Publications by authors named "Ricardo Ramirez-Gonzalez"

38 Publications

Rust expression browser: an open source database for simultaneous analysis of host and pathogen gene expression profiles with expVIP.

BMC Genomics 2021 Mar 9;22(1):166. Epub 2021 Mar 9.

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Background: Transcriptomics is being increasingly applied to generate new insight into the interactions between plants and their pathogens. For the wheat yellow (stripe) rust pathogen (Puccinia striiformis f. sp. tritici, Pst) RNA-based sequencing (RNA-Seq) has proved particularly valuable, overcoming the barriers associated with its obligate biotrophic nature. This includes the application of RNA-Seq approaches to study Pst and wheat gene expression dynamics over time and the Pst population composition through the use of a novel RNA-Seq based surveillance approach called "field pathogenomics". As a dual RNA-Seq approach, the field pathogenomics technique also provides gene expression data from the host, giving new insight into host responses. However, this has created a wealth of data for interrogation.

Results: Here, we used the field pathogenomics approach to generate 538 new RNA-Seq datasets from Pst-infected field wheat samples, doubling the amount of transcriptomics data available for this important pathosystem. We then analysed these datasets alongside 66 RNA-Seq datasets from four Pst infection time-courses and 420 Pst-infected plant field and laboratory samples that were publicly available. A database of gene expression values for Pst and wheat was generated for each of these 1024 RNA-Seq datasets and incorporated into the development of the rust expression browser ( http://www.rust-expression.com ). This enables for the first time simultaneous 'point-and-click' access to gene expression profiles for Pst and its wheat host and represents the largest database of processed RNA-Seq datasets available for any of the three Puccinia wheat rust pathogens. We also demonstrated the utility of the browser through investigation of expression of putative Pst virulence genes over time and examined the host plants response to Pst infection.

Conclusions: The rust expression browser offers immense value to the wider community, facilitating data sharing and transparency and the underlying database can be continually expanded as more datasets become publicly available.
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http://dx.doi.org/10.1186/s12864-021-07488-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941908PMC
March 2021

Multiple wheat genomes reveal global variation in modern breeding.

Nature 2020 12 25;588(7837):277-283. Epub 2020 Nov 25.

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome, and the lack of genome-assembly data for multiple wheat lines. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm1, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.
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http://dx.doi.org/10.1038/s41586-020-2961-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759465PMC
December 2020

A haplotype-led approach to increase the precision of wheat breeding.

Commun Biol 2020 Nov 25;3(1):712. Epub 2020 Nov 25.

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Crop productivity must increase at unprecedented rates to meet the needs of the growing worldwide population. Exploiting natural variation for the genetic improvement of crops plays a central role in increasing productivity. Although current genomic technologies can be used for high-throughput identification of genetic variation, methods for efficiently exploiting this genetic potential in a targeted, systematic manner are lacking. Here, we developed a haplotype-based approach to identify genetic diversity for crop improvement using genome assemblies from 15 bread wheat (Triticum aestivum) cultivars. We used stringent criteria to identify identical-by-state haplotypes and distinguish these from near-identical sequences (~99.95% identity). We showed that each cultivar shares ~59 % of its genome with other sequenced cultivars and we detected the presence of extended haplotype blocks containing hundreds to thousands of genes across all wheat chromosomes. We found that genic sequence alone was insufficient to fully differentiate between haplotypes, as were commonly used array-based genotyping chips due to their gene centric design. We successfully used this approach for focused discovery of novel haplotypes from a landrace collection and documented their potential for trait improvement in modern bread wheat. This study provides a framework for defining and exploiting haplotypes to increase the efficiency and precision of wheat breeding towards optimising the agronomic performance of this crucial crop.
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http://dx.doi.org/10.1038/s42003-020-01413-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689427PMC
November 2020

New insights into homoeologous copy number variations in the hexaploid wheat genome.

Plant Genome 2021 03 5;14(1):e20069. Epub 2020 Nov 5.

