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P. Boccacci, A. Akkak, D. Torello Marinoni, G. Bounous, and R. Botta

Microsatellite or simple sequence repeat (SSR) markers show many characteristics of the ideal molecular marker, and recent studies have shown that loci developed in one species may allow analysis in taxonomically related species. In this study, 52 primer pairs developed in two oak species—Quercus robur L. and Quercus petraea (Matt.) Lieb.—were used to amplify DNA of 5 chestnut cultivars; 28 of them yielded amplicons and 12 polymorphic loci were selected and used to fingerprint 12 european chestnut (Castanea sativa Mill.) cultivars grown in the Piedmont region of northwestern Italy. The number of alleles per locus ranged from 3 to 8, mean expected heterozygosity was 0.592 (range: 0.288 to 0.868), and mean observed heterozygosity was 0.667 (range: 0.333 to 1.000). The results demonstrate the usefulness of some SSR markers isolated in Quercus for the fingerprinting and genetic mapping of Castanea cultivars.

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M.C. Scott, G. Caetano-Anollés, and R.N. Trigiano

DNA amplification fingerprinting (DAF) was used to study genetic relationships between closely related chrysanthemum cultivars (Dendranthema grandiflora Tzvelev.). Twenty-one cultivars were examined that belonged to the Anne, Blush, Boaldi, Charm, Davis, and Pomona series (families). The genetic variability of cultivars within and between series was evaluated using eleven arbitrary octamer primers. A few polymorphic characters uniquely identified closely related cultivars within each of the series. In contrast, many DNA polymorphisms were observed between members of the different series. Phenetic patterns were established by unweighted pair group cluster analysis using arithmetic means (UPGMA) and principal coordinate analysis (PCO). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series were also bulked to generate profiles containing unique amplified products for each series. Cluster analysis and PCO of bulked DNA clearly grouped Charm and Pomona together. However, series grouping did not correspond to morphology of inflorescence types. The results demonstrate the utility of the DAF technique in distinguishing clonal materials and its potential use for patent protection, phylogenetic studies, and for identifying useful markers in breeding applications.

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Daniel J. Bell, Lisa J. Rowland, James J. Polashock, and Frank A. Drummond

). For a variety of reasons, including better reproducibility than RAPDs and derivation from inferred coding regions, we asked whether we could reliably import these EST-PCR markers originally developed for highbush blueberry to fingerprint and estimate

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Xiaomeng Li, Rangjin Xie, Zhenhua Lu, and Zhiqin Zhou

present study, we analyzed three plastid genome regions ( psb H – pet B, trn L – trn F, and trn S – trn G), nuclear ITS sequences, and AFLP fingerprints of 30 citrus accessions in an attempt to construct the cytoplasmic phylogeny of the cultivated

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U. Lavi, J. Hillel, A. Vainstein, E. Lahav, and D. Sharon

Application of four DNA fingerprint probes to avocado (Persea americana Mill.) resulted in identification of various cultivars, characterization of the three avocado races, and a genetic analysis of family structure. Genomic DNA from 14 cultivars was probed with four DNA fingerprint probes. Three of the probes gave well-resolved bands. The individual-specific patterns obtained for each cultivar validate the use of this technique for definitive cultivar characterization, with the probability of obtaining a similar pattern for two different cultivars being 2 × 10-9. DNA mixes representing either Mexican, Guatemalan, or West-Indian avocado races were hybridized with the DNA fingerprint probes, and a band pattern characteristic for each race was obtained. Progeny of a cross between the cultivars Ettinger and Pinkerton were analyzed. Their DNA fingerprints revealed one pair of linked bands and another band allelic to one of them. The application of these observations to identification, evolutionary studies, and breeding is discussed.

