QTLs for malting flavour component associated with pre-harvest sprouting susceptibility in barley (Hordeum vulgare L.)

Lipoxygenase (LOX) is a key factor affecting quality of beer in terms of foam stability and flavour. Low LOX content is a desirable trait for malting quality. A doubled haploid (DH) population was made from a cross of Australian malting barley Stirling and Canadian malting barley Harrington and mapped with 513 molecular markers. The 120 DH lines with their parents were planted in field trials and the harvested grains were micro-malted for analysis of LOX content in two consecutive years. LOX content was controlled by both genetic effects and environment conditions. Three QTLs were consistently detected. One QTL flanked by the markers E6216 and SCssr03907 at the telomere region of chromosome 5HL contributed 39% of genetic variation in LOX content. The second QTL close to the centromere region of chromosome 5H accounted for 17% of genetic variation. A minor QTL on chromosome 2H explained 6% of genetic variation but was significant in both years. The Australian variety Stirling contributed to higher LOX content for the three QTLs. The two QTLs mapped at chromosome 5H for LOX content coincided with the QTLs for seed dormancy/pre-harvest sprouting from the same population. The pre-harvest sprouting susceptible alleles were associated with low LOX content, which indicated that the low LOX QTL from the Canadian malting barleys are only useful in the barley growing areas where the pre-harvest sprouting risk is low. New genetic sources for low LOX should be exploited in different germplasm with different mechanisms

Sequence variation and haplotypes of lipoxygenase gene LOX-1 in the Australian barley varieties

Background Lipoxygenases are a family of enzymes which catalyse the hydroperoxidation of polyunsaturated fatty acids with a cis, cis-1,4-pentadiene to form conjugated hydroperoxydienes. Lipoxygenase-1 (LOX-1) in barley worsens the flavour and foam stability of beer. It has become a major selection criteria for malting quality in the last few years. Results Lipoxygenase activity was investigated in 41 Australian barley cultivars and advanced breeding lines released since the 1950s; the cultivars differed markedly, ranging from 22.3 to 46.5 U/g. The structural gene and its promoter of lipoxygenase-1 were sequenced from the barley varieties representing different levels of LOX. Based on the analysis of nucleotide and deduced amino acid sequences, two major haplotypes were identified. Barley varieties with lower LOX were classified into three categories based on their pedigrees and sequence variations in the structural gene: (1) barley varieties derived from Canadian varieties with the pre-harvest sprouting susceptible allele, (2) Skiff and Hindmarsh with unique haplotype in the structural gene, and (3) Gairdner and Onslow with an unknown mechanism. Conclusion Lipoxygenase activity has been reduced in the malting barley cultivars in the last 60 years although it is only recognized as a malting quality trait recently. There are clear haplotypes of the lipoxygenase structual gene. The polymorphisms detected in the structural gene can be used to design molecular markers for selection of low LOX haplotype. Other mechanisms also existed for controlling lipoxygenase activity. The results suggest that it is possible to develop barley varieties with lower LOX by combination of low LOX-1 haplotype and other trans-regulation factors

