Roberto A. Barrero

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Cellular dissection of the degradation pattern of cortical cell death during aerenchyma formation of rice roots

Abstract. Cellular events which occur prior to cell collapse were examined in the root cortex of rice (Oryza sativa L.) during aerenchyma formation. Cell collapse started at a specific position in the mid cortex. These cells were distinct in shape from those located towards the periphery. Furthermore, cell collapse was preceded by acidification and the loss of plasma-membrane integrity in cells of the mid cortex. Subsequent death of neighboring cells followed a radial path. Microinjection of molecules of different sizes conjugated with fluorescein isothiocyanate (FITC) showed a molecular exclusion limit of between 9.3 and 19.6 kDa in the root cortex. Furthermore, large molecules, i.e. those around 9.3 kDa, were predominantly transferred in a radial direction, which coincided with the path of sequential cell death.

High-Throughput SuperSAGE for Digital Gene Expression Analysis of Multiple Samples Using Next Generation Sequencing

We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined “High-Throughput (HT-) SuperSAGE”. SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes.

Genome sequence of the pathogenic intestinal spirochete brachyspira hyodysenteriae reveals adaptations to its lifestyle in the porcine large intestine.

Brachyspira hyodysenteriae is an anaerobic intestinal spirochete that colonizes the large intestine of pigs and causes swine dysentery, a disease of significant economic importance. The genome sequence of B. hyodysenteriae strain WA1 was determined, making it the first representative of the genus Brachyspira to be sequenced, and the seventeenth spirochete genome to be reported. The genome consisted of a circular 3,000,694 base pair (bp) chromosome, and a 35,940 bp circular plasmid that has not previously been described. The spirochete had 2,122 protein-coding sequences. Of the predicted proteins, more had similarities to proteins of the enteric Escherichia coli and Clostridium species than they did to proteins of other spirochetes. Many of these genes were associated with transport and metabolism, and they may have been gradually acquired through horizontal gene transfer in the environment of the large intestine. A reconstruction of central metabolic pathways identified a complete set of coding sequences for glycolysis, gluconeogenesis, a non-oxidative pentose phosphate pathway, nucleotide metabolism, lipooligosaccharide biosynthesis, and a respiratory electron transport chain. A notable finding was the presence on the plasmid of the genes involved in rhamnose biosynthesis. Potential virulence genes included those for 15 proteases and six hemolysins. Other adaptations to an enteric lifestyle included the presence of large numbers of genes associated with chemotaxis and motility. B. hyodysenteriae has diverged from other spirochetes in the process of accommodating to its habitat in the porcine large intestine.

DNA-Based Faecal Dietary Analysis: A Comparison of qPCR and High Throughput Sequencing Approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

Arabidopsis CAP Regulates the Actin Cytoskeleton Necessary for Plant Cell Elongation and Division

An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated. Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern. The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast. Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles. Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells. Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis. Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo. Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division.

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes.

BackgroundEuphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds.ResultsIn this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (> = 75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin.ConclusionThe resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients.

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

BackgroundThe Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.ResultsWe screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.ConclusionWe have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431

BackgroundPaclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer.ResultsThe ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely.ConclusionsOur findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

The complete genome sequence of the pathogenic intestinal spirochete Brachyspira pilosicoli and comparison with other Brachyspira genomes

Background The anaerobic spirochete Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes “intestinal spirochetosis”, a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence. Methodology/Principal Findings The genome of B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence. Conclusions/Significance The availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.