CHARACTERIZATION OF BARLEY (Hordeum vulgare L.) WITH ALTERED CARBOHYDRATE COMPOSITION
The concentrations of storage constituents in barley (Hordeum vulgare L.) grain impact its use for malt, food, feed, and fuel. Characterization of major grain constituents such as carbohydrates (total dietary fiber, beta-glucan, starch and its components amylose and amylopectin, starch granule size and physical properties), protein and fat and their interaction during starch enzymatic hydrolysis will facilitate diversification of barley grain utilization for improved malt, food or feed applications. Improved understanding of genes and their structure associated with beneficial grain constituents will help in barley improvement program to develop novel cultivars with desirable grain constituents. Nine barley genotypes with varying amount of amylose in grain starch were used to: (i) determine grain carbohydrate (dietary fiber, beta-glucan and starch), protein and fat concentrations, and starch composition and structure on its enzymatic hydrolysis; (ii) characterize allelic variation in granule bound starch synthase 1 (Gbss1) and starch branching enzyme 2b (Sbe2b) to understand the molecular basis for variation in grain starch amylose concentration. Nine barley genotypes included, one normal (~25% amylose), three near waxy (< 5% amylose), two waxy (undetected amylose) and three increased amylose (> 38%) starch. Total starch concentration showed significant positive correlation with thousand grain weight (TGW) and negative correlation with amylose, total dietary fiber and protein concentrations. Starch granule sizes varied with percent amylose where the increased amylose genotypes produced significantly lower volume percentage of C-type granules (< 5 µm diameter) but significantly higher medium sized B granules (5-15 µm diameter). Amylopectin chain length distribution (CLD) was categorized into F-I type chains (6-11 dp); F-II (12-18 dp); F-III (19-36 dp) and F-IV chains (37-45 dp). 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 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 enzymatic hydrolysis of meal samples. 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 amylopectin FIII (19-36 DP) fraction. Gbss1 nucleotide sequences revealed considerable heterogeneity, with three genotypes with severely reduced GBSSI proteins and low amylose concentration had a 403 bp deletion in the promoter region. One previously described amino acid substitution D287V in CDC Alamo was confirmed and two new amino acid substitutions, G513W in CDC Fibar and Q312H in near waxy genotype SB94912 were identified as the likely causes of inactive GBSSI resulting in no amylose in starch granules. In the increased amylose genotype SB94983 A250T substitution was also observed, which can alter GBSS 1 enzyme specificity and could be a possible reason for increased amylose concentration. To facilitate studies of GbssI expression, positive assays for four allele variants were developed in this study. These markers may also be useful for monitoring introgression of respective GbssI alleles in barley improvement programs. The barley Sbe2b gene sequence analysis of the normal (CDC McGwire), near waxy (SB94912) and increased amylose (SH99250) genotypes, revealed 22 exons and 21 introns. The three Sbe2b alleles showed 21 polymorphic sites, present only in the introns, predicting that the processed SBE IIb transcripts to be identical in the three genotypes. The longest second intron of Sbe2b, which is known to regulate promoter activity in barley was identical between SH99250 and CDC McGwire, but SB94912 allele differed at 12 sites. The remaining nine polymorphic sites were present in introns 17, 18, 19 and 21. It is speculated that allelic variation in Sbe 2b second intron could be a reason for increased amylose concentration in SB94912. In summary reduction in amylose concentration can be due to changes in GBSS1 polypeptide, but the reasons for increased amylose starch are not yet clear. The DNA polymorphisms identified in this study can be used in a marker assisted selection strategy to follow the introgression of respective alleles in a barley improvement program.
Barley, starch, amylose, amylopectin, chain length distribution beta-glucan, BGSSI, SBEIIb
Doctor of Philosophy (Ph.D.)