Selection of favorable alleles of genes controlling flowering and senescence improves malt barley quality.

Malt barley (Hordeum vulgare L.) is an important cash crop with stringent grain quality standards. Timing of the switch from vegetative to reproductive growth and timing of whole-plant senescence and nutrient remobilization are critical for cereal grain yield and quality. Understanding the genetic variation in genes associated with these developmental traits can streamline genotypic selection of superior malt barley germplasm. Here, we determined the effects of allelic variation in three genes encoding a glycine-rich RNA-binding protein (HvGR-RBP1) and two NAC transcription factors (HvNAM1 and HvNAM2) on malt barley agronomics and quality using previously developed markers for HvGR-RBP1 and HvNAM1 and a novel marker for HvNAM2. Based on a single-nucleotide polymorphism (SNP) in the first intron, the utilized marker differentiates NAM2 alleles of low-grain protein variety 'Karl' and of higher protein variety 'Lewis'. We demonstrate that the selection of favorable alleles for each gene impacts heading date, senescence timing, grain size, grain protein concentration, and malt quality. Specifically, combining 'Karl' alleles for the two NAC genes with the 'Lewis' HvGR-RBP1 allele extends grain fill duration, increases the percentage of plump kernels, decreases grain protein, and provides malt quality stability. Molecular markers for these genes are therefore highly useful tools in malt barley breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01331-7.

A critical review of 25 years of glomalin research: a better mechanical understanding and robust quantification techniques are required.

Arbuscular mycorrhizal fungi (AMF) are important contributors to both plant and soil health. Twenty-five years ago, researchers discovered 'glomalin', a soil component potentially produced by AMF, which was unconventionally extracted from soil and bound by a monoclonal antibody raised against Rhizophagus irregularis spores. 'Glomalin' can resist boiling, strong acids and bases, and protease treatment. Researchers proposed that 'glomalin' is a 60 kDa heat shock protein produced by AMF, while others suggested that it is a mixture of soil organic materials that are not unique to AMF. Despite disagreements on the nature of 'glomalin', it has been consistently associated with a long list of plant and soil health benefits, including soil aggregation, soil carbon storage and enhancing growth under abiotic stress. The benefits attributed to 'glomalin' have caused much excitement in the plant and soil health community; however, the mechanism(s) for these benefits have yet to be established. This review provides insights into the current understanding of the identity of 'glomalin', 'glomalin' quantification, and the associated benefits of 'glomalin'. We invite the community to think more critically about how glomalin-associated benefits are generated. We suggest a series of experiments to test hypotheses regarding the nature of 'glomalin' and associated health benefits.

Combined effects of a glycine-rich RNA-binding protein and a NAC transcription factor extend grain fill duration and improve malt barley agronomic performance.

KEY MESSAGE: Two key barley genes independently control anthesis and senescence timing, enabling the manipulation of grain fill duration, grain size/plumpness, and grain protein concentration. Plant developmental processes such as flowering and senescence have direct effects on cereal yield and quality. Previous work highlighted the importance of two tightly linked genes encoding a glycine-rich RNA-binding protein (HvGR-RBP1) and a NAC transcription factor (HvNAM1), controlling barley anthesis timing, senescence, and percent grain protein. Varieties that differ in HvGR-RBP1 expression, 'Karl'(low) and 'Lewis'(high), also differ in sequence 1 KB upstream of translation start site, including an ~ 400 bp G rich insertion in the 5'-flanking region of the 'Karl' allele, which could disrupt gene expression. To improve malt quality, the (low-grain protein, delayed-senescence) 'Karl' HvNAM1 allele was introgressed into Montana germplasm. After several seasons of selection, the resulting germplasm was screened for the allelic combinations of HvGR-RBP1 and HvNAM1, finding lines combining 'Karl' alleles for both genes (-/-), lines combining 'Lewis' (functional, expressed) HvGR-RBP1 with 'Karl' HvNAM1 alleles ( ±), and lines combining 'Lewis' alleles for both genes (+ / +). Field experiments indicate that the functional ('Lewis,' +) HvGR-RBP1 allele is associated with earlier anthesis and with slightly shorter plants, while the 'Karl' (-) HvNAM1 allele delays maturation. Genotypes carrying the ± allele combination therefore had a significantly (3 days) extended grain fill duration, leading to a higher percentage of plump kernels, slightly enhanced test weight, and lower grain protein concentration when compared to the other allele combinations. Overall, our data suggest an important function for HvGR-RBP1 in the control of barley reproductive development and set the stage for a more detailed functional analysis of this gene.

Correction to: A large-scale multiomics analysis of wheat stem solidness and the wheat stem sawfly feeding response, and syntenic associations in barley, Brachypodium, and rice.

The original version of this article contained a mistake. The word "RefSeq v.1" was incorrectly inserted on page 7. The correct sentence should be: To identify the differentially regulated transcripts, clean RNA-Seq reads were mapped onto the T. aestivum Chinese Spring chromosome 3B pseudomolecule.

