Genome-wide association and targeted transcriptomic analyses reveal loci and candidate genes regulating preharvest sprouting in barley.

KEY MESSAGE: Genome-wide association study of diverse barley genotypes identified loci, single nucleotide polymorphisms and candidate genes that control seed dormancy and therefore enhance resistance to preharvest sprouting. Preharvest sprouting (PHS) causes significant yield and quality loss in barley and it is strongly associated with the level of seed dormancy. This study performed genome-wide association study using a collection of 255 diverse barley genotypes grown over four environments to identify loci controlling dormancy/PHS. Our phenotypic analysis revealed substantial variation in germination index/dormancy levels among the barley genotypes. Marker-trait association and linkage disequilibrium (LD) decay analyses identified 16 single nucleotide polymorphisms (SNPs) and two QTLs associated with dormancy/PHS, respectively, on chromosome 3H and 5H explaining 6.9% to 11.1% of the phenotypic variation. QTL.5H consist of 14 SNPs of which 12 SNPs satisfy the FDR threshold of α = 0.05, and it may represent the SD2 locus. The QTL on 3H consists of one SNP that doesn't satisfy FDR (α = 0.05). Genes harbouring the significant SNPs were analyzed for their expression pattern in the seeds of selected dormant and non-dormant genotypes. Of these genes, HvRCD1, HvPSRP1 and HvF3H exhibited differential expression between the dormant and non-dormant seed samples, suggesting their role in controlling seed dormancy/PHS. Three SNPs located within the differentially expressed genes residing in QTL.5H explained considerable phenotypic variation (≥ 8.6%), suggesting their importance in regulating PHS resistance. Analysis of the SNP marker data in QTL.5H identified a haplotype for PHS resistance. Overall, the study identified loci, SNPs and candidate genes that control dormancy and therefore play important roles in enhancing PHS resistance in barley through marker-assisted breeding.

Ethylene regulates post-germination seedling growth in wheat through spatial and temporal modulation of ABA/GA balance.

This study aimed to gain insights into the molecular mechanisms underlying the role of ethylene in regulating germination and seedling growth in wheat by combining pharmacological, molecular, and metabolomics approaches. Our study showed that ethylene does not affect radicle protrusion but controls post-germination endospermic starch degradation through transcriptional regulation of specific α-amylase and α-glucosidase genes, and this effect is mediated by alteration of endospermic bioactive gibberellin (GA) levels, and GA sensitivity via expression of the GA signaling gene, TaGAMYB. Our data implicated ethylene as a positive regulator of embryo axis and coleoptile growth through transcriptional regulation of specific TaEXPA genes. These effects were associated with modulation of GA levels and sensitivity, through expression of GA metabolism (TaGA20ox1, TaGA3ox2, and TaGA2ox6) and signaling (TaGAMYB) genes, respectively, and/or the abscisic acid (ABA) level and sensitivity, via expression of specific ABA metabolism (TaNCED2 or TaCYP707A1) and signaling (TaABI3) genes, respectively. Ethylene appeared to regulate the expression of TaEXPA3 and thereby root growth through its control of coleoptile ABA metabolism, and root ABA signaling via expression of TaABI3 and TaABI5. These results show that spatiotemporal modulation of ABA/GA balance mediates the role of ethylene in regulating post-germination storage starch degradation and seedling growth in wheat.

Effects of plant growth regulator application on the malting quality of barley.

BACKGROUND: Lodging can negatively affect yield and quality of barley grain. Synthetic plant growth regulators (PGRs) reduce lodging by producing shorter, thicker, and stronger stems. However, the impact of applying PGRs on malting performance of barley is not known. The objective of this work was to assess the effect of application of three PGRs (ethephon, chlormequat chloride, and trinexapac-ethyl) in combination with different seeding rates on the malting quality of barley grown in several locations and years in western Canada. RESULTS: The kernel weight in PGR-treated barley was reduced by 1.7% to 6.5% compared with the nontreated grain. Application of PGRs had no effect on the concentration of proteins and germination energy. Seeding rates significantly affected kernel weight, protein content, and germination index (GI), but no interactions between PGRs and seeding rates were observed. The smaller kernels of ethephon- and trinexapac-treated barley showed good hydration and grain modification during malting, as indicated by high levels of starch-converting enzymes, high Kolbach indices, and low levels of wort ß-glucans. Overall, the fine extract of malt from PGR-treated barley was slightly lower than that of the control malt; however, the extract reduction was statistically significant only for chlormequat- and trinexapac-treated barley. CONCLUSIONS: The application of PGRs had significant effects on kernel plumpness and kernel weight, but the effects of PGR application on the malting quality were generally small and insignificant. The decision of PGRs application on malting barley needs to be considered in combination with potential benefits of PGRs in mitigating lodging and their effects on the agronomic performance of barley. © Her Majesty the Queen in Right of Canada 2019.

Whole-Grain Fiber Composition Influences Site of Nutrient Digestion, Standardized Ileal Digestibility of Amino Acids, and Whole-Body Energy Utilization in Grower Pigs.

