Selection of favorable alleles of genes controlling flowering and senescence improves malt barley quality.

Malt barley (Hordeum vulgare L.) is an important cash crop with stringent grain quality standards. Timing of the switch from vegetative to reproductive growth and timing of whole-plant senescence and nutrient remobilization are critical for cereal grain yield and quality. Understanding the genetic variation in genes associated with these developmental traits can streamline genotypic selection of superior malt barley germplasm. Here, we determined the effects of allelic variation in three genes encoding a glycine-rich RNA-binding protein (HvGR-RBP1) and two NAC transcription factors (HvNAM1 and HvNAM2) on malt barley agronomics and quality using previously developed markers for HvGR-RBP1 and HvNAM1 and a novel marker for HvNAM2. Based on a single-nucleotide polymorphism (SNP) in the first intron, the utilized marker differentiates NAM2 alleles of low-grain protein variety 'Karl' and of higher protein variety 'Lewis'. We demonstrate that the selection of favorable alleles for each gene impacts heading date, senescence timing, grain size, grain protein concentration, and malt quality. Specifically, combining 'Karl' alleles for the two NAC genes with the 'Lewis' HvGR-RBP1 allele extends grain fill duration, increases the percentage of plump kernels, decreases grain protein, and provides malt quality stability. Molecular markers for these genes are therefore highly useful tools in malt barley breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01331-7.

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

KEY MESSAGE: 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 1 KB upstream of translation start site, including an ~ 400 bp 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 (3 days) 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.

Estimation of the net energy value of barley for finishing beef steers.

The objective of this study was to identify barley grain characteristics measured by laboratory procedures that could be used to predict barley energy content for finishing beef steers. Twenty-eight different barley genotypes were evaluated including 18 cultivars and 10 experimental lines. Laboratory analysis of barley samples included bulk density, particle size, N, ADF, starch, and ISDMD (in situ DM disappearance after 3 h of ruminal incubation). Animal performance data (BW, DMI, ADG, steer NEm, and NEg requirements) were collected from 26 feedlot experiments conducted in Montana and Idaho during a 10-yr period and were used to estimate barley NEm and NEg content. A total of 80 experimental units were available with each experimental unit being a diet mean from an individual feedlot experiment. Fifty-eight of the 80 experimental units were randomly selected and used in the development data set and the remaining 22 experimental units were used in the validation data set. Forward, backward, and stepwise selection methods were used to identify variables to be included in regression equations for NEm using PROC REG of SAS. Barley samples in the model development data set represented a wide range in concentrations (DM basis): N (1.6% to 2.8%), ISDMD (25.7% to 58.7%), ADF (3.6% to 8.0%), starch (44.1% to 62.4%), particle size (1,100 to 2,814 µm), and bulk density (50.8 to 69.4 kg/hL). The barley grain characteristics of particle size, ISDMD, starch, and ADF were the most important variables in six successful models (R 2 = 0.48 to 0.60; P = 0.001). The six prediction equations gave mean predicted values for NEm ranging from 1.99 to 2.05 Mcal/kg (average 2.04 Mcal/kg; 0.45% CV). The mean actual NEm values from animal performance trials ranged from 1.75 to 2.48 Mcal/kg (average 2.03 Mcal/kg; 6.5% CV). The mean bias or difference in predicted vs. actual values ranged from -0.001 to 0.005 Mcal/kg. Barley NEg values calculated from animal performance ranged from 1.13 to 1.78 Mcal/kg (average 1.39 Mcal/kg; 8.4% CV). Average predicted barley NEm and NEg were 0.02 and 0.01 Mcal/kg less, respectively, than the 2.06 Mcal/kg NEm and 1.40 Mcal/kg NEg reported by NRC. Barley NE can be predicted from simple laboratory procedures which will aid plant breeders developing new feed varieties and nutritionists formulating finishing rations for beef cattle.

Structural Basis of Phosphatidic Acid Sensing by APH in Apicomplexan Parasites.

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular parasites that belong to the phylum of Apicomplexa and cause major human diseases. Their access to an intracellular lifestyle is reliant on the coordinated release of proteins from the specialized apical organelles called micronemes and rhoptries. A specific phosphatidic acid effector, the acylated pleckstrin homology domain-containing protein (APH) plays a central role in microneme exocytosis and thus is essential for motility, cell entry, and egress. TgAPH is acylated on the surface of the micronemes and recruited to phosphatidic acid (PA)-enriched membranes. Here, we dissect the atomic details of APH PA-sensing hub and its functional interaction with phospholipid membranes. We unravel the key determinant of PA recognition for the first time and show that APH inserts into and clusters multiple phosphate head-groups at the bilayer binding surface.

