Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.

Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae) of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG. The results from the application of the 18S probe confirmed the previous data obtained by silver nitrate staining, identifying a simple nucleolar organizing region system for these species. However, the labeling results from the 5S rDNA probe demonstrated a difference in the number and localization of these sites between the analyzed species. The obtained data allowed inferences on the possible processes involved in the karyotypic evolution of this genus.

Non-coding RNAs in schistosomes: an unexplored world

Non-coding RNAs (ncRNAs) were recently given much higher attention due to technical advances in sequencing which expanded the characterization of transcriptomes in different organisms. ncRNAs have different lengths (22 nt to >1, 000 nt) and mechanisms of action that essentially comprise a sophisticated gene expression regulation network. Recent publication of schistosome genomes and transcriptomes has increased the description and characterization of a large number of parasite genes. Here we review the number of predicted genes and the coverage of genomic bases in face of the public ESTs dataset available, including a critical appraisal of the evidence and characterization of ncRNAs in schistosomes. We show expression data for ncRNAs in Schistosoma mansoni. We analyze three different microarray experiment datasets: (1) adult worms' large-scale expression measurements; (2) differentially expressed S. mansoni genes regulated by a human cytokine (TNF-α) in a parasite culture; and (3) a stage-specific expression of ncRNAs. All these data point to ncRNAs involved in different biological processes and physiological responses that suggest functionality of these new players in the parasite's biology. Exploring this world is a challenge for the scientists under a new molecular perspective of host-parasite interactions and parasite development.

Solvation simplified

The effects of solvents on chemical phenomena is complex because there are various solute-solvent interaction mechanisms. Solvatochromism refers to the effects of solvents on the spectra of probes. The study of this phenomenon sheds light on the relative importance of the solvation mechanisms. Solvation in pure solvents is quantitatively analyzed in terms of a multi-parameter equation. In binary solvent mixtures, solvation is analyzed by considering the organic solvent, S, water, W, and a 1:1 hydrogen bonded species (S-W). The applications of solvatochromism to understand distinct chemical phenomena, reactivity and swelling of cellulose, is briefly discussed.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

The ability of the BANA test to detect different levels of P. gingivalis, T. denticola and T. forsythia

The aim of this study was to evaluate the ability of the BANA Test to detect different levels of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia or their combinations in subgingival samples at the initial diagnosis and after periodontal therapy. Periodontal sites with probing depths between 5-7 mm and clinical attachment level between 5-10 mm, from 53 subjects with chronic periodontitis, were sampled in four periods: initial diagnosis (T0), immediately (T1), 45 (T2) and 60 days (T3) after scaling and root planing. BANA Test and Checkerboard DNA-DNA hybridization identified red complex species in the subgingival biofilm. In all experimental periods, the highest frequencies of score 2 (Checkerboard DNA-DNA hybridization) for P. gingivalis, T. denticola and T. forsythia were observed when strong enzymatic activity (BANA) was present (p < 0.01). The best agreement was observed at initial diagnosis. The BANA Test sensitivity was 95.54% (T0), 65.18% (T1), 65.22% (T2) and 50.26% (T3). The specificity values were 12.24% (T0), 57.38% (T1), 46.27% (T2) and 53.48% (T3). The BANA Test is more effective for the detection of red complex pathogens when the bacterial levels are high, i.e. in the initial diagnosis of chronic periodontitis.

Multifractality in plasma edge electrostatic turbulence

Plasma edge turbulence in Tokamak Chauffage Alfven Bresilien (TCABR) [R. M. O. Galvao et al., Plasma Phys. Contr. Fusion 43, 1181 (2001)] is investigated for multifractal properties of the fluctuating floating electrostatic potential measured by Langmuir probes. The multifractality in this signal is characterized by the full multifractal spectra determined by applying the wavelet transform modulus maxima. In this work, the dependence of the multifractal spectrum with the radial position is presented. The multifractality degree inside the plasma increases with the radial position reaching a maximum near the plasma edge and becoming almost constant in the scrape-off layer. Comparisons between these results with those obtained for random test time series with the same Hurst exponents and data length statistically confirm the reported multifractal behavior. Moreover, the persistence of these signals, characterized by their Hurst exponent, present radial profile similar to the deterministic component estimated from analysis based on dynamical recurrences. (C) 2008 American Institute of Physics.

Gene expression profile of the plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans

The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.

