Genome architecture and diverged selection shaping pattern of genomic differentiation in wild barley.

Divergent selection of populations in contrasting environments leads to functional genomic divergence. However, the genomic architecture underlying heterogeneous genomic differentiation remains poorly understood. Here, we de novo assembled two high-quality wild barley (Hordeum spontaneum K. Koch) genomes and examined genomic differentiation and gene expression patterns under abiotic stress in two populations. These two populations had a shared ancestry and originated in close geographic proximity but experienced different selective pressures due to their contrasting micro-environments. We identified structural variants that may have played significant roles in affecting genes potentially associated with well-differentiated phenotypes such as flowering time and drought response between two wild barley genomes. Among them, a 29-bp insertion into the promoter region formed a cis-regulatory element in the HvWRKY45 gene, which may contribute to enhanced tolerance to drought. A single SNP mutation in the promoter region may influence HvCO5 expression and be putatively linked to local flowering time adaptation. We also revealed significant genomic differentiation between the two populations with ongoing gene flow. Our results indicate that SNPs and small SVs link to genetic differentiation at the gene level through local adaptation and are maintained through divergent selection. In contrast, large chromosome inversions may have shaped the heterogeneous pattern of genomic differentiation along the chromosomes by suppressing chromosome recombination and gene flow. Our research offers novel insights into the genomic basis underlying local adaptation and provides valuable resources for the genetic improvement of cultivated barley.

Manipulating GA-Related Genes for Cereal Crop Improvement.

The global population is projected to experience a rapid increase in the future, which poses a challenge to global food sustainability. The "Green Revolution" beginning in the 1960s allowed grain yield to reach two billion tons in 2000 due to the introduction of semi-dwarfing genes in cereal crops. Semi-dwarfing genes reduce the gibberellin (GA) signal, leading to short plant stature, which improves the lodging resistance and harvest index under modern fertilization practices. Here, we reviewed the literature on the function of GA in plant growth and development, and the role of GA-related genes in controlling key agronomic traits that contribute to grain yield in cereal crops. We showed that: (1) GA is a significant phytohormone in regulating plant development and reproduction; (2) GA metabolism and GA signalling pathways are two key components in GA-regulated plant growth; (3) GA interacts with other phytohormones manipulating plant development and reproduction; and (4) targeting GA signalling pathways is an effective genetic solution to improve agronomic traits in cereal crops. We suggest that the modification of GA-related genes and the identification of novel alleles without a negative impact on yield and adaptation are significant in cereal crop breeding for plant architecture improvement. We observed that an increasing number of GA-related genes and their mutants have been functionally validated, but only a limited number of GA-related genes have been genetically modified through conventional breeding tools and are widely used in crop breeding successfully. New genome editing technologies, such as the CRISPR/Cas9 system, hold the promise of validating the effectiveness of GA-related genes in crop development and opening a new venue for efficient and accelerated crop breeding.

Multi-locus genome-wide association studies reveal novel alleles for flowering time under vernalisation and extended photoperiod in a barley MAGIC population.

KEY MESSAGE: Key genes controlling flowering and interactions of different photoperiod alleles with various environments were identified in a barley MAGIC population. A new candidate gene for vernalisation requirements was also detected. Optimal flowering time has a major impact on grain yield in crop species, including the globally important temperate cereal crop barley (Hordeum vulgare L.). Understanding the genetics of flowering is a key avenue to enhancing yield potential. Although bi-parental populations were used intensively to map genes controlling flowering, their lack of genetic diversity requires additional work to obtain desired gene combinations in the selected lines, especially when the two parental cultivars did not carry the genes. Multi-parent mapping populations, which use a combination of four or eight parental cultivars, have higher genetic and phenotypic diversity and can provide novel genetic combinations that cannot be achieved using bi-parental populations. This study uses a Multi-parent advanced generation intercross (MAGIC) population from four commercial barley cultivars to identify genes controlling flowering time in different environmental conditions. Genome-wide association studies (GWAS) were performed using 5,112 high-quality markers from Diversity Arrays Technology sequencing (DArT-seq), and Kompetitive allele-specific polymerase chain reaction (KASP) genetic markers were developed. Phenotypic data were collected from fifteen different field trials for three consecutive years. Planting was conducted at various sowing times, and plants were grown with/without additional vernalisation and extended photoperiod treatments. This study detected fourteen stable regions associated with flowering time across multiple environments. GWAS combined with pangenome data highlighted the role of CEN gene in flowering and enabled the prediction of different CEN alleles from parental lines. As the founder lines of the multi-parental population are elite germplasm, the favourable alleles identified in this study are directly relevant to breeding, increasing the efficiency of subsequent breeding strategies and offering better grain yield and adaptation to growing conditions.

