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Arthur Q. Villordon and Don R. LaBonte

Polymorphism analysis and yield tests were conducted among `Jewel' sweetpotato clones [Ipomoea batatas (L.) Lam] obtained from eight state foundation seed programs. Initially, 38 arbitrary primers generated a total of 110 scorable DNA fragments in a sample of virus-indexed plants from each clone source. The number of marker loci scored for each primer varied from one to eight with an average of 2.89. Twenty-one bands (19.1%) were scored as putative polymorphic markers based on the presence or absence of amplified products. Further estimation of variability within each clone source was accomplished by an assay of 10 sample plants per clone group by 14 marker loci generated by four selected primers. Polymorphic bands ranged from 7.1% to 35.7 % in five of eight clone groups. Field studies show variation in nearly all yield grades measured. In three tests during the 1991 and 1992 seasons, yield differences ranged from 27% to 46% within the economically important U.S. no. 1 root grade. The results suggest the usefulness of arbitrarily-primed markers in detecting intra-clonal sweetpotato DNA polymorphisms and indicate an underlying genetic cause for phenotypic variability in the crop.

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Terri Woods Starman and Shane Abbitt

Our objective was to distinguish between eight cultivars of two geranium species, Pelargonium ×hortorum L.H. Bailey (cutting and seed geranium) and Pelargonium peltatum (L.) L'Hér. ex Ait. (ivy geranium), and evaluate their genetic relationships using the nucleic acid scanning techniques of DNA amplification fingerprinting (DAF) and/or arbitrary signatures from amplification profiles (ASAP). Cultivars used in the study represented three commercial types: cutting, seed, and ivy geranium. Two seed geranium cultivars from each of the Dynamo and Orbit series were included. Cutting geranium cultivars were `Designer Lilac Chiffon' and `Starburst Red' and the ivy geraniums were `Bernardo Guiber' and `Vinco Guivin'. The ASAP amplification protocol used one of two arbitrary octamer primers, followed by reamplification with one of four different minihairpin primers. ASAP profiles were complex, with 66% of bands being polymorphic and useful in distinguishing between cultivars. Genetic relationships were evaluated by principal coordinate analysis and cluster analysis based on the Jaccard distance estimator. This analysis grouped cultivars by species according to commercial type, i.e., seed geraniums were in one large group, the cutting geraniums were grouped together, and the ivy geraniums were a separate branch.

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Terri Woods Starman, Xiangrong Duan, and Shane Abbitt

DNA amplification fingerprinting (DAF) was used to evaluate the genetic relationships among 11 cultivars of poinsettia (Euphorbia pulcherrima Willd.). Amplification was with 10 octamer oligonucleotide primers that generated 336 DNA bands. Thirty-one percent of the bands were polymorphic and distinguished among cultivars. Genetic relationships were evaluated by cluster analysis, and the resulting dendrogram closely agreed with published cultivar relationships. Arbitrary signatures from amplification profiles (ASAP) were further used to characterize two cultivars, `Nutcracker Red' and `Peterstar Red', that were previously found to be genetically and morphologically similar, as well as five cultivars in the “Freedom” series. The DAF products generated with arbitrary octamer primers were reamplified with mini-hairpin decamer primers in these experiments. The ASAP profiles were complex and yielded a total of 231 bands, 38% of which were polymorphic and capable of distinguishing each Freedom cultivar. Five of the eight primer combinations distinguished `Nutcracker Red' from `Peterstar Red'. Thus, closely related cultivars of poinsettia can be separated using new and improved molecular fingerprinting protocols.

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Arthur Q. Villordon and Don R. LaBonte

Genetic uniformity was assessed among sweetpotato (Ipomoea batatas) clones propagated through adventitious and nodal procedures. A single sprout each of `Jewel,' `Sumor,' and L87-95 was used as source of clonal plants that were simultaneously propagated through conventional adventitious procedures and a tissue culture-based nodal culture technique. A sample of 15 decamer primers generated 64 scorable amplified fragments in a PCR-based assay, 29 of which were putatively polymorphic across n = 60 samples (10 each of nodal and adventitiously derived plants/genotype). Within adventitiously derived materials, putative polymorphisms ranged from 4.7% to 31.3% depending on the genotypic class. In contrast, putative polymorphisms ranged from 0.0% to 3.1% among nodally derived samples. Marker loci differentiated genotypes as well as putative marker phenotype variants through a multidimensional scaling analysis of the genetic similarity matrix. An `analysis of molecular variance' shows that genotypic effects accounted for 88.7% of the total molecular marker variability, while propagation effects (within genotypic groups) accounted for 11.3%. Results confirm that clonal plants derived from preexisting meristematic regions are more genetically uniform than plants propagated from adventitious origins.

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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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Marissa Moses and Pathmanathan Umaharan

within C. chinense . POPGENE Version 1.31 ( Yeh and Boyle, 1997 ) was used to calculate three indices of diversity (i.e., Nei’s diversity index, Shannon’s index, and percentage of polymorphic loci). Results The nine arbitrary primers used generated 138

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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

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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Patricia M. Sweeney and T. Karl Danneberger

The usefulness of random amplified polymorphic DNA (RAPD) in characterizing two perennial ryegrass (Lolium perenne L.) synthetic cultivars, `Accolade' and `Caravelle', was tested. Two out of 10 arbitrary primers produced three RAPD markers that distinguished bulk samples of 30 seedlings. Additional fragments were apparent when DNA from individual seedlings was amplified. Amplification products from bulk samples were not simply the sum of amplification products of individual seedlings and did not reflect all the diversity within or between the cultivars. The study illustrates the need to screen individuals to accurately evaluate the genotypic composition of a synthetic cultivar or heterogeneous population.

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H.L. Ko, R.J. Henry, P.R. Beal, J.A. Moisander, and K.A. Fisher

An assessment was made to determine the suitability of RAPD analysis for identification of the Australian wildflower Ozothamnus diosmifolius (Vent.) DC [syn. Helichrysum diosmifolium (Vent.) Sweet] cultivars and lines. Of 19 arbitrary primer sequences tested, 16 revealed a high degree of polymorphism between the six most important genotypes with commercial significance, producing a total of 166 markers, of which 70% were polymorphic. Several primers (such as OPD-03 and OPM-07) were able to distinguish all tested genotypes from one another, showing an intracultivar consistency. These results indicate that RAPD analysis is a useful tool for establishing genetic diversity in this species as well as assisting in commercial protection of plant breeders' rights.