Proanthocyanidin biosynthesis in the developing wheat seed coat investigated by chemical and RNA-Seq analysis.

The composition of proanthocyanidins in the testa (seed coat) of bread wheat was analyzed by thiolysis of PA oligomers from developing grain and found to consist of (+)-catechin monomers, with a small amount of (+)-gallocatechin. The average chain length of soluble PA stayed relatively constant between 10 and 20 days post-anthesis, whereas that of unextractable PA increased over the same period, suggesting that increases in chain length might account for the insolubility of PAs from mature wheat grain. We carried out RNA-Seq followed by differential expression analysis from dissected tissues of developing grain from red- and white-grained near-isogenic lines differing in the presence of an active R gene that encodes a MYB transcription factor involved in control of PA biosynthesis. In addition to genes already identified encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and dihydroxyflavonoid 4-reductase, we showed that wheat genes encoding phenylalanine ammonia lyase, flavonoid 3',5'-hydroxylase, leucoanthocyanidin reductase, and a glutathione S-transferase (the orthologue of maize Bronze-2) were more highly expressed in the red NIL. We also identified candidate orthologues of other catalytic and regulatory components of flavonoid biosynthesis in wheat.

CerealsDB: A Whistle-Stop Tour of an Open Access SNP Resource.

The CerealsDB website, created by members of the Functional Genomics Group at the University of Bristol, provides access to a database containing SNP and genotyping data for hexaploid wheat and, to a lesser extent, its progenitors and several of its relatives. The site is principally aimed at plant breeders and research scientists who wish to obtain information regarding SNP markers; for example, obtain primers used for their identification or the sequences upon which they are based. The database underpinning the website contains circa one million putative varietal SNPs of which several hundreds of thousands have been experimentally validated on a range of common genotyping platforms. For each SNP marker, the site also hosts the allelic scores for thousands of elite wheat varieties, landrace cultivars, and wheat relatives. Tools are available to help negotiate and visualize the datasets. The website has been designed to be simple and straightforward to use and is completely open access.

Inflammatory pathways are central to posterior cerebrovascular artery remodelling prior to the onset of congenital hypertension.

Cerebral artery hypoperfusion may provide the basis for linking ischemic stroke with hypertension. Brain hypoperfusion may induce hypertension that may serve as an auto-protective mechanism to prevent ischemic stroke. We hypothesised that hypertension is caused by remodelling of the cerebral arteries, which is triggered by inflammation. We used a congenital rat model of hypertension and examined age-related changes in gene expression of the cerebral arteries using RNA sequencing. Prior to hypertension, we found changes in signalling pathways associated with the immune system and fibrosis. Validation studies using second harmonics generation microscopy revealed upregulation of collagen type I and IV in both tunica externa and media. These changes in the extracellular matrix of cerebral arteries pre-empted hypertension accounting for their increased stiffness and resistance, both potentially conducive to stroke. These data indicate that inflammatory driven cerebral artery remodelling occurs prior to the onset of hypertension and may be a trigger elevating systemic blood pressure in genetically programmed hypertension.

Conversion of array-based single nucleotide polymorphic markers for use in targeted genotyping by sequencing in hexaploid wheat (Triticum aestivum).

Wheat breeders and academics alike use single nucleotide polymorphisms (SNPs) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP-based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5-10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP-based capture probes from several thousand existing and publicly available probes from Axiom® and KASP™ genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP™ genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/sequence_capture.php).

High-density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat.

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high-density and high-throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.

High-Density SNP Genotyping Array for Hexaploid Wheat and Its Relatives.

A lack of genetic diversity between wheat breeding lines has been recognized as a significant block to future yield increases. Wheat breeding and prebreeding strategies are increasingly using material from wheat ancestors or wild relatives to reintroduce diversity. Where molecular markers are polymorphic between the host and introgressed material, they may be used to track the size and location of the introgressed material through generations of backcrossing. To generate markers for this purpose, sequence capture targeted resequencing was carried out for a range of wheat varieties, wheat relatives, and wheat progenitors. From these sequences, putative SNPs were identified and used to generate the Axiom® Wheat HD array. A selection of varieties representing a selection of elite wheat breeding material, progenitor species, and wild relatives were used to validate the array. The procedures used are described here in detail.

16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer's Post-Mortem Brain.

The neurological deterioration associated with Alzheimer's disease (AD), involving accumulation of amyloid-beta peptides and neurofibrillary tangles, is associated with evident neuroinflammation. This is now seen to be a significant contributor to pathology. Recently the tenet of the privileged status of the brain, regarding microbial compromise, has been questioned, particularly in terms of neurodegenerative diseases. It is now being considered that microbiological incursion into the central nervous system could be either an initiator or significant contributor to these. This is a novel study using 16S ribosomal gene-specific Next generation sequencing (NGS) of extracted brain tissue. A comparison was made of the bacterial species content of both frozen and formaldehyde fixed sections of a small cohort of Alzheimer-affected cases with those of cognitively unimpaired (normal). Our findings suggest an increase in bacterial populations in Alzheimer brain tissue compared with normal.