Crop Updates 2007 - Cereals

This session covers twenty six papers from different authors: CEREAL BREEDING 1. Strategies for aligning producer and market imperatives in cereal breeding in Western Australia, R. Loughman, R. Lance, I. Barclay, G. Crosbie, S. Harasymow, W. Lambe, C. Li, R. McLean, C. Moore, K. Stefanova, A. Tarr and R. Wilson, Department of Agriculture and Food 2. LongReach plant breeders wheat variety trials – 2006, Matu Peipi and Matt Whiting, LongReach Plant Breeders WHEAT AGRONOMY 3. Response of wheat varieties to sowing time in the northern agricultural region in 2006, Christine Zaicou, Department of Agriculture and Food 4. Response of wheat varieties to sowing time in the central agricultural region in 2006, Shahajahan Miyan, Department of Agriculture and Food 5. Response of wheat varieties to sowing time in the Great Southern and Lakes region, Brenda Shackleyand Ian Hartley, Department of Agriculture and Food 6. Response of wheat varieties to time of sowing time in Esperance region in 2006, Christine Zaicou, Ben Curtis and Ian Hartley, Department of Agriculture and Food 7. Performance of wheat varieties in National Variety Testing (NVT) WA: Year 2, Peter Burgess, Agritech Crop Research 8. Flowering dates of wheat varieties in Western Australia in 2006, Darshan Sharma, Brenda Shackley and Christine Zaicou, Department of Agriculture and Food 9. Prospects for perennial wheat: A feasibility study, Len J. Wade, Lindsay W. Bell, Felicity Byrne (nee Flugge) and Mike A. Ewing, School of Plant Biology and CRC for Plant-based Management of Dryland Salinity, The University of Western Australia BARLEY AGRONOMY 10. Barley agronomy highlights: Time of sowing x variety, Blakely Paynter and Andrea Hills, Department of Agriculture and Food 11. Barley agronomy highlights: Weeds and row spacing, Blakely Paynter and Andrea Hills, Department of Agriculture and Food 12. Barley agronomy highlights: Weeds and barley variety, Blakely Paynter and Andrea Hills, Department of Agriculture and Food OAT AGRONOMY 13. Agronomic performance of dwarf potential milling oat varieties in varied environments of WA, Raj Malik, Blakely Paynter and Kellie Winfield, Department of Agriculture and Food 14. Sourcing oat production information in 2007, Kellie Winfield, Department of Agriculture and Food HERBICIDE TOLERANCE 15. Response of new wheat varieties to herbicides, Harmohinder Dhammu, Department of Agriculture and Food 16. Herbicide tolerance of new barley varieties, Harmohinder Dhammu, Vince Lambert and Chris Roberts, Department of Agriculture and Food 17. Herbicide tolerance of new oat varieties, Harmohinder Dhammu, Vince Lambert and Chris Roberts, Department of Agriculture and Food NUTRITION 18. Nitrogen Decision Tools – choose your weapon, Jeremy Lemon, Department of Agriculture and Food DISEASES 19. Barley agronomy highlights: Canopy management, Andrea Hills and Blakely Paynter, Department of Agriculture and Food 20. Barley agronomy highlights: Leaf diseases and spots, Andrea Hills and Blakely Paynter, Department of Agriculture and Food 21. Fungicide applications for stripe rust management in adult plant resistant (APR) wheat varieties, Geoff Thomas, Rob Loughman, Ian Hartley and Andrew Taylor; Department of Agriculture and Food 22. Effect of seed treatment with Jockey on time of onset and disease severity of stripe rust in wheat, Manisha Shankar, John Majewski and Rob Loughman, Department of Agriculture and Food 23. Rotations for management of Cereal Cyst Nematode, Vivien Vanstone, Department of Agriculture and Food 24. Occurrence of Wheat Streak Mosaic Virus in Western Australian grainbelt during the 2006 growing season, Brenda Coutts, Monica Kehoe and Roger Jones, Department of Agriculture and Food 25. Development of a seed test for Wheat Streak Mosaic Virus in bulk samples of wheat, Geoffrey Dwyer, Belinda Welsh, Cuiping Wang and Roger Jones, Department of Agriculture and Food MARKETS 26. Developing the Australian barley value chain, Linda Price, Barley Australi

Standardization of a small-scale hot water extract method for application in barley breeding programs

Hot water extract (HWE) is one of the key quality attributes considered when determining the malting performance of barley. The international malting and brewing industries utilise standard procedures for measuring HWE, including European Brewery Convention (Analytica - EBC 1998), Institute of Brewing (IOB - Methods of Analysis 1997), and American Society of Brewing Chemists (ASBC - Methods of Analysis 1992). EBC and ASBC procedures are similar and use a multi-temperature programmed mashing profile, while the IOB procedure uses a constant temperature infusion mashing profile. In Australia, maltsters and brewers generally use the EBC procedure to determine extract levels on commercial samples. Specific quality goals for developing Australian commercial barley varieties include a high extract carbohydrate potential with a desired level in excess of 82% EBC fine grind, dry basis (MBIBTC, 2001). Malting quality testing protocols vary in public Australian barley breeding programs especially in early generation testing where significant quantities of grain may not be available. Different methodologies are used including NIR and small-scale assays developed in-house. A review of the barley quality evaluation laboratories in 1995 by the Grains Research and Development Corporation (GRDC) included a recommendation of implementing standardised methodology for micromalting and quality testing of advanced material (Enright et al., 1995). The Australian Barley Chemists Group (ABCG) incorporating members of barley quality evaluation laboratories in Perth, Adelaide, Horsham, Wagga Wagga and Toowoomba, meet formally on an annual basis to discuss testing protocols and quality issues. The objective of method standardisation is to develop small-scale methods to provide breeding programs and industry groups with MBIBTC equivalent results from within the breeding programs. In 1999, a diastase method was standardised by the ABCG and correlated to the EBC industry method (Fox et al., 1999). Due to sample quantity limitations in some stages of a barley breeding program, a small-scale EBC HWE procedure has been developed and collaboratively evaluated by the participants of the ABCG

Variation in maltose in sweet wort from barley malt and rice adjuncts with differences in amylose structure

