Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Leaf transcriptome of two highly divergent genotypes of Urochloa humidicola (Poaceae), a tropical polyploid forage grass adapted to acidic soils and temporary flooding areas.

BACKGROUND: Urochloa humidicola (Koronivia grass) is a polyploid (6x to 9x) species that is used as forage in the tropics. Facultative apospory apomixis is present in most of the genotypes of this species, although one individual has been described as sexual. Molecular studies have been restricted to molecular marker approaches for genetic diversity estimations and linkage map construction. The objectives of the present study were to describe and compare the leaf transcriptome of two important genotypes that are highly divergent in terms of their phenotypes and reproduction modes: the sexual BH031 and the aposporous apomictic cultivar BRS Tupi. RESULTS: We sequenced the leaf transcriptome of Koronivia grass using an Illumina GAIIx system, which produced 13.09 Gb of data that consisted of 163,575,526 paired-end reads between the two libraries. We de novo-assembled 76,196 transcripts with an average length of 1,152 bp and filtered 35,093 non-redundant unigenes. A similarity search against the non-redundant National Center of Biotechnology Information (NCBI) protein database returned 65 % hits. We annotated 24,133 unigenes in the Phytozome database and 14,082 unigenes in the UniProtKB/Swiss-Prot database, assigned 108,334 gene ontology terms to 17,255 unigenes and identified 5,324 unigenes in 327 known metabolic pathways. Comparisons with other grasses via a reciprocal BLAST search revealed a larger number of orthologous genes for the Panicum species. The unigenes were involved in C4 photosynthesis, lignocellulose biosynthesis and flooding stress responses. A search for functional molecular markers revealed 4,489 microsatellites and 560,298 single nucleotide polymorphisms (SNPs). A quantitative real-time PCR analysis validated the RNA-seq expression analysis and allowed for the identification of transcriptomic differences between the two evaluated genotypes. Moreover, 192 unannotated sequences were classified as containing complete open reading frames, suggesting that the new, potentially exclusive genes should be further investigated. CONCLUSION: The present study represents the first whole-transcriptome sequencing of U. humidicola leaves, providing an important public information source of transcripts and functional molecular markers. The qPCR analysis indicated that the expression of certain transcripts confirmed the differential expression observed in silico, which demonstrated that RNA-seq is useful for identifying differentially expressed and unique genes. These results corroborate the findings from previous studies and suggest a hybrid origin for BH031.

Using genetic diversity information to establish core collections of Stylosanthes capitata and Stylosanthes macrocephala.

Stylosanthes species are important forage legumes in tropical and subtropical areas. S. macrocephala and S. capitata germplasm collections that consist of 134 and 192 accessions, respectively, are maintained at the Brazilian Agricultural Research Corporation Cerrados (Embrapa-Cerrados). Polymorphic microsatellite markers were used to assess genetic diversity and population structure with the aim to assemble a core collection. The mean values of H(O) and H(E) for S. macrocephala were 0.08 and 0.36, respectively, whereas the means for S. capitata were 0.48 and 0.50, respectively. Roger's genetic distance varied from 0 to 0.83 for S. macrocephala and from 0 to 0.85 for S. capitata. Analysis with STRUCTURE software distinguished five groups among the S. macrocephala accessions and four groups among those of S. capitata. Nei's genetic diversity was 27% in S. macrocephala and 11% in S. capitata. Core collections were assembled for both species. For S. macrocephala, all of the allelic diversity was represented by 23 accessions, whereas only 13 accessions were necessary to represent all allelic diversity for S. capitata. The data presented herein evidence the population structure present in the Embrapa-Cerrados germplasm collections of S. macrocephala and S. capitata, which may be useful for breeding programs and germplasm conservation.

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas.

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.

Intracellular lipid binding protein family diversity from Oyster Crassostrea gigas: genomic and structural features of invertebrate lipid transporters.

Intracellular lipid binding proteins (iLBPs) play a role in the transport and cellular uptake of fatty acids and gene expression regulation. The aim of this work was to characterize the iLBP gene family of the Pacific oyster Crassostrea gigas, one of the most cultivated marine bivalves in the world, using bioinformatics and molecular biology approaches. A total of 26 different iLBPs transcripts were identified in the Pacific oyster genome, including alternative splicing and gene duplication events. The oyster iLBP gene family seems to be more expanded than in other invertebrates. Furthermore, 3D structural modeling and molecular docking analysis mapped the main amino acids involved in ligand interactions, and comparisons to available protein structures from vertebrate families revealed new binding cavities. Ten different CgiLBPs were analyzed by quantitative PCR in various tissues of C. gigas, which suggested differential prevalent gene expression of CgiLBPs among tissue groups. The data indicate a wider repertoire of iLBPs in labial palps, a food-sorting tissue. The different gene transcription profiles and reported docking systems suggest that the iLBPs are a non-generalist ligand binding protein family with specific functions.