Grain-Filling Rate Improves Physical Grain Quality in Barley Under Heat Stress Conditions During the Grain-Filling Period.

Heat stress is a primary constraint to Australia's barley production. In addition to impacting grain yield, it adversely affects physical grain quality (weight and plumpness) and market value. The incidence of heat stress during grain filling is rising with global warming. However, breeding for new superior heat-tolerant genotypes has been challenging due to the narrow window of sensitivity, the unpredictable nature of heat stress, and its frequent co-occurrence with drought stress. Greater scientific knowledge regarding traits and mechanisms associated with heat tolerance would help develop more efficient selection methods. Our objective was to assess 157 barley varieties of contrasting genetic backgrounds for various developmental, agro-morphological, and physiological traits to examine the effects of heat stress on physical grain quality. Delayed sowing (i.e., July and August) increased the likelihood of daytime temperatures above 30°C during grain-filling. Supplementary irrigation of field trials ensured a reduced impact of drought stress. Heat tolerance appeared to be the primary factor determining grain plumpness. A wide variation was observed for heat tolerance, particularly among the Australian varieties. Genotypic variation was also observed for grain weight, plumpness, grain growth components, stay-green and stem water-soluble carbohydrates (WSC) content, and mobilisation under normal and delayed sown conditions. Compared to normal sowing, delayed sowing reduced duration of developmental phases, plant height, leaf size, head length, head weight, grain number, plumpness, grain width and thickness, stem WSC content, green leaf area retention, and harvest index (HI), and increased screenings, grain length, grain-filling rate (GFR), WSC mobilisation efficiency (WSCME), and grain protein content. Overall, genotypes with heavier and plumper grains under high temperatures had higher GFR, longer grain-filling duration, longer green leaf area retention, higher WSCME, taller stature, smaller leaf size, greater HI, higher grain weight/plumpness potentials, and earlier flowering. GFR played a significant role in determining barley grain weight and plumpness under heat-stress conditions. Enhancing GFR may provide a new avenue for improving heat tolerance in barley.

Opportunities for Improving Waterlogging Tolerance in Cereal Crops-Physiological Traits and Genetic Mechanisms.

Waterlogging occurs when soil is saturated with water, leading to anaerobic conditions in the root zone of plants. Climate change is increasing the frequency of waterlogging events, resulting in considerable crop losses. Plants respond to waterlogging stress by adventitious root growth, aerenchyma formation, energy metabolism, and phytohormone signalling. Genotypes differ in biomass reduction, photosynthesis rate, adventitious roots development, and aerenchyma formation in response to waterlogging. We reviewed the detrimental effects of waterlogging on physiological and genetic mechanisms in four major cereal crops (rice, maize, wheat, and barley). The review covers current knowledge on waterlogging tolerance mechanism, genes, and quantitative trait loci (QTL) associated with waterlogging tolerance-related traits, the conventional and modern breeding methods used in developing waterlogging tolerant germplasm. Lastly, we describe candidate genes controlling waterlogging tolerance identified in model plants Arabidopsis and rice to identify homologous genes in the less waterlogging-tolerant maize, wheat, and barley.

Genomic structural equation modelling provides a whole-system approach for the future crop breeding.

