QTL mapping for resistance to Ceratocystis wilt in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.     

Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.     

Invertebrate drift along a longitudinal gradient in a Neotropical stream in Serra do Cipó National Park,Brazil

The diversity and composition of drift invertebrate assemblages were evaluated along a longitudinal gradient of an altitudinal stream in southeastern Brazil. The main goal of this study was to evaluate the influence of seasonality, stream order, and some abiotic factors on invertebrate drift and the use of drifting invertebrate assemblages to assess aquatic invertebrate diversity. Drift samples were collected over a 24 h period using nets (open area of 0.08 m²; mesh 0.250 mm), partially submerged (60%) in the water column. Taxonomic richness, Pielou evenness (J), Shannon–Wiener diversity (H), and total density of drift invertebrate assemblages were used in unpaired t-tests, Kruskal–Wallis and stepwise multiple regression analysis. The results showed a high taxonomic richness of aquatic invertebrates, with 91 taxa found. Chironomidae and Ephemeroptera represented together c. 80% of the total density of drift organisms. The drift approach allowed the collection of new and rare taxa, besides the knowledge of pupae stage of several chironomid genera. Significant differences in the taxonomic richness and diversity of drift invertebrate assemblages were found between the rainy and dry periods, indicating a significant influence of seasonality. An increase in water flow and electrical conductivity were associated with the increase in the taxonomic richness and diversity in the rainy period. No significant differences were found among the other abiotic variables among the stream orders.     

Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana

l ‐Tyrosine is an essential aromatic amino acid required for the synthesis of proteins and a diverse array of plant natural products; however, little is known on how the levels of tyrosine are controlled in planta and linked to overall growth and development. Most plants synthesize tyrosine by TyrA arogenate dehydrogenases, which are strongly feedback‐inhibited by tyrosine and encoded by TyrA1 and TyrA2 genes in Arabidopsis thaliana. While TyrA enzymes have been extensively characterized at biochemical levels, their in planta functions remain uncertain. Here we found that TyrA1 suppression reduces seed yield due to impaired anther dehiscence, whereas TyrA2 knockout leads to slow growth with reticulate leaves. The tyra2 mutant phenotypes were exacerbated by TyrA1 suppression and rescued by the expression of TyrA2, TyrA1 or tyrosine feeding. Low‐light conditions synchronized the tyra2 and wild‐type growth, and ameliorated the tyra2 leaf reticulation. After shifting to normal light, tyra2 transiently decreased tyrosine and subsequently increased aspartate before the appearance of the leaf phenotypes. Overexpression of the deregulated TyrA enzymes led to hyper‐accumulation of tyrosine, which was also accompanied by elevated aspartate and reticulate leaves. These results revealed that TyrA1 and TyrA2 have distinct and overlapping functions in flower and leaf development, respectively, and that imbalance of tyrosine, caused by altered TyrA activity and regulation, impacts growth and development of Arabidopsis. The findings provide critical bases for improving the production of tyrosine and its derived natural products, and further elucidating the coordinated metabolic and physiological processes to maintain tyrosine levels in plants.     

On the identification of ionic species of neutral halogen dimers, monomers and pincer type palladacycles in solution by electrospray mass and tandem mass spectrometry

Electrospray (ESI) mass spectra analysis of acetonitrile solutions of a series of neutral chloro dimers, pincer type, and monomeric palladacycles has enabled the detection of several of their derived ionic species. The monometallic cationic complexes Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (1a) and [Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)]⁺ (1b) and the bimetallic cationic complex [κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]Pd-Cl-Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (1c) were detected from an acetonitrile solution of the pincer palladacycles Pd[κ¹-C,κ¹-N,κ¹-S-C(CH₃S-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Cl) 1. For the dimeric compounds {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](μ-Cl)}₂ (2, Y=H and 3, CF₃), highly electronically unsaturated palladacycles [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (2d, 3d) and their mono and di-acetonitrile adducts, namely, [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)]⁺ (2e, 3e) and [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)₂]⁺ (2f and 3f) were detected together with the bimetallic complex [Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]-Cl-Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N](CH₃)₂]⁺ (2a, 3a) and its acetonitrile adducts [κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)Pd-Cl-Pd[ κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂]⁺ (2b, 3b) and [κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)Pd-Cl-Pd[κ¹-C, κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂(CH₃CN)]⁺ (2c, 3c). The dimeric palladacycle {Pd[κ¹-C,κ¹-N-C(CH₃O-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](μ-Cl)}₂ (4) is unique as it behaves as a pincer type compound with the OCH₃ substituent acting as an intramolecular coordinating group which prevents acetonitrile full coordination, thus forming the cationic complexes [(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂-κO,κC,κN)Pd]⁺ (4b), [(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂- κO,κC,κN)Pd(CH₃CN)]⁺ (4c) and [(C₆H₄ (o-MeO)CC(Cl)CH₂N(CH₃)₂-κO, κC,κN)Pd-Cl-Pd(C₆H₄(o-CH₃O)CC(Cl)CH₂N(CH₃)₂-κO,κC,κN)]⁺ (4a). ESI-MS spectra analysis of acetonitrile solutions of the monomeric palladacycles Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Cl)(Py) (5, Y=H and 6, Y=CF₃) allows the detection of some of the same species observed in the spectra of the dimeric palladacycles, i.e., monometallic cationic 2d-3d, 2e-3e and {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](Py)}⁺ (5a, 6a) and {Pd[κ¹-C,κ¹-N-C(Y-2-C₆H₄)C(Cl)CH₂N(CH₃)₂](CH₃CN)(Py)}⁺ (5b, 6b) and the bimetallic 2a, 3a, 2b, 3b, 2c and 3c. In all cationic complexes detected by ESI-MS, the cyclometallated moiety was intact indicating the high stability of the four or six electron anionic chelate ligands. The anionic (chloride) or neutral (pyridine) ligands are, however, easily replaced by the acetonitrile solvent.     

