Annual Progress Report on Malting Barley Research March, 2007

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Introduction There is general agreement that in germinating barley, the degradation of starch and production of fermentable sugars results from the concerted action of four carbohydrases - α-amylase, β-amylase, α-glucosidase and limit dextrinase. Compared to the extensive body of knowledge about barley α- and β-amylases, there is only rudimentary knowledge available about α-glucosidase and limit dextrinase. This research was conducted to increase our fundamental knowledge of α-glucosidases in germinating barley seeds by examining the genes that encode these enzymes and by characterizing the functional properties of the α-glucosidases that are expressed in germinating barley seeds. α-Glucosidases are a diverse group of enzymes that catalyze several reactions and are capable of using a wide variety of substrates. One feature that all α- glucosidases share is the ability to hydrolytically remove the terminal monosaccharide from the non-reducing end of a carbohydrate. Plant α-glucosidases have been reported to hydrolyze di, - oligo- and polysaccharides, soluble starch, starch granules with intact crystalline structure, and the carbohydrate structures attached to glycoproteins. They can hydrolyze α-D-glycosidic bonds with –1,1-, -1,2-, -1,3-, 1,4-, and -1,6- linkages. Plant α-glucosidases can hydrolyze glycosidic bonds between glucose residues, between glucose and fructose residues, between glucose and mannose residues, and between xylose residues. Additionally, they can catalyze hydrolytic, condensation and transglycosylation reactions. All plant α-glucosidases sequenced as of this time are categorized as members of the glycosyl hydrolase family 31. In most cases this categorization is based upon sequence similarity alone as enzyme function has not yet been determined. All members of the glycosyl hydrolase family 31 share the same active site sequence and have at least one additional highly conserved region. The first α-glucosidase cloned from a plant was in 1996 when Dr. Ron Skadsen’s lab cloned Agl1 from barley (Tibbot and Skadsen, 1996). Since that time, other genes encoding α-glucosidases have been identified in plants although, in almost all cases, more is known about the gene than the 116
enzyme it encodes. We found that seven α-glucosidase genes are expressed in barley seeds that have been germinated for three days (see 2004-2005 progress report). Only one of these, Agl1, has been previously reported (Tibbot and Skadsen, 1996). Four of these seven genes, including Agl1, were abundantly expressed and three had low levels of expression. The potential roles of the novel α-glucosidases were unknown as were any characteristics of their gene expression or of the expressed proteins. In this report we present results of studies to characterize expression of Agl2 , named AglX in the 2004-2005 progress report, and compare its expression to that of Agl1. Methods Quantitative RT-PCR (qtRT-PCR) Total RNA was extracted from 3 – 9 individual samples using Plant RNA reagent from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Purified RNA was treated with DNAse using RNeasy mini kits (Qiagen, Valencia, CA) according to the manufacturer’s instructions. For cDNA synthesis, three independent reactions were undertaken for each tissue, 1 µg of total RNA for each reaction was reverse- transcribed using Superscript III (Invitrogen) and random hexamers according to the manufacturer’s instructions. For qtRT-PCR, 1 µL of a 1:5 dilution of the cDNA was used in a reaction which also consisted of 5 µL of SYBR premix ExTaq (Takara mirus bio, Madison, WI), 0.4 µL each of the forward and reverse primers at 5 µM each, 0.2 µL of ROX reference dye II, and 3 µL of water. Reactions were performed on an Applied Biosystems 7300 Real-Time PCR system using the following schedule: 10 s at 95°C followed by 40 cycles of 5 s at 95°C then 31 s at 60°C. A melting curve was obtained from the product at the end of the amplification by heating from 70°C to 99°C. Cloning of the putative promoters Genomic DNA was purified from 7 day old seedling leaves using DNeasy Mini kits (Qiagen) according to the manufacturer’s instructions. Purified genomic DNA was digested with the restriction enzymes to create blunt-end fragments that were then 117
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Annual Pro
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-i- INTRODUCTION The March 2007 <st
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USDA-ARS NORTHERN CROP SCIENCE LABO
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Spring Regional over two years at D
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Table 1. 2006 UCD Two-Rowed Malting
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environments was practiced, 2) elit
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Table 4. Selected six-rowed lines c
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Other Barley Research: Feed Program
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Executive Summary AMBA Prog
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Table 1. Agronomic performance of M
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Lines M122 and M124 were rated sati
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EARLY GENERATION LINES Forty F5 pop
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have evaluated a PCR based marker,
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Edward Schiefelbein, Research Scien
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small grains pathology input to thi
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Project Personnel Small Grains Path
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Investigations on Barley Diseases a
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Minnesota barley disease survey and
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pathotypes at the seedling stage in
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germplasm with the highest level of
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Mirlohi, A., Brueggeman, R., Steffe
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Developing Improved Malt Barley Var
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Table 2. Montana’s Statewide Tria
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As a Barley CAP collaborator, our C
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3.2. A barley nursery composed of d
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5. Related Barley Projects Barley P
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Barley Breeding - Advanced Testing
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Table 3. Agronomic comparisons of N
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Preliminary Malting Barley Yield Tr
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dwarf genotypes with the convention
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STUDIES ON BARLEY DISEASES AND THEI
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inoculated plots. The inoculum used
Page 65 and 66: much of North Dakota in 2006, few f
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Page 69 and 70: oot rot. In collaboration with Scot
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Page 79 and 80: Beta-limit dextrin Alpha-Amylase Al
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Page 87 and 88: 2. Other Research (A) Predicting Se
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Page 91 and 92: PUBLICATIONS (2006-07) Schwarz, P.B
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Page 101 and 102: Kevin Hicks, Research Leader, USDA-
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Page 107 and 108: Table 1. 2006/2007 Oregon winter ba
Page 109 and 110: Table 4. Agronomic data for OSU win
Page 111 and 112: MALTING QUALITY ANALYSIS OF NEW BAR
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Page 115: 2006-2007 Progress
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Page 121 and 122: found in plant cell walls. The degr
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Page 125: Charles Karpelenia, technician Joe
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Annual Progress Report on Malting Barley Research March, 2007

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