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Accurate characterization of splice junctions as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data and enable detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset (RTD) from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the splice junctions and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared to an earlier version, BaRTv1.0. The accuracy of transcript level quantification, splice junctions and transcript start and end sites has been validated extensively using parallel technologies and analysis, including high resolution RT PCR and 5’ RACE. BaRTv2.18 contains 39,434 genes and 148,260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

Background The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. Results A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts – BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427–433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20–28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5′ and 3′ UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. Conclusion A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

A high-quality, barley gene reference transcript dataset (BaRTv1.0), was used to quantify gene and transcript abundances from 22 RNA-seq experiments, covering 843 separate samples. Using the abundance data we developed a Barley Expression Database (EORNA*) to underpin a visualisation tool that displays comparative gene and transcript abundance data on demand as transcripts per million (TPM) across all samples and all the genes. EORNA provides gene and transcript models for all of the transcripts contained in BaRTV1.0, and these can be conveniently identified through either BaRT or HORVU gene names, or by direct BLAST of query sequences. Browsing the quantification data reveals cultivar, tissue and condition specific gene expression and shows changes in the proportions of individual transcripts that have arisen via alternative splicing. TPM values can be easily extracted to allow users to determine the statistical significance of observed transcript abundance variation among samples or perform meta analyses on multiple RNA-seq experiments. * Eòrna is the Scottish Gaelic word for Barley., Measurement(s) gene expression Technology Type(s) transcription profiling assay Factor Type(s) Genotype • Abiotic Stress • Developmental stage • Tissue • Biotic stress Sample Characteristic - Organism Hordeum vulgare Sample Characteristic - Environment sodium chloride salt • drought • increased temperature • decreased temperature Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13643387

Like many cereal crops, barley is also negatively affected by drought stress. However, due to its simple genome as well as enhanced stress resilient nature compared to rice and wheat, barley has been considered as a model to decipher drought tolerance in cereals. In the present study, transcriptomic and hormonal profiles along with several biochemical features were compared between drought-tolerant (Otis) and drought-sensitive (Baronesse) barley genotypes subjected to drought to identify molecular and biochemical differences between the genotypes. The drought-induced decrease in the leaf relative water content, net photosynthesis, and biomass accumulation was relatively low in Otis compared to Baronesse. The hormonal profiles did not reveal significant differences for majority of the compounds other than the GA20 and the cis-zeatin-o-glucoside (c-ZOG), whose levels were greatly increased in Otis compared to Baronesse under drought. The major differences that emerged from the transcriptome analysis are; (1), the overall number of differentially expressed genes was relatively low in drought-tolerant Otis compared to drought-sensitive Baronesse; (2), a wax biosynthesis gene (CER1), and NAC transcription factors were specifically induced in Otis but not in Baronesse; (3), the degree of upregulation of betaine aldehyde dehydrogenase and a homeobox transcription factor (genes with proven roles in imparting drought tolerance), was greater in Otis compared to Baronesse; (4) the extent of downregulation of gene expression profiles for proteins of the reaction center photosystem II (PSII) (D1 and D2) was low in Otis compared to Baronesse; and, (5), alternative splicing (AS) was also found to differ between the genotypes under drought. Taken together, the overall transcriptional responses were low in drought-tolerant Otis but the genes that could confer drought tolerance were either specifically induced or greatly upregulated in the tolerant genotype and these differences could be important for drought tolerance in barley.

A high-quality, barley gene reference transcript dataset (BaRTv1.0), was used to quantify gene and transcript abundances from 22 RNA-seq experiments, covering 843 separate samples. Using the abundance data we developed a Barley Expression Database (EoRNA* – Expression of RNA) to underpin a visualisation tool that displays comparative gene and transcript abundance data on demand as transcripts per million (TPM) across all samples and all the genes. EoRNA provides gene and transcript models for all of the transcripts contained in BaRTV1.0, and these can be conveniently identified through either BaRT or HORVU gene names, or by direct BLAST of query sequences. Browsing the quantification data reveals cultivar, tissue and condition specific gene expression and shows changes in the proportions of individual transcripts that have arisen via alternative splicing. TPM values can be easily extracted to allow users to determine the statistical significance of observed transcript abundance variation among samples or perform meta analyses on multiple RNA-seq experiments. * Eòrna is the Scottish Gaelic word for Barley