Chromosome-based survey sequencing reveals the genome organization of wild wheat progenitor Triticum dicoccoides.

Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome-based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein-coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small-scale evolutionary rearrangements, in addition to providing evidence for the 4AL-5AL-7BS translocation in wild emmer wheat. Chromosome-based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

A large-scale multiomics analysis of wheat stem solidness and the wheat stem sawfly feeding response, and syntenic associations in barley, Brachypodium, and rice.

The wheat stem sawfly (WSS), Cephus cinctus Norton (Hymenoptera: Cephidae), is an important pest of wheat and other cereals, threatening the quality and quantity of grain production. WSS larvae feed and develop inside the stem where they are protected from the external environment; therefore, pest management strategies primarily rely on host plant resistance. A major locus on the long arm of wheat chromosome 3B underlies most of the variation in stem solidness; however, the impact of stem solidness on WSS feeding has not been completely characterized. Here, we used a multiomics approach to examine the response to WSS in both solid- and semi-solid-stemmed wheat varieties. The combined transcriptomic, proteomic, and metabolomic data revealed that two important molecular pathways, phenylpropanoid and phosphate pentose, are involved in plant defense against WSS. We also detected a general downregulation of several key defense transcripts, including those encoding secondary metabolites such as DIMBOA, tricetin, and lignin, which suggested that the WSS larva might interfere with plant defense. We comparatively analyzed the stem solidness genomic region known to be associated with WSS tolerance in wild emmer, durum, and bread wheats, and described syntenic regions in the close relatives barley, Brachypodium, and rice. Additionally, microRNAs identified from the same genomic region revealed potential regulatory pathways associated with the WSS response. We propose a model outlining the molecular responses of the WSS-wheat interactions. These findings provide insight into the link between stem solidness and WSS feeding at the molecular level.

CRISPR/Cas9 genome editing in wheat.

Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency of CRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of stress-responsive transcription factor genes, wheat dehydration responsive element binding protein 2 (TaDREB2) and wheat ethylene responsive factor 3 (TaERF3). Targeted genome editing of TaDREB2 and TaERF3 was achieved with transient expression of small guide RNA and Cas9 protein in wheat protoplast.  The effectiveness of mutagenesis in wheat protoplast was confirmed with restriction enzyme digestion assay, T7 endonuclease assay, and sequencing. Furthermore, several off-target regions for designed sgRNAs were analyzed, and the specificity of genome editing was confirmed with amplicon sequencing. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast and it has a huge potentiality for targeted manipulation of wheat genome for crop improvement purposes.

RNA Sequencing and Co-expressed Long Non-coding RNA in Modern and Wild Wheats.

There is an urgent need for the improvement of drought-tolerant bread and durum wheat. The huge and complex genome of bread wheat (BBAADD genome) stands as a vital obstruction for understanding the molecular mechanism underlying drought tolerance. However, tetraploid wheat (Triticum turgidum ssp., BBAA genome) is an ancestor of modern bread wheat and offers an important model for studying the drought response due to its less complex genome. Additionally, several wild relatives of tetraploid wheat have already shown a significant drought tolerance. We sequenced root transcriptome of three tetraploid wheat varieties with varying stress tolerance profiles, and built differential expression library of their transcripts under control and drought conditions. More than 5,000 differentially expressed transcripts were identified from each genotype. Functional characterization of transcripts specific to drought-tolerant genotype, revealed their association with osmolytes production and secondary metabolite pathways. Comparative analysis of differentially expressed genes and their non-coding RNA partners, long noncoding RNAs and microRNAs, provided valuable insight to gene expression regulation in response to drought stress. LncRNAs as well as coding transcripts share similar structural features in different tetraploid species; yet, lncRNAs slightly differ from coding transcripts. Several miRNA-lncRNA target pairs were detected as differentially expressed in drought stress. Overall, this study suggested an important pool of transcripts where their manipulations confer a better performance of wheat varieties under drought stress.

Wheat miRNA ancestors: evident by transcriptome analysis of A, B, and D genome donors.

MicroRNAs are critical players of post-transcriptional gene regulation with profound effects on the fundamental processes of cellular life. Their identification and characterization, together with their targets, hold great significance in exploring and exploiting their roles on a functional context, providing valuable clues into the regulation of important biological processes, such as stress tolerance or environmental adaptation. Wheat is a hardy crop, extensively harvested in temperate regions, and is a major component of the human diet. With the advent of the next generation sequencing technologies considerably decreasing sequencing costs per base-pair, genomic, and transcriptomic data from several wheat species, including the progenitors and wild relatives have become available. In this study, we performed in silico identification and comparative analysis of microRNA repertoires of bread wheat (Triticum aestivum L.) and its diploid progenitors and relatives, Aegilops sharonensis, Aegilops speltoides, Aegilops tauschii, Triticum monococcum, and Triticum urartu through the utilization of publicly available transcriptomic data. Over 200 miRNA families were identified, majority of which have not previously been reported. Ancestral relationships expanded our understanding of wheat miRNA evolution, while T. monococcum miRNAs delivered important clues on the effects of domestication on miRNA expression. Comparative analyses on wild Ae. sharonensis accessions highlighted candidate miRNAs that can be linked to stress tolerance. The miRNA repertoires of bread wheat and its diploid progenitors and relatives provide important insight into the diversification and distribution of miRNA genes, which should contribute to the elucidation of miRNA evolution of Poaceae family. A thorough understanding of the convergent and divergent expression profiles of miRNAs in different genetic backgrounds can provide unique opportunities to modulation of gene regulation for better crop performance.

