New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing

AbstractSurvey sequencing of the bread wheat (Triticum aestivum L.) genome (AABBDD) has been approached through different strategies delivering important information. However, the current wheat sequence knowledge is not complete. The aim of our study is to provide different and complementary set of data for chromosome 4D. A survey sequence was obtained by pyrosequencing of flow-sorted 4DS (7.2×) and 4DL (4.1×) arms. Single ends (SE) and long mate pairs (LMP) reads were assembled into contigs (223Mb) and scaffolds (65Mb) that were aligned to Aegilops tauschii draft genome (DD), anchoring 34Mb to chromosome 4. Scaffolds annotation rendered 822 gene models. A virtual gene order comprising 1973 wheat orthologous gene loci and 381 wheat gene models was built. This order was largely consistent with the scaffold order determined based on a published high density map from the Ae. tauschii chromosome 4, using bin-mapped 4D ESTs as a common reference. The virtual order showed a higher collinearity with homeologous 4B compared to 4A. Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted. The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information

Next-generation sequencing and syntenic integration of flow-sorted arms of wheat chromosome 4A exposes the chromosome structure and gene content

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding improved varieties to cope with increasing food demand under the conditions of changing global climate. However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated construction of an ordered gene map of chromosome 4A, embracing ≥85% of its total gene content, and have enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5 and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics between homoeologous chromosomes and syntenic chromosomal regions. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.This research was financially supported by the Spanish Ministry of Science and Innovation (grant numbers BIO2009–07443, BIO2011–15237 and AGL2010–17316), the German Ministry of Education and Research GABI Barlex project, the European Commission FP7-212019 Triticeae Genome grant, the Czech Science Foundation (awards 521/08/1629 and P501/10/1740), and the Czech Republic Ministry of Education, Youth and Sports/European Regional Development Fund (Operational Programme Research and Development for Innovations grant number CZ.1.05/2.1.00/01.0007).Peer Reviewe

RNA-Seqde novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole

The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers (anthelmintics). Benzimidazoles (BZ) are currently the only anthelmintic registered in the EU for use in controlling A. galli and there is an obvious risk of overuse of one drug class, selecting for resistance. Thus we developed a reference transcriptome of A. galli to investigate the response in gene expression before and after exposure to the BZ drug flubendazole (FLBZ). Transcriptional variations between treated and untreated A. galli showed that transcripts annotated as mitochondrial glutamate dehydrogenase and cytochrome P450 were significantly down-regulated in treated worms, whereas transcripts homologous to heat shock proteins (HSP), catalase, phosphofructokinase, and a multidrug resistance Pglycoprotein (PGP1) were significantly up-regulated in treated worms. Investigation of candidate transcripts responsible for anthelmintic resistance in livestock nematodes led to identification of several tubulins, including six new isoforms of beta-tubulin, and several ligandgated ionotropic receptors and ABC-transporters. We discovered several transcripts associated with drug binding and processing genes, but further characterisation using a larger set of worms exposed to BZs in functional assays is required to determine how these are involved in drug binding and metabolism.Funding Agencies|Formas [FORMAS 2013-665]; SNIC through Uppsala Multidisciplinary Centre for Advanced Computational Science (UPPMAX) [b2014242]</p

Colistin Dependence in Extensively Drug-Resistant Acinetobacter baumannii Strain Is Associated with ISAjo2 and ISAba13 Insertions and Multiple Cellular Responses

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.Funding Agencies|Medical Research Council of Southeast SwedenUK Research &amp; Innovation (UKRI)Medical Research Council UK (MRC) [FORSS-911551]</p

The Perennial Ryegrass GenomeZipper: Targeted Use of Genome Resources for Comparative Grass Genomics

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources are yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne L.) genome on the basis of conserved synteny to barley (Hordeum vulgare L.) and the model grass genome Brachypodium (Brachypodium distachyon L.), as well as rice (Oryza sativa L.) and sorghum [Sorghum bicolor (L.) Moench]. A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macro-collinearity, and was then utilised to anchor a collection of perennial ryegrass genes in silico to their predicted genome position. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci which were used for the first genomewide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.This is a manuscript of an article published as Pfeifer, Matthias, Mihaela Martis, Torben Asp, Klaus FX Mayer, Thomas Lübberstedt, Stephen Byrne, Ursula Frei, and Bruno Studer. "The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics." Plant physiology 161, no. 2 (2013): 571-582. doi: 10.1104/pp.112.207282. Copyright American Society of Plant Biologists. Posted with permission.</p

The Perennial Ryegrass GenomeZipper - targeted use of genome resources for comparative grass genomics.

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources are yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne L.) genome on the basis of conserved synteny to barley (Hordeum vulgare L.) and the model grass genome Brachypodium (Brachypodium distachyon L.), as well as rice (Oryza sativa L.) and sorghum [Sorghum bicolor (L.) Moench]. A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macro-collinearity, and was then utilised to anchor a collection of perennial ryegrass genes in silico to their predicted genome position. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species

Volcano plot of all differentially expressed transcripts.

<p>Purple dots denote significantly differentially expressed transcripts with an adjusted p-value lower than 0.001 and at least 4-fold change.</p

Classification of differentially expressed genes.

<p>Classification of differentially expressed genes.</p

Phylogenetic tree of beta-tubulin genes.

<p>The phylogram was obtained by maximum likelihood analysis of beta-tubulin genes across six species: <i>Caenorhabditis elegans</i> (Ce), <i>Haemonchus contortus</i> (Hco), <i>Parascaris equorum</i> (Peq), <i>Ascaris suum</i> (Asu), <i>Trichinella spiralis</i> (Tsp) and <i>Ascaridia galli</i> (Asg). The tree was constructed by model selection based on the BIC criterion using the software prottest3, and maximum likelihood inference using PhyML. The branch support is given as bootstrap sampling proportion (the closer to 100, the more confident). The tree was rooted using the species <i>T</i>. <i>spiralis</i> as outgroup. Beta-tubulin isoforms belonging to the same species are indicated by colour: <i>H</i>. <i>contortus</i> is shown in purple, <i>A</i>. <i>suum</i> in blue, <i>C</i>. <i>elegans</i> in orange, <i>P</i>. <i>equorum</i> in green, <i>T</i>. <i>spiralis</i> in dark green and <i>A</i>. <i>galli</i> candidates in dark red.</p