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Brassinosteroids (BR) are plant-based steroids which influence several morphogenetic and developmental processes. A barley (Hordeum vulgare L.) genotype Kinai Kyoshinkai-2 (KK-2) carrying the uzu mutation exhibited altered starch granule size distribution. Hybridizing KK-2 with a barley genotype CDC Kendall with bi-modal starch granules produced progeny lines (116, 144 and 168) with almost uni-modal starch granules. Bioassays correlated uzu mutation with defective BR perception. DNA sequence analysis of the BR receptor-1 (BRI-1) gene detected a single-nucleotide A > G substitution at the position 2612 in the kinase domain which resulted in the change of His (CAC) to Arg (CGC) at residue 857 in subdomain IV of the kinase domain of the respective polypeptide. The study focused on the development of barley grain, accumulation of starch and composition influenced by defective BR perception due to the mutation detected in KK-2 and three other barley-breeding lines (116, 144 and 168). Aberrant BRI-1 delayed grain development, amylose synthesis and starch accumulation in the endosperm. The barley breeding lines 116, 144 and 168 carrying the aberrant BRI-1, exhibited altered granule size distribution with significant shift in the diameter maxima, but insignificant differences in amylose concentration. The BRI-1 mutation also altered amylopectin fine structure in both B- and C- type small starch granules, resulting in an increased fraction of short A-type glucan chains (<10 DP) and decreased fraction of B2 chains (25-36 DP) in genotypes carrying the BRI-1 mutation. The results show the influence of BR on barley grain development, starch accumulation, granule size distribution and amylopectin structure., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: The structure of β-glucan influences its use in cereal-based foods and feed. The objective of this study was to determine the effect of environment (E) and genotype (G) on β-glucan fine structure and its genetic control in two-row spring barley with normal starch characteristics., Results: A population of 89 recombinant inbred lines, derived from the cross of two-row spring barley genotypes Merit × H93174006 (H92076F1 × TR238), was characterized for concentration and structure of grain β-glucan in two environments. Results showed that concentrations of β-glucan, DP3, DP4 and DP3 + DP4 were positively correlated with each other, suggesting no preference for DP3 or DP4 subunit production in high- or low-β-glucan lines. The concentrations of β-glucan, DP3, DP4 and DP3:DP4 ratios were significantly influenced by genotype and environment. However, only DP3:DP4 ratio showed a significant effect of G × E interaction. Association mapping of candidate markers in 119 barley genotypes showed that marker CSLF6&#95;4105 was associated with β-glucan concentration, whereas Bmac504 and Bmac211 were associated with DP3:DP4 ratio. Bmac273e was associated with both β-glucan concentration and DP3:DP4 ratio., Conclusion: The grain β-glucan concentration and DP3:DP4 ratio are strongly affected by genotype and environment. Single-marker analyses suggested that the genetic control of β-glucan concentration and DP3:DP4 ratio was linked to separate chromosomal regions on barley genome. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Microscope analysis of starches prepared from 139 barley genotypes identified a Japanese genotype, Kinai Kyoshinkai-2 (KK-2), with altered starch granule size distribution. Compared to normal barley starch, KK-2 produced consistently higher volumes of starch granules with 5-15 μm diameter and reduced volumes of starch granules with >15 μm diameter when grown in different environments. A cross between KK-2 and normal starch cultivar CDC Kendall was made and led to the production of 154 F5 lines with alterations to the normal 7:3:1 distribution for A-:B-:C-type starch granule volumes. Three F5 lines showed unimodal starch granule size distribution due to apparent lack of very small (<5.0 μm diameter) C-type starch granules, but the phenotype was accompanied by reduced grain weight and total starch concentration. Five F5 lines produced a significantly larger population of large (>15 μm diameter) A-type starch granules as compared to normal starch and showed on average a 10:4:1 distribution for A-:B-:C-type starch granule volumes. The unusual starch phenotypes displayed by the F5 lines confirm starch granule size distribution in barley can be genetically altered.