Mapping of HKT1;5 Gene in Barley Using GWAS Approach and Its Implication in Salt Tolerance Mechanism.

Sodium (Na+) accumulation in the cytosol will result in ion homeostasis imbalance and toxicity of transpiring leaves. Studies of salinity tolerance in the diploid wheat ancestor Triticum monococcum showed that HKT1;5-like gene was a major gene in the QTL for salt tolerance, named Nax2. In the present study, we were interested in investigating the molecular mechanisms underpinning the role of the HKT1;5 gene in salt tolerance in barley (Hordeum vulgare). A USDA mini-core collection of 2,671 barley lines, part of a field trial was screened for salinity tolerance, and a Genome Wide Association Study (GWAS) was performed. Our results showed important SNPs that are correlated with salt tolerance that mapped to a region where HKT1;5 ion transporter located on chromosome four. Furthermore, sodium (Na+) and potassium (K+) content analysis revealed that tolerant lines accumulate more sodium in roots and leaf sheaths, than in the sensitive ones. In contrast, sodium concentration was reduced in leaf blades of the tolerant lines under salt stress. In the absence of NaCl, the concentration of Na+ and K+ were the same in the roots, leaf sheaths and leaf blades between the tolerant and the sensitive lines. In order to study the molecular mechanism behind that, alleles of the HKT1;5 gene from five tolerant and five sensitive barley lines were cloned and sequenced. Sequence analysis did not show the presence of any polymorphism that distinguishes between the tolerant and sensitive alleles. Our real-time RT-PCR experiments, showed that the expression of HKT1;5 gene in roots of the tolerant line was significantly induced after challenging the plants with salt stress. In contrast, in leaf sheaths the expression was decreased after salt treatment. In sensitive lines, there was no difference in the expression of HKT1;5 gene in leaf sheath under control and saline conditions, while a slight increase in the expression was observed in roots after salt treatment. These results provide stronger evidence that HKT1;5 gene in barley play a key role in withdrawing Na+ from the xylem and therefore reducing its transport to leaves. Given all that, these data support the hypothesis that HKT1;5 gene is responsible for Na+ unloading to the xylem and controlling its distribution in the shoots, which provide new insight into the understanding of this QTL for salinity tolerance in barley.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

Quantitative Trait Loci Associated with the Tocochromanol (Vitamin E) Pathway in Barley.

The Genome-Wide Association Studies approach was used to detect Quantitative Trait Loci associated with tocochromanol concentrations using a panel of 1,466 barley accessions. All major tocochromanol types- α-, ß-, δ-, γ-tocopherol and tocotrienol- were assayed. We found 13 single nucleotide polymorphisms associated with the concentration of one or more of these tocochromanol forms in barley, seven of which were within 2 cM of sequences homologous to cloned genes associated with tocochromanol production in barley and/or other plants. These associations confirmed a prior report based on bi-parental QTL mapping. This knowledge will aid future efforts to better understand the role of tocochromanols in barley, with specific reference to abiotic stress resistance. It will also be useful in developing barley varieties with higher tocochromanol concentrations, although at current recommended daily consumption amounts, barley would not be an effective sole source of vitamin E. However, it could be an important contributor in the context of whole grains in a balanced diet.

Expression and functional analysis of NUCLEAR FACTOR-Y, subunit B genes in barley.

NUCLEAR FACTOR-Y, subunit B (NF-YB) comprises a multigene family in plants and has been shown to play important roles in growth, development, and response to environmental stress. In this study, five NF-YBs containing the full-length coding region were obtained from barley (Hordeum vulgare) through database sequence analysis, cloning, and sequencing. Sequence alignment and phylogenetic analysis showed that HvNF-YB3 and HvNF-YB1 were clustered with NF-YB2 and NF-YB3 in Arabidopsis, suggesting these NF-YBs are evolutionary and functionally related. To test this hypothesis, HvNF-YB3 and HvNF-YB1 were overexpressed in Arabidopsis. Overexpression of HvNF-YB1 greatly promoted early flowering in Arabidopsis, supporting that HvNF-YB1may have conserved gene function in flowering time control as NF-YB2 and NF-YB3 in Arabidopsis. Overexpression of HvNF-YB3 in Arabidopsis had no effect on flowering time. An analysis of barley single-nucleotide polymorphism (SNP) data, however, revealed a significant association between an HvNF-YB3 SNP and heading date. While it is unknown whether HvNF-YB3 directly contributes to heading date regulation, the results implied that HvNF-YB3 may also have conserved function in flowering time (heading date in barley) control. Further studies are needed to directly verify these gene functions in barley. Barley NF-YBs showed different expression patterns associated with tissue types, developmental stages, and response to different stress treatments, suggesting that barley NF-YBs may be involved in other physiological processes.

