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Patricia Sweeney, Robert Golembiewski, and Karl Danneberger

Random amplified polymorphic DNA (RAPD) markers from leaf tissue extractions are effective for discrimination of turfgrass varieties. The usefulness of RAPD markers for turfgrass variety identification can be enhanced by use of seed rather than leaf tissue for DNA extraction. To determine whether DNA extracted from turfgrass seed was suitable for amplification, DNA was extracted from bulk samples and individual seeds of bermudagrass [Cynodon dactylon (L.) Pers.], chewings fescue (Festuca rubra var. commutata Gaud.), Poa annua L., Poa supina Schrad., creeping bentgrass [Agrostis stolonifera L. var. palustrus (Huds.) Farw.], Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.). All samples were successfully amplified using an arbitrary primer. Amplification intensity varied among species. With an almost infinite number of arbitrary primers available, it is likely that suitable primers can be found to amplify DNA from most turfgrass species. Amplification of turfgrass seed DNA, whether bulk or individual seed, is possible and should prove more useful than amplification of leaf tissue DNA for discrimination of turfgrass varieties.

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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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Channapatna S. Prakash, Guohao He, and Robert L. Jarret

Highly polymorphic DNA markers were identified in sweetpotato (Ipomoea batatas) using PCR amplification and arbitrary primers. More than 100 accessions representing US cultivars and their progenitors, and germplasm lines from around the world were analyzed. Sweetpotato germplasm exhibited high genetic variability and individual-specific profiles were obtained for all accessions. US cultivars formed a tight cluster in the principal coordinate analysis suggesting a narrow genetic base. The genetic relationship data of US cultivars and their progenitors based on DNA polymorphisms was in agreement with their known pedigree. The putative paternal parents of certain cultivars selected through open pollination were identified based on shared polymorphisms. The PCR-based markers are valuable in the characterization of sweetpotato germplasm and in ensuring a broad genetic base for future cultivars.

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Kristin Schneider and James D. Kelly

Common beans, considered sensitive to moisture stress, are an important commodity in developing countries such as the Mexican Highlands where intermittent drought conditions are prevalent during the growing season. The selection and development of high performing cultivars under drought stress is confounded by the quantitative nature of drought tolerance. To employ indirect selection in earlier generations, RAPD markers were identified that associated with QTLs controlling performance under drought stress. RAPD markers are preferred for use in Phaseolus vulgaris, over RFLPs, because they generate polymorphisms between genetically related germplasm. 48% of 620 arbitrary primers screened against three parents of two F6 derived recombinant inbred pinto populations were polymorphic for one or more bands. These polymorphisms were screened against RILs in each population and associations were determined using one-way ANOVAs and Mapmaker. Yield data used for determination of associations was collected over five years in MI and Mexico where both stress and non stress treatments were applied.

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Reynato P. Umali, Nanako Kameya, and Ikuo Nakamura

The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid-subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A133.0 and A131.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 (`Morado' from `Red' and `Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A0.8 and Ba1.3A0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.

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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.

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Rose E. Palumbo, Wai-Foong Hong, Jinguo Hu, Charles Krause, James Locke, Richard Craig, David Tay, and Guo-Liang Wang

Pelargonium is one of the priority genera collected by the Ornamental Plant Germplasm Center (OPGC). In order to protect future breeders from a loss of genetic diversity, the OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Our project was designed to analyze the current collection in order to facilitate the maintenance of a more-diverse core collection. We have expanded our TRAP (Target Region Amplified Polymorphism) analysis from 120 plants with one primer set to include 780 plants with four primer sets. Each primer set consists of a labeled arbitrary primer paired with a gene-specific primer, and two different fluorescent labels were used to allow multiplexed PCR reactions. We scored about 90 markers in each of the first two primer sets and about 60 markers in each of the second two. In comparisons between the phylogeny and the morphology and taxonomy of these plants, we show some matching clusters that may be explained by the breeding history of the plants.