KEY MESSAGE: Using genomic structural equation modelling, this research demonstrates an efficient way to identify genetically correlating traits and provides an effective proxy for multi-trait selection to consider the joint genetic architecture of multiple interacting traits in crop breeding. Breeding crop cultivars with optimal value across multiple traits has been a challenge, as traits may negatively correlate due to pleiotropy or genetic linkage. For example, grain yield and grain protein content correlate negatively with each other in cereal crops. Future crop breeding needs to be based on practical yet accurate evaluation and effective selection of beneficial trait to retain genes with the best agronomic score for multiple traits. Here, we test the framework of whole-system-based approach using structural equation modelling (SEM) to investigate how one trait affects others to guide the optimal selection of a combination of agronomically important traits. Using ten traits and genome-wide SNP profiles from a worldwide barley panel and SEM analysis, we revealed a network of interacting traits, in which tiller number contributes positively to both grain yield and protein content; we further identified common genetic factors affecting multiple traits in the network of interaction. Our method demonstrates an efficient way to identify genetically correlating traits and underlying pleiotropic genetic factors and provides an effective proxy for multi-trait selection within a whole-system framework that considers the joint genetic architecture of multiple interacting traits in crop breeding. Our findings suggest the promise of a whole-system approach to overcome challenges such as the negative correlation of grain yield and protein content to facilitating quantitative and objective breeding decisions in future crop breeding.

A global barley panel revealing genomic signatures of breeding in modern Australian cultivars.

The future of plant cultivar improvement lies in the evaluation of genetic resources from currently available germplasm. Today's gene pool of crop genetic diversity has been shaped during domestication and more recently by breeding. Recent efforts in plant breeding have been aimed at developing new and improved varieties from poorly adapted crops to suit local environments. However, the impact of these breeding efforts is poorly understood. Here, we assess the contributions of both historical and recent breeding efforts to local adaptation and crop improvement in a global barley panel by analysing the distribution of genetic variants with respect to geographic region or historical breeding category. By tracing the impact that breeding had on the genetic diversity of Hordeum vulgare (barley) released in Australia, where the history of barley production is relatively young, we identify 69 candidate regions within 922 genes that were under selection pressure. We also show that modern Australian barley varieties exhibit 12% higher genetic diversity than historical cultivars. Finally, field-trialling and phenotyping for agriculturally relevant traits across a diverse range of Australian environments suggests that genomic regions under strong breeding selection and their candidate genes are closely associated with key agronomic traits. In conclusion, our combined data set and germplasm collection provide a rich source of genetic diversity that can be applied to understanding and improving environmental adaptation and enhanced yields.

Genome-wide association mapping reveals novel genes associated with coleoptile length in a worldwide collection of barley.

BACKGROUND: Drought is projected to become more frequent and severe in a changing climate, which requires deep sowing of crop seeds to reach soil moisture. Coleoptile length is a key agronomic trait in cereal crops such as barley, as long coleoptiles are linked to drought tolerance and improved seedling establishment under early water-limited growing conditions. RESULTS: In this study, we detected large genetic variation in a panel of 328 diverse barley (Hordeum vulgare L.) accessions. To understand the overall genetic basis of barley coleoptile length, all accessions were germinated in the dark and phenotyped for coleoptile length after 2 weeks. The investigated barleys had significant variation for coleoptile length. We then conducted genome-wide association studies (GWASs) with more than 30,000 molecular markers and identified 8 genes and 12 intergenic loci significantly associated with coleoptile length in our barley panel. The Squamosa promoter-binding-like protein 3 gene (SPL3) on chromosome 6H was identified as a major candidate gene. The missense variant on the second exon changed serine to alanine in the conserved SBP domain, which likely impacted its DNA-binding activity. CONCLUSION: This study provides genetic loci for seedling coleoptile length along with candidate genes for future potential incorporation in breeding programmes to enhance early vigour and yield potential in water-limited environments.

Genome-Wide Association Study of Salinity Tolerance During Germination in Barley (Hordeum vulgare L.).