Effect of population size and unbalanced data sets on QTL detection using genome-wide association mapping in barley breeding germplasm.

Over the past two decades many quantitative trait loci (QTL) have been detected; however, very few have been incorporated into breeding programs. The recent development of genome-wide association studies (GWAS) in plants provides the opportunity to detect QTL in germplasm collections such as unstructured populations from breeding programs. The overall goal of the barley Coordinated Agricultural Project was to conduct GWAS with the intent to couple QTL detection and breeding. The basic idea is that breeding programs generate a vast amount of phenotypic data and combined with cheap genotyping it should be possible to use GWAS to detect QTL that would be immediately accessible and used by breeding programs. There are several constraints to using breeding program-derived phenotype data for conducting GWAS namely: limited population size and unbalanced data sets. We chose the highly heritable trait heading date to study these two variables. We examined 766 spring barley breeding lines (panel #1) grown in balanced trials and a subset of 384 spring barley breeding lines (panel #2) grown in balanced and unbalanced trials. In panel #1, we detected three major QTL for heading date that have been detected in previous bi-parental mapping studies. Simulation studies showed that population sizes greater than 384 individuals are required to consistently detect QTL. We also showed that unbalanced data sets from panel #2 can be used to detect the three major QTL. However, unbalanced data sets resulted in an increase in the false-positive rate. Interestingly, one-step analysis performed better than two-step analysis in reducing the false-positive rate. The results of this work show that it is possible to use phenotypic data from breeding programs to detect QTL, but that careful consideration of population size and experimental design are required.

Genetic analysis of the function of major leaf proteases in barley (Hordeum vulgare L.) nitrogen remobilization.

Most of the nitrogen harvested with the seeds of annual crops is remobilized and retranslocated within the plant between anthesis and plant death. While chloroplasts contain most of the reduced nitrogen present in photosynthetically active leaf cells, the (major) pathway(s) involved in the degradation of their proteins prior to the retranslocation of the resulting amino acids are unknown. In this study, a population of 146 recombinant inbred barley lines (RIL), derived from the cross between two varieties with a highly inheritable difference in grain protein concentration, was used to map quantitative trait loci (QTL) for leaf amino-, carboxy- and endopeptidase activities relative to previously determined QTL for grain protein, leaf N storage, and remobilization. The results strongly suggested that major endopeptidases, assayed at both acidic and slightly alkaline pH values (favouring vacuolar and extravacuolar enzymes, respectively) are not instrumental in leaf N remobilization or the control of grain protein accumulation. Similarly, QTL determined for aminopeptidases (relative to QTL for N remobilization) indicated no functional role for the enzyme(s) assayed in plant N recycling. By contrast, careful evaluation of QTL data suggested that one or several carboxypeptidase isoenzymes may be involved in this physiologically and economically important process. As these proteases (in contrast to aminopeptidases) have previously been localized in vacuoles, this result appears intriguing. These data, while shedding new light on an old problem, also indicate that the described approach may prove useful in evaluating the functional roles of additional (not assayed in this study) proteolytic systems in whole-plant nitrogen recycling.

Mapping of QTL associated with nitrogen storage and remobilization in barley (Hordeum vulgare L.) leaves.

Nitrogen uptake and metabolism are central for vegetative and reproductive plant growth. This is reflected by the fact that nitrogen can be remobilized and reused within a plant, and this process is crucial for yield in most annual crops. A population of 146 recombinant inbred barley lines (F(8) and F(9) plants, grown in 2000 and 2001), derived from a cross between two varieties differing markedly in grain protein concentration, was used to compare the location of QTL associated with nitrogen uptake, storage and remobilization in flag leaves relative to QTL controlling developmental parameters and grain protein accumulation. Overlaps of support intervals for such QTL were found on several chromosomes, with chromosomes 3 and 6 being especially important. For QTL on these chromosomes, alleles associated with inefficient N remobilization were associated with depressed yield and higher levels of total or soluble organic nitrogen during grain filling and vice versa; therefore, genes directly involved in N recycling or genes regulating N recycling may be located on these chromosomes. Interestingly, the most prominent QTL for grain protein concentration (on chromosome 6) did not co-localize with QTL for nitrogen remobilization. However, QTL peaks for nitrate and soluble organic nitrogen were detected at this locus for plants grown in 2001 (but not in 2000). For these, alleles associated with low grain protein concentration were associated with higher soluble nitrogen levels in leaves during grain filling; therefore, gene(s) found at this locus might influence the nitrogen sink strength of developing barley grains.

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare).

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.