Philippa Borrill

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Chaperonins Facilitate KNOTTED1 Cell-to-Cell Trafficking and Stem Cell Function

Active transfer of a transcription factor between plant cells is required for stem cell maintenance. Cell-to-cell communication in plants includes the selective trafficking of transcription factors and other signals through plasmodesmata. The KNOTTED1 (KN1) homeobox (KNOX) family transcription factors, which use this pathway, are essential for stem cell establishment and/or maintenance. Here we show that KN1 trafficking requires the chaperonin complex, which belongs to a group of cytosolic chaperones that fold specific substrate proteins. Genetic and physical interaction data show a functional relevance for chaperonins in KNOX family-dependent stem cell maintenance. Furthermore, tissue-specific complementation assays indicate a mechanistic basis for chaperonin function during the posttranslocational refolding process. Our study shows that chaperonins are essential for the cell-to-cell trafficking of a subset of mobile transcription factors and demonstrates the importance of chaperonin-dependent protein trafficking for plant stem cell function.

Uncovering hidden variation in polyploid wheat

Significance Pasta and bread wheat are polyploid species that carry multiple copies of each gene. Therefore, loss-of-function mutations in one gene copy are frequently masked by functional copies on other genomes. We sequenced the protein coding regions of 2,735 mutant lines and developed a public database including more than 10 million mutations. Researchers and breeders can search this database online, identify mutations in the different copies of their target gene, and request seeds to study gene function or improve wheat varieties. Mutations are being used to improve the nutritional value of wheat, increase the size of the wheat grains, and generate additional variability in flowering genes to improve wheat adaptation to new and changing environments. Comprehensive reverse genetic resources, which have been key to understanding gene function in diploid model organisms, are missing in many polyploid crops. Young polyploid species such as wheat, which was domesticated less than 10,000 y ago, have high levels of sequence identity among subgenomes that mask the effects of recessive alleles. Such redundancy reduces the probability of selection of favorable mutations during natural or human selection, but also allows wheat to tolerate high densities of induced mutations. Here we exploited this property to sequence and catalog more than 10 million mutations in the protein-coding regions of 2,735 mutant lines of tetraploid and hexaploid wheat. We detected, on average, 2,705 and 5,351 mutations per tetraploid and hexaploid line, respectively, which resulted in 35–40 mutations per kb in each population. With these mutation densities, we identified an average of 23–24 missense and truncation alleles per gene, with at least one truncation or deleterious missense mutation in more than 90% of the captured wheat genes per population. This public collection of mutant seed stocks and sequence data enables rapid identification of mutations in the different copies of the wheat genes, which can be combined to uncover previously hidden variation. Polyploidy is a central phenomenon in plant evolution, and many crop species have undergone recent genome duplication events. Therefore, the general strategy and methods developed herein can benefit other polyploid crops.

Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops

Wheat, like many other staple cereals, contains low levels of the essential micronutrients iron and zinc. Up to two billion people worldwide suffer from iron and zinc deficiencies, particularly in regions with predominantly cereal-based diets. Although wheat flour is commonly fortified during processing, an attractive and more sustainable solution is biofortification, which requires developing new varieties of wheat with inherently higher iron and zinc content in their grains. Until now most studies aimed at increasing iron and zinc content in wheat grains have focused on discovering natural variation in progenitor or related species. However, recent developments in genomics and transformation have led to a step change in targeted research on wheat at a molecular level. We discuss promising approaches to improve iron and zinc content in wheat using knowledge gained in model grasses. We explore how the latest resources developed in wheat, including sequenced genomes and mutant populations, can be exploited for biofortification. We also highlight the key research and practical challenges that remain in improving iron and zinc content in wheat.

expVIP: a Customizable RNA-seq Data Analysis and Visualization Platform.

expVIP is an adaptable platform to create an integrated gene expression interface for any species with a transcriptome assembly. The majority of transcriptome sequencing (RNA-seq) expression studies in plants remain underutilized and inaccessible due to the use of disparate transcriptome references and the lack of skills and resources to analyze and visualize these data. We have developed expVIP, an expression visualization and integration platform, which allows easy analysis of RNA-seq data combined with an intuitive and interactive interface. Users can analyze public and user-specified data sets with minimal bioinformatics knowledge using the expVIP virtual machine. This generates a custom Web browser to visualize, sort, and filter the RNA-seq data and provides outputs for differential gene expression analysis. We demonstrate expVIP’s suitability for polyploid crops and evaluate its performance across a range of biologically relevant scenarios. To exemplify its use in crop research, we developed a flexible wheat (Triticum aestivum) expression browser (www.wheat-expression.com) that can be expanded with user-generated data in a local virtual machine environment. The open-access expVIP platform will facilitate the analysis of gene expression data from a wide variety of species by enabling the easy integration, visualization, and comparison of RNA-seq data across experiments.

Wheat Grain Filling Is Limited by Grain Filling Capacity rather than the Duration of Flag Leaf Photosynthesis: A Case Study Using NAM RNAi Plants.

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.

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.

Genome-Wide Sequence and Expression Analysis of the NAC Transcription Factor Family in Polyploid Wheat

Many important genes in agriculture correspond to transcription factors (TFs) that regulate a wide range of pathways from flowering to responses to disease and abiotic stresses. In this study, we identified 5776 TFs in hexaploid wheat (Triticum aestivum) and classified them into gene families. We further investigated the NAC family exploring the phylogeny, C-terminal domain (CTD) conservation, and expression profiles across 308 RNA-seq samples. Phylogenetic trees of NAC domains indicated that wheat NACs divided into eight groups similar to rice (Oryza sativa) and barley (Hordeum vulgare). CTD motifs were frequently conserved between wheat, rice, and barley within phylogenetic groups; however, this conservation was not maintained across phylogenetic groups. Three homeologous copies were present for 58% of NACs, whereas evidence of single homeolog gene loss was found for 33% of NACs. We explored gene expression patterns across a wide range of developmental stages, tissues, and abiotic stresses. We found that more phylogenetically related NACs shared more similar expression patterns compared to more distant NACs. However, within each phylogenetic group there were clades with diverse expression profiles. We carried out a coexpression analysis on all wheat genes and identified 37 modules of coexpressed genes of which 23 contained NACs. Using gene ontology (GO) term enrichment, we obtained putative functions for NACs within coexpressed modules including responses to heat and abiotic stress and responses to water: these NACs may represent targets for breeding or biotechnological applications. This study provides a framework and data for hypothesis generation for future studies on NAC TFs in wheat.

Impact of transposable elements on genome structure and evolution in bread wheat

BackgroundTransposable elements (TEs) are major components of large plant genomes and main drivers of genome evolution. The most recent assembly of hexaploid bread wheat recovered the highly repetitive TE space in an almost complete chromosomal context and enabled a detailed view into the dynamics of TEs in the A, B, and D subgenomes.ResultsThe overall TE content is very similar between the A, B, and D subgenomes, although we find no evidence for bursts of TE amplification after the polyploidization events. Despite the near-complete turnover of TEs since the subgenome lineages diverged from a common ancestor, 76% of TE families are still present in similar proportions in each subgenome. Moreover, spacing between syntenic genes is also conserved, even though syntenic TEs have been replaced by new insertions over time, suggesting that distances between genes, but not sequences, are under evolutionary constraints. The TE composition of the immediate gene vicinity differs from the core intergenic regions. We find the same TE families to be enriched or depleted near genes in all three subgenomes. Evaluations at the subfamily level of timed long terminal repeat-retrotransposon insertions highlight the independent evolution of the diploid A, B, and D lineages before polyploidization and cases of concerted proliferation in the AB tetraploid.ConclusionsEven though the intergenic space is changed by the TE turnover, an unexpected preservation is observed between the A, B, and D subgenomes for features like TE family proportions, gene spacing, and TE enrichment near genes.

Systematic Investigation of FLOWERING LOCUS T-Like Poaceae Gene Families Identifies the Short-Day Expressed Flowering Pathway Gene, TaFT3 in Wheat (Triticum aestivum L.)

To date, a small number of major flowering time loci have been identified in the related Triticeae crops, bread wheat (Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare). Natural genetic variants at these loci result in major phenotypic changes which have adapted crops to the novel environments encountered during the spread of agriculture. The polyploid nature of bread and durum wheat means that major flowering time loci in which recessive alleles confer adaptive advantage in related diploid species have not been readily identified. One such example is the PPD-H2 flowering time locus encoded by FLOWERING LOCUS T 3 (HvFT3) in the diploid crop barley, for which recessive mutant alleles confer delayed flowering under short day (SD) photoperiods. In autumn-sown barley, such alleles aid the repression of flowering over the winter, which help prevent the development of cold-sensitive floral organs until the onset of inductive long day (LD) photoperiods the following spring. While the identification of orthologous loci in wheat could provide breeders with alternative mechanisms to fine tune flowering time, systematic identification of wheat orthologs of HvFT3 has not been reported. Here, we characterize the FT gene families in six Poaceae species, identifying novel members in all taxa investigated, as well as FT3 homoeologs from the A, B and D genomes of hexaploid (TaFT3) and tetraploid wheat. Sequence analysis shows TaFT3 homoeologs display high similarity to the HvFT3 coding region (95–96%) and predicted protein (96–97%), with conservation of intron/exon structure across the five cereal species investigated. Genetic mapping and comparative analyses in hexaploid and tetraploid wheat find TaFT3 homoeologs map to the long arms of the group 1 chromosomes, collinear to HvFT3 in barley and FT3 orthologs in rice, foxtail millet and brachypodium. Genome-specific expression analyses show FT3 homoeologs in tetraploid and hexaploid wheat are upregulated under SD photoperiods, but not under LDs, analogous to the expression of HvFT3. Collectively, these results indicate that functional wheat orthologs of HvFT3 have been identified. The molecular resources generated here provide the foundation for engineering a novel major flowering time locus in wheat using forward or reverse genetics approaches.