Evolution of the Grain Dispersal System in Barley.

About 12,000 years ago in the Near East, humans began the transition from hunter-gathering to agriculture-based societies. Barley was a founder crop in this process, and the most important steps in its domestication were mutations in two adjacent, dominant, and complementary genes, through which grains were retained on the inflorescence at maturity, enabling effective harvesting. Independent recessive mutations in each of these genes caused cell wall thickening in a highly specific grain "disarticulation zone," converting the brittle floral axis (the rachis) of the wild-type into a tough, non-brittle form that promoted grain retention. By tracing the evolutionary history of allelic variation in both genes, we conclude that spatially and temporally independent selections of germplasm with a non-brittle rachis were made during the domestication of barley by farmers in the southern and northern regions of the Levant, actions that made a major contribution to the emergence of early agrarian societies.

The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

A GDSL-motif Esterase/Lipase Affects Wax and Cutin Deposition and Controls Hull-Caryopsis Attachment in Barley.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in ß-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

A guide to barley mutants.

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments. RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections. CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.

BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables 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 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 SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction 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.

The evolutionary patterns of barley pericentromeric chromosome regions, as shaped by linkage disequilibrium and domestication.

The distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene-rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco-geographical ranges. We defined and compared variant data across the pericentromeric and non-pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single-nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome-wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two-row and six-row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non-synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be 'highly deleterious'. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.

Major chromosome 5H haplotype switch structures the European two-rowed spring barley germplasm of the past 190 years.

KEY MESSAGE: Selection over 70 years has led to almost complete fixation of a haplotype spanning ~ 250 Mbp of chomosome 5H in European two-rowed spring barleys, possibly originating from North Africa. Plant breeding and selection have shaped the genetic composition of modern crops over the past decades and centuries and have led to great improvements in agronomic and quality traits. Knowledge of the genetic composition of breeding germplasm is essential to make informed decisions in breeding programs. In this study, we characterized the structure and composition of 209 barley cultivars representative of the European two-rowed spring barley germplasm of the past 190 years. Utilizing high-density SNP marker data, we identified a distinct centromeric haplotype spanning a ~ 250 Mbp large region on chromosome 5H which likely was first introduced into the European breeding germplasm in the early to mid-twentieth century and has been non-recombining and under strong positive selection over the past 70 years. Almost all cultivars in our panel that were released after 2000 carry this new haplotype, suggesting that this region carries one or several genes conferring highly beneficial traits. Using the global barley collection of the German Federal ex situ gene bank at IPK Gatersleben, we found the new haplotype at high frequencies in six-rowed spring-type landraces from Northern Africa, from which it may have been introduced into modern European barley germplasm via southern European landraces. The presence of a 250 Mbp genomic region characterized by lack of recombination and high levels of fixation in modern barley germplasm has substantial implications for the genetic diversity of the modern barley germplasm and for barley breeding.

APETALA2 control of barley internode elongation.

Many plants dramatically elongate their stems during flowering, yet how this response is coordinated with the reproductive phase is unclear. We demonstrate that microRNA (miRNA) control of APETALA2 (AP2) is required for rapid, complete elongation of stem internodes in barley, especially of the final 'peduncle' internode directly underneath the inflorescence. Disrupted miR172 targeting of AP2 in the Zeo1.b barley mutant caused lower mitotic activity, delayed growth dynamics and premature lignification in the peduncle leading to fewer and shorter cells. Stage- and tissue-specific comparative transcriptomics between Zeo1.b and its parent cultivar showed reduced expression of proliferation-associated genes, ectopic expression of maturation-related genes and persistent, elevated expression of genes associated with jasmonate and stress responses. We further show that applying methyl jasmonate (MeJA) phenocopied the stem elongation of Zeo1.b, and that Zeo1.b itself was hypersensitive to inhibition by MeJA but less responsive to promotion by gibberellin. Taken together, we propose that miR172-mediated restriction of AP2 may modulate the jasmonate pathway to facilitate gibberellin-promoted stem growth during flowering.

The proteome of developing barley anthers during meiotic prophase I.

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.

Grain dispersal mechanism in cereals arose from a genome duplication followed by changes in spatial expression of genes involved in pollen development.

KEY MESSAGE: Grain disarticulation in wild progenitor of wheat and barley evolved through a local duplication event followed by neo-functionalization resulting from changes in location of gene expression. One of the most critical events in the process of cereal domestication was the loss of the natural mode of grain dispersal. Grain dispersal in barley is controlled by two major genes, Btr1 and Btr2, which affect the thickness of cell walls around the disarticulation zone. The barley genome also encodes Btr1-like and Btr2-like genes, which have been shown to be the ancestral copies. While Btr and Btr-like genes are non-redundant, the biological function of Btr-like genes is unknown. We explored the potential biological role of the Btr-like genes by surveying their expression profile across 212 publicly available transcriptome datasets representing diverse organs, developmental stages and stress conditions. We found that Btr1-like and Btr2-like are expressed exclusively in immature anther samples throughout Prophase I of meiosis within the meiocyte. The similar and restricted expression profile of these two genes suggests they are involved in a common biological function. Further analysis revealed 141 genes co-expressed with Btr1-like and 122 genes co-expressed with Btr2-like, with 105 genes in common, supporting Btr-like genes involvement in a shared molecular pathway. We hypothesize that the Btr-like genes play a crucial role in pollen development by facilitating the formation of the callose wall around the meiocyte or in the secretion of callase by the tapetum. Our data suggest that Btr genes retained an ancestral function in cell wall modification and gained a new role in grain dispersal due to changes in their spatial expression becoming spike specific after gene duplication.