Granule bound starch synthase 1 (GBSS1) accumulation within starch granules and structure of Gbss1 alleles were determined for nine barley ( Hordeum vulgare L.) genotypes producing amylose-free (undetectable), near-waxy (1.6-4.5%), normal (25.8%), and increased (38.0-40.8%) amylose grain starches. Compared to normal starch granules, GBSS1 accumulation was severely reduced in three near-waxy, slightly reduced in two waxy, and slightly elevated in three increased amylose starches. Gbss1 nucleotide sequence analysis for the nine genotypes distinguished them into three Gbss1 groups with several single-nucleotide polymorphisms. A new unique Q312H substitution within GBSS1 was discovered in near-waxy genotype SB94912 with reduced amylose (1.6%) concentration relative to the other two near-waxy lines, CDC Rattan and CDC Candle (4.5%). The two waxy genotype GBSS1 showed a previously described D287V change for CDC Alamo and a new G513W change for CDC Fibar. Both amino acid alterations are conserved residues within starch synthase domains involved in glucan interaction. The increased amylose genotypes showed several unique nucleotide changes within the second and fourth Gbss1 introns, but only SB94893 GBSS1 showed a unique amino acid substitution, A250T in exon 6. The Gbss1 nucleotide differences were used to design genetic markers to monitor Gbss1 alleles in genotypes with various amylose grain starches.

The relationship between starch physical properties and enzymatic hydrolysis was determined using ten different hulless barley genotypes with variable carbohydrate composition. The ten barley genotypes included one normal starch (CDC McGwire), three increased amylose starches (SH99250, SH99073, and SB94893), and six waxy starches (CDC Alamo, CDC Fibar, CDC Candle, Waxy Betzes, CDC Rattan, and SB94912). Total starch concentration positively influenced thousand grain weight (TGW) (r(2) = 0.70, p < 0.05). Increase in grain protein concentration was not only related to total starch concentration (r(2) = -0.80, p < 0.01) but also affected enzymatic hydrolysis of pure starch (r(2) = -0.67, p < 0.01). However, an increase in amylopectin unit chain length between DP 12-18 (F-II) was detrimental to starch concentration (r(2) = 0.46, p < 0.01). Amylose concentration influenced granule size distribution with increased amylose genotypes showing highly reduced volume percentage of very small C-granules (<5 μm diameter) and significantly increased (r(2) = 0.83, p < 0.01) medium sized B granules (5-15 μm diameter). Amylose affected smaller (F-I) and larger (F-III) amylopectin chains in opposite ways. Increased amylose concentration positively influenced the F-III (DP 19-36) fraction of longer DP amylopectin chains (DP 19-36) which was associated with resistant starch (RS) in meal and pure starch samples. The rate of starch hydrolysis was high in pure starch samples as compared to meal samples. Enzymatic hydrolysis rate both in meal and pure starch samples followed the order waxy > normal > increased amylose. Rapidly digestible starch (RDS) increased with a decrease in amylose concentration. Atomic force microscopy (AFM) analysis revealed a higher polydispersity index of amylose in CDC McGwire and increased amylose genotypes which could contribute to their reduced enzymatic hydrolysis, compared to waxy starch genotypes. Increased β-glucan and dietary fiber concentration also reduced the enzymatic hydrolysis of meal samples. An average linkage cluster analysis dendrogram revealed that variation in amylose concentration significantly (p < 0.01) influenced resistant starch concentration in meal and pure starch samples. RS is also associated with B-type granules (5-15 μm) and the amylopectin F-III (19-36 DP) fraction. In conclusion, the results suggest that barley genotype SH99250 with less decrease in grain weight in comparison to that of other increased amylose genotypes (SH99073 and SH94893) could be a promising genotype to develop cultivars with increased amylose grain starch without compromising grain weight and yield.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.