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Genome resequencing uncovers genome-wide DNA polymorphisms that are useful for the development of high-density InDel markers between two barley cultivars. Discovering genomic variations and developing genetic markers are crucial for genetics studies and molecular breeding in cereal crops. Although InDels (insertions and deletions) have become popular because of their abundance and ease of detection, discovery of genome-wide DNA polymorphisms and development of InDel markers in barley have lagged behind other cereal crops such as rice, maize and wheat. In this study, we re-sequenced two barley cultivars, Golden Promise (GP, a classic British spring barley variety) and Hua30 (a Chinese spring barley variety), and mapped clean reads to the reference Morex genome, and identified in total 13,933,145 single nucleotide polymorphisms (SNPs) and 1,240,456 InDels for GP with Morex, 11,297,100 SNPs and 781,687 InDels for Hua30 with Morex, and 13,742,399 SNPs and 1,191,597 InDels for GP with Hua30. We further characterized distinct types, chromosomal distribution patterns, genome location, functional effect, and other features of these DNA polymorphisms. Additionally, we revealed the functional relevance of these identified SNPs/InDels regarding different flowering times between Hua30 and GP within 17 flowering time genes. Furthermore, we developed a series of InDel markers and validated them experimentally in 43 barley core accessions, respectively. Finally, we rebuilt population structure and phylogenetic tree of these 43 barley core accessions. Collectively, all of these genetic resources will facilitate not only the basic research but also applied research in barley.

The presence of β-glucan in barley grains is one of its important quality traits. Lower β-glucan content is required for the barley used in beer and feed production, while higher β-glucan content is beneficial for food barley. Although intensive research has been carried out on the genotypic and environmental differences in β-glucan content in barley grains, little information is available on the molecular mechanisms underlying their genotypic differences and genetic regulation of β-glucan synthesis and accumulation. In this study, RNA sequencing analysis was conducted to compare the transcriptome profiles of two barley genotypes (BCS192 and BCS297) that greatly differ in grain β-glucan content, in order to identify the key genes responsible for β-glucan synthesis and accumulation during grain development. The results showed that carbohydrate metabolic processes and starch and sucrose metabolism play significant roles in β-glucan synthesis. The identified differently expressed genes (DEGs), which are closely associated with grain β-glucan content, are mainly involved in hydrolase activity and glucan metabolic processes. In addition, β-glucan accumulation in barley grains is predominantly regulated by photosynthesis and carbon metabolism. The DEGs identified in this study and their functions may provide new insights into the molecular mechanisms of β-glucan synthesis and genotypic differences in barley grains.

Barley is the fourth largest cereal crop in the world. It is mainly used for feeding, beer production and food. Barley is receiving more attention from both agricultural and food scientists because of its special chemical composition and health benefits. In comparison with other cereal crops, including wheat, rice and maize, barley grains are rich in dietary fiber (such as β-glucan) and tocols, which are beneficial to human health. It is well proved that diets rich in those chemicals can provide protection against hypertension, cardiovascular disease, and diabetes. Barley has been widely recognized to have great potential as a healthy or functional food. In this review, we present information about studies on the physical structure of the barley grain and the distribution of its main chemical components, nutrient and functional composition of barley grain and their health benefits, and the approaches of improving and utilizing the nutrient and functional chemicals in barley grain. With the development of processing technologies, functional components in barley grains, especially β-glucan, can be efficiently extracted and concentrated. Moreover, nutrient and functional components in barley grains can be efficiently improved by precise breeding and agronomic approaches. The review highlights the great potential of barley used as healthy and functional foods, and may be instructive for better utilization of barley in food processing.

