Thermostability variation in alleles of barley beta-amylase

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Journal of CerealScience28 (1998) 301-309 Article No. jc980209

Thermostability Variation in Alleles of Barley

Beta-Amylase J. K. Eglinton, P. Langridgeand D. E. Evans The Univers#yof Adelaide, Waite Campus, Departmentof PlantScience, Glen Osmond, SA 5064, Australia Received 19 May, 1998 ABSTRACT Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sdl, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmyl-Sd3) in an accession of wild barley Hordeum vu~garessp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have Ts0 temperatures of 56-8°C for the Sd2L enzyme, 58"5°C for the Sdl enzyme, and 60'8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley. © 1998 Academic Press

K(ywords: beta-amylase, thermostability, mapping, fermentability.

INTRODUCTION

Beta-amylase (1,4-0t-glucan maltohydrolase; EC 3.2.1.2) is a key enzyme in the degradation of starch in germinated barley (Hordeum vulgare L.), and catalyses the liberation of [3,maltose from the non-reducing ends of 1,4-0t-glucans. Beta-amylase is synthesised during the development of the barley grain j, in contrast to other major hydrolytic enzymes which are synthesised de novo during germination. The primary structure of barley beta-amylase has been deduced from full length cDNA clones2'3. In mature grain the enzyme consists of a single

ABBREVIATIONSUSED: A A L = a p p a r e n t attenuation limit; EBC = E u r o p e a n Brewing Convention; I O B = Institute o f Brewing; I E F = i s o e l e c t r i c focusing; B S A - - b o v i n e serum albumin; Q T L = q u a n t i t a t i ' v e trait loci; S D S = sodium dodecyl sulphate; P A G E = p o l y a c r y l a m i d e gel electrophoresis; D P = diastatic power. 0733-5210/98/060301 + 09 $30.00/0

polypeptide chain of Mr 59"7 kDa which is converted during germination to an isoform of Mr 56"0 kDa, and the reduction in molecular weight is accompanied by an increase in pI of the complex band pattern of beta-amylase detected by isoelectric focusing (IEF) 4. The conversion of isoforms is mediated by limited proteolysis of the C-terminal region of the enzyme by malt endopeptidase 5. Two alleles for the beta-amylase gene (Bmyl) have been identified in H. vulgare by electrophoretic techniques and are termed Bmyl-Sdl and BmylSd26'7. The corresponding enzymes, referred to as Sdl and Sd2, have the same apparent molecular mass and exhibit similar patterns of charge heterogeneity, although the Sd2 enzyme pattern is more basic. The two forms of beta-arfiylase can be differentiated by IEF in both the intact enzyme and the proteolytically cleaved form of the enzyme generated during germination 7. Starch hydrolysis during germination is achieved primarily by the action of four major © 1998 Academic Press

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enzymes collectively measured as diastatic power (DP). Internal cleavage of starch molecules by alpha-amylase yields branched and linear dextrins. The a-l,6 branch points are removed by limit dextrinase and the exo-action of beta-amylase degrades the linear dextrins to maltose. Gt-Glucosidase acts on both dextrins and raw starch granules releasing glucose, having a synergistic effect with alpha-amylasea. The hydrolysis of starch produces fermentable sugars required for yeast nutrition in brewing. The yield of fermentable sugars affects the level of alcohol produced, and is a critical quality parameter for brewing referred to as fermentability. The level of fermentable sugar is typically assessed in malt extracts by determining the change in specific gravity after a small scale fermentation, and is referred to as the apparent attenuation limit (AAL) 9'1°. Beta-amylase is a relatively thermolabile enzyme compared to alpha-amylase and limit dextrinase ll. During brewing, extraction of soluble malt components (mashing) is performed either isothermally at approximately 65°C, or using a ramped temperature profile from approximately 48°C to 70°C. These temperatures are required for starch gelatinisation which is necessary for rapid and complete starch degradation. Beta-amylase retains maximum activity up to 55°C, but its stability diminishes rapidly as temperature increases above 55°C ~-~.The potential for barley beta-amylase with enhanced thermal stability has been recognised with the construction of a recombinant barley betaamylase with increased thermostability by random and site directed mutagenesis ~3. In this study, natural variation in beta-amylase thermostability was investigated, and the genetic and biochemical basis for this variation determined. The impact of beta-amylase variation on wort fermentability and its significance to brewing was examined, and the application of this work to barley breeding considered.