Université Clermont Auvergne, INRAE, GDEC, Clermont-Ferrand, 63000, France.

Bread wheat is an allohexaploid species originating from two successive and recent rounds of hybridization between three diploid species that were very similar in terms of chromosome number, genome size, TE content, gene content and synteny. As a result, it has long been considered that most of the genes were in three pairs of homoeologous copies. However, these so-called triads represent only one half of wheat genes, while the remaining half belong to homoeologous groups with various number of copies across subgenomes. In this study, we examined and compared the distribution, conservation, function, expression and epigenetic profiles of triads with homoeologous groups having undergone a deletion (dyads) or a duplication (tetrads) in one subgenome. We show that dyads and tetrads are mostly located in distal regions and have lower expression level and breadth than triads. Moreover, they are enriched in functions related to adaptation and more associated with the repressive H3K27me3 modification. Altogether, these results suggest that triads mainly correspond to housekeeping genes and are part of the core genome, while dyads and tetrads belong to the Triticeae dispensable genome. In addition, by comparing the different categories of dyads and tetrads, we hypothesize that, unlike most of the allopolyploid species, subgenome dominance and biased fractionation are absent in hexaploid wheat. Differences observed between the three subgenomes are more likely related to two successive and ongoing waves of post-polyploid diploidization, that had impacted A and B more significantly than D, as a result of the evolutionary history of hexaploid wheat.
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http://dx.doi.org/10.1002/tpg2.20069DOI Listing
March 2021

Optimizing legume symbioses by simultaneous measurement of rhizobial competitiveness and N fixation in nodules.

Proc Natl Acad Sci U S A 2020 05 21;117(18):9822-9831. Epub 2020 Apr 21.

Department of Plant Sciences, University of Oxford, OX1 3RB Oxford, United Kingdom;

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple -legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.
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http://dx.doi.org/10.1073/pnas.1921225117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211974PMC
May 2020

A roadmap for gene functional characterisation in crops with large genomes: Lessons from polyploid wheat.

Elife 2020 03 24;9. Epub 2020 Mar 24.

John Innes Centre, Norwich Research Park, Norwich, United Kingdom.

Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite their importance, a lack of genomic information and resources has hindered the functional characterisation of genes in major crops. The recent release of high-quality reference sequences for these crops underpins a suite of genetic and genomic resources that support basic research and breeding. For wheat, these include gene model annotations, expression atlases and gene networks that provide information about putative function. Sequenced mutant populations, improved transformation protocols and structured natural populations provide rapid methods to study gene function directly. We highlight a case study exemplifying how to integrate these resources. This review provides a helpful guide for plant scientists, especially those expanding into crop research, to capitalise on the discoveries made in and other plants. This will accelerate the improvement of crops of vital importance for food and nutrition security.
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http://dx.doi.org/10.7554/eLife.55646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093151PMC
March 2020

The NLR-Annotator Tool Enables Annotation of the Intracellular Immune Receptor Repertoire.

Plant Physiol 2020 06 17;183(2):468-482. Epub 2020 Mar 17.

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat () cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low expression and high intrafamily sequence homology hinder their accurate annotation. Here, we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny, and expression profile of the gene family. We detected 3,400 full-length NLR loci, of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper function. Most (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress, we found up-regulation of 266 of the To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.
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http://dx.doi.org/10.1104/pp.19.01273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271791PMC
June 2020

RNA-seq, de novo transcriptome assembly and flavonoid gene analysis in 13 wild and cultivated berry fruit species with high content of phenolics.

BMC Genomics 2019 Dec 19;20(1):995. Epub 2019 Dec 19.

Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Background: Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds.

Results: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige.

Conclusions: We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.
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http://dx.doi.org/10.1186/s12864-019-6183-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924045PMC
December 2019

A Co-Expression Network in Hexaploid Wheat Reveals Mostly Balanced Expression and Lack of Significant Gene Loss of Homeologous Meiotic Genes Upon Polyploidization.

