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S. Arulsekar and F. A. Bliss

Studies of genetic variation at the DNA level in the tree fruit and nut crop species of Prunus have been very limited. Recently molecular markers based on random amplified polymorphic DNA (RAPD) markers have been shown to be highly useful and efficient gene markers in other plant and animal species. We have used a total of 50 primers (10-mers) with arbitrary nucleotide sequence to identify cultivars of cherry, plum, apricot, peach and almond. A total of 120 accessions of different cultivars were assayed. The variation revealed by RAPD markers was highly species specific in the five Prunus species examined. High levels of polymorphism were observed for almond cultivars whereas sweet cherry revealed the lowest levels of polymorphism for the RAPD primers examined. The implications of these results in the germplasm diversity in the cultivated species of Prunus will be discussed.

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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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M.R. Foolad, S. Arulsekar, and F. Bliss

A genetic linkage map of Prunus has been constructed using an interspecific F2 population generated from self-pollinating a single F1 plant of a cross between a dwarf peach selection (54P455) and an almond cultivar (Padre). This map consists of approximately 80 markers including 10 isozymes. 12 plum genomic, 19 almond genomic and 40 peach mesocarp specific cDNA clones. The backbone map will be used for identifying the genomic locations and characterization of genes governing important economic traits in the genus Prunus. Of particular interests are those genes associated with fruit ripening and mesocarp development in peach and almond.

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F. Bartolozzi, M.L. Warburton, S. Arulsekar, and T.M. Gradziel

Almond [Prunus dulcis (Mill.) D.A. Webb, syn. P. amygdalus, Batsch; P. communis (I.) Archangeli] represents a morphologically and physiologically variable group of populations that evolved primarily in central and southwest Asia. California cultivars have been developed from highly selected subgroups of these populations, while new breeding lines have incorporated germplasm from wild almond and closely related peach species. The genetic relatedness among 17 almond genotypes and 1 peach genotype was estimated using 37 RAPD markers. Genetic diversity within almond was found to be limited despite its need for obligate outcrossing. Three groupings of cultivar origins could be distinguished by RAPD analysis: bud-sport mutations, progeny from interbreeding of early California genotypes, and progeny from crosses to genotypes outside the California germplasm. A similarity index based on the proportion of shared fragments showed relatively high levels of 0.75 or greater within the almond germplasm. The level of similarity between almond and the peach was 0.424 supporting the value of peach germplasm to future almond genetic improvement.

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

Randomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.

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

DNA from 9 cultivars and 5 `Pinot noir' clones were isolated with either the Delaporta or cTAB methods Twenty five 32P label led cloned probes were constructed with the pUC18 plasmid and Hind-III digested `Pinot noir' DNA. Standard methods of isolation and labelling were used. The probes were tested for efficacy of `fingerprinting' the 14 selections. rDNA and cloroplast a/h binding protein probes were also tested. The non-specific probes were not found to be useful as they bound to an excess number of sites and could not be removed from the southern blots, rendering them useless for further analysis. Grape specific probes bound at multiple sites, indicating that multiple fragments were incorporated into the plasmid vectors during library construction. With the greater variability observable with these multi locus probes, significant polymorphism was observed between cultivars, including `Cabernet sauvignon' and `Pinot noir' which were not distinguishable with GPI or PGM isozymes. Variability between clones of `Pinot noir' was observed with several probes, indicating that these selections are different. No variability had been observed at isozyme loci of the `Pinot noir' clones

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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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P. Martínez-Gómez, S. Arulsekar, D. Potter, and T.M. Gradziel

The genetic relationships among peach [Prunus persica (L.) Batsch], almond [P. dulcis (Mill.) D.A. Webb or P. amygdalus (L.) Batsch] and 10 related Prunus species within the subgenus Amygdalus were investigated using simple sequence repeat (SSR) markers. P. glandulosa Pall. was included as an outgroup. Polymorphic alleles were scored as present or absent for each accession. The number of alleles revealed by the SSR analysis in peach and almond cultivars ranged from one to three whereas related Prunus species showed a range of one to 10 alleles. Results demonstrated an extensive genetic variability within this readily intercrossed germplasm as well as the value of SSR markers developed in one species of Prunus for the characterization of related species. Mean character difference distances were calculated for all pairwise comparisons and were used to construct an unrooted dendogram depicting the phenetic relationships among species. Four main groups were distinguished. Peach cultivars clustered with accessions of P. davidiana (Carr.) Franch. and P. mira Koehne. The second group contained almond cultivars. A third group included accessions of P. argentea (Lam) Rehd., P. bucharica Korschinsky, P. kuramica Korschinsky, P. pedunculata Pall, P. petunikowii Lits., P. tangutica (Spach) Batal., and P. webbii (Spach) Vieh.. P. glandulosa and P. scoparia Batal. were included in a fourth group.