MATERIALSAND METHODS Barley Unless otherwise stated barley varieties were grown in experimental plots during the 1996/ 1997 season at Weetulta, South Australia. H. spontaneum and Clipper were grown in a glasshouse. Samples of Universe and Hiproly were obtained from the Australian Winter Cereals Collection, Tamworth, NSW. Haruna Nijo barley was from

the Waite barley collection. The accession C.P.I. 77146/33 of H. spontaneum was provided by Dr A. H.D. Brown, CSIRO Division of Plant Industry, Canberra, Australia.

Malt Micromahing and standard malt quality analyses were performed by the Waite Malting Quality Evaluation Laboratory. Samples of 100 g of the barley variety were micromalted in duplicate in an automated micromalting unit (Phoenix Systems, Adelaide, Australia) employing a standard malting program 7. Samples of 42 commercial malts with fermentability determined by the EBC method 1° and DP by the IOB method 9 were provided by Barrett Burston Malting Company Pty Ltd, Melbourne, Australia. The malts were made from the varieties Parwan, Schooner, Franklin, Galaxy and Arapiles. Haruna Nijo malt was supplied by Mr Ken Fukuda, PBRL, Sapporo Breweries Ltd, Japan.

Isoelectric focusing (IEF) Extracts were prepared from 20 mg flour extracted for 30 min with 1"0 mL i % glycine containing 143 mM 2-mercaptoethanol. IEF was performed essentially as described 7, using a non-linear gradient of pH 4-6"5. Beta-amylase activity was detected by starch staining5 and pI values were determined relative to native IEF markers (BioRad, Richmond, California).

Beta-amylaseactivity Seed and malt extracts were prepared by incubating 100 mg samples with 1"0 mL extraction buffer containing 100 mM maleic acid, 1 mM disodium EDTA, 0"02% sodium azide and 1 m g / mL BSA (Sigma, St Louis, U.S.A.) for 2 h at room temperature. Extracts were centrifuged at 10 000 g for 10 min and the supernatant retained. Enzyme activity was determined using the substrate p-nitrophenyl maltopentaoside ~4 (PNPG5, Megazyme, Ireland). One unit of beta-amylase activity is defined as the amount of enzyme required to release 1 ~tmol of p-nitrophenol per min in the presence of excess 0t-glucosidase under the defined assay conditions.

Barley beta-amylase thermostability

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Thermal inactivation Extracts were divided into aliquots for initial assay and heat treatment. After heating, samples were chilled on ice and centrifuged at 10 000g for 5 min at 4 °C. The rate of enzyme inactivation was monitored both by incubating samples for 5 min at temperatures from 40 to 65 °C, and also during a time course at 60 °C. Relative thermostability was also determined using a two-point assay where initial activity and residual activity after 10 min incubation at 60 °C were measured. Purified enzymes were heat treated in extraction buffer supplemented with 2"8 m g / m L BSA. This was the mean protein concentration of the crude extracts as determined by the Bradford assay (BioRad, Richmond, California). Beta-amylase was added at a rate of 100 gg/mL.

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F i g u r e 1 Isoelectric focusing of barley extracts starch stained to show beta-amylase activity. 5 ~tL barley extract loaded. Lane 1, Sdl; 2, Sd2; 3, Sd3.

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Genetic analysis A population of 95 double haploid lines from the cross Galleon X Haruna Nijo (generated through anther culture by Dr S. Logue, University of Adelaide) were analysed in duplicate for beta-amylase activity and relative thermostability using the two-point assay described above. Data from the mapping population was applied to a linkage map of barley 15 using MapManager Q T (Version 19) software'6 and the Kosambi mapping function~k Associations between molecular markers and Q.TLs for beta-amylase thermostability were tested using interval analysis ~8. A graphical display of the Q.TL associations was generated using Q.GENE '9.

