Laura Maria Mariscal Ottoboni

Genetic distance of inbred lines and prediction of maize single-cross performance using RAPD markers

Abstract To evaluate the genetic diversity of 18 maize inbred lines, and to determine the correlation between genetic distance and single-cross hybrid performance, we have used random amplified polymorphic DNA (RAPD), a PCR-based technique. Eight of these lines came from a Thai synthetic population (BR-105), and the others derived from a Brazilian composite population (BR-106). Thirty two different primers were used giving a total of 325 reproducible amplification products, 262 of them being polymorphic. Genetic divergence was determinated using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method with arithmetical averages (UPGMA). Cluster analysis divided the samples into three distinct groups (GI, GII and GIII) that were confirmed by principal-coordinate analysis. The genetic distances (GD) were correlated with important agronomic traits for single-cross hybrids and heterosis. No correlation was found when group division was not considered, but significant correlations were detected between GI×GII and GI×GIII GDs with their respective single-cross hybrid grain-yield values. Three groups were identified; that is, the BR-106 population was divided in two different groups and the BR-105 population remained mostly as one group. The results indicated that RAPD can be used as a tool for determining the extent of genetic diversity among tropical maize inbred lines, for allocating genotypes into different groups, and also to aid in the choice of the superior crosses to be made among maize inbred lines, so reducing the number of crosses required under field evaluation.

Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination

BackgroundEscherichia coli strains are commonly found in the gut microflora of warm-blooded animals. These strains can be assigned to one of the four main phylogenetic groups, A, B1, B2 and D, which can be divided into seven subgroups (A0, A1, B1, B22, B23, D1 and D2), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Distinct studies have demonstrated that these phylo-groups differ in the presence of virulence factors, ecological niches and life-history. Therefore, the aim of this work was to analyze the distribution of these E. coli phylo-groups in 94 human strains, 13 chicken strains, 50 cow strains, 16 goat strains, 39 pig strains and 29 sheep strains and to verify the potential of this analysis to investigate the source of fecal contamination.ResultsThe results indicated that the distribution of phylogenetic groups, subgroups and genetic markers is non-random in the hosts analyzed. Strains from group B1 were present in all hosts analyzed but were more prevalent in cow, goat and sheep samples. Subgroup B23 was only found in human samples. The diversity and the similarity indexes have indicated a similarity between the E. coli population structure of human and pig samples and among cow, goat and sheep samples. Correspondence analysis using contingence tables of subgroups, groups and genetic markers frequencies allowed the visualization of the differences among animal samples and the identification of the animal source of an external validation set. The classifier tools Binary logistic regression and Partial least square -- discriminant analysis, using the genetic markers profile of the strains, differentiated the herbivorous from the omnivorous strains, with an average error rate of 17%.ConclusionsThis is the first work, as far as we are aware, that identifies the major source of fecal contamination of a pool of strains instead of a unique strain. We concluded that the analysis of the E. coli population structure can be useful as a supplementary bacterial source tracking tool.

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like α-coixin, the α-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like α-coixin genes designated α-3A, α-3B and α-3C were found in the 15 kb α-3 genomic clone. The α-3A and α-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the α-3B gene, suggesting that the three α-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of α-coixin clones with the published sequences of 22 kDa α-zein and 22 kDa-like α-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like α-prolamins and the 19 kDa α-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5′ and 3′ flanking regions of α-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. −300 prolamin box are present at conserved positions in α-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in α-3B that occupies approximately the same positions as those identified for the 22 kDa α-zein and α-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like α-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.

Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction

Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium that plays an important role in metal bioleaching processes. Despite the high level of tolerance to heavy metals shown by A. ferrooxidans, the genetic basis of copper resistance in this species remains unknown. We investigated the gene expression in response to copper in A. ferrooxidans LR using RNA arbitrarily primed polymerase chain reaction (RAP‐PCR). One hundred and four differentially expressed genes were identified using eight arbitrary primers. Differential gene expression was confirmed by DNA slot blot hybridization, and approximately 70% of the RAP‐PCR products were positive. The RAP‐PCR products that presented the highest levels of induction or repression were cloned, sequenced and the sequences were compared with those in databases using the BLAST search algorithm. Seventeen sequences were obtained. The RAP‐PCR product with the highest induction ratio showed similarity with the A. ferrooxidans cytochrome c. A high similarity with the thiamin biosynthesis gene thiC from Caulobacter crescentus was observed for another RAP‐PCR product induced by copper. An RAP‐PCR product repressed by copper showed significant similarity with the carboxysome operon that includes the ribulose‐1,5‐bisphosphate carboxylase/oxygenase complex from A. ferrooxidans and another copper‐repressed product was significantly similar to the XyIN outer membrane protein from Pseudomonas putida. Finally, RAP‐PCR products of unknown similarities were also present.

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Abstract Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1.

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and two‐dimensional gel electrophoresis (2‐DE) were used to identify iron‐responsive proteins in the white‐rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron‐sufficient and iron‐deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS‐PAGE for P. chrysosporium and twelve for L. edodes. In 2‐DE, the numbers of iron‐restricted induced proteins were 12 and 9, respectively, in a resolution range of 15–60 kDa and pI 4.5–8.1. SDS‐PAGE for the plasma membrane protein did not show differences, whereas the outer‐membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron‐complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

Changes in the Bacterial Community of Soil from a Neutral Mine Drainage Channel

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.

RAPD Genomic Fingerprinting Differentiates Thiobacillus ferrooxidans Strains

Summary The PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was optimized and used for assessing genomic variability among eight Thiobacillus ferrooxidans strains. RAPD fingerprints presented variation for the thirty primers used, giving a total of 269 polymorphic bands. Similarity coefficients between the strains were calculated, and UPGMA cluster analysis was used to generate a dendrogram showing relationships among them. Most primers divided T. ferrooxidans strains in two distinct groups - Group 1: S, SSP, V3, AMF and Group 2: CMV, FG-460,1-35, LR. We observed that the T. ferrooxidans strains used in this work have a high degree of genomic diversity and that RAPD is a powerful method to differentiate them.

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil.

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.