Nonsense mediated RNA decay (NMD) is an evolutionary conserved RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed in depth comparative analysis of Arabidopsis mutants lacking key proteins involved in different steps of NMD. We observed that UPF3, UPF1, and SMG7 have different impacts on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly using stringent pipeline in UPF1-null plants revealed genome wide changes in alternative splicing, including switches in mRNA variants, suggesting a role for UPF1 in splicing. We further found that UPF1 inactivation leads to translational repression, manifested by a global shift in mRNAs from polysomes to monosomes and a downregulation of genes involved in translation and ribosome biogenesis. Despite this global change, NMD targets and low-expressed mRNAs with short half-lives were enriched in polysomes, indicating that UPF1 specifically suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide-binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational de-repression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

Background The changing climate is altering timing of key fruit ripening processes and increasing the occurrence of fruit defects. To improve our understanding of the genetic control of raspberry fruit development an enhanced genetic linkage map was developed and used to examine ripening phenotypic data. Results In this study we developed an enhanced genetic linkage map for the raspberry cvs. Glen Moy x Latham reference mapping population using genotyping by sequencing (GbS). Alignment to a newly sequenced draft reference genome of red raspberry, cultivar (cv.) Glen Moy, identified 8019 single nucleotide polymorphisms (SNPs). After stringent filtering to take account of read coverage over all the progeny individuals, association with a single chromosome, heterozygosity and marker regression mapping, 2348 high confidence SNPs were retained and integrated with an existing raspberry genetic map. The linkage map contained many more SNPs segregating in Latham than in Glen Moy. This caused difficulties in quantitative trait loci (QTL) mapping with standard software and a novel analysis based on a hidden Markov model was used to improve the mapping. QTL mapping using the newly generated dense genetic map not only corroborated previously identified genetic locations but also provided additional genetic elements controlling fruit ripening in raspberry. Conclusion The high-density GbS map located the QTL peaks more precisely than in earlier studies, aligned the QTLs with Glen Moy genome scaffolds, narrowed the range of potential candidate genes to these regions that can be utilised in other populations or in gene expression studies to confirm their role and increased the repertoire of markers available to understand the genetic control of fruit ripening traits. Electronic supplementary material The online version of this article (10.1186/s12863-018-0666-z) contains supplementary material, which is available to authorized users.

How alternative splicing (AS) is regulated in plants has not yet been elucidated. Previously, we have shown that the nuclear cap-binding protein complex (AtCBC) is involved in AS in Arabidopsis thaliana. Here we show that both subunits of AtCBC (AtCBP20 and AtCBP80) interact with SERRATE (AtSE), a protein involved in the microRNA biogenesis pathway. Moreover, using a high-resolution reverse transcriptase-polymerase chain reaction AS system we have found that AtSE influences AS in a similar way to the cap-binding complex (CBC), preferentially affecting selection of 5' splice site of first introns. The AtSE protein acts in cooperation with AtCBC: many changes observed in the mutant lacking the correct SERRATE activity were common to those observed in the cbp mutants. Interestingly, significant changes in AS of some genes were also observed in other mutants of plant microRNA biogenesis pathway, hyl1-2 and dcl1-7, but a majority of them did not correspond to the changes observed in the se-1 mutant. Thus, the role of SERRATE in AS regulation is distinct from that of HYL1 and DCL1, and is similar to the regulation of AS in which CBC is involved.

Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species.

Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species.

SummaryFor plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Chloroplasts trigger retrograde signals that control alternative splicing in leaves and roots in response to light. Here we provide evidence suggesting that sugars, derived from photosynthesis, act as mobile signals controlling alternative splicing in roots. The inhibition of TOR kinase activity diminishes the alternative splicing response to light and/or sugars in roots, showing the relevance of the TOR pathway in this signaling mechanism. Furthermore, disrupting the function of the mitochondria abolishes alternative splicing changes supporting a key role for these organelles in this signaling axis. We conclude that sugars can act as mobile signals coordinating alternative splicing responses to light throughout the whole plant, exerting this function in roots by activating the TOR pathway.Graphical AbstractArt by Dr. Luciana Giono.

