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

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.

An identification tool for the Australian weedy Sporobolus species based on random amplified polymorphic DNA (RAPD) profiles

Based on morphological features alone, there is considerable difficulty in identifying the 5 most economically damaging weed species of Sporobolus [ viz. S. pyramidalis P. Beauv., S. natalensis ( Steud.) Dur and Schinz, S. fertilis ( Steud.) Clayton, S. africanus (Poir.) Robyns and Tourney, and S. jacquemontii Kunth.] found in Australia. A polymerase chain reaction (PCR)-based random amplified polymorphic DNA ( RAPD) technique was used to create a series of genetic markers that could positively identify the 5 major weeds from the other less damaging weedy and native Sporobolus species. In the initial RAPD pro. ling experiment, using arbitrarily selected primers and involving 12 species of Sporobolus, 12 genetic markers were found that, when used in combination, could consistently identify the 5 weedy species from all others. Of these 12 markers, the most diagnostic were UBC51(490) for S. pyramidalis and S. natalensis; UBC43(310,2000,2100) for S. fertilis and S. africanus; and OPA20(850) and UBC43(470) for S. jacquemontii. Species-specific markers could be found only for S. jacquemontii. In an effort to understand why there was difficulty in obtaining species-specific markers for some of the weedy species, a RAPD data matrix was created using 40 RAPD products. These 40 products amplified by 6 random primers from 45 individuals belonging to 12 species, were then subjected to numerical taxonomy and multivariate system (NTSYS pc version 1.70) analysis. The RAPD similarity matrix generated from the analysis indicated that S. pyramidalis was genetically more similar to S. natalensis than to other species of the 'S. indicus complex'. Similarly, S. jacquemontii was more similar to S. pyramidalis, and S. fertilis was more similar to S. africanus than to other species of the complex. Sporobolus pyramidalis, S. jacquemontii, S. africanus, and S. creber exhibited a low within-species genetic diversity, whereas high genetic diversity was observed within S. natalensis, S. fertilis, S. sessilis, S. elongates, and S. laxus. Cluster analysis placed all of the introduced species ( major and minor weedy species) into one major cluster, with S. pyramidalis and S. natalensis in one distinct subcluster and S. fertilis and S. africanus in another. The native species formed separate clusters in the phenograms. The close genetic similarity of S. pyramidalis to S. natalensis, and S. fertilis to S. africanus may explain the difficulty in obtaining RAPD species-specific markers. The importance of these results will be within the Australian dairy and beef industries and will aid in the development of integrated management strategy for these weeds.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

P>Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

Phenetic studies on randomly amplified polymorphic DNA-polymerase chain reaction-variability of four geographical populations of Lutzomyia whitmani (Diptera: Psychodidae) in Brazil

Previous evaluation of the genetic variability of four biogeographical populations of Lutzomyia whitmani from known foci of cutaneous leishmaniasis in Brazil demonstrated two main spatial clusters: Corte de Pedra-BA, Ilhéus-BA and Serra de Baturité-CE in the first cluster, and Martinho Campos-MG in the second. Further analysis showed a high degree of homogeneity in Corte de Pedra population but not in the others, which presented a significant percentage of specimens displaced from their phenon of origin (discrepant individuals). In the present work we analyzed the frequencies of association coefficients in the matrixes of similarity per population of Lutzomyia whitmani from both sexes and the general phenograms obtained, in a more detailed study of those discrepant specimens. Populational stability was observed for Corte de Pedra population, whereas the three remaining populations showed varying degrees of heterogeneity and different displacements according to sex. Our results strongly suggested the existence of a genetic flow between the lineages North-South/North-East and Ilhéus/Serra do Baturité of Lutzomyia whitmani.

Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model

INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA). RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100% of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system's redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.

Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD) analysis

Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.