RAPD

About: RAPD is a research topic. Over the lifetime, 15960 publications have been published within this topic receiving 360391 citations.

Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant.

Abstract: Molecular typing of bacteria has been widely used in epidemiological studies but not as extensively for tracing the transmission of pathogenic bacteria in food plants. This study was conducted to examine the potential use of two molecular typing methods, random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE), to trace Listeria monocytogenes contamination in a shrimp processing plant. Ribotyping and phase typing were also performed on a select number of strains. One hundred fifteen strains of L. monocytogenes collected in different areas of a shrimp processing plant were first serotyped and then subtyped by molecular typing. RAPD and PFGE showed great promise for typing L. monocytogenes isolates since distinguishable and reproducible DNA polymorphisms were obtained. When the composite profile from both (RAPD and PFGE) methods was generated, there was an increase in the discriminatory power to discern differences between strains of L. monocytogenes. The results indicated that environmental strains all fell into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp isolates belonging to one profile group were found in different areas of the processing line. This same profile group was also present in food handlers from the processing and packaging areas of the plant.

Random amplified polymorphic DNA analysis of olive (Olea europaea L.) cultivars

Abstract: Seventeen olive (Olea europaea L.) cultivars, including oil and table olive cultivars originating from throughout the Mediterranean area, were screened using random amplified polymorphic DNA (RAPD) markers. The results indicate that a high degree of polymorphism is evident in the olive germplasm reexamined. Forty random decamer primers were screened; seventeen of these produced 47 reproducible amplification fragments useful as polymorphic markers. Each of the 17 cultivars can be discriminated with a few primers. Results were analyzed for similarity among the cultivars and a cluster analysis was performed. These analyses revealed two main groups: one comprising primarily small-fruited cultivars grown mainly for oil production, and the other characterized by having large fruit. There was no apparent clustering of olive cultivars according to their geographic origins. Olive (Olea europaea) is one of the most ancient cultivated fruit tree species in the Mediterranean basin. It is the only Mediterranean representative of the genus Olea, which includes 35-40 species distributed over tropical and southern Africa, south Asia, eastern Australia, New Caledonia, and New Zealand (Zohary and Hopf 1993). Traditionally, morphological and phonological traits are used to identify olive cultivars. More recently, workers have found isozymes to be useful for cultivar identification and determining patterns of relatedness among cultivars. Pontikis et al. (1980) identified 27 olive cultivars, mostly of Greek origin, using 16 enzyme systems in pollen. Trujillo et al. (1990) found that by using five pollen enzyme systems they could distinguish 134 of 155 cultivars. Ouazzani et al. (1993) distinguished 33 of 44 cultivars using 9 enzyme systems in leaf tissue. Although isozyme analysis has proved useful in olive, mainly due to the high level of isozyme polymorphism present in the species, direct analysis of DNA could increase considerably the number of markers produced. Furthermore, because isozymes are products of gene expression, differential expression by environment, tissue-specificit y, and other factors is common and may make interpretation of results difficult. Random amplified polymorphic DNA (RAPD) analysis, first described by Williams et al. (1990) and Welsh and McClelland (1990), has proven to be a useful tool for genetic typing and mapping. Bogani et al. (1994) described preliminary results of RAPD analyses of olive cultivars. They used 5 decamer nucleotide primers to generate RAPD polymorphisms among 11 olive cultivars, but found no consistent relationships. In this paper, we report the use of RAPD markers to distinguish among 17 olive cultivars, and we discuss the use of RAPDs to study relationships among cultivars of this species. Materials and Methods Plant materials. Seventeen Olea europaea cultivars were used in this study (Table 1). These were obtained from a collection mainor publication 26 Sept. 94. Accepted for publication 11 Jan 1995. We acknowledge K. Pinney for her capable technical assistance and S. for his advice on technique, J. Hormaza was supported by an INIA rom the Spanish Ministry of Agriculture, The cost of publishing this paper d in part by the payment of page charges. Under postal regulations, this fore must be hereby marked advertisement solely to indicate this fact. om Universita degli Studi di Catania, Istituto di Coltivazioni Arboree, Via 5, Catania Italy 95123. To whom reprint requests should be addressed. dress: Dept. Fruticultura, S.I.A.-D.G.A., Apartado 727, Campus de 0080 Zaragoza, Spain. tained in experimental orchards at the Univ. of California, Davis. DNA extraction and RAPD analysis. Young olive leaves were collected in Spring 1993 and stored at –70C before DNA extraction. Total DNA was extracted from leaf tissue following the CTAB method of Doyle and Doyle (1987) with minor modifications. Young leaves (5.0 g) were ground in liquid nitrogen and mixed with 20 ml of CTAB buffer (100 mM Tris-HCl pH 8, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2% β− mercaptoethanol, 0.1 % NaHSO4). Samples were incubated at 65C for 1 h, mixed with an equal volume of chloroform–isoamyl alcohol (24:1), and centrifuged at 2000× g for 15 min. The aqueous phase was recovered and mixed with two-thirds volume of isopropanol. The nucleic acid precipitate was recovered with a glass hook, washed with 10 ml of 10 mM ammonium acetate in 76% ethanol, dried overnight, and resuspended in 1 ml of modified TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA). Extracted DNA was diluted to 10 ng·μl and used for PCR amplification. Forty decamer oligonucleotides (Operon Technologies, Alameda, Calif.) were used for PCR amplifications following the procedure of Williams et al. (1990), with some modifications. Amplification reactions were carried out in 25-μl volumes containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.9 mM MgCl2, 0.00 1% gelatin, 100 μM each of dATP, dGTP, dCTP, and dTTP (Promega, Madison, Wis.), 0.4 μM primer, 0.75 units of AmpliTaq DNA polymerase (Perkin-Elmer-Cetus, Norwolk, Conn.) and 50 ng of genomic DNA. Each reaction mixture was overlaid with 25 μl of mineral oil to prevent evaporation. To destroy putative carryover products, reaction mixtures and mineral oil were placed on a UV (300 nm) transilluminator before addition of template DNA and DNA polymerase (Sarkar and Sommer, 1993). DNA amplification reactions were performed in a thermal cycler (PTC100; MJ Research, Watertown, Mass.) programmed for 1 cycle of 2 min at 94C followed by 40 cycles of 45 sec at 92C, 1 min at 36C, 2 min at 72C, for denaturing, annealing and primer extension, respectively. The last cycle was followed by incubation for 5 min at 72C; PCR products were stored at 4C before analysis. Amplification products were analyzed by gel electrophoresis in 2910 agarose (SeaKem; FMC, Rockland, Maine) in 1 x TBE buffer, stained with ethidium bromide, and photographed under UV light using Type 57 Polaroid film. Molecular sizes of amplification products were estimated using a 123 bp DNA ladder (Sigma Chemical Company, St. Louis). All reactions were repeated at least three times using different lots of Taq polymerase and only reproducible bands were used in further analyses. Each amplification fragment J. AMER. SOC. HORT. SCI. 120(3):538–542. 1995. Table 1. Olive cultivars included in this study and their country of origin.

