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The important worldwide forage crop red clover (Trifolium pratense L.) is widely cultivated as cattle feed and for soil improvement. Wild populations and landraces have great natural diversity that could be used to improve cultivated red clover. However, to date, there is still insufficient knowledge about the natural genetic and phenotypic diversity of the species. Here, we developed a low-cost complexity reduced mRNA analysis (mRNA-GBS) and compared the results with population genetic (GBS) and previously published mRNA-Seq data, to assess whether analysis of intraspecific variation within and between populations and transcriptome responses is possible simultaneously. The mRNA-GBS approach was successful. SNP analyses from the mRNA-GBS approach revealed comparable patterns to the GBS results, but due to site-specific multifactorial influences of environmental responses as well as conceptual and methodological limitations of mRNA-GBS, it was not possible to link transcriptome analyses with reduced complexity and sequencing depth to previously published greenhouse and field expression studies. Nevertheless, the use of short sequences upstream of the poly(A) tail of mRNA to reduce complexity are promising approaches that combine population genetics and expression profiling to analyze many individuals with trait differences simultaneously and cost-effectively, even in non-model species. Nevertheless, our study design across different regions in Germany was also challenging. The use of reduced complexity differential expression analyses most likely overlays site-specific patterns due to highly complex plant responses under natural conditions.

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

Background The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. Results To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected. Conclusions In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-020-07200-x.

BACKGROUND: Acute myeloid leukemia (AML) patients with a high allelic burden of an internal tandem duplication (ITD)-mutated FMS-like Tyrosine Kinase-3 (FLT3) have a dismal outcome. FLT3ITD triggers the proliferation of the quiescent hematopoietic stem cell (HSC) pool but fails to directly transform HSCs. While the inflammatory transcription factor nuclear factor of activated T-cells 2 (NFAT2, NFATC1) is overexpressed in AML, it is unknown whether it plays a role in FLT3ITD-induced HSC transformation. METHODS: We generated a triple transgenic mouse model, in which tamoxifen-inducible Cre-recombinase targets expression of a constitutively nuclear transcription factor NFATC1 to FLT3ITD positive HSC. Emerging genotypes were phenotypically, biochemically, and also transcriptionally characterized using RNA sequencing. We also retrospectively analyzed the overall survival of AML patients with different NFATC1 expression status. RESULTS: We find that NFATC1 governs FLT3ITD-driven precursor cell expansion and transformation, causing a fully penetrant lethal AML. FLT3ITD/NFATC1-AML is re-transplantable in secondary recipients and shows primary resistance to the FLT3ITD-kinase inhibitor quizartinib. Mechanistically, NFATC1 rewires FLT3ITD-dependent signaling output in HSC, involving augmented K-RAS signaling and a selective de novo recruitment of key HSC-transforming signaling pathways such as the Hedgehog- and WNT/B-Catenin signaling pathways. In human AML, NFATC1 overexpression is associated with poor overall survival. CONCLUSIONS: NFATC1 expression causes FLT3ITD-induced transcriptome changes, which are associated with HSC transformation, quizartinib resistance, and a poor prognosis in AML. peerReviewed

Additional file 2: Table S2 Retroviral-related Pol polyproteins identified in Yarrowia-like yeast genomes. We identified a total of 15 genes that are annotated as retrovirus-related Pol polyproteins within the genome of the five YLYs analyzed. The proteins belong to three different categories which are ‘Line-1’, ‘transposon 297’ and ‘opus’. While the clade II genomes of Y. strains B02, F05 and H10 do only encode for one such protein per genome, genomes of the clade I genomes of Y. strains C11 and E02 encode for several retrovirus-related Pol polyproteins. Thereby, it has to be noted that the type ‘opus’ is only present in the genome of strain E02 and that all respective genes belong to the set of singleton genes (*) of E02.

