Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.

Genetic variation between several species of sections Extranervosae, Caulorrhizae, Heteranthae, and Triseminatae (genus Arachis) estimated by DNA polymorphism

Genetic variation within and among accessions of the genus Arachis representing sections Extranervosae, Caulorrhizae, Heteranthae, and Triseminatae was evaluated using RFLP and RAPD markers. RAPD markers revealed a higher level of genetic diversity than did RFLP markers, both within and among the species evaluated. Phenograms based on various band-matching algorithms revealed three major clusters of similarity among the sections evaluated. The first group included the species from section Extranervosae, the second group consisted of sections Triseminatae, Caulorrhizae, and Heteranthae, and the third group consisted of one accession of Arachis hypogaea, which had been included as a representative of section Arachis. The phenograms obtained from the RAPD and RFLP data were similar but not identical. Arachis pietrarellii, assayed only by RAPD, showed a high degree of genetic similarity with Arachis villosulicarpa. This observation supported the hypothesis that these two species are closely related. It was also shown that accession V 7786, previously considered to be Arachis sp. aff. pietrarellii, and assayed using both RFLPs and RAPDs, was possibly a new species from section Extranervosae, but very distinct from A. pietrarellii.

Taenia solium and Taenia saginata: Identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies. © 2007 Elsevier Inc. All rights reserved.

Isolamento e caracterização de marcadores de DNA associados ao sexo e segmentos de DNA repetitivo em espécies do gênero Brycon (Characidae, Bryconinae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genetic variability among Commelina weed species from the states of Paraná and São Paulo, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Frequência de microrganismos em conteúdo vulvovaginal de vacas tratadas com dispositivos intravaginais de progesterona

The dissertation was divided in two studies. With the first, aimed to evaluate the occurrence of microorganisms present in the vulvovaginal region of cows that received intravaginal progesterone devices during the fixed-time artificial insemination (FTAI) programs, and correlate the results with pregnancy rates. Samples were collected from vulvovaginal region of 30 beef cows Guzerá and 30 crossbred dairy cows, and also intravaginal devices, randomly. Of the 120 samples of cows, 60 corresponded to the collections of the period prior to the introduction device (D0) and 60 to the subsequent withdrawal of it (D9); it yielded 100% of bacterial growth, whereas, in most samples, it was found more than one isolated. In D0, the most frequent agent was Escherichia coli (52%), and in D9, Proteus spp and E. coli were the most frequent (32% and 28%, respectively). Regarding intravaginal progesterone devices, in D0 were isolated 37 microorganisms, being predominant those of the genus Bacillus (35%); in D9, 41 colony forming units (CFU) were isolated, of which 36.6% corresponded to Proteus spp. For the analysis of the antimicrobial profile, susceptibility testing was performed by diffusion agar disk, and cows that did not became pregnant after FTAI program were selected, as a future treatment. There resistance 100% to penicillin, and sensitivity, approximately, 90% to gentamicin, both isolates obtained from samples of beef cows and obtained of dairy cows. Regarding pregnancy rate, the 30 beef cows, 11 were diagnosed pregnant (36.7%), 4 (36.4%) treated with reused devices and 7 (63.6%) with new devices, which showed more effective. Of the 30 dairy cows, 15 were pregnant (50%), 8 (53.3%) were implanted with reused devices and 7 (46.7%) with new devices, with no significant differences in pregnancy rates. Because it is a research, females were chosen at random, and factors such as body condition, nutritional management and health weren't priority. With the second study, aimed to analyze the similarity between strains, conducted by technical Random Amplified Polymorphic DNA (RAPD -PCR). Presence of E. coli and the absence of pregnancy were selection criteria used. From the results, it was observed that most of the isolates wasn't phylogenetically similar, since they showed lower than 85% similarity. The study stressed the importance of E. coli in vulvovaginal microbiota of cows and the presence of phenotypic and genotypic characters of this bacterium on possible reproductive problems.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Correction of the misclassification of species in the Portuguese collection of Cucurbita pepo L. using DNA markers

In this study, the genetic variability among 130 accessions of the Portuguese germplasm collection of Cucurbita pepo L. maintained at the Banco Portugues de Germoplasma Vegetal was assessed using AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) techniques for the identification of a genetically diverse core group of accessions for field phenotypic analysis. The surprisingly completely different molecular patterns exhibited by multiple accessions was later confirmed in the distribution of the putative C. pepo plants into two clusters drastically separated at a very low level of genetic similarity (DICE coefficient = 0.37). Additional analyses with RAPD and ISSR (inter single sequence repeat) markers and the introduction of standard genotypes of C. maxima L. and C. moschata L. into the analyses allowed the identification of multiple accessions of the last species wrongly included in the C. pepo collection. This study is a good example of the usefulness of DNA markers in the establishment and management of plant germplasm collections.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Correction of the misclassification of species in the Portuguese collection of Cucurbita pepo L. using DNA markers

In this study, the genetic variability among 130 accessions of the Portuguese germplasm collection of Cucurbita pepo L. maintained at the Banco Portugues de Germoplasma Vegetal was assessed using AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) techniques for the identification of a genetically diverse core group of accessions for field phenotypic analysis. The surprisingly completely different molecular patterns exhibited by multiple accessions was later confirmed in the distribution of the putative C. pepo plants into two clusters drastically separated at a very low level of genetic similarity (DICE coefficient = 0.37). Additional analyses with RAPD and ISSR (inter single sequence repeat) markers and the introduction of standard genotypes of C. maxima L. and C. moschata L. into the analyses allowed the identification of multiple accessions of the last species wrongly included in the C. pepo collection. This study is a good example of the usefulness of DNA markers in the establishment and management of plant germplasm collections.