Genetic diversity among watermelon (Citrullus lanatus and Citrullus colocynthis) accessions

Abstract: Genetic diversity was estimated among 42 U.S. PlantIntroduction (PI) accessions of the genusCitrullus (of these, 34 PIs are reported tohave disease resistance), and 5 watermelon cultivars, using 30RAPD primers. These primers produced 662 RAPD markers that could berated with high confidence. Based on these markers, geneticsimilarity coefficients were calculated and a dendrogram wasconstructed using the unweighted pair-group method witharithmetic average (UPGMA). The analysis delineated threemajor clusters. The first cluster consisted of a group of fivewatermelon cultivars, a group of C.lanatus var. lanatusaccessions, and a group of C.lanatus var. lanatusaccessions that contained some C.lanatus var. citroidesgenes. The second cluster consisted of the C.lanatus var. citroidesaccessions, while the third cluster consisted of theC. colocynthis accessions.The two C. lanatus clustersdifferentiated from each other and from the C.colocynthis cluster at the level of 58.8%and 38.9% genetic similarity, respectively. Assessment ofgenetic diversity among accessions that have been reported to havedisease resistance indicated that resistance to either anthracnose,downy mildew, powdery mildew, or watermelon mosaic virus is foundamong all major groups of Citrullus PIs.Additionally, resistance to gummy stem blight or Fusarium wilt mayexist among C. lanatus var.citroides PIs. This study demonstrates thatmolecular markers can be useful in assessing genetic diversity, andin sorting Citrullus PIs into phylogeneticgroups prior to their evaluation for disease or pestresistance.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce

Abstract: Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.

Predicting quantitative variation within rice germplasm using molecular markers

Abstract: Diverse Asian rice (Oryza sativa) germplasm has been used to identify associations between various quantitative traits and RAPD molecular markers using multiple regression analysis. This has allowed us to predict for other samples of germplasm their performance for traits such as culm length and number, days to flowering, grain width, and panicle and leaf length using only RAPD marker data. Such predictive capability is possible because of the availability of extensive diversity held in genebanks, and can be used in the future to facilitate the exploitation of that biodiversity. More specifically the methodology could facilitate crop improvement by rapid ideotype prediction. For the mapping and isolation of QTLs (genes controlling quantitative traits) the method would provide information to guide the selection of parental material for hybridization and markers expected to show linkage to QTLs. It may also be possible that these associations could lead the way towards marker-assisted selection during breeding programs. In the future, this demonstration of association between markers and easily measured traits could also be extended to the study of important adaptive traits, such as stress tolerance, found either within germplasm collections or in natural populations.