Transcriptomic and co-expression network analyses on diverse wheat landraces identifies candidate master regulators of the response to early drought.

Introduction: Over four billion people around the world rely on bread wheat (Triticum aestivum L.) as a major constituent of their diet. The changing climate, however, threatens the food security of these people, with periods of intense drought stress already causing widespread wheat yield losses. Much of the research into the wheat drought response has centred on the response to drought events later in development, during anthesis or grain filling. But as the timing of periods of drought stress become increasingly unpredictable, a more complete understanding of the response to drought during early development is also needed. Methods: Here, we utilized the YoGI landrace panel to identify 10,199 genes which were differentially expressed under early drought stress, before weighted gene co-expression network analysis (WGCNA) was used to construct a co-expression network and identify hub genes in modules particularly associated with the early drought response. Results: Of these hub genes, two stood out as novel candidate master regulators of the early drought response - one as an activator (TaDHN4-D1; TraesCS5D02G379200) and the other as a repressor (uncharacterised gene; TraesCS3D02G361500). Discussion: As well as appearing to coordinate the transcriptional early drought response, we propose that these hub genes may be able to regulate the physiological early drought response due to potential control over the expression of members of gene families well-known for their involvement in the drought response in many plant species, namely dehydrins and aquaporins, as well as other genes seemingly involved in key processes such as, stomatal opening, stomatal closing, stomatal morphogenesis and stress hormone signalling.

Co-expression network analysis of diverse wheat landraces reveals markers of early thermotolerance and a candidate master regulator of thermotolerance genes.

Triticum aestivum L. (bread wheat) is a crop relied upon by billions of people around the world, as a major source of both income and calories. Rising global temperatures, however, pose a genuine threat to the livelihood of these people, as wheat growth and yields are extremely vulnerable to damage by heat stress. Here we present the YoGI wheat landrace panel, comprising 342 accessions that show remarkable phenotypic and genetic diversity thanks to their adaptation to different climates. We quantified the abundance of 110 790 transcripts from the panel and used these data to conduct weighted co-expression network analysis and to identify hub genes in modules associated with abiotic stress tolerance. We found that the expression of three hub genes, all heat-shock proteins (HSPs), were significantly correlated with early thermotolerance in a validation panel of landraces. These hub genes belong to the same module, with one (TraesCS4D01G207500.1) being a candidate master-regulator potentially controlling the expression of the other two hub genes, as well as a suite of other HSPs and heat-stress transcription factors (HSFs). In this work, therefore, we identify three validated hub genes, the expression of which can serve as markers of thermotolerance during early development, and suggest that TraesCS4D01G207500.1 is a potential master regulator of HSP and HSF expression - presenting the YoGI landrace panel as an invaluable tool for breeders wishing to determine and introduce novel alleles into modern varieties, for the production of climate-resilient crops.

Identification of candidate regulators of the response to early heat stress in climate-adapted wheat landraces via transcriptomic and co-expression network analyses.

Introduction: Climate change is likely to lead to not only increased global temperatures but also a more variable climate where unseasonal periods of heat stress are more prevalent. This has been evidenced by the observation of spring-time temperatures approaching 40°C in some of the main spring-wheat producing countries, such as the USA, in recent years. With an optimum growth temperature of around 20°C, wheat is particularly prone to damage by heat stress. A warming climate with increasingly common fluctuations in temperature therefore threatens wheat crops and subsequently the lives and livelihoods of billions of people who depend on the crop for food. To futureproof wheat against a variable climate, a better understanding of the response to early heat stress is required. Methods: Here, we utilised DESeq2 to identify 7,827 genes which were differentially expressed in wheat landraces after early heat stress exposure. Candidate hub genes, which may regulate the transcriptional response to early heat stress, were identified via weighted gene co-expression network analysis (WGCNA), and validated by qRT-PCR. Results: Two of the most promising candidate hub genes (TraesCS3B02G409300 and TraesCS1B02G384900) may downregulate the expression of genes involved in the drought, salinity, and cold responses-genes which are unlikely to be required under heat stress-as well as photosynthesis genes and stress hormone signalling repressors, respectively. We also suggest a role for a poorly characterised sHSP hub gene (TraesCS4D02G212300), as an activator of the heat stress response, potentially inducing the expression of a vast suite of heat shock proteins and transcription factors known to play key roles in the heat stress response. Discussion: The present work represents an exploratory examination of the heat-induced transcriptional change in wheat landrace seedlings and identifies several candidate hub genes which may act as regulators of this response and, thus, may be targets for breeders in the production of thermotolerant wheat varieties.

