Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A Bayesian approach for constructing genetic maps when markers are miscoded

The advent of molecular markers has created opportunities for a better understanding of quantitative inheritance and for developing novel strategies for genetic improvement of agricultural species, using information on quantitative trait loci (QTL). A QTL analysis relies on accurate genetic marker maps. At present, most statistical methods used for map construction ignore the fact that molecular data may be read with error. Often, however, there is ambiguity about some marker genotypes. A Bayesian MCMC approach for inferences about a genetic marker map when random miscoding of genotypes occurs is presented, and simulated and real data sets are analyzed. The results suggest that unless there is strong reason to believe that genotypes are ascertained without error, the proposed approach provides more reliable inference on the genetic map.

Characterization of Rhizoctonia solani associated with soybean in Brazil

Rhizoctonia solani causes pre- and post-emergence damping-off, root and hypocotyl rot and foliar blight in soybean. Foliar blight has resulted in yield losses of 31-60% in north and northeast Brazil. The aim of this study was to characterize isolates of R. solani associated with soybean in Brazil. Among 73 Rhizoctonia isolates examined, six were binucleate and 67 were multinucleate. The multinucleate isolates were characterized according to hyphal anastomosis reaction, mycelial growth rate, thiamine requirement, sclerotia production, and RAPD molecular markers. Four isolates that caused hypocotyl rot belonged to AG-4 and using RAPD analysis they grouped together with the HGI subgroup. Another isolate that caused root and hypocotyl rots was thiamine auxotrophic, grew at 35 °C, and belonged to AG-2-2 IIIB. All 62 isolates that caused foliar blight belonged to AG-1 IA. RAPD analysis of R. solani AG-1 IA soybean isolates showed high genetic similarity to a tester strain of AG-1 IA, confirming their classification. The teleomorph of R. solani, Thanatephorus cucumeris was produced in vitro by one AG-1 IA isolate from soybean. The AG-4 and AG-2-2 IIIB isolates caused damping-off and root and hypocotyl rots of soybean seedlings cv. 'FT-Cristalina', under greenhouse conditions. The AG-2-2 IIIB isolate caused large lesions on the cortex tissue, that was distinct from the symptoms caused by AG-4 isolates. The AG-1 IA isolates caused foliar blight in adult soybean plants cv. 'Xingu' under the greenhouse and also in a detached-leaf assay.

Genetic variation within and among species of five sections of the genus Arachis L. (Leguminosae) using RAPDs

Some Arachis species are widely used as commercial plants, e.g. the groundnut A. hypogaea, an important source of good quality protein and oil, and A. pintoi and A. glabrata, that are utilized as forage species. Germplasm of most Arachis species is available in germplasm banks. However, little it is known about the genetic attributes of this germplasm, and mainly about its genetic variability, which is very important for its maintenance. In the present study RAPDs were used to assay the genetic variation within and among 48 accessions of five sections of the genus Arachis and to establish the genetic relationships among these accessions. Ten of 34 primers tested were selected for DNA amplification reactions since they yielded the largest numbers of polymorphic loci. A dendrogram was constructed based on data from the 10 primers selected. Eighty RAPD polymorphic bands were analyzed among the accessions studied. The relationships among species based on RAPDs were similar to those previously reported based on morphological, cytological and crossability data; demonstrating that RAPDs can be used to determine the genetic relationships among species of the different sections of the genus Arachis. In general, wide variation was found among accessions and low variation was found within the accessions that had two or more plants analyzed. However, higher polymorphism was found in the section Trierectoides and in one accession of A. major, indicating that generalizations should be avoided and each species should be analyzed in order to establish collection and maintenance strategies.

Kappa-casein gene study with molecular markers in female buffaloes (Bubalus bubalis)

Caseins comprise make up about 80% of the total protein content of milk and present polymorphism with change in the amino acid sequence. Within this abundance of proteins, kappa-casein is noteworthy, since it has been associated with differences in milk yield, composition and processing. The objective of this study was to observe the existence of polymorphism in the kappa-casein gene in female buffaloes. For this purpose, blood samples from 115 female buffaloes, collected with vacutainer by needle punctionure of the jugular vein, were used. for genomic DNA extraction was done from blood samples. The PCR-RFLP and SSCP techniques demonstrated that the studied animals were monomorphic for the kappa-casein gene. Only allele B was observed in these animals, which was present in homozygosis. Therefore, it was not possible to quantify the gene action on milk yield and its constituents. The monomorphism observed in the population studied would allow the development of a method to identify mixtures of cow and buffalo milk in mozzarella cheese production, especially because, in cattle, the kappa-casein gene is polymorphic. Copyright by the Brazilian Society of Genetics.