Abiotic stress miRNomes in the Triticeae.

The continued growth in world population necessitates increases in both the quantity and quality of agricultural production. Triticeae members, particularly wheat and barley, make an important contribution to world food reserves by providing rich sources of carbohydrate and protein. These crops are grown over diverse production environments that are characterized by a range of environmental or abiotic stresses. Abiotic stresses such as drought, heat, salinity, or nutrient deficiencies and toxicities cause large yield losses resulting in economic and environmental damage. The negative effects of abiotic stresses have increased at an alarming rate in recent years and are predicted to further deteriorate due to climate change, land degradation, and declining water supply. New technologies have provided an important tool with great potential for improving crop tolerance to the abiotic stresses: microRNAs (miRNAs). miRNAs are small regulators of gene expression that act on many different molecular and biochemical processes such as development, environmental adaptation, and stress tolerance. miRNAs can act at both the transcriptional and post-transcriptional levels, although post-transcriptional regulation is the most common in plants where miRNAs can inhibit the translation of their mRNA targets via complementary binding and cleavage. To date, expression of several miRNA families such as miR156, miR159, and miR398 has been detected as responsive to environmental conditions to regulate stress-associated molecular mechanisms individually and/or together with their various miRNA partners. Manipulation of these miRNAs and their targets may pave the way to improve crop performance under several abiotic stresses. Here, we summarize the current status of our knowledge on abiotic stress-associated miRNAs in members of the Triticeae tribe, specifically in wheat and barley, and the miRNA-based regulatory mechanisms triggered by stress conditions. Exploration of further miRNA families together with their functions under stress will improve our knowledge and provide opportunities to enhance plant performance to help us meet global food demand.

MicroRNA nomenclature and the need for a revised naming prescription.

A central environment and interface for microRNA (miRNA) registry and repository and a general standardized framework for their systematic annotation was established over a decade ago. However, the numbers of experimentally and computationally identified miRNAs are swiftly accumulating, and new aspects of miRNA-mediated gene regulation are being revealed. Currently, it is of great significance that the annotation framework should be redefined to include newly discovered miRNA species such as the variants of mature miRNAs (isomiRNAs), and organellar miRNAs: cipomiRNAs and mitomiRNAs. It is also of great importance that key terminology referring to the novelty, evolutionary history or biogenesis of miRNAs, as well as the confidence of their identification are standardized in the literature and disseminated in a central miRNA registry. Here, we review the status of miRNA nomenclature, curation and critical points of need for a revision of miRNA nomenclature and terminology.

A Comprehensive Prescription for Plant miRNA Identification.

microRNAs (miRNAs) are tiny ribo-regulatory molecules involved in various essential pathways for persistence of cellular life, such as development, environmental adaptation, and stress response. In recent years, miRNAs have become a major focus in molecular biology because of their functional and diagnostic importance. This interest in miRNA research has resulted in the development of many specific software and pipelines for the identification of miRNAs and their specific targets, which is the key for the elucidation of miRNA-modulated gene expression. While the well-recognized importance of miRNAs in clinical research pushed the emergence of many useful computational identification approaches in animals, available software and pipelines are fewer for plants. Additionally, existing approaches suffers from mis-identification and annotation of plant miRNAs since the miRNA mining process for plants is highly prone to false-positives, particularly in cereals which have a highly repetitive genome. Our group developed a homology-based in silico miRNA identification approach for plants, which utilizes two Perl scripts "SUmirFind" and "SUmirFold" and since then, this method helped identify many miRNAs particularly from crop species such as Triticum or Aegliops. Herein, we describe a comprehensive updated guideline by the implementation of two new scripts, "SUmirPredictor" and "SUmirLocator," and refinements to our previous method in order to identify genuine miRNAs with increased sensitivity in consideration of miRNA identification problems in plants. Recent updates enable our method to provide more reliable and precise results in an automated fashion in addition to solutions for elimination of most false-positive predictions, miRNA naming and miRNA mis-annotation. It also provides a comprehensive view to genome/transcriptome-wide location of miRNA precursors as well as their association with transposable elements. The "SUmirPredictor" and "SUmirLocator" scripts are freely available together with a reference high-confidence plant miRNA list.