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.

A Trypsin Family Protein Gene Controls Tillering and Leaf Shape in Barley.

Tillering or branching is an important agronomic trait in plants, especially cereal crops. Previously, in barley (Hordeum vulgare) 'Vlamingh', we identified the high number of tillers1 (hnt1) mutant from a γ-ray-treated segregating population. hnt1 exhibited more tillers per plant, narrower leaves, and reduced plant height compared with the wild-type parent. In this study, we show that the hnt1-increased tiller number per plant is caused by accelerated outgrowth of tiller buds and that hnt1 narrower leaves are caused by a reduction in vascular tissue and cell number. Genetic analysis revealed that a 2-bp deletion in the gene HORVU2Hr1G098820 (HvHNT1), encoding a trypsin family protein, was responsible for the hnt1 mutant phenotype. Gene function was further confirmed by transgenic complementation with HvHNT1 and RNA interference experiments. HvHNT1 was expressed in vascular tissue, leaf axils, and adventitious root primordia and shown to negatively regulate tiller development. Mutation of HvHNT1 led to the accumulation of a putative cyclophilin-type peptidyl-prolyl cis/trans-isomerase (HvPPIase), which physically interacts with the HvHNT1 protein in the nucleus of plant cells. Our data suggest that HvHNT1 controls tiller development and leaf width through HvPPIase, thus contributing to understanding of the molecular players that control tillering in barley.

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.

Characterization of the sdw1 semi-dwarf gene in barley.

BACKGROUND: The dwarfing gene sdw1 has been widely used throughout the world to develop commercial barley varieties. There are at least four different alleles at the sdw1 locus. RESULTS: Mutations in the gibberellin 20-oxidase gene (HvGA20ox2) resulted in multiple alleles at the sdw1 locus. The sdw1.d allele from Diamant is due to a 7-bp deletion in exon 1, while the sdw1.c allele from Abed Denso has 1-bp deletion and a 4-bp insertion in the 5' untranslated region. The sdw1.a allele from Jotun resulted from a total deletion of the HvGA20ox2 gene. The structural changes result in lower gene expression in sdw1.d and lack of expression in sdw1.a. There are three HvGA20ox genes in the barley genome. The partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1and HvGA20ox3. A diagnostic molecular marker was developed to differentiate between the wild-type, sdw1.d and sdw1.a alleles and another molecular marker for differentiation of sdw1.c and sdw1.a. The markers were further tested in 197 barley varieties, out of which 28 had the sdw1.d allele and two varieties the sdw1.a allele. To date, the sdw1.d and sdw1.a alleles have only been detected in the modern barley varieties and lines. CONCLUSIONS: The results provided further proof that the gibberellin 20-oxidase gene (HvGA20ox2) is the functional gene of the barley sdw1 mutants. Different deletions resulted in different functional alleles for different breeding purposes. Truncated protein could maintain partial function. Partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1 and HvGA20ox3.

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.

A new allele of acid soil tolerance gene from a malting barley variety.

BACKGROUND: Acid soil is a serious limitation to crop production all over the world. Toxic aluminium (Al) cations in acid soil inhibit root growth and reduce yield. Although a gene tolerant to acid soil has been identified, it has not been used in malting barley breeding, which is partly due to the acid soil tolerance gene being linked to unfavorable malting quality traits. RESULTS: A Brazilian malting barley variety Br2 was identified as tolerant to acid soil. A doubled haploid (DH) population was developed from a cross between Br2 and the Australian acid-sensitive cultivar Hamelin. The DH population was tested for acid soil tolerance in native acid soil and a hydroponic system with pH 4.2, pH 4.2 + Al or pH 6.5, and genotyped using SSR, DArT and gene-specific markers. A single QTL was detected for all parameters related to acid soil tolerance. The QTL was mapped to the known HvMATE location on chromosome 4H. Sequence alignment of the HvMATE gene identified 13 INDELs and 87 SNPs, where one SNP coded for a single amino acid difference between the two varieties. A gene-specific marker was developed to detect the single nucleotide polymorphism between Hamelin and Br2. This marker co-segregated with aluminium tolerance and accounted for 79 % of phenotypic variation for acid soil tolerance. CONCLUSION: The present study identified a novel source of acid soil/Al tolerance from a Brazilian malting barley cultivar Br2. This variety tolerated Al toxicity but was sensitive to low pH which is similar to most other Al-tolerant varieties. A gene-specific marker Cit7 was developed based on the HvMATE gene sequence. Cit7 will improve the efficiency of molecular-assisted selection of new barley varieties with tolerance to acid soil. Multiple alleles exist for the acid soil tolerance gene on chromosome 4H, so a malting barley variety that tolerates acid soil could be developed by selecting suitable tolerant alleles. Tolerance to low pH may play an important role for barley to adapt to acid soils.