The tribe Triticeae provides important staple cereal crops and contains elite wild species with wide genetic diversity and high tolerance to abiotic stresses. Sea barleygrass (Hordeum marinum Huds.), a wild Triticeae species, thrives in saline marshlands and is well known for its high tolerance to salinity and waterlogging. Here, a 3.82-Gb high-quality reference genome of sea barleygrass is assembled de novo, with 3.69 Gb (96.8%) of its sequences anchored onto seven chromosomes. In total, 41 045 high-confidence (HC) genes are annotated by homology, de novo prediction, and transcriptome analysis. Phylogenetics, non-synonymous/synonymous mutation ratios (Ka/Ks), and transcriptomic and functional analyses provide genetic evidence for the divergence in morphology and salt tolerance among sea barleygrass, barley, and wheat. The large variation in post-domestication genes (e.g. IPA1 and MOC1) may cause interspecies differences in plant morphology. The extremely high salt tolerance of sea barleygrass is mainly attributed to low Na

β-glucan is an important trait to be improved in barley breeding programs, as it greatly affects the quality of the end products when barley grains are used as raw material of feed or malt production or consumed as food for human. Although the genes associated with β-glucan synthesis have been identified, genetic regulation of β-glucan accumulation in barley grains is still completely unclear. In this study, 100 accessions from International Barley Core Selected Collection (BCS) were planted in two environments for two consecutive years to determine the genotypic variation of grain β-glucan content. A genome-wide association study (GWAS) identified 14 stable marker-trait associations (MTAs) (−Log10(P)> 4) for grain β-glucan content. Significantly positive correlation was found between grain β-glucan content and the number of favorable alleles of 14 stable MTAs. Seven putative candidate genes encoding some enzymes in glucose metabolism were found to be associated with β-glucan content. One of the putative genes, HORVU6Hr1G088380, could be an important gene controlling barely β-glucan content, with the SNPs being closely linked in all tested accessions and divided into two haplotypes. High resolution melting (HRM) analysis of the first SNP suggested that the HRM-SNP marker is valid for marker-assisted selection in barley breeding. This study provides useful information for the genes and markers related to grain β-glucan content in barley. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-020-01199-5.

A low level of available potassium (K) in soils is a major factor restricting crop growth and production. In earlier studies we found that Tibetan wild barley has a wider variation in low K stress tolerance than cultivated barley. In this study, we performed a genome-wide association analysis of 179 Tibetan wild barley (Hordeum vulgare L.) accessions exposed to low K stress and investigated the associations of K uptake rate (KUR), K translocation rate (KTR) and other K metabolism traits with 11,013 diversity arrays technology markers. The results showed that there were significant differences in tissue K concentration and content, KUR and KTR among the wild barley accessions analyzed. Scores for all traits showed a normal distribution. Three significant quantitative trait loci (QTLs) for KUR and KTR were identified and located on 6H and 1H. These QTLs were found to be associated with the cation/H(+) antiporter and cyclic nucleotide gated channel. A number of unique candidate genes associated with K-related signals, including K channels, K transporters and ethylene-related genes, were also identified. The K channels and K transporters involved in K uptake and translocation, respectively, were closely associated with KUR in response to low K stress. The present study highlights the potential of Tibetan wild barley to provide elite candidate genes for the improvement of K use efficiency of 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.

Figure S2. Hierarchical cluster (a) and gene functional categories analyses (b) of low-P tolerance related DEGs. (PPTX 54 kb)

Table S1. Primes used in quantitative Real-time PCR. (PPTX 22 kb)

Figure S4. The hypothetic pathways underlying low-P tolerant mechanisms in L138. ABA, Abscisic Acid; ARF, Auxin Responsefactor; LPR, Low Phosphate Root; PDR, Phosphate Deficiency Response; PHF, Phosphate Transporter Traffic Facilitator; PHO, Phosphate; PHR, Phosphate Starvation Response; PHT, Phosphate Transporter; PstB, Phosphate Import ATP-Binding Protein; SCR, Scarecrow; SPX, SYG1/PHO81/XPR1; SPX-MFS, SYG1/PHO81/XPR1-Major Facilitator Superfamily; TIR, Transport Inhibitor Response (PPTX 25 kb)

Figure S3. Hierarchical cluster analyses of nitrate transcporter1/peptide transporter (NRT1/PTR) identified in differentially expressed genes (DEGs). (PPTX 491 kb)