Natural Variation in OASC Gene for Mitochondrial O-Acetylserine Thiollyase Affects Sulfate Levels in Arabidopsis.

Sulfur plays a vital role in the primary and secondary metabolism of plants, and carries an important function in a large number of different compounds. Despite this importance, compared to other mineral nutrients, relatively little is known about sulfur sensing and signalling, as well as about the mechanisms controlling sulfur metabolism and homeostasis. Sulfur contents in plants vary largely not only among different species, but also among accessions of the same species. We previously used associative transcriptomics to identify several genes potentially controlling variation in sulfate content in the leaves of Brassica napus, including an OASC gene for mitochondrial O-acetylserine thiollyase (OAS-TL), an enzyme involved in cysteine synthesis. Here, we show that loss of OASC in Arabidopsis thaliana lowers not only sulfate, but also glutathione levels in the leaves. The reduced accumulation is caused by lower sulfate uptake and translocation to the shoots; however, the flux through the pathway is not affected. In addition, we identified a single nucleotide polymorphism in the OASC gene among A. thaliana accessions that is linked to variation in sulfate content. Both genetic and transgenic complementation confirmed that the exchange of arginine at position 81 for lysine in numerous accessions resulted in a less active OASC and a lower sulfate content in the leaves. The mitochondrial isoform of OAS-TL is, thus, after the ATPS1 isoform of sulfurylase and the APR2 form of APS reductase 2, the next metabolic enzyme with a role in regulation of sulfate content in Arabidopsis.

Validation of an Associative Transcriptomics platform in the polyploid crop species Brassica juncea by dissection of the genetic architecture of agronomic and quality traits.

The development of more productive crops will be key to addressing the challenges that climate change, population growth and diminishing resources pose to global food security. Advanced 'omics techniques can help to accelerate breeding by facilitating the identification of genetic markers for use in marker-assisted selection. Here, we present the validation of a new Associative Transcriptomics platform in the important oilseed crop Brassica juncea. To develop this platform, we established a pan-transcriptome reference for B. juncea, to which we mapped transcriptome data from a diverse panel of B. juncea accessions. From this panel, we identified 355 050 single nucleotide polymorphism variants and quantified the abundance of 93 963 transcripts. Subsequent association analysis of functional genotypes against a number of important agronomic and quality traits revealed a promising candidate gene for seed weight, BjA.TTL, as well as additional markers linked to seed colour and vitamin E content. The establishment of the first full-scale Associative Transcriptomics platform for B. juncea enables rapid progress to be made towards an understanding of the genetic architecture of trait variation in this important species, and provides an exemplar for other crops.

Dissecting the complex regulation of lodging resistance in Brassica napus.

Lodging continues to be a major cause of yield loss in important crop species such as Brassica napus. Understanding the genetic regulation of lodging resistance is therefore of key interest to breeders worldwide. Current strategies aimed at minimising lodging risk involve the incorporation of dwarfing genes or the application of plant growth regulators. However, despite these efforts, lodging continues to be a persistent problem and it is therefore of high interest that novel, complimentary strategies for lodging control are implemented. One approach would be to focus on understanding the genetic properties underlying stem mechanical strength. With this in mind, we screened a training genetic diversity panel of B. napus accession for variation in stem mechanical strength and related traits. Using Associative Transcriptomics, we identified molecular markers for a suite of valuable traits. Using an independent test genetic diversity panel, we show that the methods employed are robust for identification of predictive markers. Furthermore, based on conserved synteny with Arabidopsis thaliana, we are able to provide a biological context to the marker associations detected and provide evidence for a role in pectin methylesterification in contributing to stem mechanical strength in Brassicaceae.