Additional file 7: Table S1. Overview of the sampling locations for the plant material. Names of the fields belonging to the Biodiversity Exploratory or greenhouse populations are shown. As well as the location, coordinates and conditions (mown/cut and not mown/uncut). Table S3. Sources and basis for description and classification of the top 20 DEG. Table S4. Number of reads for each sequenced library (transcriptome ID) before and after trimming. Table S5. General features of the transcriptome of T. pratense. Table S6. Overall alignment rate of the single transcriptomes to the references transcriptome, values above 80% are good. Table S7. Annotation of T. pratense plant-specific against different databases. Table S8. Quality of the replicates. For each library replicate the number of transcripts above TPM of 1 is shown. Further the number of transcripts shared between to related replica is shown, as well as the number of transcripts unique for a replica. The similarity of the replicas was evaluated by calculating he percentage of the shared transcripts compared to the total number of transcripts of a replica. Table S10. Main classes based on the DE contigs. 16 main classes were developed to group the DE contigs. Table S11. GO Terms specifically enriched in the individual transcriptomes. Table S12. Shared contigs with corresponding annotation. Table S13. Detailed information about transcripts described during the discussion part. Table S14. Primer sequences.

Additional file 1: Table S1 Singleton genes of Yarrowia-like yeast genomes. Singleton genes were calculated with EDGAR [31]. Singletons are defined as genes without a reasonable BLAST hit against any gene within the other genomes in the comparison. We identified eight singletons for Y. strain C11, nine singletons for Y. strain E02 and one singleton for Y. strain H10.

Additional file 6: Figure S6. Plant architectural characteristics and growth habit of gibberellin treated plants. A-E: Measured, counted or calculated plant characteristics during phenotypic monitoring experiments. Gibberellin treated plants, blue; control plants, orange. Graphs show average values and 95% confidence intervals. Time is shown in weeks. Growth habit of control plants (left side) vs. gibberellin treated plants (right side), after approximately 2 weeks of gibberellin treatment and regrowth (F), and after 4 weeks (G).

Additional file 4: Figure S4. Enrichment analysis of the DEG GO terms. A: Enriched GO terms in mown and non-mown greenhouse samples. B: Enriched GO terms in mown and non-mown field B samples. C: Enriched GO terms in mown and non-mown field A samples. Information about the respective GO number can be found in Table S11.

Additional file 6: Figure S6. Plant architectural characteristics and growth habit of gibberellin treated plants. A-E: Measured, counted or calculated plant characteristics during phenotypic monitoring experiments. Gibberellin treated plants, blue; control plants, orange. Graphs show average values and 95% confidence intervals. Time is shown in weeks. Growth habit of control plants (left side) vs. gibberellin treated plants (right side), after approximately 2 weeks of gibberellin treatment and regrowth (F), and after 4 weeks (G).

Additional file 5: Figure S5. Expressed transcription factor family members in mown and not mown T. pratense plants. The y-axis shows the number of upregulated transcription factors that are members of the respective transcription factor family. Names of the transcriptomes (GM, GNM, FaM, FaNM; FbM, FbNM) and transcription factor families are given on the x-axis. Expression of transcription factor members were compared in a pairwise manner (GM vs GNM, FaM vs FaNM, FbM vs FbNM). Comparisons that resulted in a difference of more than 10% of the contigs significantly upregulated in either the mown or the unmown condition were marked red, differences between 5 and 9 % were marked orange.

Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.

Additional file 3: Figure S3. qRT-PCR analysis of selected T. pratense genes: A) tdn_146439/ENGase85A (encodes a cytosolic beta-endo-N-acetyglucosaminidase (ENGase), B) tdn_69411/LTP (Lipid transfer protein, a pathogenesis-related protein), C) tdn85889/PME44 (pectin methylesterase 44), D) K65_9861 P5CS (DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1). Two additional T. pratense tissues (axial meristem (AM) and leaves (L)) were included and also the six RNA extractions also used for constructing the transcriptome libraries. Transcriptome data are marked with TPM, all samples are from mown (M) or not mown (NM) plants. Gene expression levels were normalized to the housekeeping contig k65_5754 (not annotated, but with similar expression throughout the transcriptomes). Shown are mean values of the fold change of the respective contig in relation to the expression of contig K65_5754 with error bars that represent standard deviation. On the right side of each graph, the log2 values of the respective gene in each transcriptome are shown for better comparison of expression patterns.