Characterization of a Wheat Breeders' Array suitable for high-throughput SNP genotyping of global accessions of hexaploid bread wheat (Triticum aestivum).

Targeted selection and inbreeding have resulted in a lack of genetic diversity in elite hexaploid bread wheat accessions. Reduced diversity can be a limiting factor in the breeding of high yielding varieties and crucially can mean reduced resilience in the face of changing climate and resource pressures. Recent technological advances have enabled the development of molecular markers for use in the assessment and utilization of genetic diversity in hexaploid wheat. Starting with a large collection of 819 571 previously characterized wheat markers, here we describe the identification of 35 143 single nucleotide polymorphism-based markers, which are highly suited to the genotyping of elite hexaploid wheat accessions. To assess their suitability, the markers have been validated using a commercial high-density Affymetrix Axiom® genotyping array (the Wheat Breeders' Array), in a high-throughput 384 microplate configuration, to characterize a diverse global collection of wheat accessions including landraces and elite lines derived from commercial breeding communities. We demonstrate that the Wheat Breeders' Array is also suitable for generating high-density genetic maps of previously uncharacterized populations and for characterizing novel genetic diversity produced by mutagenesis. To facilitate the use of the array by the wheat community, the markers, the associated sequence and the genotype information have been made available through the interactive web site 'CerealsDB'.

RNA sequencing analysis of human podocytes reveals glucocorticoid regulated gene networks targeting non-immune pathways.

Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or 'podocytes', the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes.

CerealsDB 3.0: expansion of resources and data integration.

BACKGROUND: The increase in human populations around the world has put pressure on resources, and as a consequence food security has become an important challenge for the 21st century. Wheat (Triticum aestivum) is one of the most important crops in human and livestock diets, and the development of wheat varieties that produce higher yields, combined with increased resistance to pests and resilience to changes in climate, has meant that wheat breeding has become an important focus of scientific research. In an attempt to facilitate these improvements in wheat, plant breeders have employed molecular tools to help them identify genes for important agronomic traits that can be bred into new varieties. Modern molecular techniques have ensured that the rapid and inexpensive characterisation of SNP markers and their validation with modern genotyping methods has produced a valuable resource that can be used in marker assisted selection. CerealsDB was created as a means of quickly disseminating this information to breeders and researchers around the globe. DESCRIPTION: CerealsDB version 3.0 is an online resource that contains a wide range of genomic datasets for wheat that will assist plant breeders and scientists to select the most appropriate markers for use in marker assisted selection. CerealsDB includes a database which currently contains in excess of a million putative varietal SNPs, of which several hundreds of thousands have been experimentally validated. In addition, CerealsDB also contains new data on functional SNPs predicted to have a major effect on protein function and we have constructed a web service to encourage data integration and high-throughput programmatic access. CONCLUSION: CerealsDB is an open access website that hosts information on SNPs that are considered useful for both plant breeders and research scientists. The recent inclusion of web services designed to federate genomic data resources allows the information on CerealsDB to be more fully integrated with the WheatIS network and other biological databases.

High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site.

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

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.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

BACKGROUND: The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. RESULTS: The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1ß-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. CONCLUSIONS: The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.

Discovery and development of exome-based, co-dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.).

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)-based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP-based wheat markers have been made available via the use of next-generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial-scale breeding programmes. To identify exome-based co-dominant SNP-based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re-sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co-dominant. Validation of a subset of these putative co-dominant markers confirmed that 96% were true polymorphisms and 65% were co-dominant SNP assays. The new co-dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web-based database to facilitate their use on genotyping programmes worldwide.

CerealsDB 2.0: an integrated resource for plant breeders and scientists.

BACKGROUND: Food security is an issue that has come under renewed scrutiny amidst concerns that substantial yield increases in cereal crops are required to feed the world's booming population. Wheat is of fundamental importance in this regard being one of the three most important crops for both human consumption and livestock feed; however, increase in crop yields have not kept pace with the demands of a growing world population. In order to address this issue, plant breeders require new molecular tools to help them identify genes for important agronomic traits that can be introduced into elite varieties. Studies of the genome using next-generation sequencing enable the identification of molecular markers such as single nucleotide polymorphisms that may be used by breeders to identify and follow genes when breeding new varieties. The development and application of next-generation sequencing technologies has made the characterisation of SNP markers in wheat relatively cheap and straightforward. There is a growing need for the widespread dissemination of this information to plant breeders. DESCRIPTION: CerealsDB is an online resource containing a range of genomic datasets for wheat (Triticum aestivum) that will assist plant breeders and scientists to select the most appropriate markers for marker assisted selection. CerealsDB includes a database which currently contains in excess of 100,000 putative varietal SNPs, of which several thousand have been experimentally validated. In addition, CerealsDB contains databases for DArT markers and EST sequences, and links to a draft genome sequence for the wheat variety Chinese Spring. CONCLUSION: CerealsDB is an open access website that is rapidly becoming an invaluable resource within the wheat research and plant breeding communities.