Anther Culture Protocols for Barley and Wheat.

Doubled haploid (DH) techniques remain valuable tools for wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) genetic improvement, and DH populations are used extensively in breeding and research endeavors. Several techniques are available for DH production in wheat and barley. Here, we describe two simple, robust anther culture methods used to produce more than 15,000 DH wheat and barley lines annually in Australia.

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.

Highly efficient and genotype-independent barley gene editing based on anther culture.

Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have typically been used as explants for transformation. However, the genotype dependence of this approach limits the genetic modification of commercial varieties. Here, we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in Golden Promise and four Australian varieties, which differed in phenology, callus induction, and green plant regeneration responses. Agrobacterium-mediated transformation was performed on microspore-derived callus to target the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In 51 analyzed T0 individuals, Cas9 induced a large proportion (69%) of single-base indels and two-base deletions in the target sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerant plants within a similar time frame. It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties.

The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture.

Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caffeine or trifluralin was applied at the start of the induction phase to improve early genome doubling. Caffeine treatment at 0.5 mM for 24 h significantly improved green plant production in two of the six spring wheat crosses but had no effect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caffeine had no significant effect on chromosome doubling, despite a higher frequency of doubling in several caffeine treatments in the first experiment (67-68%) compared to the control (56%). In contrast, trifluralin significantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. However, trifluralin had a significant negative effect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9-25 green plants per 20 anthers in the trifluralin treatments. Further work is required to identify a treatment regime with caffeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration.

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.

Early growth stages salinity stress tolerance in CM72 x Gairdner doubled haploid barley population.

A doubled haploid (DH) population of barley (Hordeum vulgare L.) generated from salinity tolerant genotype CM72 and salinity sensitive variety Gairdner was studied for salinity stress tolerance at germination, seedling emergence and first leaf full expansion growth stages. Germination study was conducted with deionized water, 150 mM and 300 mM NaCl treatments. Seedling stage salinity tolerance was conducted with three treatments: control, 150 mM NaCl added at seedling emergence and first leaf full expansion growth stages. Results from this study revealed transgressive phenotypic segregations for germination percentage and biomass at seedling stage. Twelve QTL were identified on chromosomes 2H-6H each explaining 10-25% of the phenotypic variations. A QTL located at 176.5 cM on chromosome 3H was linked with fresh weight per plant and dry weight per plant in salinity stress induced at first leaf full expansion growth stage, and dry weight per plant in salinity stress induced at seedling emergence. A stable QTL for germination at both 150 and 300 mM salinity stress was mapped on chromosome 2H but distantly located from a QTL linked with seedling stage salinity stress tolerance. QTL, associated markers and genotypes identified in this study play important roles in developing salinity stress tolerant barley varieties.

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.

Genome-Wide Association Mapping of Acid Soil Resistance in Barley (Hordeum vulgare L.).

Genome-wide association studies (GWAS) based on linkage disequilibrium (LD) have been used to detect QTLs underlying complex traits in major crops. In this study, we collected 218 barley (Hordeum vulgare L.) lines including wild barley and cultivated barley from China, Canada, Australia, and Europe. A total of 408 polymorphic markers were used for population structure and LD analysis. GWAS for acid soil resistance were performed on the population using a general linkage model (GLM) and a mixed linkage model (MLM), respectively. A total of 22 QTLs (quantitative trait loci) were detected with the GLM and MLM analyses. Two QTLs, close to markers bPb-1959 (133.1 cM) and bPb-8013 (86.7 cM), localized on chromosome 1H and 4H respectively, were consistently detected in two different trials with both the GLM and MLM analyses. Furthermore, bPb-8013, the closest marker to the major Al(3+) resistance gene HvAACT1 in barley, was identified to be QTL5. The QTLs could be used in marker-assisted selection to identify and pyramid different loci for improved acid soil resistance in barley.

In vitro culture for doubled haploids: tools for molecular breeding.

Doubled haploid (DH) techniques using immature microspores or embryos rescued in vitro following interspecific hybridization are important tools for breeding wheat and barley and for generating populations for gene mapping purposes. Here, we describe methods being used for the successful production of spring wheat and barley DHs in Australia.

Comparison of genetic and cytogenetic maps of hexaploid wheat (Triticum aestivum L.) using SSR and DArT markers.

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20-34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome.