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.

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.

Uncovering the evolutionary origin of blue anthocyanins in cereal grains.

Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only Hordeum vulgare (barley), Triticum aestivum (wheat) and Secale cereale (rye) accumulate delphinidin-derived (blue) anthocyanins in the aleurone layer of grains, whereas Oryza sativa (rice), Zea mays (maize) and Sorghum bicolor (sorghum) do not. The underlying genetic basis for this natural occurrence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleurone to an approximately 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue-coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co-evolved together. We also identified a Pooideae-specific flavonoid 3',5'-hydroxylase (F3'5'H) lineage, termed here Mo_F35H2, which has a higher amino acid similarity with eudicot F3'5'Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 that underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, through the interrogation of barley germplasm there is evidence that HvMYB4H and HvMYC4H have undergone human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.

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.

Fine mapping QSc.VR4, an effective and stable scald resistance locus in barley (Hordeum vulgare L.), to a 0.38-Mb region enriched with LRR-RLK and GLP genes.

KEY MESSAGE: An effective and stable quantitative resistance locus, QSc.VR4, was fine mapped, characterized and physically anchored to the short arm of 4H, conferring adult plant resistance to the fungus Rhynchosporium commune in barley. Scald caused by Rhynchosporium commune is one of the most destructive barley diseases worldwide. Accumulation of adult plant resistance (APR) governed by multiple resistance alleles is predicted to be effective and long-lasting against a broad spectrum of pathotypes. However, the molecular mechanisms that control APR remain poorly understood. Here, quantitative trait loci (QTL) analysis of APR and fine mapping were performed on five barley populations derived from a common parent Vlamingh, which expresses APR to scald. Two QTLs, designated QSc.VR4 and QSc.BR7, were detected from a cross between Vlamingh and Buloke. Our data confirmed that QSc.VR4 is an effective and stable APR locus, residing on the short arm of chromosome 4H, and QSc.BR7 derived from Buloke may be an allele of reported Rrs2. High-resolution fine mapping revealed that QSc.VR4 is located in a 0.38 Mb genomic region between InDel markers 4H2282169 and 4H2665106. The gene annotation analysis and sequence comparison suggested that a gene cluster containing two adjacent multigene families encoding leucine-rich repeat receptor kinase-like proteins (LRR-RLKs) and germin-like proteins (GLPs), respectively, is likely contributing to scald resistance. Adult plant resistance (APR) governed by QSc.VR4 may confer partial levels of resistance to the fungus Rhynchosporium commune and, furthermore, be an important resource for gene pyramiding that may contribute broad-based and more durable resistance.

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.

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.

Marker development using SLAF-seq and whole-genome shotgun strategy to fine-map the semi-dwarf gene ari-e in barley.

BACKGROUND: Barley semi-dwarf genes have been extensively explored and widely used in barley breeding programs. The semi-dwarf gene ari-e from Golden Promise is an important gene associated with some agronomic traits and salt tolerance. While ari-e has been mapped on barley chromosome 5H using traditional markers and next-generation sequencing technologies, it has not yet been finely located on this chromosome. RESULTS: We integrated two methods to develop molecular markers for fine-mapping the semi-dwarf gene ari-e: (1) specific-length amplified fragment sequencing (SLAF-seq) with bulked segregant analysis (BSA) to develop SNP markers, and (2) the whole-genome shotgun sequence to develop InDels. Both SNP and InDel markers were developed in the target region and used for fine-mapping the ari-e gene. Linkage analysis showed that ari-e co-segregated with marker InDel-17 and was delimited by two markers (InDel-16 and DGSNP21) spanning 6.8 cM in the doubled haploid (DH) Dash × VB9104 population. The genetic position of ari-e was further confirmed in the Hindmarsh × W1 DH population which was located between InDel-7 and InDel-17. As a result, the overlapping region of the two mapping populations flanked by InDel-16 and InDel-17 was defined as the candidate region spanning 0.58 Mb on the POPSEQ physical map. CONCLUSIONS: The current study demonstrated the SLAF-seq for SNP discovery and whole-genome shotgun sequencing for InDel development as an efficient approach to map complex genomic region for isolation of functional gene. The ari-e gene was fine mapped from 10 Mb to 0.58 Mb interval.

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.

A nonsense mutation in a putative sulphate transporter gene results in low phytic acid in barley.

Low phytic acid grains can provide a solution to dietary micronutrient deficiency and environmental pollution. A low phytic acid 1-1 (lpa1-1) barley mutant was identified using forward genetics and the mutant gene was mapped to chromosome 2HL. Comparative genomic analysis revealed that the lpa1-1 gene was located in the syntenic region of the rice Os-lpa-MH86-1 gene on chromosome 4. The gene ortholog of rice Os-lpa-MH86-1 (designated as HvST) was isolated from barley using polymerase chain reaction and mapped to chromosome 2HL in a doubled haploid population of Clipper×Sahara. The results demonstrate the collinearity between the rice Os-lpa-MH86-1 gene and the barley lpa1-1 region. Sequence analysis of HvST revealed a single base pair substitution (C→T transition) in the last exon of the gene in lpa1-1 (M422), which resulted in a nonsense mutation. These results will facilitate our understanding of the molecular mechanisms controlling the low phytic acid phenotype and assist in the development of a diagnostic marker for the selection of the lpa1-1 gene in barley.

Expression level of a gibberellin 20-oxidase gene is associated with multiple agronomic and quality traits in barley.

The use of dwarfing genes has resulted in the most significant improvements in yield and adaptation in cereal crops. The allelic dwarfing gene sdw1/denso has been used throughout the world to develop commercial barley varieties. The sdw1 gene has never been used successfully for malting barley, but only for a large number of feed varieties. One of the gibberellin 20-oxidase genes (Hv20ox2) was identified as the candidate gene for sdw1/denso. Semi-quantitative real-time RT-PCR revealed that Hv20ox2 was expressed at different levels in various organs of barley. Transcriptional levels were reduced in leaf blade, sheath, stem and rachis tissue in the barley variety Baudin with the denso gene. Subsequently, the relative expression levels of Hv20ox2 were determined by quantitative real-time RT-PCR in a doubled haploid population and mapped as a quantitative trait. A single expression quantitative trait locus (eQTL) was identified and mapped to its structural gene region on chromosome 3H. The eQTL was co-located with QTLs for yield, height, development score, hectolitre weight and grain plumpness. The expression level of Hv20ox2 was reduced fourfold in the denso mutant, but around 60-fold in the sdw1 mutant, compared to the control variety. The reduced expression level of Hv20ox2 enhanced grain yield by increasing the number of effective tillers, but had negative effects on grain and malting quality. The sdw1 gene can be used only in feed barley due to its severe reduction of Hv20ox2 expression. The gene expression marker for Hv20ox2 can be used to distinguish different alleles of sdw1/denso.

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley.

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin x AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley.

Comparative analysis of genetic diversity between Qinghai-Tibetan wild and Chinese landrace barley.

Fifty-two SSR markers were used to evaluate the genetic diversity of 33 Qinghai-Tibetan wild barley accessions, 56 landraces collected primarily from other parts of China, and 1 Israeli wild barley accession. At the 52 SSR loci, 206 alleles were detected for the 90 accessions, among which 111 were common alleles. The number of alleles per locus ranged from 1 to 9, with an average of 4.0. Polymorphism information content (PIC) values ranged from 0 to 0.856 among all the markers, with an average of 0.547. The PIC value of Qinghai-Tibetan wild barley varied from 0 to 0.813 with an average of 0.543, while in landraces, the markers showed a range of 0 to 0.790 with an average of 0.490. The SSR markers could clearly differentiate the Qinghai-Tibetan wild barley from the landraces. Twenty-four unique alleles were observed in Qinghai-Tibetan wild barley, and the frequency of unique alleles in Qinghai-Tibetan wild barley was about 2.1 times higher than that in the landraces, on average. Five of the 7 chromosomes had more unique alleles in the Qinghai-Tibetan wild barley, but chromosome 2H had more unique alleles in the landraces. The presence of many unique alleles may reflect the adaptation of this barley germplasm to diverse environments and production systems.

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.

Towards the identification of a gene for prostrate tillers in barley (Hordeum vulgare L.).

Tiller angle, an important agronomic trait, contributes to crop production and plays a vital role in breeding for plant architecture. A barley line V-V-HD, which has prostrate tillers during vegetative growth and erect tillers after booting, is considered the ideal type for repressing weed growth and increasing leaf area during early growth. Genetic analysis identified that the prostrate trait in V-V-HD is controlled by a single gene. A double haploid population with 208 lines from V-V-HD × Buloke was used to map the prostrate growth gene. Ninety-six SNP markers were used for primary mapping, and subsequently, SSR and InDel markers were used for fine mapping. The gene was fine-mapped to a 3.53 Mb region on chromosome 3HL between the markers InDelz3028 and InDelz3032 with 52 candidate genes located in this region. Gene annotation analysis of the 52 genes within the target region indicated that a gene involved in zinc-ion binding (gene ID HORVU3Hr1G090910) is likely to be the candidate gene for prostrate growth in V-V-HD, and is linked to the denso/sdw gene. Association analysis showed that prostrate plants were shorter, flowered later.

