Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing.

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non-coding regions in the gene promoter and 5'UTR regions of genes. Poly-A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non-crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non-homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Polar Interactions at the Dimer-Dimer Interface of Methionine Adenosyltransferase MAT I Control Tetramerization.

Catalytic MATα1 subunits associate into kinetically distinct homo-dimers (MAT III) and homo-tetramers (MAT I) that synthesize S-adenosylmethionine in the adult liver. Pathological reductions in S-adenosylmethionine levels correlate with MAT III accumulation; thus, it is important to know the determinants of dimer-dimer associations. Here, polar interactions (<3.5 Å) at the rat MAT I dimer-dimer interface were disrupted by site-directed mutagenesis. Heterologous expression rendered decreased soluble mutant MATα1 levels that appeared mostly as dimers. Substitutions at the B1-B2 or B3-C1 ß-strand loops, or changes in charge on helix α2 located behind, induced either MAT III or MAT I accumulation. Notably, double mutants combining neutral changes on helix α2 with substitutions at either ß-strand loop further increased MAT III content. Mutations had negligible impact on secondary or tertiary protein structure, but induced changes of 5-10 °C in thermal stability. All mutants preserved tripolyphosphatase activity, although AdoMet synthesis was only detected in single mutants. Kinetic parameters were altered in all purified proteins, their AdoMet synthesis Vmax and methionine affinities correlating with the association state induced by the corresponding mutations. In conclusion, polar interactions control MATα1 tetramerization and kinetics, diverse effects being induced by changes on opposite ß-sheet loops putatively leading to subtle variations in central domain ß-sheet orientation.

The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

Distribution, position and genomic characteristics of crossovers in tomato recombinant inbred lines derived from an interspecific cross between Solanum lycopersicum and Solanum pimpinellifolium.

We determined the crossover (CO) distribution, frequency and genomic sequences involved in interspecies meiotic recombination by using parent-assigned variants of 52 F6 recombinant inbred lines obtained from a cross between tomato, Solanum lycopersicum, and its wild relative, Solanum pimpinellifolium. The interspecific CO frequency was 80% lower than reported for intraspecific tomato crosses. We detected regions showing a relatively high and low CO frequency, so-called hot and cold regions. Cold regions coincide to a large extent with the heterochromatin, although we found a limited number of smaller cold regions in the euchromatin. The CO frequency was higher at the distal ends of chromosomes than in pericentromeric regions and higher in short arm euchromatin. Hot regions of CO were detected in euchromatin, and COs were more often located in non-coding regions near the 5' untranslated region of genes than expected by chance. Besides overrepresented CCN repeats, we detected poly-A/T and AT-rich motifs enriched in 1-kb promoter regions flanking the CO sites. The most abundant sequence motifs at CO sites share weak similarity to transcription factor-binding sites, such as for the C2H2 zinc finger factors class and MADS box factors, while InterPro scans detected enrichment for genes possibly involved in the repair of DNA breaks.

Correcting palindromes in long reads after whole-genome amplification.

BACKGROUND: Next-generation sequencing requires sufficient DNA to be available. If limited, whole-genome amplification is applied to generate additional amounts of DNA. Such amplification often results in many chimeric DNA fragments, in particular artificial palindromic sequences, which limit the usefulness of long sequencing reads. RESULTS: Here, we present Pacasus, a tool for correcting such errors. Two datasets show that it markedly improves read mapping and de novo assembly, yielding results similar to these that would be obtained with non-amplified DNA. CONCLUSIONS: With Pacasus long-read technologies become available for sequencing targets with very small amounts of DNA, such as single cells or even single chromosomes.

AIL and HDG proteins act antagonistically to control cell proliferation.

Aintegumenta-like (AIL) transcription factors are key regulators of cell proliferation and meristem identity. Although AIL functions have been well described, the direct signalling components of this pathway are largely unknown. We show that baby boom (BBM) and other AIL proteins physically interact with multiple members of the L1-expressed homeodomain glabrous (HDG) transcription factor family, including HDG1, HDG11 and HDG12. Overexpression of HDG1, HDG11 and HDG12 restricts growth due to root and shoot meristem arrest, which is associated with reduced expression of genes involved in meristem development and cell proliferation pathways, whereas downregulation of multiple HDG genes promotes cell overproliferation. These results suggest a role for HDG proteins in promoting cell differentiation. We also reveal a transcriptional network in which BBM and HDG1 regulate several common target genes, and where BBM/AIL and HDG regulate the expression of each other. Taken together, these results suggest opposite roles for AIL and HDG proteins, with AILs promoting cell proliferation and HDGs stimulating cell differentiation, and that these functions are mediated at both the protein-protein interaction and transcriptional level.

