Barley Gene Research Articles

The Barley (Hordeum vulgare ssp. vulgare) Respiratory Burst Oxidase Homolog (HvRBOH) Gene Family and Their Plausible Role on Malting Quality.

Controlled generation of reactive oxygen species (ROS) is pivotal for normal plant development and adaptation to changes in the external milieu. One of the major enzymatic sources of ROS in plants are the plasma-membrane localized NADPH oxidases, also called as Respiratory Burst Oxidase Homologs (RBOH). In addition to the six previously reported, seven new members of RBOH gene family were identified in barley using in silico analysis. Conservation of genomic structure and key residues important for catalytic activity and co-factor binding was observed in barley RBOH genes. Phylogenetic analysis of plant RBOHs revealed distinct clades for monocot and dicot RBOH proteins. Hence, we propose to use the rice nomenclature for naming barley RBOH genes. Temporal changes in ROS profiles were observed during barley malting and was accompanied by changes in protein carbonylation, lipid peroxidation, and antioxidant capacity. Among the nine differentially expressed HvRBOHs during various malting stages, HvRBOHA and HvRBOHC showed most significant sustained changes in expression. RNAi knockdown lines with reduced expression of HvRBOHA/C gene exhibited genetic compensation via inducible expression of other gene family members during malting. However, the physiological consequence of reduced expression of HvRBOHA/C manifested as a poor malting quality profile attributable to low alpha-amylase activity and high levels of beta-glucan. We propose that the HvRBOHs play a critical role in modulating the redox milieu during the early stages of malting, which in turn can significantly impact carbohydrate metabolism.

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non‐selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP‐tagged PQL proteins localized to intracellular membrane structures, both when transiently over‐expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non‐stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

Using Genome-Wide Predictions to Assess the Phenotypic Variation of a Barley (Hordeum sp.) Gene Bank Collection for Important Agronomic Traits and Passport Information

Genome-wide predictions are a powerful tool for predicting trait performance. Against this backdrop we aimed to evaluate the potential and limitations of genome-wide predictions to inform the barley collection of the Federal ex situ Genebank for Agricultural and Horticultural Crops with phenotypic data on complex traits including flowering time, plant height, thousand grain weight, as well as on growth habit and row type. We used previously published sequence data, providing information on 306,049 high-quality SNPs for 20,454 barley accessions. The prediction abilities of the two unordered categorical traits row type and growth type as well as the quantitative traits flowering time, plant height and thousand grain weight were investigated using different cross validation scenarios. Our results demonstrate that the unordered categorical traits can be predicted with high precision. In this way genome-wide prediction can be routinely deployed to extract information pertinent to the taxonomic status of gene bank accessions. In addition, the three quantitative traits were also predicted with high precision, thereby increasing the amount of information available for genotyped but not phenotyped accessions. Deeply phenotyped core collections, such as the barley 1,000 core set of the IPK Gatersleben, are a promising training population to calibrate genome-wide prediction models. Consequently, genome-wide predictions can substantially contribute to increase the attractiveness of gene bank collections and help evolve gene banks into bio-digital resource centers.

New insights on key genes involved in drought stress response of barley: gene networks reconstruction, hub, and promoter analysis

BackgroundBarley (Hordeum vulgare L.) is one of the most important cereals worldwide. Although this crop is drought-tolerant, water deficiency negatively affects its growth and production. To detect key genes involved in drought tolerance in barley, a reconstruction of the related gene network and discovery of the hub genes would help. Here, drought-responsive genes in barley were collected through analysis of the available microarray datasets (− 5 ≥ Fold change ≥ 5, adjusted p value ≤ 0.05). Protein-protein interaction (PPI) networks were reconstructed. ResultsThe hub genes were identified by Cytoscape software using three Cyto-hubba algorithms (Degree, Closeness, and MNC), leading to the identification of 17 and 16 non-redundant genes at vegetative and reproductive stages, respectively. These genes consist of some transcription factors such as HvVp1, HvERF4, HvFUS3, HvCBF6, DRF1.3, HvNAC6, HvCO5, and HvWRKY42, which belong to AP2, NAC, Zinc-finger, and WRKY families. In addition, the expression pattern of four hub genes was compared between the two studied cultivars, i.e., “Yousef” (drought-tolerant) and “Morocco” (susceptible). The results of real-time PCR revealed that the expression patterns corresponded well with those determined by the microarray. Also, promoter analysis revealed that some TF families, including AP2, NAC, Trihelix, MYB, and one modular (composed of two HD-ZIP TFs), had a binding site in 85% of promoters of the drought-responsive genes and of the hub genes in barley. ConclusionsThe identified hub genes, especially those from AP2 and NAC families, might be among key TFs that regulate drought-stress response in barley and are suggested as promising candidate genes for further functional analysis.