Genetic Mapping and Evolutionary Analyses of the Black Grain Trait in Barley.

Barley occupies the widest ecological area among the major cereal crops, thereby generating a high potential for adaptive genetic diversity against various environmental factors. Colored barley such as black grain barley has been suggested to result from environmental adaptation to biotic and abiotic stresses. Using one double haploid population (433 lines), plus three F5 recombinant inbred line (RIL) populations (1,009 lines), the black lemma and pericarp (Blp) gene was mapped between two Insertion/deletion (Indel) markers MC_1570156 and MC_162350 with a physical distance of 0.807 Mb, containing 21 annotated genes in the mapped interval. Whole-genome re-sequencing was performed on two Tibetan wild barley lines (X1 and W1) with black grain phenotype. The probable candidate genes for Blp were discussed based on gene functional annotation and gene sequence variation analyses. Thirteen polymorphic Indel markers covering the target genetic region were used to analyze 178 barley accessions including 49 black husk entries. Genotype-based clustering analyses showed that the black landraces of different geographical background may have evolved from a single origin. Our study represents a significant improvement on the genetic mapping of Blp and would facilitate future study on the characterization of the genetic basis underlying this interesting agronomic trait.

Characterization of a Thermo-Inducible Chlorophyll-Deficient Mutant in Barley.

Leaf color is an important trait for not only controlling crop yield but also monitoring plant status under temperature stress. In this study, a thermo-inducible chlorophyll-deficient mutant, named V-V-Y, was identified from a gamma-radiated population of the barley variety Vlamingh. The leaves of the mutant were green under normal growing temperature but turned yellowish under high temperature in the glasshouse experiment. The ratio of chlorophyll a and chlorophyll b in the mutant declined much faster in the first 7-9 days under heat treatment. The leaves of V-V-Y turned yellowish but took longer to senesce under heat stress in the field experiment. Genetic analysis indicated that a single nuclear gene controlled the mutant trait. The mutant gene (vvy) was mapped to the long arm of chromosome 4H between SNP markers 1_0269 and 1_1531 with a genetic distance of 2.2 cM and a physical interval of 9.85 Mb. A QTL for grain yield was mapped to the same interval and explained 10.4% of the yield variation with a LOD score of 4. This QTL is coincident with the vvy gene interval that is responsible for the thermo-inducible chlorophyll-deficient trait. Fine mapping, based on the barley reference genome sequence, further narrowed the vvy gene to a physical interval of 0.428 Mb with 11 annotated genes. This is the first report of fine mapping a thermo-inducible chlorophyll-deficient gene in barley.

Discovery of novel Bmy1 alleles increasing ß-amylase activity in Chinese landraces and Tibetan wild barley for improvement of malting quality via MAS.

China has a large barley germplasm collection which has not been well characterized and is therefore underutilized. The Bmy1 locus encoding the ß-amylase enzyme on chromosome 4H has been well characterized in the worldwide barley germplasm collections due to its importance in the malting and brewing industry. The Bmy1 locus was chosen as an indicator to understand genetic potential for improvement of malting quality in Chinese landraces and Tibetan wild barley. The genetic diversity of 91 barley accessions was assessed using allele specific Multiplex-ready molecular markers. Eight accessions were further sequenced, based on the Multiplex-ready marker diversity for Bmy1 in the germplasm. Six of the eight accessions clustered together in a unique group, and showed similarities to 'Haruna Nijo', wild barley accession PI296896 and 'Ashqelon'. Sequence comparisons with the known Bmy1 alleles identified not only the existing 13 amino acid substitutions, but also a new substitution positioned at A387T from a Chinese landrace W127, which has the highest ß-amylase activity. Two new alleles/haplotypes namely Bmy1-Sd1c and Bmy1-Sd5 were designated based on different amino acid combinations. We identified new amino acid combination of C115, D165, V233, S347 and V430 in the germplasm. The broad variation in both ß-amylase activity and amino acid composition provides novel alleles for the improvement of malting quality for different brewing styles, which indicates the high potential value of the Chinese landraces and Tibetan wild barley.