompatible and six self-compatible cultivars were PCR-amplified by oligonucleotide primers designed from conserved regions of Prunus S-RNases. Self-compatible cultivars exhibited a common band of ≈1.5 kbp. Self-compatible cultivars also showed a common band of ≈12.1 kbp when genomic DNA digested with HindIII was probed with the cDNA encoding S 2-RNase of sweet cherry (Prunus avium L.). These results suggest that self-compatible cultivars of Japanese apricot have a common S-RNase allele that can be used as a molecular marker for self-compatibility.

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Zsolt Galli, Gábor Halász, Erzsébet Kiss, László Heszky, and Judit Dobránszki

A collection of 66 commercial apple (Malus ×domestica Borkh.) cultivars was screened with six previously described SSR (Simple Sequence Repeat) markers for molecular identification. In total, 55 polymorphic alleles were detected at the 6 SSR loci (average 9.2 alleles per locus) and the polymorphism information content (PIC) averaged 0.72. Successful differentiation of all apple genotypes except for somatic mutants was accomplished by using only four (CH03g07, CH04e03, CH05d11, and CH05e03) SSR markers. Sport mutants proved to be indistinguishable from each other and their progenitors. The cumulative probability of obtaining an identical SSR profile for two randomly chosen apple genotypes was 1.79 × 10–4, which confirms the high potential of simple sequence repeats (SSRs) for cultivar identification.

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M.J. Striem, G. Ben-Hayyim, and P. Spiegel-Roy

Excluding seeded offspring at an early stage could be of great value to the breeder concerned with the development of seedless grapes (Vitis vinifera L.). We used the random amplified polymorphic DNA (RAPD) technique to identify molecular genetic markers, analyzing 82 individuals of a progeny resulting from a cross between `Early Muscat' (seeded) and `Flame Seedless'. Seven variables representing the traits of seedlessness were analyzed: mean fresh weight of one seed, total fresh weight of seeds per berry, perception of seed content, seed size categories evaluated visually, degree of hardness of the seed coat, degree of development of the endosperm, and degree of development of the embryo. Among 160 10mer primers, 110 gave distinct band patterns. Twelve markers yielded significant correlations with several subtraits of seedlessness, mainly with the mean fresh weight of one seed and the total fresh weight of seeds per berry. Multiple linear regression analysis resulted in high coefficients, such as R = 0.779 for fresh weight of seeds per berry, when the seven markers were included as independent variables in the model. Most of the seeded individuals, about 44% of the progeny, could be excluded using a two-step process of marker assisted selection.

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Y. Gogorcena, S. Arulsekar, and D.E. Parfitt

The work reported here is an extension of studies reported in 1990. The general objective was to develop molecular markers for genotype `fingerprinting', with specific reference to possible clonal differences among `Pinot noir' clones. Leaf DNA from 8 cultivars and 9 `Pinot noir' clones were isolated. RFLP and RAPD markers were identified and used to characterize the genotypes. 65 32-P labelled cloned probes were constructed with the pUC18 plasmid and Hind-III digested `Pinot noir' DNA. The probes were tested for their ability to discriminate among the 8 cultivars. 3 probes pGAD10, pGAD15, and pGAD44 showed polymorphisms among the cultivars. pGAD15 was most useful, with 5 polymorphisms for the 8 cultivars. RAPD makers were also tested for `fingerprinting'. Several primers were tested and polymorphisms were identified among cultivars. However, significant problems with repeatability for some bands were observed. Therefore, a series of experiments were conducted to test the effect of season and extraction method. These factors did not account for the inconsistancy which seemed to be more a function of the primer used. None of these studies showed clear evidence that the `Pinot noir' clones tested were geetically different.

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

about relatedness between genotypes based on phenotypic markers are not always reliable. More appropriate are molecular markers, which appear to be more stable. In the genus Pyrus , several studies have been carried out using molecular markers to

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Eric T. Stafne, John R. Clark, and Kim S. Lewers

A tetraploid blackberry population that segregates for two important morphological traits, thornlessness and primocane fruiting, was tested with molecular marker analysis. Both randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to screen a population of 98 genotypes within the population plus the two parents, `Arapaho' and `Prime-Jim' (APF-12). RAPD analysis averaged 3.4 markers per primer, whereas SSR analysis yielded 3.0 markers per primer pair. Similarity coefficient derived from the Dice index averaged over all individuals was 63% for RAPD markers, 73% for SSR markers, and 66% for RAPD and SSR markers together. The average similarity coefficients ranged from a high of 72% to a low of 38% for RAPD markers, 80% to 57% for SSR markers, and 73% to 55% for both. Comparison of the parents indicated a similarity of 67% for RAPD markers, 62% for SSR markers, and 67% for both. This is similar to a previous study that reported the similarity coefficient at 66%. Although inbreeding exists within the population, the level of heterozygosity is high. Also, evidence of tetrasomic inheritance was uncovered within the molecular marker analysis. This population will be used to identify potential markers linked to both morphological traits of interest. Further genetic linkage analysis and mapping is needed to identify any putative markers.

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David Byrne

Fruit and ornamental breeders were surveyed about their use of molecular markers in either their breeding programs or in their related research programs. Responses were obtained from over 100 fruit and ornamental breeding programs from throughout the world. Of these, less than 50% used molecular markers in their programs. The two most common uses of these markers were for studies in plant identification and diversity. These were followed by the use of markers in developing molecular maps, in discovering molecular tags and/or trying to identify the genes for specific plant traits, for marker assisted selection, and finally, for the elucidation of plant taxonomy. In conclusion, although there is much research in this area, few programs are actually using markers in the context of an applied breeding program. The major reason for this situation is the lack of available markers and the cost of using these markers to screen large numbers of progeny. Those that use markers in their breeding tend to use them to verify the genotype of the parents or confirm the genotype of selected seedlings rather than screen unselected seedlings.

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James D. Kell

55 COLLOQUIUM 2 (Abstr. 995-999) Classical and Molecular Approaches to Breeding Horticultural Plants for Disease Resistance

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Vance Baird

Prunus, which includes peach/nectarine, almond, apricot, cherry, and plum, is a large and economically important genus in the family Rosaceae. The size and long generation time of these tree crops have hampered improvement through classical breeding and long-term selection programs. With the advent of DNA-based molecular diagnostics, an exciting era in germplasm improvement has dawned. Efforts are underway, notably in the United States (e.g., California, Michigan, North Carolina, and South Carolina) and the European Community (e.g., England, France, Italy, and Spain), to apply the tools of molecular mapping and marker-assisted selection to this important genus. The objective of these projects is to develop molecular linkage maps of sufficient marker density to tag phenotypic trait loci of agronomic importance. These include traits controlled by single genes (e.g., flower color, compatibility, flesh color, pest resistance), as well as more-complex, quantitative traits (e.g., cold hardiness, tree architecture, sugar content). An immediate outcome of these mapping efforts has been the development of DNA “fingerprints,” allowing for the discrimination of cultivars—both scion and rootstock. The maps will be used by breeders and molecular biologists to monitor gene introgression from wild species into elite lines, for marker-assisted selection of desired trait combinations, and for map-based cloning of specific genes. The molecular markers used in these mapping projects include RFLPs, RAPDs, and microsatellites. Each has their appropriate applications and advantages depending upon the resources at hand and the project's specific goals.