Starch from malt and solid adjuncts provides the majority of fermentable sugars for fermentation. However, there is no current data on the variation in starch structure (particularly long chained amylose) and its impact on the final wort composition of fermentable sugars, specifically maltose. This is the first study to report variation in amylose structure from barley malt and rice used as an adjunct and how this impacts the production of maltose. We compared four commercial malts with two rice adjuncts mashes, in solid and liquid additions, with an all-malt mash used as a control. All combinations of malt and rice adjuncts were tested under two grist-to-liquor (G:L) ratios (1:3 and 1:4) in a 65°C ramped mash. After mashing, the wort original gravity and maltose concentration were measured. The commercial malts had different malt quality but very similar gelatinisation temperatures (~65°C). The malts varied in starch and amylose contents but had only minor variations in average amylose chain lengths. The two rice adjuncts also had similar average amylose chains lengths, but quite different amylose contents, and hence different gelatinisation temperatures. The results showed that liquid adjunct mashes had higher original gravity and maltose concentration for both G:L ratios. However, there was no consistent result in original gravity or maltose between G:L ratio or adjunct type, suggesting interactions between each malt and rice adjunct. Knowing amylose chain length could improve understanding of the potential maltose levels of the sweet wort prior to fermentation

MALDI-TOF mass spectrometry provides an efficient approach to monitoring protein modification in the malting process

Proteins in barley grains are determinants of beer quality, which are modified during malting to provide nutrition for yeast during brewing. Different malting barley varieties behave differently during malting. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to characterize storage proteins including albumin, globulin, hordein and glutelin in grains and malt of parental varieties and a doubled haploid (DH) population. Each parental variety produces malt with particular quality profiles. The protein profiles of analyzed extracts in seed and malt were compared to find the most influenced proteins during the malting process. Our results revealed that malting procedure dramatically affected the composition and amounts of albumin, then hordein, glutelin and globulin in barley. Moreover, hordein and glutelin profiles in the DH population exhibited high levels of polymorphism. The genetic information represented by individual hordein and glutelin profiles was successfully mapped as molecular markers in a genetic linkage map. Twelve and 8 (hordein in seed and malt, respectively) or 10 and 8 (glutelin in seed and malt, respectively) segregating peaks were scored as polymorphic across the population, and all peaks were located to the chromosome 1H. The MALDI-TOF MS provides a method with high resolution and throughput to characterize mass patterns of extracted storage proteins in barley and malt

Improved prediction of malt fermentability by measurement of the diastatic power enzymes beta-amylase, alpha-amylase, and limit dextrinase: II. Impact of barley genetics, growing environment, and gibberellin on levels of alpha-amylase

The determination of the levels of the diastatic power enzymes (DPEs) beta-amylase, limit dextrinase, and alpha-amylase has previously been shown to predict barley malt fermentability. Using micromalted samples of barley from different genotypes and growing environments, it was demonstrated that both these factors were important in determining the level of DPEs in malt. In terms of genotypic effects, a trial of breeders’ lines and varieties showed substantial variation in the levels of total beta-amylase (means 455–914 U/g), total limit dextrinase (means 268–603 U/kg), and alpha-amylase (means 154–316 U/g). The application of gibberellin (GA) during malting resulted in substantial increases in the levels of total limit dextrinase and alpha-amylase and the extent of modification (KI). However, the level of total beta-amylase was relatively unchanged. It was also observed that the levels of total limit dextrinase and alpha-amylase and extent of KI generally were highly correlated (r » 0.6–0.7), which was attributed to the sensitivity of each of these malt quality factors to GA. The results are discussed in terms of their practical importance to barley breeders and maltsters seeking to supply malt that satisfies the malt fermentability requirements of their brewing customers.D. Evan Evans, Chengdao Li, Stefan Harasymow, Sophia Roumeliotis and Jason K. Eglinto

Refining the prediction of potential malt fermentability by including an assessment of limit dextrinase thermostability and additional measures of malt modification, using two different methods for multivariate model development