Front Plant Sci 2019 18;10:1325. Epub 2019 Oct 18.

John Innes Centre, Norwich Research Park, Norwich, United Kingdom.

Polyploidization has played an important role in plant evolution. However, upon polyploidization, the process of meiosis must adapt to ensure the proper segregation of increased numbers of chromosomes to produce balanced gametes. It has been suggested that meiotic gene (MG) duplicates return to a single copy following whole genome duplication to stabilize the polyploid genome. Therefore, upon the polyploidization of wheat, a hexaploid species with three related (homeologous) genomes, the stabilization process may have involved rapid changes in content and expression of MGs on homeologous chromosomes (homeologs). To examine this hypothesis, sets of candidate MGs were identified in wheat using co-expression network analysis and orthology informed approaches. In total, 130 RNA-Seq samples from a range of tissues including wheat meiotic anthers were used to define co-expressed modules of genes. Three modules were significantly correlated with meiotic tissue samples but not with other tissue types. These modules were enriched for GO terms related to cell cycle, DNA replication, and chromatin modification and contained orthologs of known MGs. Overall, 74.4% of genes within these meiosis-related modules had three homeologous copies which was similar to other tissue-related modules. Amongst wheat MGs identified by orthology, rather than co-expression, the majority (93.7%) were either retained in hexaploid wheat at the same number of copies (78.4%) or increased in copy number (15.3%) compared to ancestral wheat species. Furthermore, genes within meiosis-related modules showed more balanced expression levels between homeologs than genes in non-meiosis-related modules. Taken together, our results do not support extensive gene loss nor changes in homeolog expression of MGs upon wheat polyploidization. The construction of the MG co-expression network allowed identification of hub genes and provided key targets for future studies.
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http://dx.doi.org/10.3389/fpls.2019.01325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813927PMC
October 2019

A carbohydrate-binding protein, B-GRANULE CONTENT 1, influences starch granule size distribution in a dose-dependent manner in polyploid wheat.

J Exp Bot 2020 01;71(1):105-115

NIAB, Cambridge, UK.

In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.
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http://dx.doi.org/10.1093/jxb/erz405DOI Listing
January 2020

Global transcriptome analysis uncovers the gene co-expression regulation network and key genes involved in grain development of wheat (Triticum aestivum L.).

Funct Integr Genomics 2019 Nov 21;19(6):853-866. Epub 2019 May 21.

College of Life Sciences, Northwest A & F University, Yangling, 712100, Shaanxi, China.

Wheat grain development is a robust biological process that largely determines grain quality and yield. In this study, we investigated the grain transcriptome of winter wheat cv. Xiaoyan-6 at four developmental stages (5, 10, 15, and 20 days post-anthesis), using high-throughput RNA sequencing (RNA-Seq). We identified 427 grain-specific transcription factors (TFs) and 1653 differentially expressed TFs during grain development as well as a grain co-expression regulation network (GrainNet) of the TFs and their predicted co-expressed genes. Our study identified ten putative key TFs and the predicted regulatory genes of these TFs in wheat grain development of Xiaoyan-6. The analysis was given a firm basis through the study of additional wheat tissues, including root, stem, leaf, flag leaf, grain, spikes (from wheat plants at booting or heading stages) to provide a dataset of 92,478 high-confidence protein-coding genes that were mostly evenly distributed among subgenomes, but unevenly distributed across each of the chromosomes or each of the seven homeologous groups. Within this larger framework of the transcriptomes, we identified 4659 grain-specific genes (SEGs) and 26,500 differentially expressed genes (DEGs) throughout grain development stages tested. The SEGs identified mainly associate with regulation and signaling-related biological processes, while the DEGs mainly involve in cellular component organization or biogenesis and nutrient reservoir activity during grain development of Xiaoyan-6. This study establishes new targets for modifying genes related to grain development and yield, to fine-tune expression in different varieties and environments.
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http://dx.doi.org/10.1007/s10142-019-00678-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797667PMC
November 2019

BrAPI-an application programming interface for plant breeding applications.

