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D.E. Parfitt

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R. Fjellstrom and D.E. Parfitt

RFLP probes were developed to determine the degree of genetic diversity both within and between 12 walnut species (Juglans spp.), including the widely cultivated English walnut (J. regia). One to three kilobase DNA fragments from Pst I digested J. regia nuclear DNA were cloned using the vector pUC18. Inserts corresponding to low copy number walnut genomic sequences were used to assess the genetic variability among walnut species. Extensive polymorphism was found between species and limited polymorphism within species. The inheritances of the RFLP loci are being analyzed to provide a genetic basis for the polymorphisms detected and to establish a RFLP based linkage map in walnuts

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Y. Gogorcena and D.E. Parfitt

A number of 10 base primers were screened to identify RAPD polymorphisms among a population of semi wild apricot genotypes that had been collected by Maxine Thompson in 1988. 30 families collected from trees at 6 locations were analyzed. DNA from leaf tissue of 180 plants, ca. 6 genotypes per family, were isolated and tested against 20 primers. Seven primers were identified that produced consistent results with relatively few (thus, scoreable) and consistent bands. DNA was isolated using the cTAB method and the effects of additional CsCl centrifugation isolation were tested. No differences were found. Reaction conditions were tested to ensure consistent results. Considerable RAPD polymorphism was observed in this population. Parsimony analysis is being conducted to assess the relative variation among and within populations and to determine whether collection location had a more significant effect on DNA variation than other factors such as outcrossing or level of heterogeneity within populations.

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R. Fjellstrom and D. E. Parfitt

RFLP analysis was employed to study the inheritance of and genetic diversity identified by cloned walnut genomic probes. An interspecific backcross population of (J. hindsii × J. regia) × J. regia was used to determine the inheritance of thirty low copy number RFLP cloned probes. Of these probes, approximately 20% correspond to single copy loci, 40% correspond to single major loci with multiple minor loci, and 40% correspond to two major loci. Twenty of these probes were used to analyze variability within and between 13 walnut species (Juglans spp.). Substantial genetic variation was identified within many wild walnut species, while limited variation was identified within butternut (J. cinerea) and the widely cultivated English walnut (J. regia). Extensive polymorphism was found between walnut species, allowing a phylogenetic relationship of walnuts based upon RFLP markers to be developed. Identification of clonally propagated walnut cultivars by RFLP typing was readily performed in black walnut (J. nigra) accessions, was more difficult in English walnut accessions, and rarely possible in butternut accessions.

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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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D.E. Parfitt and A.A. Almehdi

An improved medium for the in-vitro micropropagation of P. vera genotypes was developed. Several basal medium preparations were tested and DKW (Driver-Kuniyuki-Walnut) medium was selected. Macro and micronutrients were adjusted to provide optimum growth and multiplication of shoots, some of which have been grown and multiplied for more than 2 years. The use of thidiazuron as a growth regulator was tested, but was detrimental to the explants at all tested concentrations. Initial experiments were hindered by significant bacterial internal contaminants. This problem was eliminated through the use of plexiglass chambers to provide up to a 20,000ppm CO2 atmosphere, increased light levels to promote photosynthesis, and the elimination of all carbon sources (sugars) from the substrate. An additional benefit was better shoot growth, better survival when rooting and better acclimation. 3/7 plants were rooted using 2.5 ppm IBA. Continuing experiments are focused on the effect of support medium on growth and multiplication as well as media development for other Pistacia species.

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D.E. Parfitt, N. Arjmand, and T.J. Michailides

Procedures were developed to permit screening pistachio for resistance to infection by Botryosphaeria dothidia panicle and shoot blight. A method of growing B. dothidea in liquid culture was developed and two inoculation procedures, direct injection of inoculum into shoots at the base of leaf petioles and a leaf scratch assay, were used to test selected pistachio (Pistacia vera L.) clones for resistance to B. dothidea. Both the direct injection and the scratch test procedures provided easily scored symptoms. Both solid and liquid cultures produced visible infections. Sources of resistance were identified in an F1 interspecific P. vera cv. Sfax × P. integerrima hybrid cross and P. integerrima genotypes. P. integerrima may be a valuable source of resistance for cultivar improvement, but resistant P. vera genotypes were not found. No correlation between Alternaria alternata resistance and B. dothidea resistance was found in P. vera.

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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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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