Transferability and use of microsatellite markers for the genetic analysis of the germplasm of some Arachis section species of the genus Arachis

The Arachis section is the most important of the nine sections of the genus Arachis because it includes the cultivated peanut, Arachis hypogaea. The genetic improvement of A. hypogaea using wild relatives is at an early stage of development in spite of their potential as sources of genes, including those for disease and pests resistance, that are not found in the A. hypogaea primary gene pool. Section Arachis species germplasm has been collected and maintained in gene banks and its use and effective conservation depends on our knowledge of the genetic variability contained in this material. Microsatellites are routinely used for the analysis of genetic variability because they are highly polymorphic and codominant. The objective of this study was to evaluate the transferability of microsatellite primers and the assay of genetic variability between and within the germplasm of some species of the Arachis section. Fourteen microsatellite loci developed for three different species of Arachis were analyzed and 11 (78%) were found to be polymorphic. All loci had transferability to all the species analyzed. The polymorphic loci were very informative, with expected heterozygosity per locus ranging from 0.70 to 0.94. In general, the germplasm analyzed showed wide genetic variation. © 2006 Sociedade Brasileira de Genética.

RAPD optimization for genetic studies with Citrulus lanatus and Sesamum indicum genotypes

The Random Amplified Polymorphic DNA (RAPD) technique is powerful for DNA polymorphism determinations and is widely used in research involving different organisms, but it is known that RAPD can be affected by many factors that may result in false positive bands and non-reproducible assays. In this study, we analyzed the effect of several factors such as DNA template, primer and Taq DNA polymerase concentrations to optimize and standardize the RAPD technique for further genetic studies with Citrulus lanattus and Sesamum indicum L. The best combination of DNA, Taq DNA polymerase enzyme and primer concentrations in RAPD amplification procedures for sesame and watermelon genotypes was established.

In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.

Development of SCAR marker linked to stem canker resistance gene in soybean

Stem canker caused by the fungus Diaporthe phaseolorum f. sp. meridionalis is a disease that limits soybean cultivation. Phenotypic evaluations aiming at disease resistance require labor-intensive processes, as for instance handling and transport of phytopathogens. The use of DNA markers in the selective procedures eases certain phases, besides being practical, safe and reliable. A RAPD fragment of 588pb was identified among bulks of resistant and susceptible plants in the cross BR92-15454 (R) x IAC-11 (S). Through co-segregation, the distance between the resistance locus and the fragment was estimated at 7.4 ± 2.1 cM, with a Lodmax. of 23.072 (first year) and at 6.0 ± 3.4 cM with a Lodmax. of 7.806 (second year). The fragment was converted into a SCAR marker and digested with enzyme Hinc II, which made the classification in homozygous resistant, heterozygous resistant and susceptible plants possible. This SCAR marker is suitable for use in the improvement program conducted in Jaboticabal.

QTL identification for tolerance to fruit set in tomato by FAFLP markers

This study aimed to detect quantitative trait loci (QTL) by fALP (Fluorescent Amplified Fragment Length Polymorphism) markers associated to the trait tomato fruit set at high temperatures. A biparental cross between line Jab-95 (heat-tolerant) and cultivar Caribe (heat-susceptible) was made. A total of 192 plants of the F2 generation were evaluated, generating 172 polymorphic markers through six primer combinations previously identified by the Bulked Segregant Analysis technique. To construct the genetic map, 106 of the 172 markers that segregated in the expected Mendelian segregation proportion (3:1) were used. The map covered 1191.46 cM of the genome. Six trait-linked QTL were identified in the analysis of simple markers and three others by the interval-mapping methodology. These results could be highly useful in improvement programs, since heat-tolerant plants can be selected rapidly, which improves tomato fruit set.

Associations between microsatellite markers and traits related to performance, carcass and organs in chickens

Associations between four microsatellite markers on chromosome 11 and five on chromosome 13 with performance, carcass and organs traits were investigated in chickens using a least-squares approach applied to single-marker analysis. Three hundred and twenty seven F 2 chickens from the EMBRAPA broiler×layer experimental population were evaluated for 16 traits: five related to performance, five to carcass and five to organs, plus the hematocrit. Two significance thresholds were considered: p<0.05 and p<0.0056; the last value resulted from the application of a multiple tests analyses correction. On chromosome 11, six associations (p<0.05) between the genotypes of two markers with four growth related and one carcass trait were found. On chromosome 13, six associations (p<0.05) between marker genotypes and three performance traits, eight associations (p<0.05) between marker genotypes and two carcass traits and eight associations (p<0.05) between marker genotypes and four organs traits were detected. These associations were indications of the presence of quantitative trait loci on these chromosomes, especially on chromosome 13. In this chromosome, the strongest evidence was for body weight at 41 days of age and percentage of carcass because the p-values exceeded the multiple test threshold (p<0.0056), but also for breast percentage and heart weight due to the large number of markers (four) on chromosome 13 associated with each one of these traits. These associations should be further investigated by interval mapping analyses to find QTL positions and to allow the estimation of their effects. © Asian Network for Scientific Information, 2009.