Bioinformatics 2019 10;35(20):4147-4155

LeafNode Technology, Auckland, New Zealand.

Motivation: Modern genomic breeding methods rely heavily on very large amounts of phenotyping and genotyping data, presenting new challenges in effective data management and integration. Recently, the size and complexity of datasets have increased significantly, with the result that data are often stored on multiple systems. As analyses of interest increasingly require aggregation of datasets from diverse sources, data exchange between disparate systems becomes a challenge.

Results: To facilitate interoperability among breeding applications, we present the public plant Breeding Application Programming Interface (BrAPI). BrAPI is a standardized web service API specification. The development of BrAPI is a collaborative, community-based initiative involving a growing global community of over a hundred participants representing several dozen institutions and companies. Development of such a standard is recognized as critical to a number of important large breeding system initiatives as a foundational technology. The focus of the first version of the API is on providing services for connecting systems and retrieving basic breeding data including germplasm, study, observation, and marker data. A number of BrAPI-enabled applications, termed BrAPPs, have been written, that take advantage of the emerging support of BrAPI by many databases.

Availability And Implementation: More information on BrAPI, including links to the specification, test suites, BrAPPs, and sample implementations is available at https://brapi.org/. The BrAPI specification and the developer tools are provided as free and open source.
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http://dx.doi.org/10.1093/bioinformatics/btz190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792114PMC
October 2019

Genome-Wide Transcription During Early Wheat Meiosis Is Independent of Synapsis, Ploidy Level, and the Locus.

Front Plant Sci 2018 4;9:1791. Epub 2018 Dec 4.

John Innes Centre, Norwich, United Kingdom.

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat-rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis.
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http://dx.doi.org/10.3389/fpls.2018.01791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288783PMC
December 2018

Hotspots in the genomic architecture of field drought responses in wheat as breeding targets.

Funct Integr Genomics 2019 Mar 16;19(2):295-309. Epub 2018 Nov 16.

Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Alameda del Obispo s/n, 14004, Córdoba, Spain.

Wheat can adapt to most agricultural conditions across temperate regions. This success is the result of phenotypic plasticity conferred by a large and complex genome composed of three homoeologous genomes (A, B, and D). Although drought is a major cause of yield and quality loss in wheat, the adaptive mechanisms and gene networks underlying drought responses in the field remain largely unknown. Here, we addressed this by utilizing an interdisciplinary approach involving field water status phenotyping, sampling, and gene expression analyses. Overall, changes at the transcriptional level were reflected in plant spectral traits amenable to field-level physiological measurements, although changes in photosynthesis-related pathways were found likely to be under more complex post-transcriptional control. Examining homoeologous genes with a 1:1:1 relationship across the A, B, and D genomes (triads), we revealed a complex genomic architecture for drought responses under field conditions, involving gene homoeolog specialization, multiple gene clusters, gene families, miRNAs, and transcription factors coordinating these responses. Our results provide a new focus for genomics-assisted breeding of drought-tolerant wheat cultivars.
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http://dx.doi.org/10.1007/s10142-018-0639-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6394720PMC
March 2019

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research.

Nat Protoc 2018 12;13(12):2944-2963

Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, Australia.

'Speed breeding' (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants' daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber-based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.
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http://dx.doi.org/10.1038/s41596-018-0072-zDOI Listing
December 2018

Shifting the limits in wheat research and breeding using a fully annotated reference genome.

Science 2018 08 16;361(6403). Epub 2018 Aug 16.

GDEC (Genetics, Diversity and Ecophysiology of Cereals), INRA, Université Clermont Auvergne (UCA), 5 chemin de Beaulieu, 63039 Clermont-Ferrand, France.

An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue- and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.
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http://dx.doi.org/10.1126/science.aar7191DOI Listing
August 2018

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

Genome Biol 2018 08 17;19(1):103. Epub 2018 Aug 17.