Validation of an updated Associative Transcriptomics platform for the polyploid crop species Brassica napus by dissection of the genetic architecture of erucic acid and tocopherol isoform variation in seeds.

An updated platform was developed to underpin association genetics studies in the polyploid crop species Brassica napus (oilseed rape). Based on 1.92 × 1012 bases of leaf mRNAseq data, functional genotypes, comprising 355 536 single-nucleotide polymorphism markers and transcript abundance were scored across a genetic diversity panel of 383 accessions using a transcriptome reference comprising 116 098 ordered coding DNA sequence (CDS) gene models. The use of the platform for Associative Transcriptomics was first tested by analysing the genetic architecture of variation in seed erucic acid content, as high-erucic rapeseed oil is highly valued for a variety of applications in industry. Known loci were identified, along with a previously undetected minor-effect locus. The platform was then used to analyse variation for the relative proportions of tocopherol (vitamin E) forms in seeds, and the validity of the most significant markers was assessed using a take-one-out approach. Furthermore, the analysis implicated expression variation of the gene Bo2g050970.1, an orthologue of VTE4 (which encodes a γ-tocopherol methyl transferase converting γ-tocopherol into α-tocopherol) associated with the observed trait variation. The establishment of the first full-scale Associative Transcriptomics platform for B. napus enables rapid progress to be made towards an understanding of the genetic architecture of trait variation in this important species, and provides an exemplar for other crops.

Variation across a wheat genetic diversity panel for saccharification of hydrothermally pretreated straw.

BACKGROUND: Wheat straw forms an important, reliable source of lignocellulosic biomass for use in second-generation ethanol production. However, there is limited understanding of the variation in quality of straw from current breeding cultivars, and studies on such variation have generally employed suboptimal pretreatments. There is also a degree of confusion regarding phenotypic characteristics relevant to optimising the enzymatic saccharification of cellulose after suitable pretreatments for biorefining compared with those which determine good ruminant digestibility. The aim of this study has been to (a) evaluate and compare the levels of glucose enzymatically released from straw obtained from 89 cultivars of winter wheat after optimised hydrothermal pretreatments and (b) identify the underlying phenotypic characteristics relevant to enhanced glucose production with special reference to the ratios of constituent tissue types. RESULTS: Optimised pretreatment involved hydrothermal extraction at 210 °C for 10 min. Using excess cellulases, quantitative saccharification was achieved within 24 h. The amount of glucose released ranged from 192 to 275 mg/g. The extent of glucose release was correlated with (a) the level of internode tissue (R = 0.498; p = 6.84 × 10-7), (b) stem height (R = 0.491; p = 1.03 × 10-6), and (c) chemical characteristics particular to stem tissues including higher levels of cellulose (R = 0.552; p = 2.06 × 10-8) and higher levels of lignin R = 0.494; p = 8.67 × 10-7. CONCLUSIONS: In order to achieve maximum yields of cellulosic glucose for second-generation ethanol production, a predisposition for wheat to produce cellulose-enriched internode stem tissue, particularly of longer length, would be beneficial. This contrasts with the ideotype for ruminant nutrition, in which an increased proportion of leaf tissue is preferable.

Carbohydrate microarrays and their use for the identification of molecular markers for plant cell wall composition.