RING-Type E3 Ubiqitin Ligase Barley Genes (HvYrg1-2) Control Characteristics of Both Vegetative Organs and Seeds as Yield Components.

Previously, studies on RING-type E3 ubiquitin ligases in cereals were preferentially focused on GW2 genes primarily controlling seed parameters in rice and wheat. Here we report cloning two HvYrg genes from barley that share significant homology with rice GW2 gene. In antisense genotypes efficiency of gene silencing varied between genes and transgenic lines: ASHvYrg1: 30–50% and ASHvYrg2: 20–27%. Reduced activity of both genes altered shoot system with increasing number of side shoots. Changes in leaf width, weight, or plant weight and height reached significant levels in some transgenic lines. Lowering expression of the two barley HvYrg genes caused opposite responses in spike development. Plants with ASHvYrg1 gene construct showed earlier heading time and prolonged grain-filling period, while plants from ASHvYrg2 genotype flowered in delay. Digital imaging of root development revealed that down-regulation of HvYrg1 gene variant stimulated root growth, while ASHvYrg2 plants developed reduced root system. Comparison of seed parameters indicated an increase in thousand grain weight accompanied with longer and wider seed morphology. In summary we conclude that in contrast to inhibition of GW2 genes in rice and wheat plants, down-regulation of the barely HvYrg genes caused substantial changes in vegetative organs in addition to alteration of seed parameters.

Genome-wide diversity analysis of TCP transcription factors revealed cases of selection from wild to cultivated barley.

Plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTORS 1/2 (TCP) transcription factors have known roles in inflorescence architecture. In barley, there are two family members INTERMEDIUM-C (INT-c/HvTB1-1) and COMPOSITUM 1 (COM1/HvTCP24) which are involved in the manipulation of spike architecture, whereas the participation of TCP family genes in selection from wild (Hordeum vulgare subsp. spontaneum, Hs) to cultivated barley (Hordeum vulgare subsp. vulgare, Hv) remains poorly investigated. Here, by conducting a genome-wide survey for TCP-like sequences in publicly-released datasets, 22 HsTCP and 20 HvTCP genes encoded for mature proteins were identified and assigned into two classes (I and II) based on their functional domains and the phylogenetic analysis. Each counterpart of the orthologous gene in wild and cultivated barley usually represented a similarity on the transcriptional profile across the tissues. The diversity analysis of TCPs in 90 wild barley accessions and 137 landraces with geographically-referenced passport information revealed the detectable selection at three loci including INT-c/HvTB1-1, HvPCF2, and HvPCF8. Especially, the HvPCF8 haplotypes in cultivated barley were found correlating with their geographical collection sites. There was no difference observed in either transactivation activity in yeast or subcellular localization in Nicotiana benthamiana among these haplotypes. Nevertheless, the genome-wide diversity analysis of barley TCP genes in wild and cultivated populations provided insight for future functional characterization in plant development such as spike architecture.

Investigation of Heat-Induced Changes in the Grain Yield and Grains Metabolites, with Molecular Insights on the Candidate Genes in Barley

Heat stress is one of the abiotic stresses that cause a significant reduction in barley yield. Climate change will increase the number of heatwaves, which will result in more deterioration in the agricultural sector. Therefore, understanding the physiological changes that occur in the plant to tolerate heat stress is very important. A collection of 60 Egyptian spring barley genotypes has been tested for heat stress under field conditions. To quantify the changes in yield-related traits and the grain-reserve parameters as indicators for heat tolerance, several traits were scored. The causative genes that regulate the variation of all traits of interest were identified via single-marker analysis using 16,966 single nucleotide polymorphisms (SNP). Heat stress reduced yield-related traits, while some physiological traits (chlorophyll index, soluble carbohydrates, amino acids, and proline contents) increased. The genotypes were classified into four classes, A, B, C, and D, based on a reduction in grain yield per spike (GYPS) of 10%, 20%, 30%, and 40%, respectively. The physiological aspects were extensively studied in each group. The tolerant genotypes (class A) retained high yield-related traits as well as high reserved metabolites relative to the sensitive class D. The single-marker analysis and gene annotations revealed that the most effective markers and genes resided on chromosomes 1H and 4H. One of these markers, S4_250499621, was found to be associated with increased proline content, increased chlorophyll content, and decreased reduction in grain yield per spike and thousand kernel weight. This study is a part of our extended evaluation of this collection under various abiotic stresses at different developmental stages to develop climate-resilient crops.

