De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

Microsatellite Markers For Urochloa Humidicola (poaceae) And Their Transferability To Other Urochloa Species.

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species. A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species. This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction.

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes.

Development And Characterization Of Microsatellite Loci For Tabebuia Cassinoides (bignoniaceae).

Tabebuia cassinoides (Lam.) DC., popularly known as caxeta, is a tree species that belongs to the plant family Bignoniaceae. This species is endemic to the Brazilian Atlantic Forest and is widely exploited commercially. To date, little is known about its genetic structure, preventing the establishment of adequate management plans for this taxon. The objective of this study was to construct a microsatellite-enriched genomic library for T. cassinoides to select polymorphic loci, and standardize polymerase chain reaction amplification conditions. Of the 15 loci examined, 5 were polymorphic. The number of alleles per locus ranged from 2 to 8, with a mean of 4.4. The microsatellite loci described here represent the basis for detailed population genetic studies of this species, which will greatly contribute for the development of better conservation strategies for this taxon.

Convergence Of A Specialized Root Trait In Plants From Nutrient-impoverished Soils: Phosphorus-acquisition Strategy In A Nonmycorrhizal Cactus.

In old, phosphorus (P)-impoverished habitats, root specializations such as cluster roots efficiently mobilize and acquire P by releasing large amounts of carboxylates in the rhizosphere. These specialized roots are rarely mycorrhizal. We investigated whether Discocactus placentiformis (Cactaceae), a common species in nutrient-poor campos rupestres over white sands, operates in the same way as other root specializations. Discocactus placentiformis showed no mycorrhizal colonization, but exhibited a sand-binding root specialization with rhizosheath formation. We first provide circumstantial evidence for carboxylate exudation in field material, based on its very high shoot manganese (Mn) concentrations, and then firm evidence, based on exudate analysis. We identified predominantly oxalic acid, but also malic, citric, lactic, succinic, fumaric, and malonic acids. When grown in nutrient solution with P concentrations ranging from 0 to 100 μM, we observed an increase in total carboxylate exudation with decreasing P supply, showing that P deficiency stimulated carboxylate release. Additionally, we tested P solubilization by citric, malic and oxalic acids, and found that they solubilized P from the strongly P-sorbing soil in its native habitat, when the acids were added in combination and in relatively low concentrations. We conclude that the sand-binding root specialization in this nonmycorrhizal cactus functions similar to that of cluster roots, which efficiently enhance P acquisition in other habitats with very low P availability.

Microsatellite Loci For Orthophytum Ophiuroides (bromelioideae, Bromeliaceae) Species Adapted To Neotropical Rock Outcrops.

• Microsatellite primers were developed for Orthophytum ophiuroides, a rupicolous bromeliad species endemic to neotropical rocky fields. These microsatellite loci will be used to investigate population differentiation and species cohesion in such fragmented environments. The loci were tested for cross-amplification in related bromeliad species. • Eleven polymorphic microsatellite markers were isolated and characterized from an enriched library of O. ophiuroides. The loci were tested on 42 individuals from two populations of this species. The number of alleles per locus ranged from three to nine and the expected and observed heterozygosities ranged from 0.167 to 0.870 and from 0.369 to 0.958, respectively. Seven loci successfully amplified in other related bromeliad species. • Our results suggest that the microsatellite loci developed here will be useful to assess genetic diversity and gene flow in O. ophiuroides for the investigation of population differentiation and species cohesion in neotropical mountainous habitats.

Development And Characterization Of 32 Microsatellite Loci In Genipa Americana (rubiaceae).

• Microsatellite primers were developed for the tree species Genipa americana (Rubiaceae) for further population genetic studies. • We identified 144 clones containing 65 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 32 microsatellite loci and validated in 40 individuals of two natural G. americana populations. Seventeen loci were polymorphic, revealing from three to seven alleles per locus. The observed and expected heterozygosities ranged from 0.24 to 1.00 and from 0.22 to 0.78, respectively. • The 17 primers identified as polymorphic loci are suitable to study the genetic diversity and structure, mating system, and gene flow in G. americana.

[factors That Influence Bone Mass Of Healthy Children And Adolescents Measured By Quantitative Ultrasound At The Hand Phalanges: A Systematic Review].

To analyze the main factors that influence bone mass in children and teenagers assessed by quantitative ultrasound (QUS) of the phalanges. A systematic literature review was performed according to the PRISMA method with searches in databases Pubmed/Medline, SciELO and Bireme for the period 2001-2012, in English and Portuguese languages, using the keywords: children, teenagers, adolescent, ultrasound finger phalanges, quantitative ultrasound of phalanges, phalangeal quantitative ultrasound. 21 articles were included. Girls had, in QUS, Amplitude Dependent Speed of Sound (AD-SoS) values higher than boys during pubertal development. The values of the parameters of QUS of the phalanges and dual-energy X-ray Absorptiometry (DXA) increased with the increase of the maturational stage. Anthropometric variables such as age, weight, height, body mass index (BMI), lean mass showed positive correlations with the values of QUS of the phalanges. Physical activity has also been shown to be positively associated with increased bone mass. Factors such as ethnicity, genetics, caloric intake and socioeconomic profile have not yet shown a conclusive relationship and need a larger number of studies. QUS of the phalanges is a method used to evaluate the progressive acquisition of bone mass during growth and maturation of individuals in school phase, by monitoring changes that occur with increasing age and pubertal stage. There were mainly positive influences in variables of sex, maturity, height, weight and BMI, with similar data when compared to the gold standard method, the DXA.

New Loci Of Lychnophora Ericoides And Transferability To Lychnophora Pinaster, Endangered Medicinal Species From Brazil.

Lychnophora ericoides and Lychnophora pinaster are species used in popular medicine as analgesic or anti-inflammatory agents to treat contusions, rheumatism, and insect bites. In this study, 21 simple sequence repeat loci of L. ericoides were developed and transferred to L. pinaster. Three populations of L. ericoides and 2 populations of L. pinaster were evaluated; they were collected in the State of Minas Gerais. Population parameters were estimated, and the mean values of observed and expected heterozygosity were 0.297 and 0.408 (L. ericoides) and 0.228 and 0.310 (L. pinaster), respectively. Greater genetic variability was observed within populations than between populations of L. ericoides (62 and 37%) and L. pinaster (97 and 2.8%). These results provide information for genetic conservation and taxonomic studies of these endangered species.

Flow injection green method for the quantitative analysis of ketoconazole in pharmaceutical preparations

A flow injection method for the quantitative analysis of ketoconazole in tablets, based on the reaction with iron (III) ions, is presented. Ketoconazole forms a red complex with iron ions in an acid medium, with maximum absorbance at 495 nm. The detection limit was estimated to be 1×10--4 mol L-1; the quantitation limit is about 3×10--4 mol L-1 and approximately 30 determinations can be performed in an hour. The results were compared with those obtained with a reference HPLC method. Statistical comparisons were done using the Student's t procedure and the F test. Complete agreement was found at the 0.95 significance level between the proposed flow injection and the HPLC procedures. The two methods present similar precision, i.e., for HPLC the mean relative standard deviation was ca. 1.2% and for FIA ca. 1.6%.