Additional file 7: Table S1. Overview of the sampling locations for the plant material. Names of the fields belonging to the Biodiversity Exploratory or greenhouse populations are shown. As well as the location, coordinates and conditions (mown/cut and not mown/uncut). Table S3. Sources and basis for description and classification of the top 20 DEG. Table S4. Number of reads for each sequenced library (transcriptome ID) before and after trimming. Table S5. General features of the transcriptome of T. pratense. Table S6. Overall alignment rate of the single transcriptomes to the references transcriptome, values above 80% are good. Table S7. Annotation of T. pratense plant-specific against different databases. Table S8. Quality of the replicates. For each library replicate the number of transcripts above TPM of 1 is shown. Further the number of transcripts shared between to related replica is shown, as well as the number of transcripts unique for a replica. The similarity of the replicas was evaluated by calculating he percentage of the shared transcripts compared to the total number of transcripts of a replica. Table S10. Main classes based on the DE contigs. 16 main classes were developed to group the DE contigs. Table S11. GO Terms specifically enriched in the individual transcriptomes. Table S12. Shared contigs with corresponding annotation. Table S13. Detailed information about transcripts described during the discussion part. Table S14. Primer sequences.

Background Red clover (Trifolium pratense) is globally used as a fodder plant due its high nutritional value and soil improving qualities. In response to mowing, red clover exhibits specific morphological traits to compensate the loss of biomass. The morphological reaction is well described, but the underlying molecular mechanisms and its role for plants grown in the field are unclear. Results Here, we characterize the global transcriptional response to mowing of red clover by comparing plants grown under greenhouse conditions with plants growing on agriculturally used fields. Unexpectedly, we found that biotic and abiotic stress related changes of plants grown in the field overlay their regrowth related transcriptional changes and characterized transcription related protein families involved in these processes. Further, we can show that gibberellins, among other phytohormones, also contribute to the developmental processes related to regrowth after biomass-loss. Conclusions Our findings show that massive biomass loss triggers less transcriptional changes in field grown plants than their struggle with biotic and abiotic stresses and that gibberellins also play a role in the developmental program related to regrowth after mowing in red clover. Our results provide first insights into the physiological and developmental processes of mowing on red clover and may serve as a base for red clover yield improvement. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-02867-0.

Additional file 1: Figure S1. Uncropped images. Figure S2. CircRNAs identified in the exosome iCLIP analysis of S. solfataricus. Figure S3. Distribution of bases in RNA-tails detected in the aRrp41-iCLIP of the archaeal exosome. Figure S4. Poly(A) stretches in RNA are bound by the exosome. Figure S5. Global analysis of RNAs enriched by coimmunoprecipitation with the archaeal exosome by iCLIP. Figure S6. Antisense RNAs as preferred substrates of the archaeal exosome. Figure S7. Binding of the archaeal exosome to 5’and 3’parts of selected genes. Figure S8. Low affinity of the archaeal exosome to the abundant mRNA tmoA (SSO_RS06040 gene encoding toluene-4-monooxygenase system protein). Figure S9. Distribution of read counts in the genome of S. solfataricus was analyzed with respect to biotype RNA. Figure S10. Clustering of read counts at the 5′-and 3′-end of protein coding genes. Figure S11. Clustering of crosslink sites at the 5′ and 3′ end of predicted operons. Figure S12. Distribution of crosslink sites around the ends of annotated genes

Additional file 6: Nucleotide content of the linear non-mapped cDNA reads in the iCLIP analysis of S. solfataricus.