Outcrossing rate in sweet passion fruit based on molecular markers

P>Yellow and sweet passion fruit are insect-pollinated species native to the tropics. Fruits are used commercially for human consumption worldwide. The yellow passion fruit is an outcrossing species with self-incompatible flowers. However, the reproductive system of the sweet passion fruit (Passiflora alata) has not been well elucidated. The objective of this work was to characterize aspects of the mating system in the sweet passion fruit using random amplified polymorphic DNA (RAPD) and microsatellite markers, particularly the rate of outcrossing in P. alata progenies. A multilocus outcrossing rate of t(m) = 0.994 was determined from RAPD and t(m) = 0.940 from microsatellites, supporting P. alata as an outcrossing species. The fixation indices of the maternal generation (F(m)) were -0.200 and 0.071 with RAPD and microsatellite loci, respectively, indicating the absence of inbreeding in the maternal generation. The paternity correlation (r(p)) varied from -0.008 with RAPD markers to 0.208 with microsatellite markers, suggesting a low probability of finding full sibs within the progenies. The results demonstrated that all progenies assessed in this study were derived from outcrossing.

Genetics and race variability of the lucerne - Colletotrichum trifolii pathosystem in Australia

The Australian-bred lucerne cultivars, Trifecta and Sequel, were found to possess useful levels of resistance to both Colletotrichum trifolii races 1 and 2. Race 2 has only been previously observed in the United States and surveys did not reveal its presence in Australia. Multilocus fingerprinting using random amplified polymorphic DNA (RAPDs) analysis revealed low diversity (<10% dissimilarity) within Australian C. trifolii collections, and between the Australian race 1 isolates and a US race 2 isolate. Studies on the inheritance of resistance to C. trifolii race 1 in individual clones from Trifecta and Sequel revealed the presence of 2 different genetic mechanisms. One inheritance was for resistance as a recessive trait, and the other indicated that resistance was dominant. The recessive system has never been previously reported, whereas in the US, 2 completely dominant and independent tetrasomic genes Anl and Ant have been reported to condition C. trifolii resistance. It was not possible to fit the observed segregations from our studies to a single-gene model. In contrast to US studies, clones of cv. Sequel exhibiting the recessive resistance reacted differently to spray and stem injection with C. trifolii inoculum, being resistant to the former and susceptible to the latter, providing additional evidence for the presence of a different genetic mechanism conditioning resistance to those previously reported in the US. As C. trifolii is one of the most serious diseases of lucerne worldwide, the future development of molecular markers closely linked to the dominant and recessive resistances identified in these studies, and the relationships between these resistances and Anl and Ans as determined by genetic mapping, appear to be useful areas of future study.

Identification in Australia of the quarantine pathogen of sunflower Phoma macdonaldii (Teleomorph : Leptosphaeria lindquistii)

Isolations from black stem lesions of sunflower growing in south-eastern Queensland yielded fungi putatively identified as species of Phoma. Pathogenicity assays showed that these isolates were capable of killing sunflower plants under glasshouse conditions. The isolates were compared with authentic cultures of Phoma macdonaldii and other isolates of Phoma taken from sunflower from around the world. Random amplified polymorphic DNA analysis showed that all the Australian isolates examined were very similar to the holotype culture of Phoma macdonaldii from Canada. Sequencing of the internal transcribed spacer regions also revealed the relatedness of the Australian isolates to the holotype. This is the first official record of P. macdonaldii in Australia.

Differential disease reactions on lucerne genotypes inoculated with Phytophthora medicaginis isolates from lucerne and chickpea

Stem inoculation of clonally propagated lucerne genotypes was used to assess levels of host species and genotype specialisation in Phytophthora medicaginis. A quantitative assessment of pathogenic aggressiveness of 29 P. medicaginis isolates (from lucerne and chickpea) on 9 different clonally propagated lucerne genotypes revealed no significant difference in aggressiveness between isolates from lucerne and those from chickpea on all of the lucerne genotypes. This supported previous studies which showed that P. medicaginis isolates from lucerne and chickpea were indistinguishable using random amplified polymorphic DNA (RAPD) analysis. Analysis of pathogenic aggressiveness towards individual lucerne genotypes revealed, for the first time, specificity of individual P. medicaginis isolates. This has implications for breeding for resistance to P. medicaginis in lucerne, where screening should be done using the widest range of pathogen specificity obtainable.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.