Genetic monitoring of the Amazonian fish matrincha (Brycon cephalus) using RAPD markers: insights into supportive breeding and conservation programmes

The importance of genetic evaluations in aquaculture programmes has been increased significantly not only to improve effectiveness of hatchery production but also to maintain genetic diversity. In the present study, wild and captive populations of a commercially important neotropical freshwater fish, Brycon cephalus (Amazonian matrincha), were analyzed in order to evaluate the levels of genetic diversity in a breeding programme at a Brazilian research institute of tropical fish. Random Amplified Polymorphic DNA fingerprinting was used to access the genetic variability of a wild stock from the Amazon River and of three captive stocks that correspond to consecutive generations from the fishery culture. Although farmed stocks showed considerably lower genetic variation than the wild population, a significantly higher level of polymorphism was detected in the third hatchery generation. The results seem to reflect a common breeding practice on several hatchery fish programmes that use a small number of parents as broodstocks, obtaining reproductive success with few non-identified mating couples. The obtained data were useful for discussing suitable strategies for the genetic management and biodiversity conservation of this species.

Development and characterization of microsatellite loci in Phractocephalus hemioliopterus (Siluriformes: Pimelodidae) and their cross-species amplification in six related species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genetic variability in geographical populations of Aedes aegypti (Diptera, Culicidae) in Brazil elucidated by molecular markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82 x 10(-21) mol L-1. Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papaya L.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1:2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210(438)molecular marker was much better at papaya sex differentiation than other markers described in the literature.

Protein profile of Acidithiobacillus ferrooxidans strains exhibiting different levels of tolerance to metal sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe(2+) oxidation in the presence of 250 mm of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30-70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and 135) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.

Genetic variability in species of bats revealed by RAPD analysis

Random amplified polymorphic DNA molecular marker was utilized as a means of analyzing genetic variability in seven bat species: Molossus molossus, M. rufus, Eumops glaucinus, E. perotis, Myotis nigricans, Eptesicus furinalis, and Artibeus planirostris. The determination of genetic diversity was based on 741 bands produced by a 20-random primer set. Only eight bands were considered monomorphic to one species. The greatest number of bands and the most polymorphic condition were exhibited by M. molossus, followed by M. nigricans, A. planirostris, E. furinalis, E. glaucinus, M. rufus, and E. perotis. Nei's genetic diversity index in the seven species considering the 20 primers was not greater than 0.22, but some primers were capable of detecting values between 0.39 and 0.49. Nei's unbiased genetic distance values and the UPGMA clustering pattern show that M. molossus and M. rufus have a close genetic relationship, unlike that observed between E. perotis and E. glaucinus. The latter was clustered with A. planirostris and E. furinalis. The low values for genetic diversity and distance observed indicate a genetic conservatism in the seven species. The fluorescent in situ hybridization experiments did not confirm a monomorphic condition for the eight bands identified, demonstrating that the monomorphic bands obtained by random amplified polymorphic DNA are insufficient for the identification of bat species.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%. (C) 2002 Elsevier B.V. All rights reserved.

An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in São Paulo, Brazil

Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Cafe, respectively, were indistinguish able based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Evaluation of the Spreading of Isolated Bacterias from Dental Consulting-Room using RAPD Technique

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of microsatellite markers in cultivars of sweet orange (Citrus sinensis [L.] Osbeck)

Sweet orange is considered a very important species in the citrus world market and presents wide morphological variability. However, its characterization at the molecular level by random amplified polymorphic DNA (RAPD) and isozyme markers is not appropriate. Microsatellite or simple sequence repeats (SSRs) have been suggested as ideal for studies in cultures of vegetative propagation and as value markers for mapping in several species. However, information on microsatellite polymorphism in citrus species is scarce. In this work, microsatellite markers (AG-repeats) were developed from an enrichment library of genomic DNA of sweet orange cv. Pera (Citrus sinensis [L.] Osbeck), and 31 cultivars of sweet orange were evaluated. Evaluation of 18 microsatellite primers did not permit differentiation of the varieties studied. New microsatellite primers are being evaluated with the aim of detecting polymorphisms among the cultivars and closely related species to be used in genetic mapping programs.