The Effect of Pesticide Application on QTLs Controlling Traits in Barley

Among cereals, barley (Hordeum vulgare L.) ranks fourth in consumption worldwide. Among barley breeding goals, one can refer to gene mapping, studying their inheritance, and saturated genetic linkage maps. Problems with pesticide applications include reduced genetic diversity, reduced nitrogen fixation, and destruction of the habitat of especially endangered species. The effect of pesticide application on the emergence of QTLs expressing traits in experimental barley was investigated using 104 barley F2:4 families from Badia × Kavir cross. A total of 25 QTLs were mapped for all traits. In non-using pesticides, 12 QTLs were identified for peduncle length, stem diameter, flag leaf length, and awn length. It was found that qFL-4 has major effects on flag leaf length. or using the pesticide, 13 QTLs were detected that QTLs related to stem diameter, grain weight, flag leaf length explained a high percentage of phenotypic variation. The results of this study showed that pesticide application affects the expression of some genes in barley. Besides, major-effect trait-controller QTLs and their associated markers can be used in marker-assisted selection (MAS) programs.

Genome Wide Identification and Comparative Analysis of the Serpin Gene Family in Brachypodium and Barley.

Serpins (serine protease inhibitors) constitute one of the largest and most widely distributed superfamilies of protease inhibitors and have been identified in nearly all organisms. To gain significant insights, a comprehensive in silico analysis of the serpin gene family was carried out in the model plant for temperate grasses Brachypodium distachyon and barley Hordeum vulgare using bioinformatic tools at the genome level for the first time. We identified a total of 27 BdSRPs and 25 HvSRP genes in Brachypodium and barley, respectively, showing an unexpectedly high gene number in these model plants. Gene structure, conserved motifs and phylogenetic comparisons of serpin genes supported the role of duplication events in the expansion and evolution of serpin gene family. Further, purifying selection pressure was found to be a main driving force in the evolution of serpin genes. Genome synteny analysis indicated that BdSRP genes were present in syntenic regions of barley, rice, sorghum and maize, suggesting that they evolved before the divergence of these species from common ancestor. The distinct expression pattern in specific tissues further suggested a specialization of functions during development and in plant defense. These results suggest that the LR serpins (serpins with Leu-Arg residues at P2–P1′) identified here can be utilized as candidates for exploitation in disease resistance, pest control and preventing stress-induced cell death. Additionally, serpins were identified that could lead to further research aimed at validating and functionally characterizing the role of potential serpin genes from other plants.

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

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

Identification, gene expression and genetic polymorphism of zinc finger A20/AN1 stress-associated genes, HvSAP, in salt stressed barley from Kazakhstan

BackgroundA family of genes designated as the Zinc finger A20/AN1 Transcription factors encoding stress-associated proteins (SAP) are well described in Arabidopsis and rice, and include 14 AtSAP and 18 OsSAP genes that are associated with variable tolerances to multiple abiotic stresses. The SAP gene family displays a great diversity in its structure and across different plant species. The aim of this study was to identify all HvSAP genes in barley (Hordeum vulgare L.), to analyse the expression of selected genes in response to salinity in barley leaves and develop SNP marker for HvSAP12 to evaluate the association between genotypes of barley plants and their grain yield in field trials.ResultsIn our study, 17 HvSAP genes were identified in barley, which were strongly homologous to rice genes. Five genes, HvSAP5, HvSAP6, HvSAP11, HvSAP12 and HvSAP15, were found to be highly expressed in leaves of barley plants in response to salt stress in hydroponics compared to controls, using both semi-quantitative RT-PCR and qPCR analyses. The Amplifluor-like SNP marker KATU-B30 was developed and used for HvSAP12 genotyping. A strong association (R2 = 0.85) was found between KATU-B30 and grain yield production per plant of 50 F3 breeding lines originating from the cross Granal × Baisheshek in field trials with drought and low to moderate salinity in Northern and Central Kazakhstan.ConclusionsA group of HvSAP genes, and HvSAP12 in particular, play an important role in the tolerance of barley plants to salinity and drought, and is associated with higher grain yield in field trials. Marker-assisted selection with SNP marker KATU-B30 can be applied in barley breeding to improve grain yield production under conditions of abiotic stress.