Figure S1. Quantitative real-time PCR validation of 5 differentially expressed genes (DEGs) detected in L138 and L73 with low-P stress. Transcript levels of (a) PHO1-3, (b) ARF2, (c) SPX-MFS1, (d) SPX-MFS2, (e) sugar transporter 7 and the corresponding expression data of RNA-Seq are displayed by the columns and lines, respectively. (f) Comparison between the relative expressions (fold changes) obtained from RNA-Seq data and qPCR. (PPTX 399 kb)

The content and composition of starch in cereal grains are closely related to yield. Few studies have been done on the identification of the genes or loci associated with these traits in barley. This study was conducted to identify the genes or loci controlling starch traits in barley grains, including total starch (TS), amylose (AC) and amylopectin (AP) contents. A large genotypic variation was found in all examined starch traits. GWAS analysis detected 13, 2, 10 QTLs for TS, AC and AP, respectively, and 5 of them were commonly shared by AP and TS content. qTS-3.1, qAC-6.2 and qAP-5.1 may explain the largest variation of TS, AC and AP, respectively. Four putative candidate genes, i.e., HORVU6Hr1G087920, HORVU5Hr1G011230, HORVU5Hr1G011270 and HORVU5Hr1G011280, showed the high expression in the developing barley grains when starch accumulates rapidly. The examined 100 barley accessions could be divided into two groups based on the polymorphism of the marker S5H_29297679, with 93 accessions having allele GG and seven accessions having AA. Moreover, significantly positive correlation was found between the number of favorable alleles of the identified QTLs and TS, AC, AP content. In conclusion, the identified loci or genes in this study could be useful for genetic improvement of grains starch in barley.

Limit dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley.

Haze formation in beer is a trait closely related to beer quality and it is largely affected by the haze active proteins (HAPs) in barley (Hordeum vulgare L.). Up to date, little is known about the genetics of HAPs and relevant genes. In this study, we obtained the beer samples from a Franklin/Yerong double haploid (DH) population and the two parents using micro-malting and micro-brewing, and determined tannin-related HAPs. It was found that there was a wide difference in HAPs among all lines of the DH population, and Yerong had a higher HAPs content than Franklin. Quantitative trait locus (QTL) analysis identified five QTLs associated with HAPs in beer, being located on chromosomes 1HS, 5HL and 6HS, respectively. The loci QHAP1.FrYe-1H and QHAP2.FrYe-1H were overlapped in the short arm of chromosome 1H, and they controlled HAP1 and HAP2, respectively. Moreover, the candidate genes were also predicted based on published whole barley genome sequence and corresponding gene annotations.

Our previous study found that the critical protein in SE (silica eluted) proteins is BTI-CMe, and assumed that SE-ve malt for brewing may improve the haze stability in beer. In this study, we investigated the difference in gene sequence and corresponding amino acid sequence of BTI-CMe between SE+ve and SE-ve types. The results showed that there were 7 amino acid differences between Yerong (SE-ve) and Franklin (SE+ve). Two types BTI-CMe were expressed in vitro and purified successfully. By adding the purified BTI-CMe into commercial beer, we found that both original turbidity and alcohol chill haze degree of beer were increased. BTI-CMe of SE-ve haplotype showed a lower level of haze formation in beer than SE+ve haplotype. Response surface methodology (RSM) was conducted to determine the relationship between BTI-CMe and tannic acid, and their effects on haze formation. It was found that (1) higher content of BTI-CMe and/or tannic acid in beer would give rise to higher turbidity; (2) there was a significant interaction between BTI-CMe and tannic acid; (3) haze activity disparity of BTI-CMe between two types was significantly and positively correlated with the tannic acid concentration.

Limit dextrinase (LD) is a unique de-branching enzyme involved in starch mobilization of barley grains during malting, and closely related to malt quality. Genotypic variation of LD activity is controlled by genetic factors and also affected by environmental conditions. Correlation analysis between LD activity and four malt quality parameters showed that LD activity was positively correlated with diastatic power, Kolbach index and the quality of malt extract, while negatively correlated with viscosity. The structure-based association analysis demonstrated that HvLDI, a gene encoding limit dextrinase inhibitor, was a major determinant of LD activity and malt quality. The single nucleotide polymorphisms associated with LD activity could be used in early generation selection for barley breeding.

