Stephen J. Robinson

Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea

BackgroundBrassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus.ResultsWe generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event.ConclusionsDifferential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.

The emerging biofuel crop Camelina sativa retains a highly undifferentiated hexaploid genome structure

Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop.

Polyploid Evolution of the Brassicaceae during the Cenozoic Era

This study identified multiple whole-genome duplication (WGD) events among Brassicaceae species. Remarkably, these events, as well as previously identified WGD events, are synchronized in age, coincident with epoch transitions, adding to the evidence suggesting the environmental instability associated with these transitions favors polyploidy and rapid species diversification. The Brassicaceae (Cruciferae) family, owing to its remarkable species, genetic, and physiological diversity as well as its significant economic potential, has become a model for polyploidy and evolutionary studies. Utilizing extensive transcriptome pyrosequencing of diverse taxa, we established a resolved phylogeny of a subset of crucifer species. We elucidated the frequency, age, and phylogenetic position of polyploidy and lineage separation events that have marked the evolutionary history of the Brassicaceae. Besides the well-known ancient α (47 million years ago [Mya]) and β (124 Mya) paleopolyploidy events, several species were shown to have undergone a further more recent (∼7 to 12 Mya) round of genome multiplication. We identified eight whole-genome duplications corresponding to at least five independent neo/mesopolyploidy events. Although the Brassicaceae family evolved from other eudicots at the beginning of the Cenozoic era of the Earth (60 Mya), major diversification occurred only during the Neogene period (0 to 23 Mya). Remarkably, the widespread species divergence, major polyploidy, and lineage separation events during Brassicaceae evolution are clustered in time around epoch transitions characterized by prolonged unstable climatic conditions. The synchronized diversification of Brassicaceae species suggests that polyploid events may have conferred higher adaptability and increased tolerance toward the drastically changing global environment, thus facilitating species radiation.

An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches

BackgroundFunctional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenised plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.ResultsWe have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T3 generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.ConclusionThis publicly available population provides an additional tool for plant researchers to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.

Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature

BackgroundAbiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses. The wide range of physiological, biochemical and molecular changes that occur in plants exposed to low temperature require a robust global approach to studying the response. We have employed Serial Analysis of Gene Expression (SAGE) to uncover changes in the transcriptome of Arabidopsis thaliana over a time course of low temperature stress.ResultsFive SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Interpretation of these data was facilitated by representing the SAGE data by gene identifier, allowing more robust statistical analysis, cross-platform comparisons and the identification of genes sharing common expression profiles. Simultaneous statistical calculations across all five libraries identified 920 low temperature responsive genes, only 24% of which overlapped with previous global expression analysis performed using microarrays, although similar functional categories were affected. Clustering of the differentially regulated genes facilitated the identification of novel loci correlated with the development of freezing tolerance. Analysis of their promoter sequences revealed subsets of genes that were independent of CBF and ABA regulation and could provide a mechanism for elucidating complementary signalling pathways. The SAGE data emphasised the complexity of the plant response, with alternate pre-mRNA processing events increasing at low temperatures and antisense transcription being repressed.ConclusionAlternate transcript processing appears to play an important role in enhancing the plasticity of the stress induced transcriptome. Novel genes and cis-acting sequences have been identified as compelling targets to allow manipulation of the plants ability to protect against low temperature stress. The analyses performed provide a contextual framework for the interpretation of quantitative sequence tag based transcriptome analysis which will prevail with the application of next generation sequencing technology.

Maximizing the Efficacy of SAGE Analysis Identifies Novel Transcripts in Arabidopsis

The efficacy of using Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of the model dicotyledonous plant Arabidopsis was assessed. We describe an iterative tag-to-gene matching process that exploits the availability of the whole genome sequence of Arabidopsis. The expression patterns of 98% of the annotated Arabidopsis genes could theoretically be evaluated through SAGE and using an iterative matching process 79% could be identified by a tag found at a unique site in the genome. A total of 145,170 reliable experimental tags from two Arabidopsis leaf tissue SAGE libraries were analyzed, of which 29,632 were distinct. The majority (93%) of the 12,988 experimental tags observed greater than once could be matched within the Arabidopsis genome. However, only 78% were matched to a single locus within the genome, reflecting the complexities associated with working in a highly duplicated genome. In addition to a comprehensive assessment of gene expression in Arabidopsis leaf tissue, we describe evidence of transcription from pseudo-genes as well as evidence of alternative mRNA processing and anti-sense transcription. This collection of experimental SAGE tags could be exploited to assist in the on-going annotation of the Arabidopsis genome.