GDEC, INRA, UCA (Université Clermont Auvergne), Clermont-Ferrand, France.

Background: Transposable 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.

Results: The 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.

Conclusions: Even 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.
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http://dx.doi.org/10.1186/s13059-018-1479-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097303PMC
August 2018

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

Genome Res 2017 05;27(5):885-896

Earlham Institute, Norwich, NR4 7UZ, United Kingdom.

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.
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http://dx.doi.org/10.1101/gr.217117.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411782PMC
May 2017

Uncovering hidden variation in polyploid wheat.

Proc Natl Acad Sci U S A 2017 02 17;114(6):E913-E921. Epub 2017 Jan 17.

Department of Plant Sciences, University of California, Davis, CA 95616;

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.
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http://dx.doi.org/10.1073/pnas.1619268114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5307431PMC
February 2017

Genome sequence and genetic diversity of European ash trees.

Nature 2017 01 26;541(7636):212-216. Epub 2016 Dec 26.

School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK.

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.
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http://dx.doi.org/10.1038/nature20786DOI Listing
January 2017

transPLANT Resources for Triticeae Genomic Data.

Plant Genome 2016 03;9(1)

The genome sequences of many important Triticeae species, including bread wheat ( L.) and barley ( L.), remained uncharacterized for a long time because their high repeat content, large sizes, and polyploidy. As a result of improvements in sequencing technologies and novel analyses strategies, several of these have recently been deciphered. These efforts have generated new insights into Triticeae biology and genome organization and have important implications for downstream usage by breeders, experimental biologists, and comparative genomicists. transPLANT () is an EU-funded project aimed at constructing hardware, software, and data infrastructure for genome-scale research in the life sciences. Since the Triticeae data are intrinsically complex, heterogenous, and distributed, the transPLANT consortium has undertaken efforts to develop common data formats and tools that enable the exchange and integration of data from distributed resources. Here we present an overview of the individual Triticeae genome resources hosted by transPLANT partners, introduce the objectives of transPLANT, and outline common developments and interfaces supporting integrated data access.
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http://dx.doi.org/10.3835/plantgenome2015.06.0038DOI Listing
March 2016

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

Plant Physiol 2016 04 11;170(4):2172-86. Epub 2016 Feb 11.

John Innes Centre, Norwich NR4 7UH, United Kingdom (P.B., C.U.); andGenome Analysis Centre, Norwich NR4 7UH, United Kingdom (R.R.-G.)

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.
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http://dx.doi.org/10.1104/pp.15.01667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825114PMC
April 2016

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.

PLoS One 2015 3;10(9):e0137549. Epub 2015 Sep 3.

Plant Biology and Crop Science Department, Rothamsted Research, Harpenden AL5 2JQ, United Kingdom.

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137549PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559439PMC
May 2016

Host Subtraction, Filtering and Assembly Validations for Novel Viral Discovery Using Next Generation Sequencing Data.

PLoS One 2015 22;10(6):e0129059. Epub 2015 Jun 22.

Lab of Viral Zoonotics, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB30ES, United Kingdom.

The use of next generation sequencing (NGS) to identify novel viral sequences from eukaryotic tissue samples is challenging. Issues can include the low proportion and copy number of viral reads and the high number of contigs (post-assembly), making subsequent viral analysis difficult. Comparison of assembly algorithms with pre-assembly host-mapping subtraction using a short-read mapping tool, a k-mer frequency based filter and a low complexity filter, has been validated for viral discovery with Illumina data derived from naturally infected liver tissue and simulated data. Assembled contig numbers were significantly reduced (up to 99.97%) by the application of these pre-assembly filtering methods. This approach provides a validated method for maximizing viral contig size as well as reducing the total number of assembled contigs that require down-stream analysis as putative viral nucleic acids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129059PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476701PMC
March 2016

bio-samtools 2: a package for analysis and visualization of sequence and alignment data with SAMtools in Ruby.