Red clover (Trifolium pratense) is used worldwide as a fodder plant due its high nutritional value. In response to mowing, red clover exhibits specific morphological traits to compensate the loss of biomass. The morphological reaction is well described, but knowledge of the underlying molecular mechanisms are still lacking. Here we characterize the molecular genetic response to mowing of red clover by using comparative transcriptomics in greenhouse conditions and agriculturally used field. The analysis of mown and control plants revealed candidate genes possibly regulating crucial steps of the genetic network governing the regrowth reaction. In addition, multiple identified gibberellic acid (GA) related genes suggest a major role for GA in establishing the regrowth morphology of red clover. Mown red clover plants showing this regrowth morphology were partially “rescued” by exogenous GA application, demonstrating the influence of GA during regrowth. Our findings provide insights into the physiological and genetic processes of mowing red clover, to serve as a base for red clover yield improvement.

Figure S2. Immunophenotyping of bone marrow and spleen. (A) Relative frequencies of LSK (mean ± SD. n = 6 to 16). (B) Relative frequencies of LK (mean ± SD. n = 6 to 16). (C) Relative frequencies of GMP, CMP, and MEP (mean ± SEM. n = 3 to 4). (D) Relative myeloid populations (mean ± SD. n = 6 to 16). (E) Relative frequencies of B-cells (B220+), T-cells (CD3+), erythrocytes (TER119+) and megakaryocytes (CD41+) (mean ± SEM. n = 3 to 10). (F) Relative frequencies of B-cells (B220+), T-cells (CD3+), erythrocytes (TER119+) and megakaryocytes (CD41+) (mean ± SEM. n = 3 to 10). A 1-way ANOVA or 2-way ANOVA with Tukey’s multiple comparison was used for p values. *p

Figure S5. Working model. Activation of NFATC1 by an inflammatory environment rewires the transcriptional program induced by FLT3-ITD in stem cells, which promotes leukemic transformation. The FLT3-ITD/NFATC1 synergism activates multiple oncogenic pathways such as WNT/B-Catenin, Hedgehog, and K-RAS, causing extensive proliferation and drug resistance. (PPTX 91 kb)

Figure S4. GSEA analysis from C3 H database for mutual FCN and F genes. Summary table for analysis from GSEA H database: 14 pathways enriched in both FCN and F (only values for FCN are shown). (PPTX 80 kb)

Figure S1. Breeding scheme. The genotypes used for the experiments are marked with red boxes: FLT3ITD homozygous (F+/+), SCL-HSC-CreER:NFATC1 homozygous (CN+/+), and FLT3ITD:SCL-HSC-CreER:NFATC1 homozygous (F+/+CN+/+). Note: in all the SCL-HSC-CreERâ baring animals, the zygosity of the Cre allele is unknown. (PPTX 44 kb)

Figure S3. Immunophenotyping of bone marrow and spleen after transplantation into CN or WT recipients. 107 total bone marrow cells from F or FCN donor mice were transplanted into irradiated WT recipients. FACS analysis of LSK/LK and myeloid populations are shown for each recipient mouse. (PPTX 484 kb)