Astrocyte intermediate filaments are important markers in olfactory bulb of bovine herpesvirus type 5 natural infections

Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear.

Application of molecular markers on genetic improvement and reproduction in the post-genomic Era: Emerging biotechnologies and perspectives in livestock

Background: New challenges are rising in the animal protein market, and one of the main world challenges is to produce more in shorter time, with better quality and in a sustainable way. Brazil is the largest beef exporter in volume hence the factors affecting the beef meat chain are of major concern in countrýs economy. An emerging class of biotechnological approaches, the molecular markers, is bringing new perspectives to face these challenges, particularly after the publication of the first complete livestock genome (bovine), which has triggered a massive initiative to put in practice the benefits of the so called the Post-Genomic Era. Review: This article aimed at showing the directions and insights in the application of molecular markers on livestock genetic improvement and reproduction as well at organizing the progress so far, pointing some perspectives of these emerging technologies in Brazilian ruminant production context. An overview on the nature of the main molecular markers explored in ruminant production is provided, which describes the molecular bases and detection approaches available for microsatellites (STR) and single nucleotide polymorphisms (SNP). A topic is dedicated to review the history of association studies between markers and important trait variation in livestock, showing the timeline starting on quantitative trait loci (QTL) identification using STR markers and ending in high resolution SNP panels to proceed whole genome scans for phenotype/genotype association. Also the article organizes this information to reveal how QTL prospection using STR could open ground to the feasibility of marker-assisted selection and why this approach is quickly being replaced by studies involving the application of genome-wide association using SNP research in a new concept called genomic selection. Conclusion: The world's scientific community is dedicating effort and resources to apply SNP information in livestock selection through the development of high density panels for genomic association studies, connecting molecular genetic data with phenotypes of economic interest. Once generated, this information can be used to take decisions in genetic improvement programs by selecting animals with the assistance of molecular markers.

Identification of microsatellites markers associated with yield components and quality parameters in sugarcane

Marker assisted selection depends on the identification of tightly linked association between marker and the trait of interest. In the present work, functional (EST-SSRs) and genomic (gSSRs) microsatellite markers were used to detect putative QTLs for sugarcane yield components (stalk number, diameter and height) and as well as for quality parameters (Brix, Pol and fibre) in plant cane. The mapping population (200 individuals) was derived from a bi-parental cross (IACSP95-3018 x IACSP93-3046) from the IAC Sugarcane Breeding Program. As the map is under construction, single marker trait association analysis based on the likelihood ratio test was undertaken to detect the QTLs. Of the 215 single dose markers evaluated (1:1 and 3:1), 90 (42%) were associated with putative QTLs involving 43 microsatellite primers (18 gSSRs and 25 EST-SSRs). For the yield components, 41 marker/trait associations were found: 20 for height, 6 for diameter and 15 for stalk number. An EST-SSRs marker with homology to non-phototropic hypocotyls 4 (NPH4) protein was associated with a putative QTL with positive effect for diameter as also with a negative effect for stalk number. In relation to the quality parameters, 18 marker trait associations were found for Brix, 19 for Pol, and 12 for fibre. For fibre, 58% of the QTLs detected showed a negative effect on this trait. Some makers associated with QTLs with a negative effect for fibre showed a positive effect for Pol, reflecting the negative correlation generally observed between these traits.

Historical and recent biological markers of exposure to fluoride

Recent and historical biomarkers assess chronic or subchronic exposure to fluoride. The most studied recent biomarkers are nails and hair. Both can be non-invasively obtained, although collection of nails is more accepted by the subjects. External contamination may be a problem for both biomarkers and still needs to be better evaluated. Nails have been more extensively studied. Although the available knowledge does not allow their use as predictors of dental fluorosis by individual subjects, since reference values of fluoride have not yet been established, they have a strong potential for use in epidemiological surveys. Toenails should be preferred instead of fingernails, and variables that are known to affect nail fluoride concentrations - such as age, gender and geographical area - should be considered. The main historical biomarkers that could indicate total fluoride body burden are bone and dentin. Of these, bone is more studied, but its fluoride concentrations vary according to the type of bone and subjects' age and gender. They are also influenced by genetic background, renal function and remodeling rate, variables that complicate the establishment of a normal range of fluoride levels in bone that could indicate 'desirable' exposure to fluoride. The main issue when attempting to use bone as biomarker of fluoride exposure is the difficulty and invasiveness of sample collection. In this aspect, collection of dentin, especially from 3rd molars that are commonly extracted, is advantageous. However, mean values also span a wide range and reference concentrations have not been published yet. © 2011 S. Karger AG, Basel.