Bioinformatics 2015 Aug 29;31(15):2565-7. Epub 2015 Mar 29.

The Sainsbury Laboratory and.

Motivation: bio-samtools is a Ruby language interface to SAMtools, the highly popular library that provides utilities for manipulating high-throughput sequence alignments in the Sequence Alignment/Map format. Advances in Ruby, now allow us to improve the analysis capabilities and increase bio-samtools utility, allowing users to accomplish a large amount of analysis using a very small amount of code. bio-samtools can also be easily developed to include additional SAMtools methods and hence stay current with the latest SAMtools releases.

Results: We have added new Ruby classes for the MPileup and Variant Call Format (VCF) data formats emitted by SAMtools and introduced more analysis methods for variant analysis, including alternative allele calculation and allele frequency calling for SNPs. Our new implementation of bio-samtools also ensures that all the functionality of the SAMtools library is now supported and that bio-samtools can be easily extended to include future changes in SAMtools. bio-samtools 2 also provides methods that allow the user to directly produce visualization of alignment data.
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http://dx.doi.org/10.1093/bioinformatics/btv178DOI Listing
August 2015

Field pathogenomics reveals the emergence of a diverse wheat yellow rust population.

Genome Biol 2015 Feb 25;16:23. Epub 2015 Feb 25.

Background: Emerging and re-emerging pathogens imperil public health and global food security. Responding to these threats requires improved surveillance and diagnostic systems. Despite their potential, genomic tools have not been readily applied to emerging or re-emerging plant pathogens such as the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. tritici (PST). This is due largely to the obligate parasitic nature of PST, as culturing PST isolates for DNA extraction remains slow and tedious.

Results: To counteract the limitations associated with culturing PST, we developed and applied a field pathogenomics approach by transcriptome sequencing infected wheat leaves collected from the field in 2013. This enabled us to rapidly gain insights into this emerging pathogen population. We found that the PST population across the United Kingdom (UK) underwent a major shift in recent years. Population genetic structure analyses revealed four distinct lineages that correlated to the phenotypic groups determined through traditional pathology-based virulence assays. Furthermore, the genetic diversity between members of a single population cluster for all 2013 PST field samples was much higher than that displayed by historical UK isolates, revealing a more diverse population of PST.

Conclusions: Our field pathogenomics approach uncovered a dramatic shift in the PST population in the UK, likely due to a recent introduction of a diverse set of exotic PST lineages. The methodology described herein accelerates genetic analysis of pathogen populations and circumvents the difficulties associated with obligate plant pathogens. In principle, this strategy can be widely applied to a variety of plant pathogens.
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http://dx.doi.org/10.1186/s13059-015-0590-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342793PMC
February 2015

PolyMarker: A fast polyploid primer design pipeline.

Bioinformatics 2015 Jun 2;31(12):2038-9. Epub 2015 Feb 2.

The Genome Analysis Centre (TGAC), Norwich Research Park, Norwich NR4 7UH, UK, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK and National Institute of Agricultural Botany (NIAB), Cambridge CB3 0LE, UK.

Unlabelled: The design of genetic markers is of particular relevance in crop breeding programs. Despite many economically important crops being polyploid organisms, the current primer design tools are tailored for diploid species. Bread wheat, for instance, is a hexaploid comprising of three related genomes and the performance of genetic markers is diminished if the primers are not genome specific. PolyMarker is a pipeline that generates SNP markers by selecting candidate primers for a specified genome using local alignments and standard primer design tools to test the viability of the primers. A command line tool and a web interface are available to the community.

Availability And Implementation: PolyMarker is available as a ruby BioGem: bio-polyploid-tools. Web interface: http://polymarker.tgac.ac.uk.
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http://dx.doi.org/10.1093/bioinformatics/btv069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765872PMC
June 2015

RNA-Seq bulked segregant analysis enables the identification of high-resolution genetic markers for breeding in hexaploid wheat.

Plant Biotechnol J 2015 Jun 8;13(5):613-24. Epub 2014 Nov 8.