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

<p>Overexpression of <em>alanine aminotransferase<strong> </strong></em>(<em>AlaAT</em>) gene can improve nitrogen use efficiency (NUE) in plants. The previous isolated <em>AlaAT</em> genes cannot be freely applied to generate NUE plants due to IPR restriction. Therefore, isolation of the gene from targeted monocot and dicot plants is necessary. The objectives of this study were to isolate <em>AlaAT</em> genes from barley, foxtail millet, cucumber, and tomato and analyze their homology to other isolated <em>AlaAT</em> genes in sequence databases (gene bank). Total RNA was isolated from roots of barley, foxtail millet, cucumber, and tomato, and then converted into cDNA using reverse transcription method. The cDNA was then cloned into <em>pGEM®-T Easy</em> plasmid and the verified clones were sequenced. The amino acid sequences were analyzed for their homologies using Clustal Omega software and phylogenetic tree was constructed. The results showed that the amino acids of <em>AlaAT</em> gene from barley was different from <em>AlaAT</em> genes of tomato and cucumber with homology level less than 80%. Phylogenetic tree predicted that <em>AlaAT</em> genes clustered into three groups with <em>AlaAT</em> genes of foxtail millet and barley clustered in one group together with other monocots in group I. <em>AlaAT</em> genes derived from dicots clustered into two groups<em> </em>in which<em> AlaAT</em> gene of tomato clustered in group II, while that of cucumber was in group III. The identity differences of <em>AlaAT</em> gene of tomato and that of cucumber as well as the estimates of the same enzymatic functions can open up enormous opportunities in genetic engineering research for the development of NUE rice.</p>

Genome-Wide Identification of Barley ABC Genes and Their Expression in Response to Abiotic Stress Treatment.

Adenosine triphosphate-binding cassette transporters (ABC transporters) participate in various plant growth and abiotic stress responses. In the present study, 131 ABC genes in barley were systematically identified using bioinformatics. Based on the classification method of the family in rice, these members were classified into eight subfamilies (ABCA–ABCG, ABCI). The conserved domain, amino acid composition, physicochemical properties, chromosome distribution, and tissue expression of these genes were predicted and analyzed. The results showed that the characteristic motifs of the barley ABC genes were highly conserved and there were great diversities in the homology of the transmembrane domain, the number of exons, amino acid length, and the molecular weight, whereas the span of the isoelectric point was small. Tissue expression profile analysis suggested that ABC genes possess non-tissue specificity. Ultimately, 15 differentially expressed genes exhibited diverse expression responses to stress treatments including drought, cadmium, and salt stress, indicating that the ABCB and ABCG subfamilies function in the response to abiotic stress in barley.

Genome-wide identification and expression pattern analysis of the KCS gene family in barley

β-Ketoacyl CoA synthetase (KCS) is a key rate-limiting enzyme for the synthesis of very-long-chain fatty acids (VLCFAs) in plants that determines the synthesis rate and carbon chain length of VLCFAs, ultimately affecting the stress resistance of epidermal wax. In this study, a genome-wide characterization of the KCS family was conducted using high-quality barley (Hordeum vulgare L.) genome sequences. In total, 33 KCS genes were identified and were unevenly distributed in the distal centromere region of each chromosome. These genes were divided 12 subgroups based on sequence alignment and phylogenetic analysis, with members of the same subgroup possessing similar genes and motif structures. Expression analysis showed that the KCS genes demonstrate diverse tissue expression patterns in barley and also have overlapping functions. The barley KCS gene family also exhibited different response characteristics under drought stress. These results provide insights into the evolutionary processes and potential functions of the KCS gene family, offering a theoretical reference for studies on the physiological and biochemical regulation of KCS genes during barley growth and development, as well as for the genetic breeding analysis of wheat crops.

