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

Four chrysanthemum (Dendranthema grandiflora) spontaneous and radiation-induced sports from the cultivar `Charm' and phenotypically differing only in flower color were individually characterized using arbitrary signatures from amplification profiles (ASAP). ASAP analysis is based on a two-step arbitrary primer amplification procedure that produces “fingerprints of fingerprints.” In the first step, `Charm', `Dark Charm', `Dark Bronze Charm', `Salmon Charm', and `Coral Charm' were fingerprinted by DNA amplification fingerprinting (DAF) with standard octamer arbitrary primers. Diluted products from three monomorphic fingerprints for each cultivar were subsequently reamplified using four minihairpin decamer primers. Each of the 12 ASAP profiles revealed about 30% polymorphic loci and some were used to uniquely identify cultivars and estimate genetic relationships. The ASAP technique permits identification of previously genetically indistinguishable plant material and should facilitate marker assisted breeding and protection of ownership rights.

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

The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assessed using DAF. Thirteen cultivars of chrysanthemum included in the study were members of the following series: Charm (five), Davis (four), and Pomona (four). The genetic variability within and between series were evaluated using 11 arbitrary octamer primers. A few polymorphic loci were evident that uniquely identified closely related cultivars within a series. In contrast, there were many polymorphisms between members of different series. Genetic distances between cultivars within and between series were calculated using marker comparison and UPGMA (cluster analysis). The average distance between series was 10-fold greater than between cultivars within a series. DNA from all cultivars belonging to a series also were bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique possibly could be used for patent protection and phylogenetic studies and may be useful in breeding for chrysanthemums.

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G. Caetano-Anollés, R.N. Trigiano and M.T. Windham

Fifty-one isolates of Discula destructiva obtained from various Cornus species were evaluated using arbitrary signatures from amplification profiles (ASAP). ASAP analysis is based on dual-step arbitrary primer-based amplification procedure that produces “fingerprints of fingerprints” and in many instances increases detection of polymorphic DNA. This novel technique was able to distinguish groups of isolates from the northeast, middle and southeast range of the disease as well as western United States and Canada. The data supports the contention of recent and independent introduction of the disease on both east and west coasts, a genetic “bottleneck” that has limited diversity of the pathogen, and directionality of introduction of disease from coastal ports-of-entry to interior populations of C. florida and C. nuttalli.

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

Four chrysanthemum (Dendranthema grandiflora) spontaneous and radiation-induced sports from the cultivar `Charm' and phenotypically differing only in flower color were individually characterized using arbitrary signatures from amplification profiles (ASAP). ASAP analysis is based on a two-step arbitrary primer amplification procedure that produces “fi ngerprints of fingerprints.” In the first step, `Charm', `Dark Charm', `Dark Bronze Charm', `Salmon Charm', and `Coral Charm' were fingerprinted by DNA amplification fingerprinting (DAF) with standard octamer arbitrary primers. Diluted products from three monomorphic fingerprints for each cultivar were subsequently reamplified using four minihairpin decamer primers. Each of the 12 ASAP profiles revealed polymorphic loci that were used to uniquely identify cultivars and estimate genetic relationships. The ASAP technique permits identification of previously genetically indistinguishable plant material and should facilitate marker assisted breeding and protection of ownership rights.

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

The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars `Dark Charm', `Salmon Charm', `Coral Charm' and `Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of `Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing ≈37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or 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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M.C. Scott, G. Caetano-Anollés and R.N. Trigiano

The genetic distance of closely related cultivars of Dendranthema grandiflora (chrysanthemum) was assess using DAF. Twenty-three cultivars of chrysanthemum included in the study were members of the following series: Anne (3), Blush (3), Boaldi (4), Charm (5), Davis (4), and Pomona (4). The genetic variability within and between series were evaluated using 11 arbitrary octamer primers. A few polymorphic loci were evident that uniquely identified closely related cultivars within a series. In contrast, many polymorphisms were observed between members of different series. Genetic distances between cultivars within and between series were evaluated using marker comparison and analyzed with PAUP (phylogenic analysis using parsimony) and UPGMA (unweighted pair group cluster analysis using arithmetic means). The average distance between series was 10-fold greater than between cultivars within a series. Furthermore, series with similar flower morphology, pompon or daisy-like, were more closely related than those with different phenotypes. DNA from all cultivars belonging to a series were also bulked to generate DNA profiles containing unique amplified products for each series. Polymorphic loci that were generated by the DAF technique can possibly be used for patent protection and phylogenetic studies, and may be useful in breeding chrysanthemums.

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R. N. Trigiano, G. Caetano-Anollés, B. J. Bassam and M. T. Windham

DNA Amplification Fingerprinting (DAF) was used to characterize ten isolates of Discula destructiva Redlin and three isolates of an undescribed species of Discula, the causal organisms of dogwood (Cornus species) anthracnose. Isolates were obtained throughout the disease range in the eastern United States and DAF profiles generated with ten arbitrary oligonucleotide primers. Very few polymorphic loci (27/298) were detected between isolates of D. destructiva; whereas, a greater number were observed between and among the isolates of Discula species. Relationships among and between the two fungal groups were analyzed using PAUP and UPGMA and indicate that the genome of D. destructiva is highly conserved throughout the distribution. In contrast, isolates of Discula species exhibited greater variability. This suggests that D. destructive was recently introduced to the eastern United States.