Genetic improvement of the plant cell wall has enormous potential to increase the quality of food, fibers, and fuels. However, the identification and characterization of genes involved in plant cell wall synthesis is far from complete. Association mapping is one of the few techniques that can help identify candidate genes without relying on our currently incomplete knowledge of cell wall synthesis. However, few cell wall phenotyping methodologies have proven sufficiently precise, robust, or scalable for association mapping to be conducted for specific cell wall polymers. Here, we created high-density carbohydrate microarrays containing chemically extracted cell wall polysaccharides collected from 331 genetically diverse Brassica napus cultivars and used them to obtain detailed, quantitative information describing the relative abundance of selected noncellulosic polysaccharide linkages and primary structures. We undertook genome-wide association analysis of data collected from 57 carbohydrate microarrays and identified molecular markers reflecting a diversity of specific xylan, xyloglucan, pectin, and arabinogalactan moieties. These datasets provide a detailed insight into the natural variations in cell wall carbohydrate moieties between B. napus genotypes and identify associated markers that could be exploited by marker-assisted breeding. The identified markers also have value beyond B. napus for functional genomics, facilitated by the close genetic relatedness to the model plant Arabidopsis Together, our findings provide a unique dissection of the genetic architecture that underpins plant cell wall biosynthesis and restructuring.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Extensive homoeologous genome exchanges in allopolyploid crops revealed by mRNAseq-based visualization.

Polyploidy, the possession of multiple sets of chromosomes, has been a predominant factor in the evolution and success of the angiosperms. Although artificially formed allopolyploids show a high rate of genome rearrangement, the genomes of cultivars and germplasm used for crop breeding were assumed stable and genome structural variation under the artificial selection process of commercial breeding has remained little studied. Here, we show, using a repurposed visualization method based on transcriptome sequence data, that genome structural rearrangement occurs frequently in varieties of three polyploid crops (oilseed rape, mustard rape and bread wheat), meaning that the extent of genome structural variation present in commercial crops is much higher than expected. Exchanges were found to occur most frequently where homoeologous chromosome segments are collinear to telomeres and in material produced as doubled haploids. The new insights into genome structural evolution enable us to reinterpret the results of recent studies and implicate homoeologous exchanges, not deletions, as being responsible for variation controlling important seed quality traits in rapeseed. Having begun to identify the extent of genome structural variation in polyploid crops, we can envisage new strategies for the global challenge of broadening crop genetic diversity and accelerating adaptation, such as the molecular identification and selection of genome deletions or duplications encompassing genes with trait-controlling dosage effects.

Elucidation of the genetic basis of variation for stem strength characteristics in bread wheat by Associative Transcriptomics.

BACKGROUND: The current approach to reducing the tendency for wheat grown under high fertilizer conditions to collapse (lodge) under the weight of its grain is based on reducing stem height via the introduction of Rht genes. However, these reduce the yield of straw (itself an important commodity) and introduce other undesirable characteristics. Identification of alternative height-control loci is therefore of key interest. In addition, the improvement of stem mechanical strength provides a further way through which lodging can be reduced. RESULTS: To investigate the prospects for genetic alternatives to Rht, we assessed variation for plant height and stem strength properties in a training genetic diversity panel of 100 wheat accessions fixed for Rht. Using mRNAseq data derived from RNA purified from leaves, functional genotypes were developed for the panel comprising 42,066 Single Nucleotide Polymorphism (SNP) markers and 94,060 Gene Expression Markers (GEMs). In the first application in wheat of the recently-developed method of Associative Transcriptomics, we identified associations between trait variation and both SNPs and GEMs. Analysis of marker-trait associations revealed candidates for the causative genes underlying the trait variation, implicating xylan acetylation and the COP9 signalosome as contributing to stem strength and auxin in the control of the observed variation for plant height. Predictive capabilities of key markers for stem strength were validated using a test genetic diversity panel of 30 further wheat accessions. CONCLUSIONS: This work illustrates the power of Associative Transcriptomics for the exploration of complex traits of high agronomic importance in wheat. The careful selection of genotypes included in the analysis, allowed for high resolution mapping of novel trait-controlling loci in this staple crop. The use of Gene Expression markers coupled with the more traditional sequence-based markers, provides the power required to understand the biological context of the marker-trait associations observed. This not only adds to the wealth of knowledge that we strive to accumulate regarding gene function and plant adaptation, but also provides breeders with the information required to make more informed decisions regarding the potential consequences of incorporating the use of particular markers into future breeding programmes.