Targeted re-sequencing of the allohexaploid wheat exome.

Bread wheat, Triticum aestivum, is an allohexaploid composed of the three distinct ancestral genomes, A, B and D. The polyploid nature of the wheat genome together with its large size has limited our ability to generate the significant amount of sequence data required for whole genome studies. Even with the advent of next-generation sequencing technology, it is still relatively expensive to generate whole genome sequences for more than a few wheat genomes at any one time. To overcome this problem, we have developed a targeted-capture re-sequencing protocol based upon NimbleGen array technology to capture and characterize 56.5 Mb of genomic DNA with sequence similarity to over 100 000 transcripts from eight different UK allohexaploid wheat varieties. Using this procedure in conjunction with a carefully designed bioinformatic procedure, we have identified more than 500 000 putative single-nucleotide polymorphisms (SNPs). While 80% of these were variants between the homoeologous genomes, A, B and D, a significant number (20%) were putative varietal SNPs between the eight varieties studied. A small number of these latter polymorphisms were experimentally validated using KASPar technology and 94% proved to be genuine. The procedures described here to sequence a large proportion of the wheat genome, and the various SNPs identified should be of considerable use to the wider wheat community.

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.).

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker-assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single-nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single-nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next-generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single-nucleotide polymorphisms in hexaploid bread wheat using competitive allele-specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross-section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single-nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza.

Plant responses to cold: Transcriptome analysis of wheat.

Temperature and light are important environmental stimuli that have a profound influence on the growth and development of plants. Wheat varieties can be divided on the basis of whether they require an extended period of cold to flower (vernalization). Varieties that have a requirement for vernalization also tend to be winter hardy and are able to withstand quite extreme subzero temperatures. This capacity, however, is not constitutive and plants require a period of exposure to low, non-freezing temperatures to acquire freezing tolerance: this process is referred to as cold acclimation. Cold acclimation and the acquisition of freezing tolerance require the orchestration of many different, seemingly disparate physiological and biochemical changes. These changes are, at least in part, mediated through the differential expression of many genes. Some of these genes code for effector molecules that participate directly to alleviate stress. Others code for proteins involved in signal transduction or transcription factors that control the expression of further banks of genes. In this review, we provide an overview of some of the main features of cold acclimation with particular focus on transcriptome reprogramming. In doing so, we highlight some of the important differences between cold-hardy and cold-sensitive varieties. An understanding of these processes is of great potential importance because cold and freezing stress are major limiting factors for growing crop plants and periodically account for significant losses in plant productivity.

Cold- and light-induced changes in the transcriptome of wheat leading to phase transition from vegetative to reproductive growth.

BACKGROUND: For plants to flower at the appropriate time, they must be able to perceive and respond to various internal and external cues. Wheat is generally a long-day plant that will go through phase transition from vegetative to floral growth as days are lengthening in spring and early summer. In addition to this response to day-length, wheat cultivars may be classified as either winter or spring varieties depending on whether they require to be exposed to an extended period of cold in order to become competent to flower. Using a growth regime to mimic the conditions that occur during a typical winter in Britain, and a microarray approach to determine changes in gene expression over time, we have surveyed the genes of the major pathways involved in floral transition. We have paid particular attention to wheat orthologues and functional equivalents of genes involved in the phase transition in Arabidopsis. We also surveyed all the MADS-box genes that could be identified as such on the Affymetrix genechip wheat genome array. RESULTS: We observed novel responses of several genes thought to be of major importance in vernalisation-induced phase transition, and identified several MADS-box genes that might play an important role in the onset of flowering. In addition, we saw responses in genes of the Gibberellin pathway that would indicate that this pathway also has some role to play in phase transition. CONCLUSION: Phase transition in wheat is more complex than previously reported, and there is evidence that day-length has an influence on genes that were once thought to respond exclusively to an extended period of cold.

Multiplex single nucleotide polymorphism (SNP)-based genotyping in allohexaploid wheat using padlock probes.

Single nucleotide polymorphisms are the most common polymorphism in plant and animal genomes and, as such, are the logical choice for marker-assisted selection. However, many plants are also polyploid, and marker-assisted selection can be complicated by the presence of highly similar, but non-allelic, homoeologous sequences. Despite this, there is practical and academic demand for high-throughput genotyping in several polyploid crop species, such as allohexaploid wheat. In this paper, we present such a system, which utilizes public single nucleotide polymorphisms previously identified in both agronomically important genes and in randomly selected, mapped, expressed sequence tags developed by the wheat community. To achieve relatively high levels of multiplexing, we used non-amplified genomic DNA and padlock probe pairs, together with high annealing temperatures, to differentiate between similar sequences in the wheat genome. Our results suggest that padlock probes are capable of discriminating between homoeologous sequences and hence can be used to efficiently genotype wheat varieties.