Isolation and characterization of a GCN5-interacting protein from Arabidopsis thaliana

An Arabidopsis protein, AtEML, was isolated based on its interaction with the histone acetyltransferase AtGCN5 in a yeast two-hybrid screen. RNA blot and RT-PCR analysis showed that AtEML is expressed in flowers, leaves, stems and siliques. The promoter region of AtEML has several cis-acting elements associated with response to biotic and abiotic stress conditions, and the accumulation of the AtEML transcript was found to be regulated by cold and salt treatments. In vitro and in vivo protein–protein interaction assays indicated that AtEML interacts with AtGCN5 through the N-terminal region. Furthermore, AtEML was shown to activate expression of the lacZ reporter gene in yeast through recruitment of AtGCN5. Such recruitment was accompanied by an increase in histone H3 acetylation at the promoter driving lacZ expression, as determined by chromatin immunoprecipitation. A higher level of AtEML gene expression was detected in the Arabidopsis gcn5 knockout mutant as compared to wild type Arabidopsis, indicating that AtEML expression is regulated by AtGCN5. These results suggest that AtEML may be a transcription factor that co-ordinates the expression of target stress regulated genes through involvement in recruiting AtGCN5 to their promoters.

Changes in the Sclerotinia sclerotiorum transcriptome during infection of Brassica napus

BackgroundSclerotinia sclerotiorum causes stem rot in Brassica napus, which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection.ResultsTranscript analysis revealed the temporal pattern and amplitude of the deployment of genes associated with aspects of pathogenicity or virulence during the course of S. sclerotiorum infection on Brassica napus. These genes were categorized into eight functional groups: hydrolytic enzymes, secondary metabolites, detoxification, signaling, development, secreted effectors, oxalic acid and reactive oxygen species production. The induction patterns of nearly all of these genes agreed with their predicted functions. Principal component analysis delineated gene expression patterns that signified transitions between pathogenic phases, namely host penetration, ramification and necrotic stages, and provided evidence for the occurrence of a brief biotrophic phase soon after host penetration.ConclusionsThe current observations support the notion that S. sclerotiorum deploys an array of factors and complex strategies to facilitate host colonization and mitigate host defenses. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B. napus by S. sclerotiorum and provides information for further characterization of genes involved in the S. sclerotiorum-host plant interactions.

Bridging the Gene-to-Function Knowledge Gap Through Functional Genomics

The explosion of genomics data has led to a significant knowledge gap, with thousands of genes identified having no known function. The following chapter describes the available forward and reverse genetics strategies, which can assist researchers in assigning functions to novel genes. Details of the available resources for a number of model and crop species are provided. In addition, protocols are presented for utilising T-DNA tagged populations to identify genes underlying novel phenotypes and to assist with functional characterisation of target genes.