Prediction of malt fermentability (apparent attenuation limit –AAL) by measurement of the diastatic power enzymes (DPE), α-amylase, total limit dextrinase, total β-amylase, β-amylase thermostability, and the Kolbach index (KI or free amino nitrogen – FAN) is superior to the conventional use of diastatic power (DP) alone. The thermostability of β-amylase is known to be an important factor in determining fermentability, thus the thermostability of the other relatively thermolabile enzyme, limit dextrinase, was investigated to determine if it was also useful in predicting fermentability. To facilitate this aim, methods were developed for a rapid and cost efficient assay of both β-amylase and limit dextrinase thermostability. Internationally important Australian and international malting varieties were compared for their total limit dextrinase and β-amylase activity and thermostability. Interestingly, the level of limit dextrinase thermostability was observed to be inversely correlated with total limit dextrinase activity. The prediction of malt fermentability was achieved by both forward step-wise multi-linear regression (MLR) and the partial least squares (PLS) multivariate model development methods. Both methods produced similar identifications of the parameters predicting wort fermentability at similar levels of predictive power. Both models were substantially better at predicting fermentability than the traditional use of DP on its own. The emphasis of this study was on the identification of predictive factors that can be consistently used in models to predict fermentability, because the model parameter estimates will subtly vary depending on mashing conditions, yeast strain/fermentation conditions and malt source. The application of these multivariate model development methods (PLS and MLR) enabled the identification of further potential fermentability predicting factors. The analyses divided the predictive parameters into those defined by DP enzymes and those associated with modification (KI, FAN, fine/coarse difference, wort β-glucan and friability). Surprisingly, limit dextrinase thermostability was not a substantial predictor of fermentability, presumably due to its negative correlation with total limit dextrinase activity. The application of these insights in the malting and brewing industries is expected to result in substantial improvements in brewing consistency and enable more specific quality targets for barley breeder’s progeny selection cut-off limits to be more precisely defined.D. Evan Evans, Robert Dambergs, David Ratkowsky, Chengdao Li, Stefan Harasymow, Sophia Roumeliotis and Jason K. Eglinto

Molecular marker-assisted backcrossing breeding: An example to transfer a thermostable β-amylase gene from wild barley

Molecular marker-assisted backcrossing (MABC) is widely recommended for transferring favorable alleles from a donor to an elite variety. The question remains whether MABC is an effective approach to developing a competitive commercial variety. Here, we illustrate the transfer of a thermostable β-amylase allele Sd3 from wild barley into a commercial barley variety Gairdner. The elite lines were chosen for the Regional Crop Variety Test that followed a standard conventional breeding process. The results demonstrated that the Sd3 allele not only increased enzyme thermostability but dramatically enhanced diastatic power, an important malting quality trait. The BC1F1 individuals had a fundamental impact on the comprehensive agronomic and quality traits of the final progenies, demonstrating the importance of screening at the early stage of backcrossing in MABC. There was sufficient genetic variation in the BC3F3 families to select other malting quality and agronomic traits. Ten individual breeding lines with improved β-amylase thermostability also had improved yields and grain plumpness. Three elite lines with improved malting quality and agronomic traits were selected to provide a parental line to incorporate the wild barley allele for breeding a commercial variety. A new strategy should be considered that uses marker-assisted selection and backcrossing to transfer a favorable allele from a wild parent

Refining the prediction of potential malt fermentability by including an assessment of limit dextrinase thermostability and additional measures of malt modification, using two different methods for multivariate model development

Prediction of malt fermentability (apparent attenuation limit — AAL) by measurement of the diastatic power enzymes (DPE), α-amylase, total limit dextrinase, total β-amylase, β-amylase thermostability, and the Kolbach index (KI or free amino nitrogen — FAN) is superior to the conventional use of diastatic power (DP) alone. The thermostability of β-amylase is known to be an important factor in determining fermentability, thus the thermostability of the other relatively thermolabile enzyme, limit dextrinase, was investigated to determine if it was also useful in predicting fermentability. To facilitate this aim, methods were developed for a rapid and cost efficient assay of both β-amylase and limit dextrinase thermostability. Internationally important Australian and international malting varieties were compared for their total limit dextrinase and β-amylase activity and thermostability. Interestingly, the level of limit dextrinase thermostability was observed to be inversely correlated with total limit dextrinase activity. The prediction of malt fermentability was achieved by both forward step-wise multi-linear regression (MLR) and the partial least squares (PLS) multivariate model development methods. Both methods produced similar identifications of the parameters predicting wort fermentability at similar levels of predictive power. Both models were substantially better at predicting fermentability than the traditional use of DP on its own. The emphasis of this study was on the identification of predictive factors that can be consistently used in models to predict fermentability, because the model parameter estimates will subtly vary depending on mashing conditions, yeast strain/fermentation conditions and malt source. The application of these multivariate model development methods (PLS and MLR) enabled the identification of further potential fermentability predicting factors. The analyses divided the predictive parameters into those defined by DP enzymes and those associated with modification (KI, FAN, fine/coarse difference, wort β-glucan and friability). Surprisingly, limit dextrinase thermostability was not a substantial predictor of fermentability, presumably due to its negative correlation with total limit dextrinase activity. The application of these insights in the malting and brewing industries is expected to result in substantial improvements in brewing consistency and enable more specific quality targets for barley breeder's progeny selection cut-off limits to be more precisely defined