The primers used in quantitative real-time PCR. (DOC 51Â kb)

Heat Map analysis of DEGs involved in hormone signaling in XZ149 and XZ56. (DOCX 76Â kb)

Real-time PCR analysis of the HvHRT2.1 gene in XZ149 under low N stress. (DOCX 16Â kb)

The primers used in quantitative real-time PCR. (DOC 51Â kb)

Real-time PCR analysis of the HvHRT2.1 gene in XZ149 under low N stress. (DOCX 16Â kb)

DEGs related antioxidant stress under low N stress. (DOCX 16Â kb)

DEGs related antioxidant stress under low N stress. (DOCX 16Â kb)

Heat Map analysis of DEGs involved in hormone signaling in XZ149 and XZ56. (DOCX 76Â kb)

Background In bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer. Results In this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding. Conclusions We identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1683-1) contains supplementary material, which is available to authorized users.

Sugar degradation during grain germination is important for malt quality. In malting industry, gibberellin (GA) is frequently used for improvement of malting quality. In this study, the changes of metabolite profiles and starch-degrading enzymes during grain germination, and as affected by GA and abscisic acid (ABA) were investigated using two wild barley accessions XZ72 and XZ95. Totally fifty-two metabolites with known structures were detected and the change of metabolite during germination was time- and genotype dependent. Sugars and amino acids were the most dramatically changed compounds. Addition of GA enhanced the activities of starch-degrading enzymes, and increased most metabolites, especially sugars and amino acids, whereas ABA had the opposite effect. The effect varied with the barley accessions. The current study is the first attempt in investigating the effect of hormones on metabolite profiles in germinating barley grain, being helpful for identifying the factors affecting barley germination or malt quality.

The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.

A thorough understanding of the mechanisms underlying barley salt tolerance and exploitation of elite genetic resource are essential for utilizing wild barley germplasm in developing barley varieties with salt tolerance. In order to reveal the physiological and molecular difference in salt tolerance between Tibetan wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare), profiles of 82 key metabolites were studies in wild and cultivated barley in response to salinity. According to shoot dry biomass under salt stress, XZ16 is a fast growing and salt tolerant wild barley. The results of metabolite profiling analysis suggested osmotic adjustment was a basic mechanism, and polyols played important roles in developing salt tolerance only in roots, and high level of sugars and energy in roots and active photosynthesis in leaves were important for barley to develop salt tolerance. The metabolites involved in tolerance enhancement differed between roots and shoots, and also between genotypes. Tibetan wild barley, XZ16 had higher chlorophyll content and higher contents of compatible solutes than CM72, while the cultivated barley, CM72 probably enhanced its salt tolerance mainly through increasing glycolysis and energy consumption, when the plants were exposed to high salinity. The current research extends our understanding of the mechanisms involved in barley salt tolerance and provides possible utilization of Tibetan wild barley in developing barley cultivars with salt tolerance.

The evaluation of both the genetic variation and the identification of salinity tolerant accessions of Tibetan annual wild barley (hereafter referred to as Tibetan barley) (Hordeum vulgare L. ssp. Spontaneum and H. vulgare L. ssp. agriocrithum) are essential for discovering and exploiting novel alleles involved in salinity tolerance. In this study, we examined tissue dry biomass and the Na(+) and K(+) contents of 188 Tibetan barley accessions in response to salt stress. We investigated the genetic variation of transcription factors HvCBF1, HvCBF3 and HvCBF4 within these accessions, conducting association analysis between these three genes and the respective genotypic salt tolerance. Salt stress significantly reduced shoot and root dry weight by 27.6% to 73.1% in the Tibetan barley lines. HvCBF1, HvCBF3 and HvCBF4 showed diverse sequence variation in amplicon as evident by the identification of single nucleotide polymorphisms (SNPs) and 3, 8 and 13 haplotypes, respectively. Furthermore, the decay of Linkage disequilibrium (LD) of chromosome 5 was 8.9 cM (r(2)