Molecular markers for tolerance of European ash (Fraxinus excelsior) to dieback disease identified using Associative Transcriptomics.

Tree disease epidemics are a global problem, impacting food security, biodiversity and national economies. The potential for conservation and breeding in trees is hampered by complex genomes and long lifecycles, with most species lacking genomic resources. The European Ash tree Fraxinus excelsior is being devastated by the fungal pathogen Hymenoscyphus fraxineus, which causes ash dieback disease. Taking this system as an example and utilizing Associative Transcriptomics for the first time in a plant pathology study, we discovered gene sequence and gene expression variants across a genetic diversity panel scored for disease symptoms and identified markers strongly associated with canopy damage in infected trees. Using these markers we predicted phenotypes in a test panel of unrelated trees, successfully identifying individuals with a low level of susceptibility to the disease. Co-expression analysis suggested that pre-priming of defence responses may underlie reduced susceptibility to ash dieback.

Genome distribution of differential homoeologue contributions to leaf gene expression in bread wheat.

Using a combination of de novo transcriptome assembly, a newly developed 9495-marker transcriptome SNP genetic linkage map and comparative genomics approaches, we developed an ordered set of nonredundant transcripts for each of the subgenomes of hexaploid wheat: A (47 160 unigenes), B (59 663 unigenes) and D (40 588 unigenes). We used these as reference sequences against which to map Illumina mRNA-Seq reads derived from young leaf tissue. Transcript abundance was quantified for each unigene. Using a three-way reciprocal BLAST approach, 15 527 triplet sets of homoeologues (one from each genome) were identified. Differential expression (P < 0.05) was identified for 5248 unigenes, with 2906 represented at greater abundance than their two homoeologues and 2342 represented at lower abundance than their two homoeologues. Analysis of gene ontology terms revealed no biases between homoeologues. There was no evidence of genomewide dominance effects, rather the more highly transcribed individual genes were distributed throughout all three genomes. Transcriptome display tile plot, a visualization approach based on CMYK colour space, was developed and used to assess the genome for regions of skewed homoeologue transcript abundance. Extensive striation was revealed, indicative of many small regions of genome dominance (transcripts of homoeologues from one genome more abundant than the others) and many larger regions of genome repression (transcripts of homoeologues from one genome less abundant than the others).

Variation in the chemical composition of wheat straw: the role of tissue ratio and composition.

BACKGROUND: Wheat straw is an attractive substrate for second generation ethanol production because it will complement and augment wheat production rather than competing with food production. However, like other sources of lignocellulosic biomass, even from a single species, it is heterogeneous in nature due to the different tissues and cell types, and this has implications for saccharification efficiency. The aim of this study has been to use Fourier transform infrared (FTIR) spectroscopy and Partial least squares (PLS) modelling to rapidly screen wheat cultivars for the levels of component tissues, the carbohydrate composition and lignin content, and the levels of simple cross-linking phenolics such as ferulic and diferulic acids. RESULTS: FTIR spectroscopy and PLS modelling was used to analyze the tissue and chemical composition of wheat straw biomass. Predictive models were developed to evaluate the variability in the concentrations of the cell wall sugars, cell wall phenolics and acid-insoluble lignin. Models for the main sugars, phenolics and lignin were validated and then used to evaluate the variation in total biomass composition across 90 cultivars of wheat grown over two seasons. CONCLUSIONS: Whilst carbohydrate and lignin components varied across the varieties, this mainly reflected differences in the ratios of the component tissues rather than differences in the composition of those tissues. Further analysis indicated that on a mol% basis, relative levels of sugars within the tissues varied to only a small degree. There were no clear associations between simple phenolics and tissues. The results provide a basis for improving biomass quality for biofuels production through selection of cultivars with appropriate tissue ratios.