Molecular Networking of Regulated Transcription Factors under Salt Stress in Wild Barley (H. spontaneum)

Salt stress is among environmental conditions that severely retards plant growth. Scope of this work is the detection of transcription factors that might participate in regulating salt-stressed genes in wild barley (Hordeum spontaneum). Expression profiles of important types of transcription factors (TFs) were displayed. They include WRKY and MYB, that were regulated under salt stress. WRKY19 and NAC96 are known to induce stress tolerance through activation of DREB2A (or Ap2-ERF). NAC96 concordantly upregulated with DREB2A gene under salt stress in H. spontaneum, a possible crosstalking to compensate the negative performance of WRKY19 gene. P5CS, for proline accumulation, is also known to be driven by ERF1 and genes encoding these proteins concordantly upregulated in H. spontaneum under salt stress supporting NAC96/ERF1/P5CS crosstalking towards proline accumulation under stress. Genes encoding enzymes participating in the last steps of glucose, sucrose and maltose biosyntheses concordantly upregulated with WRKY11 that is also involved in driving genes encoding free proline. B-box zinc finger protein 21 (BZF21) concordantly expressed with genes encoding catalase and SAUR40 indicating that BZF21 gene might drive expression of the two genes under salt stress. Upregulated WRKY41 and WRKY46 under salt stress in wild barley are known to exhibit enhanced stomatal closure, reactive oxygen species (ROS) scavenging, lateral roots development via regulation of ABA signaling and auxin homeostasis. The latter action is governed by GH3.8 gene that was upregulated in wild barley. MYB30 is known for being SUMOylated by SIZ1. In the present study, MYB30, MYB44 and MYB3R-2 genes were concordantly expressed with SIZ2 gene supporting their crosstalking under salt stress in H. spontaneum. Based on the regulation of WRKY19 and MYB30 genes under salt stress in H. spontaneum, we suggest that the first is a positive activator, while the second is a negative activator of FT gene that drives early flowing in plants. MYB44 that promotes stomatal closure under stress can also serve in conferring tolerance to abiotic stresses in wild barley. Several other downregulated genes under salt stress, e.g., MYB1, MYB20 and MYB73, were previously reported to negatively regulate abiotic stress tolerance in plants. We suggest that WRKY gene family participates in salt stress responses in leaves of H. spontaneum following approaches different from those of other plants. Regulation of MYB gene family is almost similar to that of other plant species under salt stress. In conclusion, the present study addresses some of the regulatory frameworks driving expression of salt-related genes in H. spontaneum that can be utilized in plant, e,g, cereals, breeding programs to improve their salt stress tolerance.

A CYC/TB1-type TCP transcription factor controls spikelet meristem identity in barley.

Barley possesses a branchless, spike-shaped inflorescence where determinate spikelets attach directly to the main axis, but the developmental mechanism of spikelet identity remains largely unknown. Here we report the functional analysis of the barley gene BRANCHED AND INDETERMINATE SPIKELET 1 (BDI1), which encodes a TCP transcription factor and plays a crucial role in determining barley inflorescence architecture and spikelet development. The bdi1 mutant exhibited indeterminate spikelet meristems that continued to grow and differentiate after producing a floret meristem; some spikelet meristems at the base of the spike formed two fully developed seeds or converted to branched spikelets, producing a branched inflorescence. Map-based cloning analysis showed that this mutant has a deletion of ~600 kb on chromosome 5H containing three putative genes. Expression analysis and virus-induced gene silencing confirmed that the causative gene, BDI1, encodes a CYC/TB1-type TCP transcription factor and is highly conserved in both wild and cultivated barley. Transcriptome and regulatory network analysis demonstrated that BDI1 may integrate regulation of gene transcription cell wall modification and known trehalose-6-phosphate homeostasis to control spikelet development. Together, our findings reveal that BDI1 represents a key regulator of inflorescence architecture and meristem determinacy in cereal crop plants.

