A genome-based approached to improving barley for the malting and distilling industries.
Up to 60% of the Scottish barley crop is used in malting for brewing and distilling. The distilling industry alone uses some 500,000 tonnes per annum with the total malt purchases in Scotland exceeding 800,000 tonnes. Scotch Whisky is the fifth largest British export and the leading food and drink commodity, earning over £2 billion per annum. Malt whisky can only be made from malted barley and is the premium end of the market. High spirit yield is probably the main quality requirement of the malt whisky distilling industry, because a 1% increase in spirit yield would lead to a saving of approximately £1.1 million in distilling production costs. Spirit yield is the product of hot water extract, i.e. the total soluble component following malting, and the fermentability of the extract since not all solubilised components are fermentable. The peak level of fermentability is achieved earlier in the malting process than the peak level of extract and malting has to be optimised to produce the maximum spirit yield. Under certain conditions a breakdown product of epi-heterodendrin, a glycosidic nitrile produced in germinating barley, can react with ethanol, catalysed by copper in stills, to produce the putative carcinogen ethyl carbamate (urethane). Barley cultivars that do not produce epi-heterodendrin are essential in grain whisky distilling and also in some malt whisky distilleries.
The development of genetic finger-printing techniques in human genetics has led to applications of the various types of molecular markers, especially in the rapid creation of genetic maps of an organism. The advantage of such maps is that regions controlling complex characters such as malting quality can be identified as Quantitative Trait Loci (QTL). This knowledge can then be applied in a targeted manner to improve plant characters for a specific end-user need. Fermentability is, as noted above, a key character for the distilling industry but its analysis is difficult to carry out in plant breeding and genetical studies. It is, however, an ideal character for exploiting molecular marker methods in plant breeding for a specific end-user requirement and is the basis for the project being reported here. The aims of the project were:
Determine the genetic control of fermentability and spirit yield and their relationships to other characters
Identify molecular markers linked to genes controlling fermentability, hot water extract and spirit yield
Combine genes for high spirit yield in a spring barley genotype suitable for Northern Britain.
We collected genetic marker and malting quality data from a spring barley population of random inbred lines that was constructed from a cross between commercially relevant parents. The parents did, however, exhibit contrasting combinations of fermentability and hot water extract and so maximised our chances of revealing QTLs for the former that could be used to improve spirit yield.
Materials & Methods
Random inbred lines were produced by doubled haploidy from the F1 of a spring barley cross between the genotypes Derkado and B83-12/21/5 to achieve the first two objectives of the project. Derkado had good malting quality and was one of the main Scottish cultivars in the 1990's, principally because it was a non-producer of epi-heterodendrin, and B83-12/21/5 was a breeding line from the Scottish Crop Research Institute (SCRI). The whole population, together with the parents and some controls, was grown in trials at SCRI from 1995 to 1997 inclusive and at a site near Sleaford, Lincs, UK in 1996 and 1997. Each trial was sown in plots at a normal commercial density, received a typical fertiliser regime, and was kept free of foliar pathogens by the application of fungicides. The fraction passing over a 2.5mm sieve from each plot was retained for phenotypic analysis of quality characters. Adverse weather conditions delayed harvest of the 1997 trial near Sleaford and there was considerable pre-harvest sprouting in a number of samples. No malting analyses were therefore carried out on this trial, leaving four trials for analysis.
A large number of molecular markers, the two major dwarfing genes sdw1 and ari-eGP, the mildew resistance gene mlo, and a gene controlling the non-production of epi-heterodendrin (eph) were used to generate a genetic map that covered most of the barley genome. A range of malting quality characters was also scored on the population, namely hot water extract, fermentability, predicted spirit yield (PSY), grain nitrogen content, soluble nitrogen content of the malt, soluble nitrogen ratio, grain b -Glucan content and quantitative production of epi-heterodendrin. In addition, malt samples from a subset of lines from two trials were used to estimate the wort contents of the sugars glucose, sucrose, maltose and malto-triose. These data were used to study the genetics of each character (Objective 1) and, when combined with the marker data, to identify QTLs controlling each character (Objective 2).
We developed a breeding population for the third objective of the project. We selected 8 lines, on the basis of their phenotypic performance, as donors of high fermentability to initiate a progamme to produce first backcross (BC1) inbred lines. The cultivar Landlord and two SCRI breeding lines, B91-47/22 and B91-99/15, were chosen as recipient parents, as there was scope to improve the fermentability of each. We found, however, that our target QTL was closely linked in coupling with the ari-eGP dwarfing gene and in repulsion to a hot water extract QTL at the ari-e locus. We would therefore need to produce recombinants between the fermentability QTL and the dwarfing gene to develop a successful cultivar within the project. For every 100 BC1DH lines that we produced, we would expect an average of 5 to be recombinants and we would therefore need to develop a very large population to generate a sufficiently large number of desired recombinants within the project. We therefore changed our strategy to a more random one by testing all the BC1DH lines that we developed. Time constraints limited the development of the breeding population so that seed was available from only 255 BC1DH plants in time for sowing in trials in 1999. Selections based on field observations made on the trials were multiplied over winter in New Zealand and returned for large plot (7m2) trials at commercial density with and without fungicide at SCRI and fungicide treated trials near Sleaford and Docking in 2000. Cleaned and sieved samples from the plots were retained for analysis of the malting quality characters hot water extract, fermentability, PSY, grain nitrogen content, soluble nitrogen content of the malt, soluble nitrogen ratio and grain b -Glucan content.