Segmental duplications are hot spots of copy number variants affecting barley gene content.

Copy number variants (CNVs) are pervasive in several animal and plant genomes and contribute to shaping genetic diversity. In barley, there is evidence that changes in gene copy number underlie important agronomic traits. The recently released reference sequence of barley represents a valuable genomic resource for unveiling the incidence of CNVs that affect gene content and for identifying sequence features associated with CNV formation. Using exome sequencing and read count data, we detected 16 605 deletions and duplications that affect barley gene content by surveying a diverse panel of 172 cultivars, 171 landraces, 22 wild relatives and other 32 uncategorized domesticated accessions. The quest for segmental duplications (SDs) in the reference sequence revealed many low-copy repeats, most of which overlap predicted coding sequences. Statistical analyses revealed that the incidence of CNVs increases significantly in SD-rich regions, indicating that these sequence elements act as hot spots for the formation of CNVs. The present study delivers a comprehensive genome-wide study of CNVs affecting barley gene content and implicates SDs in the molecular mechanisms that lead to the formation of this class of CNVs.

Targeted mutation of barley (1,3;1,4)-ß-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content.

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-ß-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-ß-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-ß-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-ß-glucan content. (1,3;1,4)-ß-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-ß-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-ß-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-ß-glucan in mature grain.

Genome-wide association study reveals the genetic complexity of fructan accumulation patterns in barley grain.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

The effect of heat stress on sugar beet recombination.

Meiotic recombination plays a crucial role in plant breeding through the creation of new allelic combinations. Therefore, lack of recombination in some genomic regions constitutes a constraint for breeding programmes. In sugar beet, one of the major crops in Europe, recombination occurs mainly in the distal portions of the chromosomes, and so the development of simple approaches to change this pattern is of considerable interest for future breeding and genetics. In the present study, the effect of heat stress on recombination in sugar beet was studied by treating F1 plants at 28 °C/25 °C (day/night) and genotyping the progeny. F1 plants were reciprocally backcrossed allowing the study of male and female meiosis separately. Genotypic data indicated an overall increase in crossover frequency of approximately one extra crossover per meiosis, with an associated increase in pericentromeric recombination under heat treatment. Our data indicate that the changes were mainly induced by alterations in female meiosis only, showing that heterochiasmy in sugar beet is reduced under heat stress. Overall, despite the associated decrease in fertility, these data support the potential use of heat stress to foster recombination in sugar beet breeding programmes.

3D RNA-seq: a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists.

RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the '3D RNA-seq' App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing.

Exome sequences and multi-environment field trials elucidate the genetic basis of adaptation in barley.

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi-environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype-by-environment (G×E) modelling. Sub-populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock-related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large-effect alleles. Our analysis supports a gene-level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.

Association mapping of malting quality traits in UK spring and winter barley cultivar collections.

KEY MESSAGE: Historical malting quality data was collated from UK national and recommended list trial data and used in a GWAS. 25 QTL were identified, with the majority from spring barley cultivar sets. In Europe, the most economically significant use of barley is the production of malt for use in the brewing and distilling industries. As such, selection for traits related to malting quality is of great commercial interest. In order to study the genetic basis of variation for malting quality traits in UK cultivars, a historical set of trial data was collated from national and recommended list trials from the period 1988 to 2016. This data was used to estimate variety means for 20 quality related traits in 451 spring barley cultivars, and 407 winter cultivars. Genotypes for these cultivars were generated using iSelect 9k and 50k genotyping platforms, and a genome wide association scan performed to identify malting quality quantitative trait loci (QTL). 24 QTL were identified in spring barley cultivars, and 2 from the winter set. A number of these correspond to known malting quality related genes but the remainder represents novel genetic variation that is accessible to breeders for the genetic improvement of new cultivars.

The pseudogenes of barley.

Pseudogenes have a reputation of being 'evolutionary relics' or 'junk DNA'. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole-genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11 015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense-response were found to be under-represented in cultivated barley, we detected several defense-related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations.

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq.

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.

RNAi-suppression of barley caffeic acid O-methyltransferase modifies lignin despite redundancy in the gene family.

Caffeic acid O-methyltransferase (COMT), the lignin biosynthesis gene modified in many brown-midrib high-digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl-to-guaiacyl ratio was reduced by ~50%, the 5-hydroxyguaiacyl (5-OH-G) unit incorporated into lignin at 10--15-fold higher levels than normal, and the amount of p-coumaric acid ester-linked to cell walls was reduced by ~50%. No brown-midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown-midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.