BACKGROUND: Variant chemical composition and physical structure of whole grains may change the site of energy digestion from the small to the large intestine. OBJECTIVE: We determined the site of nutrient digestion, standardized ileal digestibility (SID) of amino acids (AAs), and net energy (NE) value of barley cultivars that vary in nutrient composition compared with wheat. METHODS: Ileal-cannulated barrows (27.7 kg initial body weight) were fed diets containing 800 g whole grains/kg alongside a basal and nitrogen-free diet for calculations in a 6 (period) × 7 (diet) Youden square. Diets included 1 of 5 whole grains-1) high-fermentable, high-ß-glucan, hull-less barley (HFB); 2) high-fermentable, high-amylose, hull-less barley (HFA); 3) moderate-fermentable, hull-less barley (MFB); 4) low-fermentable, hulled barley (LFB); and 5) low-fermentable, hard red spring wheat (LFW). Intestine nutrient flow and whole-body energy utilization were tested and explained by using whole-grain and digesta confocal laser scanning. RESULTS: Starch apparent ileal digestibility was 14-29% lower (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW due to the unique embedding of starch within the protein-fiber matrix of HFB and the high amylose content in HFA. Starch hindgut fermentation was 50-130% higher (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW. The SID of indispensable AAs was lower (P < 0.05) in HFB and HFA than in MFB, LFB, and LFW. NE value was 18% higher (P < 0.05) for HFB than for HFA and was not different from MFB, LFB, and LFW. CONCLUSIONS: Whole grains high in fermentable carbohydrates shifted digestion from the small intestine to the hindgut. NE value depended on the concentration of fermentable fiber and starch and digestible protein, ranging from 2.12-1.76 Mcal/kg in barley to 1.94 Mcal/kg in wheat. High-fiber whole grains may be used as energy substrates for pigs; however, the reduced SID of AAs requires titration of indispensable AAs to maintain growth.

Diverging fates of cadmium and glyphosate during pasta cooking.

Durum wheat cultivars with varying abilities to accumulate cadmium were grown and treated in the field with a glyphosate-containing herbicide at different stages of maturity to produce grain with higher and lower concentrations of cadmium (0.066-0.214 mg/kg) and glyphosate (0.474-0.874 mg/kg). The grain was milled, and fractions were analysed for cadmium and glyphosate. The highest concentrations for both cadmium and glyphosate were associated with bran and shorts, although the percentage of total cadmium mass in bran (23-25%) was less than glyphosate (38%). The preparation of dried pasta from semolina and flour milling fractions reduced concentrations by a factor of 1.8 for glyphosate and 1.4 for cadmium. Dried pasta was cooked and analysed along with the cooking water for cadmium and glyphosate at seven-time points from 0 to 15 min. Concentrations of glyphosate in cooked pasta decreased significantly with cooking time; no decrease was observed for cadmium concentrations. Analysis of cooking water demonstrated that glyphosate migrated from pasta to the cooking water. After 15 min of cooking, approximately 73% of the total glyphosate mass had transferred from pasta to cooking water. Over the same time period, only 5% of the total cadmium mass had transferred from pasta to cooking water.

The dynamics of indigenous epiphytic bacterial and fungal communities of barley grains through the commercial malting process in Western Canada.

Microbial activity is present at every step of the malting process. It is, therefore, critical to manage the grain-associated microbial communities for the production of high-quality malts. This study characterized barley and malt epiphytic microbiota by metabarcoding the internal transcribed spacer (ITS) 2 region and the 16S rRNA gene V1-V4 metabarcodes, respectively. We elucidated the changes in the diversity and the compositional and functional changes of the grain-associated microbiota and inferred the impact of such changes on malting efficiency and premature yeast flocculation (PYF) of the commercial malt end product. Through the malting process, the fungal diversity decreased while bacterial community diversity increased. Lactic acid bacteria (LAB) and some mycotoxin-producing fungi (e.g. Fusarium spp.) were found to be significantly enriched in malts. Most potential fungal pathogens, however, did not change in abundance through the malting process. Fungi (e.g. Aureobasidium, Candida) and bacteria (e.g. LAB, Arthrobacter, Brachybacterium) with the potential to generate organic acids or exhibit high hydrolytic enzymatic activity for degrading the endosperm cell walls and storage proteins were detected in greater abundance in kilned malt, suggesting their contribution to malting efficiency. Bacterial and fungal operational taxonomic units (OTUs) associated with PYF-positive malt were mainly identified as Aureobasidium, Candida, and Leuconostoc, while Pleosporaceae, Steptococcus, and Leucobacter were associated with PYF-negative malt. The ecological networks of the field and steeped barley samples were found to be larger and denser, while that of the malt microbiome was smaller and less connected. A decrease in the proportion of negative interactions through the malting process suggested that malting destabilized the microbial networks. In summary, this study profiled the microbiota of commercial malting barley and malt samples in western Canada; the findings expanded our knowledge in the microbiology of malting while providing potential insights regarding the management of microbial-associated problems, such as PYF, in commercial malting.