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S. Jorge, M.C. Pedroso, D.B. Neale, and G. Brown

Random amplified polymorphic DNA (RAPD) analysis was used to estimate genetic similarities between Portuguese Camelliasinensis (L.) O. Kuntze (tea plant) accessions and those obtained from the germplasm collections from the Tea Research Foundation of Kenya and from the National Research Institute of Vegetables, Ornamental Plants, and Tea of Japan. The accessions studied are taxonomically classified as C. sinensis, var. sinensis, var. assamica, or ssp. lasiocalyx. A set of 118 ten-base arbitrary primers was tested, of which 25 produced informative, reproducible, and polymorphic banding patterns. These primers were used to amplify DNA from 71 tea plant accessions and produced a total of 282 bands, of which 195 were polymorphic. The phenotypic frequencies were calculated using Shannon's Index and employed in estimating genetic diversity within tea plant populations. Our study demonstrates that tea plant populations, including the Portuguese tea plants, show considerable genetic variability. From the UPGMA cluster analysis based on a matrix using the Jaccard coefficient, it was possible to distinguish the Portuguese tea plants from the remaining accessions. The RAPD markers discriminated the three C. sinensis varieties. Moreover, within each variety cluster, subclusters formed according to geographic distribution. The RAPD analysis also separated the commercially cultivated tea plants from the Taiwanese wild tea plants. The present results show that RAPD analysis constitutes a good method to estimate genetic diversity within C. sinensis, and to differentiate C. sinensis accessions according to taxonomic variety and geographical distribution.

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Jinguo Hu, Beiquan Mou, and Brady A. Vick

Target region amplified polymorphism (TRAP) markers were used to evaluate genetic variability among 48 accessions of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Thirty-eight accessions collected and preserved by the USDA National Plant Germplasm System (NPGS) and 10 commercial hybrids were used in the current study. For assessing genetic diversity within accessions, DNA samples were prepared from nine to 12 individual seedlings from six germplasm accessions and two hybrids. Relatively high levels of polymorphism was found within accessions based on 61 polymorphic TRAP markers generated with two fixed primers derived from the Arabidopsis-type telomere repeat sequence and two arbitrary primers. For evaluating inter-accession variability, DNA was extracted from a bulk of six to 10 seedlings of each accession. Of the 1092 fragments amplified by 14 primer combinations, 96 (8.8%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice, Nei) was 57.5% with a range from 23.2 to 85.3%. A dendrogram was constructed based on the similarity matrix. It was found that the genetic relationships were not highly correlated with the geographic locations in which the accessions were collected. However, seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities have effect on the genetic variability. This study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability of spinach germplasm.

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D.G. Ranamukhaarachchi, R.J. Henny, C.L. Guy, and Q.B. Li

Randomly amplified polymorphic DNA (RAPD) markers were utilized to determine the genetic relationships of nine morphologically similar pot plant cultivars of Anthurium sp. by developing DNA fingerprints (DFP). Of 25 arbitrary primers screened, nine generated DFPs that were used in computing the genetic distance (d) and similarity coefficient (C) values. All cultivars tested exhibited a high degree of genetic similarity. `Lady Ann' and `Lady Beth' possessed the closest relationship with d and C values of 0.06 and 0.98, respectively. The next closest genetic relationship was between `Red Hot' and `Southern Blush' (d = 0.33, C = 0.89). These two cultivars exhibited a more distant relationship to the other seven cultivars as indicated by higher `d' values. However, this study showed that the nine Anthurium cultivars examined were genetically closely related. These cultivars share specific DNA bands with three possible parental species (A. andraeanum Linden ex Andre, A. antioquens L., and A. amnicola Dressler) included in this study, which may indicate similarities in their pedigree. This study shows that RAPDs can be a useful tool to distinguish Anthurium pot plant cultivars as well as identify their genetic relationships.