Beta-amylasepurification Beta-amylase was purified from barley grain essentially as previously described 4, with the modifications for stable storage at --20 °Ck Sdl betaamylase was purified from Franklin, Sd2 from Schooner, and Sd3 from accession 77146/33 of

H. spontaneum.

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F i g u r e 2 Irreversible thermal inactivation of beta-amylase in barley extracts incubated at 60°C. - - O - - , Sdl; - - I N - - , Sd2-H; - - m - - , Sd2-L; - - A - - , Sd3. Values are the mean of five barley varieties and the standard error of the mean is shown.

responding enzymes can be differentiated by IEF 7. An additional beta-amylase allele was identified in an accession of wild barley. H. spontaneum, and named Bmyl-Sd3 (Fig. 1). This form of the enzyme exhibits charge heterogeneity characteristic of barley beta-amylase, and consists of the same general IEF band pattern as the other two forms, shifted to a more basic pI. The major bands of the Sd3 pattern are at pH 6" 1, 6"05, 5"8 and a double band at pH 5"5.

RESULTS Examination of two beta-amylase alleles from cultivated barley and an additional allele from

Identification of variation in thermostability of beta-amylaseactivity in seed extracts

Two beta-amylase alleles, Bmyl-Sdl and -Sd2, are found in different cultivated barleys and the c o r -

The rate of irreversible thermal inactivation of Sdl, Sd2 and Sd3 forms of beta-amylase were analysed in barley extracts (Fig. 2). The rate of

H. spontaneum

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thermal inactivation was independent of initial enzyme activity, therefore beta-amylase activity is expressed as a percentage of initial activity. Varieties with the Bmyl-Sdl allele display an intermediate level of thermal stability. The Bmyl-Sd2 varieties divide into three discrete groups, with high (Sd2H) and low (Sd2L) relative beta-amylase thermostabilities. In addition, the varieties Forrest and Morrell exhibit an intermediate level of stability in grain extracts (data not shown). The extracts from the wild barley H. vulgate ssp. spontaneum carrying the Bmyl-Sd3 allele show the highest level of thermostability.

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Variation in thermostabiliq persists through germination and the associated COOH-terminal proteolysis of beta-amylase Barley beta-amylase undergoes proteolytic cleavage during germination, with between 38 and 42 amino acids removed from the C-terminus of the enzym e4. To determine if the variation in enzyme thermostability persists in the forms of beta-amylase found in malt, ten barley varieties representing the range of stability within the Bmyl-Sdl and -Sd2 types were micromalted, and the level of residual enzyme activity after thermal inactivation determined. The wild barley accession carrying the Bmyl-Sd3 allele is not included due to poor germination rates under micromalting conditions. Figure 3 shows the variation in enzyme stability detected in barley extracts is maintained in the malt extracts. The Bmyl-Sd2 varieties were divided into the same high and low stability groups, with the level of residual activity ranging from approximately 25% to less than 2%, respectively. An intermediate level of residual beta-amylase was found in malt extracts of the Sd2 variety Morrell and the Bmyl-Sdl varieties, again consistent with the barley grain extracts.

QTL mapping of variation in beta-amylase activity and thermostabiliq The relative thermostability of beta-amylase was mapped to determine if the difference in the levels of residual beta-amylase activity were due to an intrinsic characteristic of the enzyme, or secondary factors modulating enzyme stability. The mapping population consisted of 95 double haploid derived individuals from a Galleon X Haruna Nijo cross. Both parental lines carry the Bmyl-Sd2 form of

Figure 3 Residual beta-amyteiseactivity in malt extracts after incubation at 60 °C for 1Qmin. Activity is expressed as a percentage of initial activity ~nd the standard deviation of four independent determination~is shown. 13.43

0

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Figure 4 Log-likelihood(LOD) values for the association of beta-amylasethermostabiity and molecular markers on chromosome 4H. the enzyme. Galleon exhibits low beta-amylase thermostability (Sd2L) and Haruna Nijo high stability (Sd2H) allowing the genetics of beta-amylase thermostability to be examined. The double haploid population fell into two nonoverlapping classes of beta-amylase thermostability. At the confidence level of P=0"0001 only one significant association between markers and the thermostability trait was detected (Fig. 4). This

Barley beta-amylase thermostabilily Q T L corresponds to the Bmyl locus on chromosome 4H and explains 55% of the observed variation, no other factors influencing beta-amylase thermostability were detected.