QTL mapping for yield components in a tropical maize population using microsatellite markers

QTL mapping provides usefull information for breeding programs since it allows the estimation of genomic locations and genetic effects of chromossomal regions related to the expression of quantitative traits. The objective of this study was to map QTL related to several agronomic important traits associated with grain yield: ear weight (EW), prolificacy (PROL), ear number (NE), ear length (EL) and diameter (ED), number of rows on the ear (NRE) and number of kernels per row on the ear (NKPR). Four hundred F-2:3 tropical maize progenies were evaluated in five environments in Piracicaba, Sao Paulo, Brazil. The genetic map was previously estimated and had 117 microssatelite loci with average distance of 14 cM. Data was analysed using Composite Interval Mapping for each trait. Thirty six QTL were mapped and related to the expression of EW (2), PROL (3), NE (2), EL (5), ED (5), NRE (10), NKPR (5). Few QTL were mapped since there was high GxE interaction. Traits EW, PROL and EN showed high genetic correlation with grain yield and several QTL mapped to similar genomic regions, which could cause the observed correlation. However, further analysis using apropriate statistical models are required to separate linked versus pleiotropic QTL. Five QTL (named Ew1, Ne1, Ed3, Nre3 and Nre10) had high genetic effects, explaining from 10.8% (Nre3) to 16.9% (Nre10) of the phenotypic variance, and could be considered in further studies.

NEW MICROSATELLITE LOCI FOR WATER YAM (DIOSCOREA ALATA, DIOSCOREACEAE) AND CROSS-AMPLIFICATION FOR OTHER DIOSCOREA SPECIES

Premise of the study: Dioscorea alata L. is one of the most widely distributed species of the genus in the humid and semihumid tropics and is associated with traditional agriculture. Only a few microsatellite markers have been developed so far for this and other Dioscorea species. Methods and Results: We isolated 14 codominant polymorphic microsatellite markers using a microsatellite-enriched genomic library technique. Ten microsatellite loci were selected, and 80 D. alata accessions from different regions in Brazil were evaluated with nine polymorphic loci. The polymorphism information content (PIC) varied from 0.39 to 0.78 and the power discrimination (PD) ranged from 0.15 to 0.91. Six of the markers showed transferability for the species D. bulbifera, D. cayenensis-D. rotundata, and D. trifida. Conclusions: The SSR markers obtained are an important tool for further studies aiming to characterize the genetic diversity in D. alata and other Dioscorea spp. accessions.

DEVELOPMENT OF MICROSATELLITE MARKERS FOR AULONEMIA ARISTULATA (POACEAE) AND CROSS-AMPLIFICATION IN OTHER BAMBOO SPECIES

Premise of the study: Microsatellite primers were developed for Aulonemia aristulata, an endangered species of economic interest, to further describe its genetic variability and population structure. We also tested cross-amplification in 18 other bamboo species. Methods and Results: Using an enrichment genomic library, 13 microsatellite loci were isolated and characterized in A. aristulata. Seven of these loci were polymorphic. Twelve markers were cross-amplified in at least ten of the tested bamboo species. Conclusions: These markers will be useful for studies on the genetic diversity and structure of A. aristulata, which are important for future conservation, management and breeding programs of this species.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus x Bos indicus)

Background : In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) x Holstein (Bos taurus) cross. Results: Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions: The experimental F2 population derived from Gyr x Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

CHARACTERIZATION OF MICROSATELLITE LOCI IN HIMATANTHUS DRASTICUS (APOCYNACEAE), A MEDICINAL PLANT FROM THE BRAZILIAN SAVANNA

Premise of the study: We developed a new set of microsatellite markers for studying the genome of the janaguba tree, Himatanthus drasticus (Mart.) Plumel, which is used in folk medicine in northeastern Brazil. These novel markers are being used to evaluate the effect of harvesting on the genetic structure and diversity of natural populations of this species. Methods and Results: Microsatellite loci were isolated from an enriched H. drasticus genomic library. Nine primer pairs successfully amplified polymorphic microsatellite regions, with an average of 8.5 alleles per locus. The average values of observed and expected heterozygosity were 0.456 and 0.601, respectively. Conclusions: The microsatellite markers described here are valuable tools for population genetics studies of H. drasticus. The majority of the primers also amplified sequences in the genome of another species of the same genus. This new set of markers may be useful in designing a genetic conservation strategy and a sustainable management plan for the species.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.