Making the difference: integrating structural variation detection tools.

From prokaryotes to eukaryotes, phenotypic variation, adaptation and speciation has been associated with structural variation between genomes of individuals within the same species. Many computer algorithms detecting such variations (callers) have recently been developed, spurred by the advent of the next-generation sequencing technology. Such callers mainly exploit split-read mapping or paired-end read mapping. However, as different callers are geared towards different types of structural variation, there is still no single caller that can be considered a community standard; instead, increasingly the various callers are combined in integrated pipelines. In this article, we review a wide range of callers, discuss challenges in the integration step and present a survey of pipelines used in population genomics studies. Based on our findings, we provide general recommendations on how to set-up such pipelines. Finally, we present an outlook on future challenges in structural variation detection.

Introgression browser: high-throughput whole-genome SNP visualization.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.

Local auxin biosynthesis regulation by PLETHORA transcription factors controls phyllotaxis in Arabidopsis.

Lateral organ distribution at the shoot apical meristem defines specific and robust phyllotaxis patterns that have intrigued biologists and mathematicians for centuries. In silico studies have revealed that this self-organizing process can be recapitulated by modeling the polar transport of the phytohormone auxin. Phyllotactic patterns change between species and developmental stages, but the processes behind these variations have remained unknown. Here we use regional complementation experiments to reveal that phyllotactic switches in Arabidopsis shoots can be mediated by PLETHORA-dependent control of local auxin biosynthesis.

Cnidaria: fast, reference-free clustering of raw and assembled genome and transcriptome NGS data.

BACKGROUND: Identification of biological specimens is a requirement for a range of applications. Reference-free methods analyse unprocessed sequencing data without relying on prior knowledge, but generally do not scale to arbitrarily large genomes and arbitrarily large phylogenetic distances. RESULTS: We present Cnidaria, a practical tool for clustering genomic and transcriptomic data with no limitation on genome size or phylogenetic distances. We successfully simultaneously clustered 169 genomic and transcriptomic datasets from 4 kingdoms, achieving 100% identification accuracy at supra-species level and 78% accuracy at the species level. CONCLUSION: CNIDARIA allows for fast, resource-efficient comparison and identification of both raw and assembled genome and transcriptome data. This can help answer both fundamental (e.g. in phylogeny, ecological diversity analysis) and practical questions (e.g. sequencing quality control, primer design).

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Perception of low red:far-red ratio compromises both salicylic acid- and jasmonic acid-dependent pathogen defences in Arabidopsis.

In dense stands of plants, such as agricultural monocultures, plants are exposed simultaneously to competition for light and other stresses such as pathogen infection. Here, we show that both salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent disease resistance is inhibited by a simultaneously reduced red:far-red light ratio (R:FR), the early warning signal for plant competition. Conversely, SA- and JA-dependent induced defences did not affect shade-avoidance responses to low R:FR. Reduced pathogen resistance by low R:FR was accompanied by a strong reduction in the regulation of JA- and SA-responsive genes. The severe inhibition of SA-responsive transcription in low R:FR appeared to be brought about by the repression of SA-inducible kinases. Phosphorylation of the SA-responsive transcription co-activator NPR1, which is required for full induction of SA-responsive transcription, was indeed reduced and may thus play a role in the suppression of SA-mediated defences by low R:FR-mediated phytochrome inactivation. Our results indicate that foraging for light through the shade-avoidance response is prioritised over plant immune responses when plants are simultaneously challenged with competition and pathogen attack.

Prioritization of candidate genes in QTL regions based on associations between traits and biological processes.

BACKGROUND: Elucidation of genotype-to-phenotype relationships is a major challenge in biology. In plants, it is the basis for molecular breeding. Quantitative Trait Locus (QTL) mapping enables to link variation at the trait level to variation at the genomic level. However, QTL regions typically contain tens to hundreds of genes. In order to prioritize such candidate genes, we show that we can identify potentially causal genes for a trait based on overrepresentation of biological processes (gene functions) for the candidate genes in the QTL regions of that trait. RESULTS: The prioritization method was applied to rice QTL data, using gene functions predicted on the basis of sequence- and expression-information. The average reduction of the number of genes was over ten-fold. Comparison with various types of experimental datasets (including QTL fine-mapping and Genome Wide Association Study results) indicated both statistical significance and biological relevance of the obtained connections between genes and traits. A detailed analysis of flowering time QTLs illustrates that genes with completely unknown function are likely to play a role in this important trait. CONCLUSIONS: Our approach can guide further experimentation and validation of causal genes for quantitative traits. This way it capitalizes on QTL data to uncover how individual genes influence trait variation.

SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis.

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

Metabolism control over growth: a case for trehalose-6-phosphate in plants.

How plants relate their requirements for energy with the reducing power necessary to fuel growth is not understood. The activated glucose forms and NADPH are key precursors in pathways yielding, respectively, energy and reducing power for anabolic metabolism. Moreover, they are substrates or allosteric regulators of trehalose-phosphate synthase (TPS1) in fungi and probably also in plants. TPS1 synthesizes the signalling metabolite trehalose-6-phosphate (T6P) and, therefore, has the potential to relate reducing power with energy metabolism to fuel growth. A working model is discussed where trehalose-6-phosphate (T6P) inhibition of SnRK1 is part of a growth-regulating loop in young and metabolically active heterotrophic plant tissues. SnRK1 is the Snf1 Related Kinase 1 and the plant homologue of the AMP-dependent protein kinase of animals, a central energy gauge. T6P accumulation in response to high sucrose levels in a cell inhibits SnRK1 activity, thus promoting anabolic processes and growth. When T6P levels drop due to low glucose-6-phosphate, uridine-diphosphoglucose, and altered NADPH or due to restricted TPS1 activity, active SnRK1 promotes catabolic processes required to respond to energy and carbon deprivation. The model explains why too little or too much T6P has been found to be growth inhibitory: Arabidopsis thaliana embryos and seedlings without TPS1 are growth arrested and Arabidopsis seedlings accumulating T6P on a trehalose medium are growth arrested. Finally, the insight gained with respect to the possible role of T6P metabolism, where it is known to alter developmental and environmental responses of plants, is discussed.

Probing the roles of LRR RLK genes in Arabidopsis thaliana roots using a custom T-DNA insertion set.

Leucine-rich repeat receptor-like protein kinases (LRR RLKs) represent the largest group of Arabidopsis RLKs with approximately 235 members. A minority of these LRR RLKs have been assigned to diverse roles in development, pathogen resistance and hormone perception. Using a reverse genetics approach, a collection of homozygous T-DNA insertion lines for 69 root expressed LRR RLK genes was screened for root developmental defects and altered response after exposure to environmental, hormonal/chemical and abiotic stress. The obtained data demonstrate that LRR RLKs play a role in a wide variety of signal transduction pathways related to hormone and abiotic stress responses. The described collection of T-DNA insertion mutants provides a valuable tool for future research into the function of LRR RLK genes.

Adapting to environmental changes using specialized paralogs.

When a bacterial species survives under changing environmental circumstances (e.g. salinity or temperature), its proteins might not function in all physicochemical conditions. We propose that prokaryotes cope with this problem by having two or more copies of the genes affected by environmental fluctuations, each one performing the same function under different conditions (i.e. ecoparalog). We identify potential examples in the bacterium Salinibacter ruber and in other species that experience wide environmental variations.

MHC class I molecules exploit the low G+C content of pathogen genomes for enhanced presentation.

Distinguishing self from nonself and pathogenic from nonpathogenic is a fundamental challenge to the immune system but whether adaptive immune systems use pathogen-specific signatures to achieve this is largely unknown. By investigating the presentation of large sets of viruses and bacteria on MHC class I molecules, we analyze whether MHC-I molecules have a preference for pathogen-derived peptides. The fraction of potential MHC-I binders in different organisms can vary up to eight-fold. We find that this variation can be largely explained by G+C content differences of the organisms, which are reflected in amino acid frequencies. A significant majority of HLA-A, but not HLA-B, molecules has a preference for peptides derived from organisms with a low G+C content. Interestingly, a low G+C content seems to be a universal signature for pathogenicity. Finally, we find the same preferences in chimpanzee and rhesus macaque MHC-I molecules. These results demonstrate that despite the fast evolution of MHC-I alleles and their extreme polymorphism and diversity in peptide-binding preferences, MHC-I molecules can acquire a preference to exploit pathogen-specific signatures.

Refolding and characterization of methionine adenosyltransferase from Euglena gracilis.

Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 µmol/min/mg and K(m) values of approximately 85 and 260 µM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed ß-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.