The variability of isozymes in nine enzyme systems was studied in 25 carob (Ceratonia siliqua L.) cultivars using starch gel electrophoresis of leaf extracts. Five enzymes (phosphoglucomutase, phosphoglucoisomerase, aspartate aminotransferase, shikimic dehydrogenase, and aconitase) were polymorphic, making it possible for the 25 cultivars to be classified into eight phenotype categories.
J. Tous, C. Olarte, M.J. Truco and P. Arús
L. Pascual, F. Perfectti, M. Gutierrez and A.M. Vargas
Isozymes have been used as genetic markers to characterize seven Spanish cherimoya (Annona cherimola Mill.) cultivars. Fifteen enzyme systems were analyzed. Ten varied [aconitase (ACO, EC 22.214.171.124), alcohol dehydrogenase (ADH, EC 126.96.36.199), glutamate oxalacetate transaminase (GOT, EC 188.8.131.52), isocitrate dehydrogenase (IDH, EC 184.108.40.206), leucine aminopeptidase (LAP, EC 220.127.116.11), malate dehydrogenase (MDH, EC 18.104.22.168), phosphoglucose isomerase (PGI, EC 22.214.171.124), phosphoghtcomutase (PGM, EC 126.96.36.199), shikimate dehydrogenase (SKDH, EC 188.8.131.52), and triose phosphate isomerase (TPI, EC 184.108.40.206)] and five did not [acid phosphatase (ACPH, EC 220.127.116.11), diaphorase (DIA, EC 18.104.22.168), malic enzyme (ME, EC 22.214.171.124), 6-phosphogluconic dehydrogenase (6PGDH, EC 126.96.36.199), and superoxide dismutase (SOD, EC 188.8.131.52)]. Two cultivars, Campa and Campa Mejorada, had identical banding patterns for all enzymes tested. All others were identified as distinct cultivars because of isozyme differences. The identical isozyme profiles of `Campa' and `Campa Mejorada' probably indicate that they are the same cultivar. A cluster analysis of isozyme profiles showed that Spanish cultivars were clearly different from Californian cultivars.
S. Mancuso and F.P. Nicese
Backpropagation neural networks (BPNNs) were used to distinguish among 10 olive (Olea europaea L.) cultivars, originating throughout the Mediterranean basin. Identification was performed on the basis of 17 phyllometric parameters resulting from image analysis. Different BPNN architectures were attempted and best performance was achieved using a 17 × 20 × 10 BPNN. Networks were tested with sets of phyllometric parameters not involved in the training phase. Results enabled identification with certainty all cultivars tested.
Johanne C. Cousineau and Danielle J. Donnelly
Isoenzyme staining was used to characterize 55 of 78 raspberry cultivars (Rubus idaeus L., R. × neglectus Peck, and R. occidentalis L.). Six enzymes were needed to achieve this characterization: isocitrate dehydrogenase, malate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, shikimic acid dehydrogenase, arid triose phosphate isomerase. The 23 cultivars that were not uniquely characterized were grouped into eight groups of two and two groups of three and four. Two of these groups comprised black raspberry cultivars, all of which were similar isozymically. Isoenzymes could not distinguish between the cultivar Willamette and a spine-free mutant of the cultivar. Analysis of cultivars obtained from several sources revealed that raspberry cultivar mislabeling exists but is not very prevalent. Regular isoenzyme analysis of raspberry cultivars held by germplasm repositories, certified and other propagators, and breeders is both feasible and advisable for early detection of cultivar mislabeling.
I. Paran, M. Horowitz, D. Zamir and S. Wolf
B. Sosinski and D.S. Douches
DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.
J. Fang, Y. Qiao, Z. Zhang and C.T. Chao
We used amplified fragment length polymorphism (AFLP) markers to analyze 14 fruiting mei cultivars from China and Japan. The levels of polymorphism and genetic relationship among cultivars were studied using two types of AFLP primer combinations [EcoR I + Mse I (E+M) and EcoR I + Taq I (E+T)] and the combined data from both types of primer combinations (E+M+T). The polymorphism among the cultivars was 57.92% based on E+M primers and 63.04% based on E+T primers. All three dendrograms generated by the three sets of data showed similar relationships among the fruiting mei cultivars. The corresponding main clusters contained the same cultivars and the subgroups correlated closely with the known geographic origins of the cultivars.
Daniel Potter, Fangyou Gao, Giovanna Aiello, Charles Leslie and Gale McGranahan
The utility of intersimple sequence repeat (ISSR) markers for identification of English or Persian walnut (Juglans regia L.) cultivars was explored. Four cultivars were screened with 47 ISSR primers; eight of these primers, which generated reproducible and informative data, were selected for further study. Two individuals from each of 48 cultivars, including many currently important in the California walnut industry as well as accessions from Europe and Asia, were then examined with the eight ISSR primers. Polymerase chain reaction (PCR) products were separated on agarose gels and stained with ethidium bromide. Fifty-four bands were scored as present or absent in each cultivar; of these, 31 (57%) were polymorphic among the 48 cultivars. Combined data from the eight ISSR primers provided a unique fingerprint for each of the cultivars tested. Fifteen of the cultivars could be distinguished from all others with just one primer, 31 with a minimum of two primers, and two required three primers. Pairwise genetic distances between the cultivars were calculated and a dendrogram was generated using the neighbor-joining algorithm. Some of the groupings in the dendrogram corresponded to groups which, based on known pedigrees, are genealogically closely related. Others included accessions from diverse genetic and/or geographic origins. These results can be attributed to a combination of the limitations of the ISSR method for inferring genetic relationships, on the one hand, and the complex history of walnut cultivar development involving extensive exchange and breeding of germplasm from different geographic regions, on the other.
Darush Struss, Riaz Ahmad, Stephen M. Southwick and Manuela Boritzki
Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evaluate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite containing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of `Bing' fruit collected from different grocery stores in the United States to that of a standard `Bing' cultivar. Results indicated that, with one exception, all `Bing'samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study.
Riaz Ahmad, Dan Potter and Stephen M. Southwick
Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine (Prunus persica) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.