Sequential solvent extraction and structural characterization of polysaccharides from the endosperm cell walls of barley grown in different environments.

The objective of this study was to examine the composition and molecular structure of the endosperm cell walls (CW) derived from barley grain grown in three environments in Canada, and differing in grain hardness, protein, and total ß-glucan contents. The endosperm CW were isolated from barley, cv. Metcalfe, grown in Davidson, SK (Sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The CW were sequentially extracted with water at 65(o)C, saturated Ba(OH)2, again with water at 25(o)C, and 1M NaOH, resulting in fractions designated WE65, BaE, Ba/WE, and NaE, respectively. The monosaccharide analysis indicated the presence of ß-glucans, arabinoxylans, and small amounts of arabinogalactans, glucomannans, and xyloglucans. Cellulose was detected in the CW remnants. The CW of sample A, exhibiting a lower grain hardness than sample B, contained the lowest amount of ß-glucans, but the highest amount of arabinoxylans and the mannose-containing polysaccharides. The CW of sample C, characterized by very high protein content in the grain, contained the highest amount of ß-glucans and the lowest amount of other polysaccharides. Polysaccharides in the CW of sample B, exhibiting the highest grain hardness, were characterized by the highest weight average molecular weights (Mw). ß-Glucans in the CW of Sample B showed the highest ratio of DP3/DP4 and the longest cellulosic fragments in the polymeric chains. Of the three barley samples, arabinoxylans in the endosperm CW of sample A exhibited the lowest degree of branching, the highest amount of unsubstituted Xyl residues, and the highest ratio of singly to doubly substituted Xylp. The highest water solubility of the CW of sample C was associated with the highest concentration of ß-glucans, the lowest DP3/DP4 ratio, and the lowest Mw of the polymeric constituents. Arabinoxylans with the lowest amount of doubly substituted but the highest amount of unsubstituted xylose residues and long sequences of unsubstituted xylan regions were found in the NaE fractions. The NaE fractions showed a high ratio of →4)-Glcp-(1→ to →3)-Glcp-(1→ linkages and some →4)-Manp-(1→ linkages, indicating a high level of long cellulosic regions in ß-glucan chains and the presence of glucomannans.

Antiglycemic Effect of Water Extractable Arabinoxylan from Wheat Aleurone and Bran.

The studies on the effects of arabinoxylan (AX) polysaccharides on postprandial glucose response have resulted in contrasting results owing to the diversity in AX structures. Four water extractable AX (WEAX) extracts obtained from wheat aleurone and bran were used to investigate (a) the effect of AX on activities of α-amylase and α-glucosidase, (b) influence of AX chemical composition on their inhibition potency, and (c) kinetics of enzyme inhibition. α-Amylase activity was not significantly affected by the presence WEAX fractions regardless of type or concentration. WEAX inhibited α-glucosidase activity only when maltose was used as a substrate but not sucrose. The IC50 values of WEAX (4.88 ± 0.3-10.14 ± 0.5 mg/mL) were highly correlated to ferulic acid content (R = -0.89), arabinose to xylose ratio (R = -0.67), and relative proportions of xylose being unsubstituted (R = 0.69), disubstituted (R = -0.63), and monosubstituted (R = -0.76). The Lineweaver-Burk plot suggested an uncompetitive enzyme inhibition mode. Thus, our results suggest that antiglycemic properties of WEAX may be derived from direct inhibition of α-glucosidase activity.

Antioxidant properties of pearled barley fractions.

Two barley varieties (Falcon and AC Metcalfe) were separated by pearling into seven fractions and subsequently extracted with 80% methanol. The extracts, after solvent removal, were evaluated for their radical scavenging efficacy using Trolox equivalent antioxidant capacity (TEAC). The radical scavenging capacity of the extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC(FL)), and superoxide radical assays and a photoinduced chemiluminescence technique. In both barley varieties the outermost fraction (F1) yielded the highest phenolic content. In general, Falcon had a significantly higher total phenolic content than AC Metcalfe. A similar trend was observed for TEAC, DPPH, and superoxide radical scavenging capacities of the extracts. The contents of water-soluble antioxidants of Falcon and AC Metcalfe were 1.15-12.98 and 2.20-12.25 micromol of Trolox equiv/(g of defatted material), while the corresponding lipid-soluble counterparts varied from 1.44 to 4.70 micromol of alpha-tocopherol equiv/(g of defatted material). Phenolic acids, namely, vanillic, caffeic, p-coumaric, ferulic, and sinapic acids, were identified by HPLC in barley fractions.

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture.

The production of pullulan from beet molasses by a pigment-free strain of Aztreobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by 13C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (Mw = 2.1-4.1 x 10(5)), root mean square of the radius of gyration (R = 30-38 nm), and polydispersity index (Mw/Mn = 1.4-2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8-10.0 (w/v), the solution's rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s(-1) at 20 degrees C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.