Genome-wide association mapping reveals putative candidate genes for drought tolerance in barley

Drought is one of the most important abiotic stresses limiting yield production in cereal crops which deleteriously affects food security worldwide. The study aimed to identify marker-trait associations (MTAs) and putative candidate genes underlying agronomic and seed related traits under post-anthesis drought stress in a set of 183 spring barley gene bank accessions from 23 countries that were genotyped with a 9 K SNP chip. Plants were grown in the field in two consecutive years and post-anthesis drought stress was simulated by spraying 1% KI ten days after anthesis. The treatment caused a significant reduction in total biomass by 10.7 %, in kernel number by 2.5 %, in kernel weight per spike by 21.4 % and in thousand kernel weight (TKW) by 16.7 %, whereby most tolerant accessions were HOR 5970, HOR 6606 and HOR 6560. GWAS analysis of best linear unbiased estimators (BLUEs) revealed 97 significant MTAs associated with kernel number and kernel weight per spike and confirmed prominent regions such as 2H (18.9 cM), 6H (54.8 cM) and 7H (70.5-71.0 cM) for drought response and were co-localized with ten putative candidate genes. Gene annotation indicates that genes for the plant respiratory burst oxidase homologs (rboh) family, the carotenoid cleavage dioxygenase 1 (CCD1) and RNA-binding proteins (RBPs) might be involved in tolerance and stress response mechanisms during post-anthesis drought stress. In conclusion, the study provides essential information about significant MTAs and candidate genes related to drought tolerance which can be further used for marker-assisted selection. More drought tolerant barley accessions may be used as pre-bred material to widen the genetic diversity of cultivated barley and to meet the demands for cultivars in world’s areas which suffer post-anthesis drought stress.

GENOME‐WIDE IDENTIFICATION AND ANALYSIS OF THE CslF GENE FAMILY IN BARLEY (Hordeum vulgare L.)

The Cellulose synthase-like (Csl) F family has been considered as one of the most crucial genes regulating β-glucan synthesis. It is a cereal cell wall component, holding advantages for human nutrition, but disadvantages in animal nutrition, malting and brewing industries. Based on a genome-wide search method, present study identified barley CslF gene family members by considering the importance of (1,3;1,4)-β-D-glucan and newly completed barley genome. A sum of Eighteen CslF genes were recognized in the barley genome. Then, phylogenetic analyses classified them into 3 groups, that shared conserved motif compositions. A new motif, D,D,WQxxD was also found, which was responsible for the cellulose synthase. Furthermore, using RNA-seq data, the HvCslFs expression profiles were systematically examined in different tissues and tissue-specific candidates were found. Lastly, interaction network analysis identified 11 CslF genes involved in the interaction network. All together, prsent task provides valuable evidence about the genomic organization and evolutionary relationship of the CslF gene family in barley, and facilitate the functional surveys of CslF genes in barley and beyond.

Characterization of Genetic Diversity and Genome-Wide Association Mapping of Three Agronomic Traits in Qingke Barley (Hordeum Vulgare L.) in the Qinghai-Tibet Plateau.

Barley (Hordeum vulgare L.) is one of the most important cereal crops worldwide. In the Qinghai-Tibet Plateau, six-rowed hulless (or naked) barley, called “qingke” in Chinese or “nas” in Tibetan, is produced mainly in Tibet. The complexity of the environment in the Qinghai-Tibet Plateau has provided unique opportunities for research on the breeding and adaptability of qingke barley. However, the genetic architecture of many important agronomic traits for qingke barley remains elusive. Heading date (HD), plant height (PH), and spike length (SL) are three prominent agronomic traits in barley. Here, we used genome-wide association (GWAS) mapping and GWAS with eigenvector decomposition (EigenGWAS) to detect quantitative trait loci (QTL) and selective signatures for HD, PH, and SL in a collection of 308 qingke barley accessions. The accessions were genotyped using a newly-developed, proprietary genotyping-by-sequencing (tGBS) technology, that yielded 14,970 high quality single nucleotide polymorphisms (SNPs). We found that the number of SNPs was higher in the varieties than in the landraces, which suggested that Tibetan varieties and varieties in the Tibetan area may have originated from different landraces in different areas. We have identified 62 QTLs associated with three important traits, and the observed phenotypic variation is well-explained by the identified QTLs. We mapped 114 known genes that include, but are not limited to, vernalization, and photoperiod genes. We found that 83.87% of the identified QTLs are located in the non-coding regulatory regions of annotated barley genes. Forty-eight of the QTLs are first reported here, 28 QTLs have pleotropic effects, and three QTL are located in the regions of the well-characterized genes HvVRN1, HvVRN3, and PpD-H2. EigenGWAS analysis revealed that multiple heading-date-related loci bear signatures of selection. Our results confirm that the barley panel used in this study is highly diverse, and showed a great promise for identifying the genetic basis of adaptive traits. This study should increase our understanding of complex traits in qingke barley, and should facilitate genome-assisted breeding for qingke barley improvement.

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.