Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

Detection of Intra-Clonal Genetic Variability in Vegetatively Propagated Tea Using RAPD Markers

The role of random amplified polymorphic DNA (RAPD) markers in detecting intra-clonal genetic variability in vegetatively propagated UPASI-9 clone of tea (Camellia sinensis) was studied. Twenty five decamer primers were used, of which three did not amplify, three gave single bands and the rest of nineteen primers generated upto twelve bands (an average of 6.3 bands per primer). Twenty one primers exhibiting amplified products gave monomorphic banding patterns. Only one primer (OPE-17) gave a unique extra band of similar size in four plants.     

The potato in Spain during the late 16th century

A study is presented of the Hospital de la Sangre account books in Seville at the Archivo Hispalense for the period 1546 to 1601, to verify purchases of potatoes (Solanum tuberosum) during that period. Potatoes were bought regularly in the Seville market from 1580 onwards, with the first record appearing in 1573 thus agreeing with Salaman’s conclusion that potatoes became established in Spain by about 1570. Purchases were confined almost entirely to December and January each year, lending weight to the hypothesis that these were short day adaptedS. tuberosum ssp.andigena, actually grown in Spain and forming tubers in the short days at the end of the year. If potatoes had been imported for direct consumption from South America shipping records indicate that they could have arrived in Seville at all times of the year. A listing of other fruits and vegetables bought in the Seville market shows a wide range of mostly mediterranean crops with some of Near Eastern origin and certain spices imported through Lisbon from the Ear East. Surprisingly, very few New World crops are mentioned.     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD marker polymorphism among commercial winery yeast strains

Random amplified polymorphic DNA (RAPD) analysis was employed to characterize commercial winery yeast strains currently used in Brazilian industries and to examine the relationship between them. Sixteen strains were subjected to RAPD analysis using eight primers, selected from the 40 primers of the kits B and X of Operon Techn. From the total of 93 scorable bands, 39 (41.9%) were polymorphics. Using the accumulated information of several reactions, all strains under investigation were differentiated. Cluster analysis revealed three groups. Most of the strains (11) were clustered in one group. The highest distance (28%) was obtained with the strain Redstar (Universal Food Corp.).     

Genetical analysis of in vitro cell and tissue culture response in potato

The genetics of tissue culture response in potato has been examined by analysing a sample of dihaploids (2n=2x=24) extracted from tetraploid parents (4n=4x=48). The genotypes were screened for rate of nodal multiplication, in vitro tuberisation, regeneration from leaf discs and protoplast plating efficiency. Significant differences were detected between dihaploids for the traits measured and this indicates that tissue culture response in the tetraploid parents must be in the heterozygous condition. Estimates of the broad sense heritabilities were calculated together with the number of genes or effective factors involved in the control of the traits. These estimates indicate that tissue culture response in potato is under relatively simple genetic control and blocks of genes may be located on specific chromosomes. The inheritance of RFLP markers in the segregating dihaploid population was also monitored and the potential of using molecular markers linked to gene(s) controlling tissue culture response is discussed.     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

A synergism between oxalic acid and polygalacturonases in the depolymerization of potato tuber tissue

Rhizoctonia bataticola produced oxalic acid in vitro and in vivo during pathogenesis of patato tuber. Polygalacturonase (PG) was also detected in culture filtrates of the rot-causing organism. Levels of maceration and cell death in tuber tissue were higher when a mixture of oxalic acid and PG was used than when either oxalic acid or PG were used alone.     

Determination of RAPD Markers in Rice and their Conversion into Sequence Tagged Sites (STSs) and STS-Specific Primers

We produced 102 randomly amplified polymorphic DNA (RAPD) markersmapped on all 12 chromosomes of rice using DNAs of cultivarsNipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers10 nucleotides long were used both singly and in random pairsand about 1,400 primer-pairs were tested. Using both agarosegel and polyacrylamide gel electrophoresis enabled us to detectpolymorphisms appearing in the range from <100 bp to 2 kb.The loci of the RAPD markers were determined onto the frameworkof our RFLP linkage map and some of these markers were mappedto regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs weredetermined by cloning, identifying and sequencing of the mappedpolymorphic fragments.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Cytoplasmic DNA variation in a potato protoclonal population

Summary Mitochondrial DNA variation was detected in potato plants (protoclones) regenerated from leaf mesophyll protoplasts. Two forms of variation were evident; (1) DNA sequence alterations within the high molecular weight mitochondrial chromosome and (2) the appearance of an additional low molecular weight mitochondrial DNA species. Variation in chloroplast DNA was not detected. The data suggests that protocloning can introduce molecular diversity into mitochondrial genomes and thereby assist in overcoming the cytoplasmic genetic uniformity prevalent in most major crops.     

Flower production,male sterility and berry setting in andigena potato

Summary Unlike tuberosum, andigena potato germ plasm exhibits a high degree of genetic variation in morphological, biochemical and reproductive traits. Sixty-five percent of the 565 genotypes comprising 145 accessions of Solanum tuberosum ssp. andigena obtained from Argentina, Bolivia, Chile, Colombia and Peru remain totally vegetative and never develop any floral bud when cultivated in northern India. In 18% of genotypes, the floral buds develop but they drop off from the plants prematurely. Thus, 83% of genotypes do not develop mature flowers. The frequency of such genotypes is maximum in the Bolivian genotypes. Whereas 17% of genotypes produce mature flowers, only 2% develop berries. The highest proportion of floral bud formation and their subsequent development and differentiation into mature flowers occur in Peruvian and Colombian genotypes. Partial to high male sterility occurs in 93% of the flowering genotypes; their pollen sterility ranges from 15% to 91%. Seven percent of the flowering genotypes are completely pollen sterile. The male sterility is expressed variously, ranging from structural to sporogenous types. The floral bud formation, its development and retention to maturity, pollen and ovule functionability and fruit development are under the control of a large number of genes, most of which are unlinked and independent. Many of these genes are polygenic in nature.     

Effect of maltose on the response of potato anthers in culture

Anthers of the Solanum tuberosum genotype H3703 were cultured on medium containing equimolar concentrations of sucrose or maltose. It was found that significantly more pollen embryos became plants after culture on maltose and hence the yield of plants per 100 anthers cultured increased significantly. Mechanisms by which carbohydrate source may influence response to anther culture are discussed.