A novel and fast approach for population structure inference using kernel-PCA and optimization.

Population structure is a confounding factor in genome-wide association studies, increasing the rate of false positive associations. To correct for it, several model-based algorithms such as ADMIXTURE and STRUCTURE have been proposed. These tend to suffer from the fact that they have a considerable computational burden, limiting their applicability when used with large datasets, such as those produced by next generation sequencing techniques. To address this, nonmodel based approaches such as sparse nonnegative matrix factorization (sNMF) and EIGENSTRAT have been proposed, which scale better with larger data. Here we present a novel nonmodel-based approach, population structure inference using kernel-PCA and optimization (PSIKO), which is based on a unique combination of linear kernel-PCA and least-squares optimization and allows for the inference of admixture coefficients, principal components, and number of founder populations of a dataset. PSIKO has been compared against existing leading methods on a variety of simulation scenarios, as well as on real biological data. We found that in addition to producing results of the same quality as other tested methods, PSIKO scales extremely well with dataset size, being considerably (up to 30 times) faster for longer sequences than even state-of-the-art methods such as sNMF. PSIKO and accompanying manual are freely available at https://www.uea.ac.uk/computing/psiko.

Dissection of the control of anion homeostasis by associative transcriptomics in Brassica napus.

To assess the variation in nutrient homeostasis in oilseed rape and to identify the genes responsible for this variation, we determined foliar anion levels in a diversity panel of Brassica napus accessions, 84 of which had been genotyped previously using messenger RNA sequencing. We applied associative transcriptomics to identify sequence polymorphisms linked to variation in nitrate, phosphate, or sulfate in these accessions. The analysis identified several hundred significant associations for each anion. Using functional annotation of Arabidopsis (Arabidopsis thaliana) homologs and available microarray data, we identified 60 candidate genes for controlling variation in the anion contents. To verify that these genes function in the control of nutrient homeostasis, we obtained Arabidopsis transfer DNA insertion lines for these candidates and tested them for the accumulation of nitrate, phosphate, and sulfate. Fourteen lines differed significantly in levels of the corresponding anions. Several of these genes have been shown previously to affect the accumulation of the corresponding anions in Arabidopsis mutants. These results thus confirm the power of associative transcriptomics in dissection of the genetic control of complex traits and present a set of candidate genes for use in the improvement of efficiency of B. napus mineral nutrition.

Associative transcriptomics study dissects the genetic architecture of seed glucosinolate content in Brassica napus.

Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed 'associative transcriptomics' (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction-based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

Genome-wide association study dissects the genetic architecture of seed weight and seed quality in rapeseed (Brassica napus L.).

Association mapping can quickly and efficiently dissect complex agronomic traits. Rapeseed is one of the most economically important polyploid oil crops, although its genome sequence is not yet published. In this study, a recently developed 60K Brassica Infinium(®) SNP array was used to analyse an association panel with 472 accessions. The single-nucleotide polymorphisms (SNPs) of the array were in silico mapped using 'pseudomolecules' representative of the genome of rapeseed to establish their hypothetical order and to perform association mapping of seed weight and seed quality. As a result, two significant associations on A8 and C3 of Brassica napus were detected for erucic acid content, and the peak SNPs were found to be only 233 and 128 kb away from the key genes BnaA.FAE1 and BnaC.FAE1. BnaA.FAE1 was also identified to be significantly associated with the oil content. Orthologues of Arabidopsis thaliana HAG1 were identified close to four clusters of SNPs associated with glucosinolate content on A9, C2, C7 and C9. For seed weight, we detected two association signals on A7 and A9, which were consistent with previous studies of quantitative trait loci mapping. The results indicate that our association mapping approach is suitable for fine mapping of the complex traits in rapeseed.