Background: Many cnidarians, including stony corals, engage in complex symbiotic associations, comprising the eukaryotic host, photosynthetic algae, and highly diverse microbial communities-together referred to as holobiont. This taxonomic complexity makes sequencing and assembling coral host genomes extremely challenging. Therefore, previous cnidarian genomic projects were based on symbiont-free tissue samples. However, this approach may not be applicable to the majority of cnidarian species for ecological reasons. We therefore evaluated the performance of an alternative method based on sequence binning for reconstructing the genome of the stony coral Porites rus from a hologenomic sample and compared it to traditional approaches. Results: Our results demonstrate that binning performs well for hologenomic data, producing sufficient reads for assembling the draft genome of P. rus. An assembly evaluation based on operational criteria showed results that were comparable to symbiont-free approaches in terms of completeness and usefulness, despite a high degree of fragmentation in our assembly. In addition, we found that binning provides sufficient data for exploratory k-mer estimation of genomic features, such as genome size and heterozygosity. Conclusions: Binning constitutes a powerful approach for disentangling taxonomically complex coral hologenomes. Considering the recent decline of coral reefs on the one hand and previous limitations to coral genome sequencing on the other hand, binning may facilitate rapid and reliable genome assembly. This study also provides an important milestone in advancing binning from the metagenomic to the hologenomic and from the prokaryotic to the eukaryotic level., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Many cnidarians, including stony corals, engage in complex symbiotic associations, comprising the eukaryotic host, photosynthetic algae, and highly diverse microbial communities—together referred to as holobiont. This taxonomic complexity makes sequencing and assembling coral host genomes extremely challenging. Therefore, previous cnidarian genomic projects were based on symbiont-free tissue samples. However, this approach may not be applicable to the majority of cnidarian species for ecological reasons. We therefore evaluated the performance of an alternative method based on sequence binning for reconstructing the genome of the stony coral Porites rus from a hologenomic sample and compared it to traditional approaches. Results Our results demonstrate that binning performs well for hologenomic data, producing sufficient reads for assembling the draft genome of P. rus. An assembly evaluation based on operational criteria showed results that were comparable to symbiont-free approaches in terms of completeness and usefulness, despite a high degree of fragmentation in our assembly. In addition, we found that binning provides sufficient data for exploratory k-mer estimation of genomic features, such as genome size and heterozygosity. Conclusions Binning constitutes a powerful approach for disentangling taxonomically complex coral hologenomes. Considering the recent decline of coral reefs on the one hand and previous limitations to coral genome sequencing on the other hand, binning may facilitate rapid and reliable genome assembly. This study also provides an important milestone in advancing binning from the metagenomic to the hologenomic and from the prokaryotic to the eukaryotic level.

Background Many cnidarians, including stony corals, engage in complex symbiotic associations, comprising the eukaryotic host, photosynthetic algae, and highly diverse microbial communities - together referred to as holobiont. This taxonomic complexity makes sequencing and assembling coral host genomes extremely challenging. Therefore, previous cnidarian genomic projects were based on symbiont-free tissue samples. However, this approach may not be applicable to the majority of cnidarian species for ecological reasons. We therefore evaluated the performance of an alternative method based on sequence binning for reconstructing the genome of the stony coral Porites rus from a hologenomic sample and compared it to traditional approaches. Results Our results demonstrate that binning performs well for hologenomic data, producing sufficient reads for assembling the draft genome of P. rus. An assembly evaluation based on operational criteria showed results that were comparable to symbiont-free approaches in terms of completeness and usefulness, despite a high degree of fragmentation in our assembly. In addition, we found that binning provides sufficient data for exploratory k-mer estimation of genomic features, such as genome size and heterozygosity. Conclusions Binning constitutes a powerful approach for disentangling taxonomically complex coral hologenomes. Considering the recent decline of coral reefs on the one hand and previous limitations to coral genome sequencing on the other hand, binning may facilitate rapid and reliable genome assembly. This study also provides an important milestone in advancing binning from the metagenomic to the hologenomic and from the prokaryotic to the eukaryotic level.

Plants are non-hosts or resistant to most pathogenic organisms. In many cases they react to pathogen attack either with a general or specific reaction leading to resistance. But what happens when a compatible pathogen attacks, leading to disease? We study the interaction of susceptible tomato (Solanum lycopersicon) with its major pathogens Phytophthora infestans, Oidium neolycopersici and Botrytis cinerea by means of RNA-seq and quantitative real time PCR (qPCR). Major question are (1) can the plant recognize and react specifically to each of the pathogens? (2) how are the regulated genes expressed within time and space? Pathogen- and mock-inoculated tomato leaf samples were subjected to Illumina transcriptome sequencing. A selection of the differentially expressed genes has been analyzed by qPCR for their spatio-temporal regulation patterns. The current state of the work will be presented.