Purification of three beta-amylasesrepresenting the range in stability observed in crude preparations Identification of a major Q T L for beta-amylase thermostability at the Bmyl locus suggests that this variation is due to differences in the beta-amylase enzyme, although the involvement of a closely linked locus could not be excluded. Three forms of beta-amylase representing the observed range in thermostability profiles were purified from barley grain. The low stability enzyme (Sd2L) was purified from the variety Schooner, and the most heat stable form of beta-amylase (Sd3) was purified from accession 77146/33 of H. spontaneum. An example of the enzyme with intermediate thermostability (Sdl) was purified from Franklin. The three betaamylases have an apparent molecular mass of 6 0 k D a and exhibit the charge heterogeneity shown in Figure 1 (data not shown). SDS-PAGE of the three preparations revealed a minor polypeptide with a slightly lower molecular weight which is probably a beta-amylase degradation product based on immunological identity (data not shown). Protein sequencing revealed all three forms to be blocked to Edman degradation consistent with the presence ofNH2-terminal N-acetylmethionine reported in barley beta-amylase from the Sdl variety Gula 4, and no secondary sequences were detected. The rate of thermal inactivation for the purified enzymes is shown in Figure 5. The Sd2L betaamylase purified from Schooner was shown to exhibit rapid thermal inactivation, consistent with results for crude extracts. The purified Sdl betaamylase shows an intermediate response to thermal inactivation, and the Sd3 beta-amylase remains the most thermostable form of the enzyme. The purified enzymes were incubated at a range of temperatures and the Ts0 temperatures determined to be S d 2 L = 5 6 " 8 ° C , S d 1 = 5 8 " 5 ° C , S d 3 = 60"8 °C. The thermostability profiles are consistent with the results found in barley extracts and, as expected, the purified enzymes are less stable under these experimental conditions. For example, after 10min at 6 0 ° C only 10% of the purified Sd3 activity remains, compared to 35% in the barley extracts.

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Figure 5 Irreversible thermal inactivation of purified barley beta-amylase enzymes incubated at 60°C. - - 0 - - , Sdl; - - . - - , Sd2-L; --A--, Sd3. Activity is expressed as a percentage of initial activity and the standard deviation of three independent determinations is shown.

Table I Amino acid substitutions reported in the Sd2 betaamylase sequence. The substitutions restricted to the high stability beta-amylaseare shown in bold Galleon* Sd2-L

Hiproly2 Sd2-L

Glu 85 Arg 85 Val 233 Val 233 Leu 347 Leu 347 Ile 527 Ile 527 * C. Li, pers. comm.

Universe* Sd2-L

Haruna Nijo 3 Sd2-H

Glu 85 Val 233 Leu 347 Ile 527

Glu 85 Ala 233 Ser 347 M e t 527

Comparison of SD2 gene sequences There is no published sequence data for Sdl or Sd3 barley beta-amylase, however Sd2 beta-amylase genes have been cloned and sequenced from four different barley varieties. The varieties Galleon, Hiproly and Universe were found to contain the low thermostability Sd2L beta-amylase allele, and Haruna Nijo the high thermostability Sd2H allele (data not shown). The reported sequences for the Sd2 beta-amylase show four amino acid differences (Table I). The three substitutions restricted to the high thermostability beta-amylase are shown in bold. The substitution of methionine for isoleucine at position

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Figure 6 Relationship in 42 commercial malts between wort fermentability (AAL %) and: (a) beta-amylasethermostability with respect to beta-amylaseallele; (b) diastatic power with respect to beta-amylaseallele. +, Sd2L varieties; O, Sdl varieties; n, Sd2H varieties