Barley seeds need to be able to germinate and establish seedlings in saline soils in Mediterranean-type climates. Despite being a major cereal crop, barley has few reported quantitative trait loci (QTL) and candidate genes underlying salt tolerance at the germination stage. Breeding programs targeting salinity tolerance at germination require an understanding of genetic loci and alleles in the current germplasm. In this study, we investigated seed-germination-related traits under control and salt stress conditions in 350 diverse barley accessions. A genome-wide association study, using ~24,000 genetic markers, was undertaken to detect marker-trait associations (MTA) and the underlying candidate genes for salinity tolerance during germination. We detected 19 loci containing 52 significant salt-tolerance-associated markers across all chromosomes, and 4 genes belonging to 4 family functions underlying the predicted MTAs. Our results provide new genetic resources and information to improve salt tolerance at germination in future barley varieties via genomic and marker-assisted selection and to open up avenues for further functional characterization of the identified candidate genes.

Gene-set association and epistatic analyses reveal complex gene interaction networks affecting flowering time in a worldwide barley collection.

Single-marker genome-wide association studies (GWAS) have successfully detected associations between single nucleotide polymorphisms (SNPs) and agronomic traits such as flowering time and grain yield in barley. However, the analysis of individual SNPs can only account for a small proportion of genetic variation, and can only provide limited knowledge on gene network interactions. Gene-based GWAS approaches provide enormous opportunity both to combine genetic information and to examine interactions among genetic variants. Here, we revisited a previously published phenotypic and genotypic data set of 895 barley varieties grown in two years at four different field locations in Australia. We employed statistical models to examine gene-phenotype associations, as well as two-way epistasis analyses to increase the capability to find novel genes that have significant roles in controlling flowering time in barley. Genetic associations were tested between flowering time and corresponding genotypes of 174 putative flowering time-related genes. Gene-phenotype association analysis detected 113 genes associated with flowering time in barley, demonstrating the unprecedented power of gene-based analysis. Subsequent two-way epistasis analysis revealed 19 pairs of gene×gene interactions involved in controlling flowering time. Our study demonstrates that gene-based association approaches can provide higher capacity for future crop improvement to increase crop performance and adaptation to different environments.

Targeted enrichment by solution-based hybrid capture to identify genetic sequence variants in barley.

In barley and other cereal crops, phenological diversity drives adaptation to different cultivation areas. Improvement of barley yield and quality traits requires adaptation to specific production areas with introgression of favorable alleles dependent upon precise identification of the underlying genes. Combining targeted sequence capture systems with next-generation sequencing provides an efficient approach to explore target genetic regions at high resolution, and allows rapid discovery of thousands of genetic polymorphisms. Here, we apply a versatile target-capture method to detect genome-wide polymorphisms in 174 flowering time-related genes, chosen based on prior knowledge from barley, rice, and Arabidopsis thaliana. Sequences were generated across a phenologically diverse panel of 895 barley varieties, resulting a high mean depth coverage of ~25x allowing reliable discovery and calling of insertion-deletion (InDel) and single nucleotide polymorphisms (SNPs). Sequences of InDel and SNPs from the targeted enrichment were utilized to develop 67 Kompetitive Allele Specific PCR (KASP) markers for validation. This work provides researchers and breeders a comprehensive molecular toolkit for the selection of phenology-related traits in barley.

Hybridisation-based target enrichment of phenology genes to dissect the genetic basis of yield and adaptation in barley.

Barley (Hordeum vulgare L.) is a major cereal grain widely used for livestock feed, brewing malts and human food. Grain yield is the most important breeding target for genetic improvement and largely depends on optimal timing of flowering. Little is known about the allelic diversity of genes that underlie flowering time in domesticated barley, the genetic changes that have occurred during breeding, and their impact on yield and adaptation. Here, we report a comprehensive genomic assessment of a worldwide collection of 895 barley accessions based on the targeted resequencing of phenology genes. A versatile target-capture method was used to detect genome-wide polymorphisms in a panel of 174 flowering time-related genes, chosen based on prior knowledge from barley, rice and Arabidopsis thaliana. Association studies identified novel polymorphisms that accounted for observed phenotypic variation in phenology and grain yield, and explained improvements in adaptation as a result of historical breeding of Australian barley cultivars. We found that 50% of genetic variants associated with grain yield, and 67% of the plant height variation was also associated with phenology. The precise identification of favourable alleles provides a genomic basis to improve barley yield traits and to enhance adaptation for specific production areas.

Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress.

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na+ and compatible organic solutes, and more K+ in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress.

Cell-Type-Specific H+-ATPase Activity in Root Tissues Enables K+ Retention and Mediates Acclimation of Barley (Hordeum vulgare) to Salinity Stress.

While the importance of cell type specificity in plant adaptive responses is widely accepted, only a limited number of studies have addressed this issue at the functional level. We have combined electrophysiological, imaging, and biochemical techniques to reveal the physiological mechanisms conferring higher sensitivity of apical root cells to salinity in barley (Hordeum vulgare). We show that salinity application to the root apex arrests root growth in a highly tissue- and treatment-specific manner. Although salinity-induced transient net Na+ uptake was about 4-fold higher in the root apex compared with the mature zone, mature root cells accumulated more cytosolic and vacuolar Na+, suggesting that the higher sensitivity of apical cells to salt is not related to either enhanced Na+ exclusion or sequestration inside the root. Rather, the above differential sensitivity between the two zones originates from a 10-fold difference in K+ efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Major factors contributing to this poor K+ retention ability are (1) an intrinsically lower H+-ATPase activity in the root apex, (2) greater salt-induced membrane depolarization, and (3) a higher reactive oxygen species production under NaCl and a larger density of reactive oxygen species-activated cation currents in the apex. Salinity treatment increased (2- to 5-fold) the content of 10 (out of 25 detected) amino acids in the root apex but not in the mature zone and changed the organic acid and sugar contents. The causal link between the observed changes in the root metabolic profile and the regulation of transporter activity is discussed.

De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure.

Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes.

Metabolomics, Standards, and Metabolic Modeling for Synthetic Biology in Plants.

Life on earth depends on dynamic chemical transformations that enable cellular functions, including electron transfer reactions, as well as synthesis and degradation of biomolecules. Biochemical reactions are coordinated in metabolic pathways that interact in a complex way to allow adequate regulation. Biotechnology, food, biofuel, agricultural, and pharmaceutical industries are highly interested in metabolic engineering as an enabling technology of synthetic biology to exploit cells for the controlled production of metabolites of interest. These approaches have only recently been extended to plants due to their greater metabolic complexity (such as primary and secondary metabolism) and highly compartmentalized cellular structures and functions (including plant-specific organelles) compared with bacteria and other microorganisms. Technological advances in analytical instrumentation in combination with advances in data analysis and modeling have opened up new approaches to engineer plant metabolic pathways and allow the impact of modifications to be predicted more accurately. In this article, we review challenges in the integration and analysis of large-scale metabolic data, present an overview of current bioinformatics methods for the modeling and visualization of metabolic networks, and discuss approaches for interfacing bioinformatics approaches with metabolic models of cellular processes and flux distributions in order to predict phenotypes derived from specific genetic modifications or subjected to different environmental conditions.

Quantitative profiling of polar primary metabolites of two chickpea cultivars with contrasting responses to salinity.

This study reports a GC-QqQ-MS method for the quantification of forty-eight primary metabolites from four major classes (sugars, sugar acids, sugar phosphates, and organic acids) which can be applied to a number of biological systems. The method was validated in terms of linearity, reproducibility and recovery, using both calibration standards and real samples. Additionally, twenty-eight biogenic amines and amino acids were quantified using an established LC-QqQ-MS method. Both GC-QqQ-MS and LC-QqQ-MS quantitative methods were applied to plant extracts from flower and pod tissue of two chickpea (Cicer arietinum L.) cultivars differing in their ability to tolerate salinity, which were grown under control and salt-treated conditions. Statistical analysis was applied to the data sets using the absolute concentrations of metabolites to investigate the differences in metabolite profiles between the different cultivars, plant tissues, and treatments. The method is a significant improvement of present methodology for quantitative GC-MS metabolite profiling of organic acids and sugars, and provides new insights of chickpea metabolic responses to salinity stress. It is applicable to the analysis of dynamic changes in endogenous concentrations of polar primary metabolites to study metabolic responses to environmental stresses in complex biological tissues.