John Innes Centre, Norwich, UK.

The identification of genetic markers linked to genes of agronomic importance is a major aim of crop research and breeding programmes. Here, we identify markers for Yr15, a major disease resistance gene for wheat yellow rust, using a segregating F2 population. After phenotyping, we implemented RNA sequencing (RNA-Seq) of bulked pools to identify single-nucleotide polymorphisms (SNP) associated with Yr15. Over 27 000 genes with SNPs were identified between the parents, and then classified based on the results from the sequenced bulks. We calculated the bulk frequency ratio (BFR) of SNPs between resistant and susceptible bulks, selecting those showing sixfold enrichment/depletion in the corresponding bulks (BFR > 6). Using additional filtering criteria, we reduced the number of genes with a putative SNP to 175. The 35 SNPs with the highest BFR values were converted into genome-specific KASP assays using an automated bioinformatics pipeline (PolyMarker) which circumvents the limitations associated with the polyploid wheat genome. Twenty-eight assays were polymorphic of which 22 (63%) mapped in the same linkage group as Yr15. Using these markers, we mapped Yr15 to a 0.77-cM interval. The three most closely linked SNPs were tested across varieties and breeding lines representing UK elite germplasm. Two flanking markers were diagnostic in over 99% of lines tested, thus providing a reliable haplotype for marker-assisted selection in these breeding programmes. Our results demonstrate that the proposed methodology can be applied in polyploid F2 populations to generate high-resolution genetic maps across target intervals.
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http://dx.doi.org/10.1111/pbi.12281DOI Listing
June 2015

Sequencing quality assessment tools to enable data-driven informatics for high throughput genomics.

Front Genet 2013 Dec 17;4:288. Epub 2013 Dec 17.

The Genome Analysis Centre Norwich, UK.

The processes of quality assessment and control are an active area of research at The Genome Analysis Centre (TGAC). Unlike other sequencing centers that often concentrate on a certain species or technology, TGAC applies expertise in genomics and bioinformatics to a wide range of projects, often requiring bespoke wet lab and in silico workflows. TGAC is fortunate to have access to a diverse range of sequencing and analysis platforms, and we are at the forefront of investigations into library quality and sequence data assessment. We have developed and implemented a number of algorithms, tools, pipelines and packages to ascertain, store, and expose quality metrics across a number of next-generation sequencing platforms, allowing rapid and in-depth cross-platform Quality Control (QC) bioinformatics. In this review, we describe these tools as a vehicle for data-driven informatics, offering the potential to provide richer context for downstream analysis and to inform experimental design.
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http://dx.doi.org/10.3389/fgene.2013.00288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865868PMC
December 2013

Identifying and classifying trait linked polymorphisms in non-reference species by walking coloured de bruijn graphs.

PLoS One 2013 25;8(3):e60058. Epub 2013 Mar 25.

The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich, United Kingdom.

Single Nucleotide Polymorphisms are invaluable markers for tracing the genetic basis of inheritable traits and the ability to create marker libraries quickly is vital for timely identification of target genes. Next-generation sequencing makes it possible to sample a genome rapidly, but polymorphism detection relies on having a reference genome to which reads can be aligned and variants detected. We present Bubbleparse, a method for detecting variants directly from next-generation reads without a reference sequence. Bubbleparse uses the de Bruijn graph implementation in the Cortex framework as a basis and allows the user to identify bubbles in these graphs that represent polymorphisms, quickly, easily and sensitively. We show that the Bubbleparse algorithm is sensitive and can detect many polymorphisms quickly and that it performs well when compared with polymorphism detection methods based on alignment to a reference in Arabidopsis thaliana. We show that the heuristic can be used to maximise the number of true polymorphisms returned, and with a proof-of-principle experiment show that Bubbleparse is very effective on data from unsequenced wild relatives of potato and enabled us to identify disease resistance linked genes quickly and easily.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060058PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607606PMC
December 2013