Climate change is impacting soft fruit crops. In raspberry, uneven bud break, greater variability in time to fruit ripening and crumbly fruit are already in evidence. Understanding the developmental process and how the environment impacts will be crucial in sustaining the industry in this changing climate against a background of biotic stresses. This chapter reviews regulation of processes leading to flowering time and fertilisation, developing fruit, ripening, colour, flavour and size. Recent developments of genomic and transcriptome tools which will have a significant role in breeding of the next generation of raspberry fruit are considered.

In this chapter, we refer to the expressed portion of the barley genome as the relatively small fraction of the total cellular DNA that either contains the genes that ultimately produce proteins, or that directly/indirectly controls the level, location and/or timing of when these genes are expressed and proteins are produced. We start by describing the dynamics of tissue and time-dependent gene expression and how common patterns across multiple samples can provide clues about gene networks involved in common biological processes. We then describe some of the complexities of how a single mRNA template can be differentially processed by alternative splicing to generate multiple different proteins or provide a mechanism to regulate the amount of functional gene product in a cell at a given point in time. We extend our analysis, using a number of biological examples, to address how diverse families of small non-coding microRNAs specifically regulate gene expression, and complete our appraisal by looking at the physical/molecular environment around genes that can result in either the promotion or repression of gene expression. We conclude by assessing some of the issues that remain around our ability to fully exploit the depth and power of current approaches for analysing gene expression and propose improvements that could be made using new but available sequencing and bioinformatics technologies.

Recent developments in genetics and genomics have advanced research in all crops including soft fruit species. Molecular markers which detect genome-wide variability in both protein coding and non-coding regions have enabled genetic mapping studies to move beyond linkages between simple morphological traits (Jennings 1967a, 1988; Ourecky 1975; Crane and Lawrence 1931; Keep 1968) to linkage maps containing numerous genetic markers which can be utilised in marker assisted breeding. Until recently, mapping in blackberry and other Rubus species has lagged behind that of red raspberry due to their more complex genetic make-up and lesser economic importance.

Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement.

QTL mapping identifies a range of underlying and unrelated genes with apparent roles in raspberry fruit ripening and softening that show characteristic developing fruit expression profiles. Fruit softening is an important agronomical trait that involves a complex interaction of plant cell processes. We have used both qualitative and quantitative scoring of fruit firmness, length, mass, and resistance to applied force to identify QTL in a raspberry mapping population. QTLs were located primarily on linkage group (LG) 3 with other significant loci on LG 1 and LG 5 and showed mostly additive effects between the two parents. The expression of key genes that underlie these QTLs with roles in cell-wall solubility, water uptake, polyamine synthesis, transcription, and cell respiration was tested across five stages of fruit development, from immature green to red ripe fruit, using real-time RT-qPCR. Gene expression patterns showed variable expression patterns across fruit development with a highly significant positive and negative correlation between genes, supporting precise regulation of expression of different cell processes throughout raspberry fruit development. Variable timing in expression was also found in some genes at different fruit development stages between soft and firm cultivars. Multiple processes have a role to play in fruit softening and this will require development of multiple marker combinations to genes that characterise raspberry fruit softening.

Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative splicing events. The use of the panel allows the quantification of changes in the proportion of splice isoforms between different samples, e.g., different time points, different tissues, genotypes, ecotypes, or treatments.

Transcript annotation in plant databases is incomplete and often inaccurate, leading to misinterpretation. As more and more RNA-seq data are generated, plant scientists need to be aware of potential pitfalls and understand the nature and impact of specific alternative splicing transcripts on protein production. A primary area of concern and the topic of this article is the (mis)annotation of open reading frames and premature termination codons. The basic message is that to adequately address expression and functions of transcript isoforms, it is necessary to be able to predict their fate in terms of whether protein isoforms are generated or specific transcripts are unproductive or degraded.

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression. This research was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K006568/1 to J.W.S.B.; BB/K006835/1 to H.G.N.), the Scottish Government Rural and Environment Science and Analytical Services division (RESAS) and by the Austrian Science Fund (FWF) (P26333) to M.K. and (DK W1207) to A.B. The authors acknowledge the European Alternative Splicing Network of Excellence (EURASNET), LSHG-CT-2005-518238 for catalysing important collaborations.