Expression profiles of selected R. solani AG2-2IIIB genes during infection of sugar beet seedlings 6 dpi. Genotypes 1 and 2 are partial resistant, while genotypes 3 and 4 are susceptible. Bars represent mean ± SD on at least 4 biological replicates. Different letters (a, b, c) indicate statistically significant differences (P ≤ 0.05) using the Tukey test (PDF 435 kb)

Number of CAZymes identified in Rhizoctonia solani (five anastomosis groups), Ustilago maydis, Pythium ulticum and Verticillium dahliae. (a) Auxiliary Activities (AA), (b) Carbohydrate Binding Modules (CBM) and (c) Glycosyl Transferases (GT). (PDF 405 kb)

Gene family phylogeny of (a) PL1, (b) GH43 and (c) CE12. Analysis was conducted using the maximum likelihood method with the WAG (for PL1) or WAG + F (for GH43 and CE12) substitution model based on CLUSTAL W alignments of catalytic domain amino acid sequences and 1000 bootstraps. Numbers at nodes indicate the bootstrap value. The protein ID accession numbers originate from the respective database entry. R. solani AG2-2IIIB proteins are indicated in red. (PDF 970 kb)

Small cysteine-rich effector proteins with a size of less than 400 amino acids predicted for 5 R. solani AGs, Ustilago maydis, Verticillium dahliae and Pythium ultimum. (DOCX 108 kb)

Distribution of CAZyme gene families of R. solani AG2-2IIIB within 6 main categories according to the CAZy database. Glysoside hydrolases (GH), Carbohydrate esterases (CE), Auxilliary activity (AA), Carbohydrate-binding modules (CBM), Glycosyl transferases (GT), and Polysaccharide lyases (PL). (PDF 1185 kb)

Background Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. Results The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. Conclusions The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2561-1) contains supplementary material, which is available to authorized users.

Statistics of the contig-length vs. read-count analysis. (DOCX 19 kb)

Sequencing and assembly statistics of R. solani AG-2IIIB EST datasets. (DOCX 50 kb)

Statistics of the completeness of the genome based on 248 core eukaryotic genes (CEGs). (DOC 25 kb)

Rhizoctonia solani, a soil-born plant pathogenic basidiomycetous fungus, affects various economically important agricultural and horticultural crops. The draft genome sequence for the R. solani AG1-IB isolate 7/3/14 as well as a corresponding transcriptome dataset (Expressed Sequence Tags--ESTs) were established previously. Development of a specific R. solani AG1-IB gene model based on GMAP transcript mapping within the eukaryotic gene prediction platform AUGUSTUS allowed detection of new genes and provided insights into the gene structure of this fungus. In total, 12,616 genes were recognized in the genome of the AG1-IB isolate. Analysis of predicted genes by means of different bioinformatics tools revealed new genes whose products potentially are involved in degradation of plant cell wall components, melanin formation and synthesis of secondary metabolites. Comparative genome analyses between members of different R. solani anastomosis groups, namely AG1-IA, AG3 and AG8 and the newly annotated R. solani AG1-IB genome were performed within the comparative genomics platform EDGAR. It appeared that only 21 to 28% of all genes encoded in the draft genomes of the different strains were identified as core genes. Based on Average Nucleotide Identity (ANI) and Average Amino-acid Identity (AAI) analyses, considerable sequence differences between isolates representing different anastomosis groups were identified. However, R. solani isolates form a distinct cluster in relation to other fungi of the phylum Basidiomycota. The isolate representing AG1-IB encodes significant more genes featuring predictable functions in secondary metabolite production compared to other completely sequenced R. solani strains. The newly established R. solani AG1-IB 7/3/14 gene layout now provides a reliable basis for post-genomics studies.

Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro cultivation as they require assimilable low sustenance [1]. Today, CHO cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins and are considered as the mammalian equivalent of E. coli in research and biotechnology [2]. The production of biopharmaceuticals in CHO cells is superior to protein production in bacteria, because mammalian cell lines procure complex folding and post-translational modifications like glycosylation. However, contrary to the increasing importance in biotechnology and industry, comprehensive genome and transcriptome information of CHO cell lines is still rare. In this study, the pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences were used to achieve a major step forward towards unraveling the transcriptome of CHO cells. CHO cDNA samples derived from different CHO cell lines and growth conditions were used for the generation of 1.84 mill. high quality sequencing reads with an average read length of 373 nt summing up to 603 Mb data. Assembly of the sequencing data resulted in 41,039 contiguous sequences. These contigs were grouped by the Newbler software into 36,383 isotigs and 28,039 isogroups. Taxonomical classification and comparison to the Mus musculus transcriptome demonstrated the actual quality of the CHO cell line sequences. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway and the central metabolism of CHO cells were detected. Only fructose-1,6-bisphosphatase (3.1.3.11) and 6-phosphogluconolactonase (3.1.1.31) were not identified within the pentose phosphate pathway. The newly sequenced CHO cell line transcriptome was the basis for the design of a customized CHO microarray. Contig sequences were used for the design of 94.580 probes. The designed probes cover 31,905 splice variants of CHO transcripts. With a Self-Self Hybridization experiment (Figure ​(Figure1)1) the functionality of the probes was demonstrated. This experiment was performed with the same RNA as which was used for sequencing. Half of this RNA was labeled with Cy3, the other half with Cy5. For this study the dye intensity, the dye ratio and the adjusted p-value (student´s t-test, FDR controlled, α=0.05) of four microarray replicates were analyzed. It is expected, that the two labeled RNA samples bind equally to the probe, if a transcript is expressed (Figure ​(Figure1:1: pink, dark blue, red, light blue). Only the probes for one transcript could be rejected, because of their dysfunctionality (Figure ​(Figure1,1, green). Figure 1 Self-Self Hybridization experiment of the customized CHO microarray. The dye ratio (M-value) is plotted against the adjusted p-value (student´s t-test, FDR controlled, α=0.05). The dye ratio was calculated using the mean intensity values ... This CHO microarray is now available for further experiments and will support transcriptional analysis of CHO cells under process conditions for cell line and process optimization. It was used already used successfully for a gene expression study of CHO DP-12 cells cultivated under sodium butyrate treatment [3].

Background Studies of non-model species are important for understanding the molecular processes underpinning phenotypic variation under natural ecological conditions. The common buzzard (Buteo buteo; Aves: Accipitriformes) is a widespread and common Eurasian raptor with three distinct plumage morphs that differ in several fitness-related traits, including parasite infestation. To provide a genomic resource for plumage polymorphic birds in general and to search for candidate genes relating to fitness, we generated a transcriptome from a single dead buzzard specimen plus easily accessible, minimally invasive samples from live chicks. Results We not only de novo assembled a near-complete buzzard transcriptome, but also obtained a significant fraction of the transcriptome of its malaria-like parasite, Leucocytozoon buteonis. By identifying melanogenesis-related transcripts that are differentially expressed in light ventral and dark dorsal feathers, but which are also expressed in other regions of the body, we also identified a suite of candidate genes that could be associated with fitness differences among the morphs. These include several immune-related genes, providing a plausible link between melanisation and parasite load. qPCR analysis of a subset of these genes revealed significant differences between ventral and dorsal feathers and an additional effect of morph. Conclusion This new resource provides preliminary insights into genes that could be involved in fitness differences between the buzzard colour morphs, and should facilitate future studies of raptors and their malaria-like parasites. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2254-1) contains supplementary material, which is available to authorized users.

Sample sizes used for qPCR according to the morph of the donor buzzard and the body region of sampling. For two light and one intermediate birds both ventral and dorsal feathers were available. (DOC 29 kb)

Sample sizes used for qPCR according to the morph of the donor buzzard and the body region of sampling. For two light and one intermediate birds both ventral and dorsal feathers were available. (DOC 29 kb)