Microvascular pressure and functional capillary density in extreme hemodilution with low- and high-viscosity dextran and a low-viscosity Hb-based O2 carrier

Blood losses are usually corrected initially by the restitution of volume with plasma expanders and subsequently by the restoration of oxygen-carrying capacity using either a blood transfusion or possibly, in the near future, oxygen-carrying plasma expanders. The present study was carried out to test the hypothesis that high-plasma viscosity hemodilution maintains perfused functional capillary density (FCD) by preserving capillary pressure. Microvascular pressure responses to extreme hemodilution with low- (LV) and high-viscosity (HV) plasma expanders and an exchange transfusion with a polymerized bovine cell-free Hb (PBH) solution were analyzed in the awake hamster window chamber model (n = 26). Systemic hematocrit was reduced from 50% to 11%. PBH produced a greater mean arterial blood pressure than the nonoxygen carriers. FCD was higher after a HV plasma expander (70 +/- 15%) vs. PBH (47 +/- 12%). Microvascular pressure spanning the capillary network was higher after a HV plasma expander (16-19 mmHg) compared with PBH (12-16 mmHg) and a LV plasma expander (11-14 mmHg) but lower than control (22-26 mmHg). FCD was found to be directly proportional to capillary pressure. The use of a HV plasma expander in extreme hemodilution maintained the number of perfused capillaries and tissue perfusion by comparison with a LV plasma expander due to increased mean arterial blood pressure and capillary pressure. The use of PBH increased mean arterial pressure but reduced capillary pressure due to vasoconstriction and did not maintain FCD.     

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (H_O?=?0.312; H_E?=?0.436) and B. subterranea presented the highest values (H_O?=?0.687; H_E?=?0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q?≥?0.976) and the low combined probability of identity (I?≤?9.91?×?10^–6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

     

Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Gondwanan radiation of the Southern Hemisphere crayfishes (Decapoda: Parastacidae): evidence from fossils and molecules

Aim The sequential break‐up of Gondwana is thought to be a dominant process in the establishment of shared biota across landmasses of the Southern Hemisphere. Yet similar distributions are shared by taxa whose radiations clearly post‐date the Gondwanan break‐up. Thus, determining the contribution of vicariance versus dispersal to seemingly Gondwanan biota is complex. The southern freshwater crayfishes (family Parastacidae) are distributed on Australia and New Guinea, South America, Madagascar and New Zealand and are unlikely to have dispersed via oceans, owing to strict freshwater limitations. We test the hypotheses that the break‐up of Gondwana has led to (1) a predominately east–west (((Australia, New Zealand: 80 Ma) Madagascar: 160–121 Ma) South America: 165–140 Ma), or (2) a southern (((Australia, South America: 52–35 Ma) New Zealand: 80 Ma) Madagascar: 160–121 Ma) pattern for parastacid crayfish. Further, we examine the evidence for a complete drowning of New Zealand and subsequent colonization by freshwater crayfish. Location Southern Hemisphere. Methods The evolutionary relationships among the 15 genera of Parastacidae were reconstructed using mitochondrial [16S, cytochrome c oxidase subunit I (COI)] and nuclear (18S, 28S) sequence data and maximum likelihood and Bayesian methods of phylogenetic reconstruction. A Bayesian (multidivtime ) molecular dating method using six fossil calibrations and phylogenetic inference was used to estimate divergence time among crayfish clades on Gondwanan landmasses. Results The South American crayfish are monophyletic and a sister group to all other southern crayfish. Australian crayfish are not monophyletic, with two Tasmanian genera, Spinastacoides and Ombrastacoides, forming a clade with New Zealand and Malagasy crayfish (both monophyletic). Divergence of crayfish among southern landmasses is estimated to have occurred around the Late Jurassic to Early Cretaceous (109–178 Ma). Main conclusions The estimated phylogenetic relationships and time of divergence among the Southern Hemisphere crayfishes were consistent with an east–west pattern of Gondwanan divergence. The divergence between Australia and New Zealand (109–160 Ma) pre‐dated the rifting at around 80 Ma, suggesting that these lineages were established prior to the break‐up. Owing to the age of the New Zealand crayfish, we reject the hypothesis that there was a complete drowning of New Zealand crayfish habitat.