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Abstract
Applications of gel electrophoresis and isozyme analysis to horticultural science were outlined more than a decade ago by Peirce and Brewbaker (15). Isozymes now are being widely used in almost all areas of plant biology, including plant breeding, plant population genetics, systematics, evolutionary genetics, and somatic cell genetics. Isozymes offer the most reliable single gene markers and they are often codominant in inheritance. Genetic studies using isozymes in tree fruit and nut crops are few. Torres and Tisserat (26) reported single gene markers using isozymes in date palm, Arulsekar et al. (4) in strawberry, Torres et al. (23) in avocado, and Arulsekar et al. (1, 3) in walnuts; perhaps the only reliable gene markers available for breeders of these crops. Torres et al. (24) have also reported isozyme genetic markers in Citrus and demonstrated their use in distinguishing nucellar seedlings from those of zygotic origin. Ben-Hayyim et al. (5) developed isozyme systems that will facilitate identification of interspecific cell hybrids and fusion products in Citrus. Arulsekar et al. (3) studied glucose phosphate isomerase (GPI) and aspartate aminotransferase (AAT) isozymes in callus tissues, juvenile leaves, and adult leaves of walnuts using the same enzyme extraction procedure and found no tissue-specific differences in isozyme patterns. McGranahan et al. (11) used isozymes to identify intergeneric hybrids generated between wingnut (Pterocarya sp.) and walnut (Juglans sp.). Parfitt et al. (13) demonstrated the use of isozymes in the identification of interspecific hybrids between peach and plum cultivars. The inheritance of MDH isozymes in peach was reported by Arulsekar et al. (2). Among 298 peach cultivars tested, variation was observed only for malate dehydrogenase (MDH) isozymes. The MDH isozymes were used by Parfitt and Miller (unpublished data) to study outcrossing rates among peach cultivars.
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.
Abstract
Leaf isozymes of 145 cultivars of grapes (Vitis vinifera L. and Vitis spp.) were separated via starch gel electrophoresis. They could be separated into 52 groups using glucosephosphate isomerase (GPI) and phosphoglucomutase (PGM) isozymes. Twenty-four cultivars had unique combinations of isozyme patterns. Two loci, Gpi-2 and Pgm-2, were identified and shown to be simply inherited with multiple alleles at each locus. The mean heterozygosity for the two loci (Gpi-2 and Pgm-2) among all the cultivars was 57.5%, indicating a high level of genetic variability among these cultivars. Indirect evidence for expressed linked lethal alleles was demonstrated by the missing classes in Gpi-2 and Pgm-2 segregation ratios (1:2:0 ratios) in several selfed progeny families. Chemical name used: methyl[1-(methylethyl)-1H-benzimidazol-2-y]-carbamate (benomyl).
Abstract
Genetic analysis of phosphoglucoisomerase and leucine amino peptidase allozymes clearly indicated that the octoploid cultivated strawberries Fragaria × ananassa Duch. are highly diploidized. A 4-locus genetic model is proposed for the PGI allozymes in the octoploid strawberries commensurate with the genomic structure. The LAP system needs further investigation in order to establish the number of loci and alleles present. The possible adaptive value of the biochemical diversity found in the Fragaria species is discussed.
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.
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.
Abstract
Starch gel electrophoresis was employed to distinguish the University of California-released strawberry cultivars. Isozyme patterns of 3 enzyme systems (PGI, LAP, and PGM) were studied. Fourteen of the 22 cultivars studied were classified uniquely by using the 3 enzyme systems. The use of electrophoresis as a tool in breeding clonally propagated crops is discussed.
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
Abstract
Two isoenzyme systems, glucose phosphate isomerase and phosphoglucomutase, were identified for use as starch gel electrophoretic markers of plum × peach (Prunus salicina × Prunus persica) interspecific hybrids. Two distinct regions of banding were associated with each enzyme system. Different unique banding patterns for each species were observed for plum and peach at 3 of 4 banding regions. Interspecific hybrid plants exhibited hybrid enzyme patterns with bands from both plum and peach in each region. Consequently, interspecific plum × peach hybrid genotypes may be distinguished from parental plum or peach genotypes. These enzyme systems may be used in breeding programs to identify plum × peach hybrid seedlings.
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.