The genetic fingerprints of the BC1DH lines entered into trials at SCRI were established by surveying them with 44 previously mapped Simple Sequence Repeat (SSR) markers, which were selected to sample the whole barley genome as well as the target QTL. In addition, allelic differences at the sdw1 and ari-eGP loci were established from observations of the juvenile growth habits of the plots. As well as developing lines of potential commercial merit, we wished to detect whether or not the donor QTL chromosomal segment altered the expression of fermentability in the recipient. We coded all the genotypic data as being either donor or recipient in origin and compared the means of the different genotypes observed in the target region. We also used regression analysis to identify markers that acted together in statistically significant associations with the characters and compared the results to those obtained from the mapping population.
Validation of laboratory tests
An essential question that this project sought to answer was the relevance of the laboratory measures to commercial practice. This applied particularly to the measures of fermentability and epi-heterodendrin. The problem is that methodology based upon commercial practice is resource consuming and cannot be applied to a large number of samples and certainly not on a scale large enough to conduct detailed genetic studies. We therefore selected a stratified set of malt samples for high gravity spirit yield (HGSY) analysis by the Scotch Whisky Research Institute (SWRI). This test gives an estimate of the likely spirit yield under distillery conditions. As the malts for both PSY and HGSY had been prepared under the same conditions, the two measures can be compared to determine the value of PSY in predicting spirit yield under distillery conditions. This test was applied to samples from both the mapping and the breeding populations. Validation of the measures of epi-heterodendrin was also carried out by SWRI using the standard distillery method.
Genetics of the traits
Derkado was generally the better parent for most of the quality characters but B83-12/21/5 had a greater fermentability. In general, DH lines that transgressed, or equalled, the parental means were apparent for all characters, indicating the presence of useful alleles in both parents that potentially could be recombined to produce superior inbred lines. There was highly significant genetic variation for all the characters apart from the wort sugar data, which indicates that most of the characters should be responsive to selection. The high amount of genetic variation found for epi-hetrodendrin reflects the segregation of the major gene controlling production of the compound but the figure is still high when the effects of the gene are excluded by restricting analysis to lines without the eph gene. Apart from glucose, there was little indication of genetic variation for the wort sugars but this may reflect the fact that there was not a proper error to test for genetic effects in the project, which may therefore have been obscured by interactions from contrasting sites.
The correlations between the means of the characters show that extract is the major determinant of PSY although fermentability does have a small but significant positive correlation with the character (Table 1). Selection for increased fermentability could improve spirit yield but would need to be applied cautiously due to its higher but negative correlation with hot water extract. QTL mapping of the two traits would identify a suitable locus for selection. The correlations of the wort sugars with hot water extract are as expected but, with the exception of glucose, the wort sugars are not correlated with fermentability. The negative correlation of glucose with fermentability is surprising but could be an indication of over-modification, particularly as there is evidence of a positive correlation between glucose and soluble nitrogen ratio.
About this project
The distilling industry utilises around 25% of the UK malting barley crop, equivalent to over 60% of the Scottish malting barley crop. Scotch Whisky is by far the leading export in the Food and Drink sector and is currently the UK's fifth highest export earner. Whilst the distilling industry purchases specified barley cultivars, little or no testing for specific distilling requirements is carried out by breeders or the testing authorities. Distillers therefore have little knowledge about the quality of newly recommended cultivars other than their extract levels. The major requirement of the distilling industry is to produce the maximum amount of spirit per tonne of malt as efficiently as possible. Not all of the components extracted during mashing from a malt are fermentable by yeast so spirit yield is the product of extract and its fermentability. Little was known about fermentability and its relationship to other malting quality characters so the aims of this project were to: understand the genetical and environmental control of fermentability and its relationship to spirit yield and other characters; identify genetic markers that could be used to select for the character without the need for expensive malting quality and fermentability assays; and initiate a programme to produce barley lines for specific use in the distilling industry.
We clearly showed that while fermentability was genetically controlled, it was also liable to be affected by environmental variation. Some fermentability genes were inversely related to some affecting extract so that increasing fermentability without careful selection could actually reduce extract and, therefore, spirit yield. We identified regions of barley chromosomes responsible for the genetic control of fermentability and a range of other malting quality characters. In doing so, we also identified genetic markers that could be used to indirectly select for these characteristics. Plant breeders could use such markers to eliminate poor malting quality lines before initiating an expensive trialling and testing scheme. An example of this was a marker linked to a gene controlling non-production of epi-heterodendrin, a characteristic required by some distilleries and found in cultivars such as Maresi, Delibes, Derkado and Decanter. This was a single major gene and the marker was found to be effective in discriminating between producers and non-producers of epiheterodendrin. Fermentability was a more complex character and a number of genes were found to be controlling the character. Markers were used to select for one gene found to have a major influence on fermentability but this was of limited effectiveness in the absence of selection for the other genes. The gene could also have a different effect when the genetic background is changed. Nevertheless, we were able to develop some lines of potential commercial value within the project and also identify more specific targets that are highly likely to lead to improved barley cultivars for use in the distilling industry.
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