The transcriptional landscape of polyploid wheat

Insights from the annotated wheat genome Wheat is one of the major sources of food for much of the world. However, because bread wheats genome is a large hybrid mix of three separate subgenomes, it has been difficult to produce a high-quality reference sequence. Using recent advances in sequencing, the International Wheat Genome Sequencing Consortium presents an annotated reference genome with a detailed analysis of gene content among subgenomes and the structural organization for all the chromosomes. Examples of quantitative trait mapping and CRISPR-based genome modification show the potential for using this genome in agricultural research and breeding. Ramírez-González et al. exploited the fruits of this endeavor to identify tissue-specific biased gene expression and coexpression networks during development and exposure to stress. These resources will accelerate our understanding of the genetic basis of bread wheat. Science, this issue p. eaar7191; see also p. eaar6089 Expression profiling of homoeologs (pairs of genes united by polyploidy) across tissues reveals expression asymmetry along wheat chromosomes. INTRODUCTION Polyploidy, arising from whole-genome duplication or interspecific hybridization, is ubiquitous across the plant and fungal kingdoms. The presence of highly related genes in polyploids, referred to as homoeologs, has been proposed to confer adaptive plasticity—for example, through neofunctionalization of duplicated genes or tissue-specific expression. This plasticity has facilitated the domestication and adaptation of major polyploid crops (e.g., wheat, cotton, and coffee). However, despite its likely importance, we have a limited understanding of the effect of polyploidy on gene expression and the extent to which homoeologs are similar or different in their expression patterns across development and tissues. RATIONALE Bread wheat is a polyploid derived from the hybridizations between three distinct diploid species and is an informative system for analyzing the effects of recent polyploidy on gene expression. Understanding the coordination of homoeologs and identifying the mechanisms associated with these processes should help define strategies to improve trait biology in a crop that provides more than 20% of the protein and caloric intake of humans. RESULTS Here we leverage 850 wheat RNA-sequencing samples, alongside the annotated genome, to determine the similarities and differences between homoeolog expression across a range of tissues, developmental stages, and cultivars. On average, ~30% of wheat homoeolog triads (composed of A, B, and D genome copies) showed nonbalanced expression patterns, with higher or lower expression from a single homoeolog with respect to the other two. These differences between homoeologs were associated with epigenetic changes affecting DNA methylation and histone modifications. Although nonbalanced homoeolog expression could be partially predicted by expression in diploid ancestors, large changes in relative homoeolog expression were observed owing to polyploidization. Our results suggest that the transposable elements in promoters relate more closely to the variation in the relative expression of homoeologs across tissues than to a ubiquitous effect across all tissues. We found that homoeologs with the highest inter-tissue variation had promoters with more frequent transposable element insertions and more varied cis-regulatory elements than homoeologs that were stable across tissues. We also identified expression asymmetry along wheat chromosomes. Homoeologs with the largest inter-tissue, inter-cultivar, and coding sequence variation were most often located in the highly recombinogenic distal ends of chromosomes. These transcriptionally dynamic homoeologs are under more relaxed selection pressure, potentially representing the first steps toward functional innovation through neo- or subfunctionalization. We generated tissue- and stress-specific coexpression networks that reveal extensive coordination of homoeolog expression throughout development. These networks, alongside detailed gene expression atlases (www.wheat-expression.com and http://bar.utoronto.ca), lay the groundwork to identify candidate genes influencing agronomic traits in wheat. CONCLUSION This study provides detailed insights into the transcriptional landscape of bread wheat, an evolutionarily young polyploid. Our work shows that homoeolog expression patterns in bread wheat have been shaped by polyploidy and are associated with both epigenetic modifications and variation in transposable elements within promoters of homoeologous genes. The extensive datasets and analyses presented here provide a framework that can help researchers and breeders develop strategies to improve crops by manipulating individual or multiple homoeologs to modulate trait responses. Homoeolog expression patterns in polyploid wheat. Seventy percent of triads (A, B, and D homoeologs) show balanced expression among homoeologs and are ubiquitously expressed (left), whereas ~30% show nonbalanced expression and are more tissue-specific (right; symbolized by three exemplar tissues). Variation in promoter elements and nonsynonymous substitution rates distinguish between individual triads with stable relative expression across tissues and triads with more inter-tissue variation (tissue-dynamic triads). The coordinated expression of highly related homoeologous genes in polyploid species underlies the phenotypes of many of the world’s major crops. Here we combine extensive gene expression datasets to produce a comprehensive, genome-wide analysis of homoeolog expression patterns in hexaploid bread wheat. Bias in homoeolog expression varies between tissues, with ~30% of wheat homoeologs showing nonbalanced expression. We found expression asymmetries along wheat chromosomes, with homoeologs showing the largest inter-tissue, inter-cultivar, and coding sequence variation, most often located in high-recombination distal ends of chromosomes. These transcriptionally dynamic genes potentially represent the first steps toward neo- or subfunctionalization of wheat homoeologs. Coexpression networks reveal extensive coordination of homoeologs throughout development and, alongside a detailed expression atlas, provide a framework to target candidate genes underpinning agronomic traits in wheat.