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Elizabeth J. Parks and James W. Moyer

Fingerprinting using molecular markers is a highly effective method of cultivar identification that is a powerful aid to traditional methods based on morphology. Amplified fragment length polymorphism (AFLP) is a robust and reliable method for generating molecular markers that has been used to evaluate many crops for a variety of applications. In this study, AFLP was used to develop and validate robust genetic fingerprints for poinsettia (Euphorbia pulcherrima Willd. ex Klotzch) cultivars. Polymorphism selection was completed to facilitate the identification of useful polymorphisms and minimize future fingerprinting costs and time. Poinsettia is a highly variable crop subject to genetic drift and variable cultivars. Validation of polymorphisms to remove those associated with intracultivar variation improved the reliability of the fingerprinting. The result was a poinsettia AFLP database that defines the genetic fingerprints of 104 cultivars. Cluster analysis illustrated differentiation of most poinsettia cultivars tested. Selection of a subset of AFLP polymorphisms resulted in clustering of cultivars according to known origin and breeding program. This method has applications not only for cultivar identification for cultivar protection, and maintenance of cultivar uniformity, but also has the potential application of developing markers for important traits.

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Viji Sitther, Dapeng Zhang, Sadanand A. Dhekney, Donna L. Harris, Anand K. Yadav, and William R. Okie

of parents, which could broaden the genetic base of resulting populations. Among various methods used for identification of cultivars, DNA-based markers are routinely used for fingerprinting and detecting genetic polymorphisms. Of these, simple

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A. Adato, D. Sharon, U. Lavi, J. Hillel, and S. Gazit

DNA fingerprint information was used for identification of mango (Mangifera indica L.) cultivars for genetic relatedness analysis of20 mango cultivars and for genetic analysis of a family structure. Genomic DNA was extracted from young leaves, digested with Hind III or Dra I, and hybridized with 10 different DNA probes. Jeffreys' minisatellite probe 33.6 was the most useful, resulting in well-resolved bands representing highly polymorphic loci. Specific patterns were obtained for each cultivar. The probability of obtaining a similar pattern for two different cultivars was 9.4 × 10-6. Based on DNA fingerprint information, genetic distances between 20 mango cultivars were evaluated and an evolutionary tree was established. Analysis of DNA fingerprint band patterns of 12 progeny resulting from a cross between `Tommy Atkins' and `Keitt' mango revealed neither linked nor allelic bands. Application of the reported results for identification, genetic analyses, and mango breeding is discussed.

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R.N. Trigiano and G. Caetano-Anollés

The primary objectives of these laboratory exercises are to familiarize advanced undergraduate and graduate students (and instructors) with the general concepts, techniques, and uses of DNA fingerprinting and to remove some of the perceived mystique underlying molecular genetics. The technique of DNA amplification fingerprinting (DAF) is partitioned into four independent laboratory exercises that include DNA isolation, DNA amplification, gel electrophoresis and silver staining, and data collection and analysis. Although the DNA amplification and gel electrophoresis exercises are emphasized, very detailed and easy-to-follow instructions and protocols are provided for all aspects of the DNA fingerprinting process. These exercises, or similar ones, have been successfully completed on the first attempt by several classes of novice graduate students and other researchers.

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Warren F. Lamboy and Christopher G. Alpha

The USDA-ARS Vitis genetic resources collections in Geneva, N.Y., and Davis, Calif., contain ≈3600 accessions of >35 species. Accurate and unambiguous identification of these grapes is essential for efficient and effective use of this germplasm. Previous workers have successfully used polymerase chain reaction (PCR)-generated SSRs to fingerprint cultivars of the wine and table grape species, V. vinifera. Building on this work, we conducted a test of five previously characterized SSR loci on 110 accessions of 25 grape taxa (21 Vitis species and 4 hybrids) to determine if they would satisfy our need for identifying cultivars within the USDA-ARS grape collections. Scorable SSR fragments were produced with all 550 primer-accession combinations, with no null loci observed. The loci were highly polymorphic, with 16 to 38 different alleles found at a locus. Heterozygosity values ranged from 0.464 to 0.818, while gene diversity values ranged from 0.875 to 0.955. Discrimination power at a locus varied from a low of 0.947 to a high of 0.987. Combined discrimination power of all loci was effectively 1.000, with 2 chances in 100,000,000 that two sexually, independently derived grape accessions would not be distinguishable using this set of five SSR loci. Two plants in the study that had previously been classified as belonging to different grape species were shown to have identical SSR fingerprints, showing that they almost certainly possessed the same genotype. Because SSR markers are codominant and highly polymorphic and SSR loci are generally conserved across a range of related species, we strongly recommend SSRs for fingerprinting not only grape, but other clonal genetic resources collections as well.