527 is probably not responsible for the observed difference in thermostability. This residue lies within the C-terminal region that is removed during germination 4, and the increased thermostability is maintained after germination. The remaining two amino acid substitutions of V233A and L347S are presumed to be the reason for the increased thermostability in the beta-amylase from Haruna Nijo. Impact of variation in

beta-amylase

thermostability on fermentability The relationship between beta-amylase thermostability and wort fermentability was investigated in 43 commercial malts [Fig. 6(a)]. The malt samples are divided into three discrete populations, consistent with their beta-amylase alleles. A similar relationship has been reported in a survey of Japanese breeding lines 2°. The same three groups are observed whether residual beta-amylase is expressed as a percentage of activity prior to heat treatment [Fig. 6(a)], or in absolute terms of Units/ g after heat treatment (not shown). The malt samples containing Sdl and Sd2H beta-amylases yield AAL between 80-84% under standard EBC ~0 mash conditions. The malt samples containing Sd2L beta-amylase show the lowest levels of fermentability and do not exhibit AAL above 81 "5%. The production of fermentable sugars from starch is accomplished by the combined activity

of the diastatic enzymes, therefore the level of DP has a significant impact upon fermentability. The relationship between DP and AAL is plotted in Figure 6(b), and shows a positive correlation (r= 0"76, P<0"01). A major contribution to the variation in this association is from the high thermostability Sd2H malts, which generate relatively high AAL from low diastatic power. The removal of the Sd2H samples from Figure 6(b) increases the correlation coefficient for DP and AAL (r= 0"87, P<0"01). Beta-amylase represents a large component of DP, and as expected the relationship between beta-amylase activity and AAL is very similar, with r=0"72 (P<0"01) for all samples, and r=0"79 (P<0"01) after removal of the Sd2H malts (data not shown). DISCUSSION

In the present study, variation in the rates of thermal inactivation of beta-amylase in barley extracts have been identified. Isoelectric focusing was used to differentiate two distinct beta-amylase alleles (Bmyl-Sdl and -Sd2) in cultivated barley and an additional allele (Bmyl-Sd3) from the wild barley H. vulgaressp. spontaneum.The Bmyl-Sd 1 and -Sd3 forms of beta-amylase behave as discrete alleles with respect to the thermostability trait. The Bmyl-Sd2 varieties were divided into three separate groups based on relative thermostability. The variation was shown to persist after proteolytic

Barley beta-amylase thermostabiJity cleavage of the enzyme during germination, with only small differences detected between the relative thermostability of the grain and malt forms of betaamylase. The level of beta-amylase activity after incubation of extracts at elevated temperatures could be modulated by a number of factors. Protein content varies between barley varieties and the level of soluble protein 2~ and carbohydrate 22 in the extract could have a significant impact on enzyme stability. More specific protein/protein interactions may also affect stability, the beta-amylase has been reported to form dimers and heterodimers with other barley proteins 23. Testing enzyme thermostability in crude extracts is also complicated by the possible action of proteolytic enzymes in the barley grain and malt 24. Molecular mapping of variation in the relative thermostability of beta-amylase between a BmylSd2 low stability and a Bmyl-Sd2 high stability line revealed a single Q T L at the Bmyl locus on chromosome 4H. This implies that variation observed in enzyme stability in crude extracts is due to differences in the beta-amylase enzyme, and is not significantly affected by other loci. This further suggests that the high and low levels of stability observed within the Sd2 beta-amylase are the result of discrete Bmyl alleles (Sd2L and Sd2H), even though these forms of the enzyme cannot be differentiated by electrophoresis. The presence of an intermediate level of thermal stability within the Sd2 phenotype (Morrell) may be the result of an additional allele or of heterogeneous samples, and will be the subject of further analysis. The relative contribution of the four Bmyl alleles to total beta-amylase activity is also an important consideration in breeding barley for improved malt quality. While the Bmyl-Sd2 allele of the variety Steptoe has been shown to contribute slightly increased beta-amylase with respect to the Bmyl-Sdl allele 25, it has not been determined if Steptoe carries the Bmyl-Sd2L or Bmyl-Sd2H allele. However, Q T L mapping typically reveals several genomic regions associated with beta-amylase activity25,26 Bmyl-Sd 1, -Sd2L and -Sd3 forms of beta-amylase were purified from barley grain and shown to exhibit variation in thermal stability consistent with the results from grain and malt extracts. Rates of thermal inactivation determined for the purified beta-amylases show Ts0 temperatures of 56"8 °C for Sd2L, 58"5°C for Sdl, and 60-8°C for the Sd3 beta-amylase. These results allow comparison of

307

the different forms of beta-amylase under defined experimental conditions. The actual rates of thermal inactivation under mashing conditions will be significantly slower due to stabilisation by the high protein concentrations 21 and the presence of carbohydrates 22. It has been shown that under stimulated mashing conditions up to 10% of betaamylase activity may remain after 60min at 65 °C II. Analysis of existing sequence data for Sd2 betaamylases has revealed two amino acid changes restricted to the Bmyl-Sd2H allele from Haruna Nijo. At position 233 valine is replaced by alanine, and at position 347 leucine is replaced by serine. The soybean beta-amylase sequence also contains these substitutions27, and the thermal stability reported for beta-amylase from soybean is higher than that of barley ~. The crystal structure of soybean beta-amylase has been determined at the 2,~ level and shows a (a/[3)8 core structure with a catalytic site consisting of a deep pocket bordered by a hinged loop 2a. By homology to this structure, the two amino acid substitutions in the Sd2H betaamylase of V233A and L347S occur in the Cterminal loops L4 and L6, respectively, which form part of the walls of the catalytic site pocket of betaamylase and exhibit higher than average chain flexibility28. This is consistent with results from site directed mutagenesis which showed that the Cterminal loops L3, L4 and L5 are key regions in stabilising barley beta-amylase~3. Both amino acid substitutions are in solvent accessible regions of the protein, and L347S will therefore decrease the surface hydrophobicity and increase the propensity for hydrogen bonding, consistent with many stabilising amino acid substitutions 29. Brewing requires high levels of fermentable sugars from starch degradation for conversion into alcohol. Beta-amylase may be the rate limiting enzyme in the degradation of starch 3°'~1, and betaamylase is also more thermolabile than the other diastatic enzymes lj. Therefore an improvement in the survival of beta-amylase during mashing may yield increased production of fermentable sugars. Analysis of 42 commercial malts revealed a strong positive correlation between diastatic power and fermentability. The malts with the high thermostability Bmyl-Sd2H allele do not strictly follow this relationship, generating high AAL from relatively low diastatic power. This suggests that the increase in thermostability results in more efficient starch degradation compared to Sdl and Sd2L malts with similar levels of DP. The use of starch-

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based ad'unctstj or temperatures higher than standard EBCI° mash conditions increases the demand on the diastatic enzymes and is likely to amplify the difference between malts from barleys with the high and low thermostability forms of betaamylase. The results of this study suggest an alternative approach for breeding barley with higher levels of fermentability. The selection for specific betaamylase alleles using the two point thermostability assay applied in this study would be a useful screening tool. Since only a small sample is needed and there is no requirement for micromalting, it is amenable to early generation screening, which is a distinct advantage over current methods of selecting for fermentability. In addition, the identification of the amino acid substitutions between the Sd2L and Sd2H forms of beta-amylase provides scope for the development of PCR markers to directly select for the allele of choice. The Sdl and Sd3 beta-amylases can be selected for directly by IEF. In this study we have identified natural variation for beta-amylase thermostability within the H. vulgare L. gene pool and from H. vulgatespp. spontaneum, and this has been linked to fermentability in malted barley. The availability of a range of selection tools for these alleles makes these findings amenable to direct application in barley breeding.

Acknowledgements The authors wish to thank Dr Lesley MacLeod and Barrett Burston Malting Co., and the Waite Malting Quality Evaluation Laboratory for the provision of malt samples, micromalting and quality data. We also thank Dr A. H. D. Brown and Dr R. C. M. Lance for their assistance, and Prof G. B. Fincher for critical reading of this manuscript. Funding for this research was provided by the Grains Research and Development Corporation.

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