Identification of barley mutants and characterisation of their mixed-linkage (1,3;1,4)-~-glucan (MLG)

Abstract

Barley is one of Australia’s main cereal crops, used for human consumption, animal feed, malting and brewing. Mixed-linkage (1,3;1,4)-β-glucan (MLG) is the major fibre constituent of barley grain that has positive impacts on human health and negative impacts on animal diet and the brewing industry. Thus, the modification of MLG content in barley grain in both high and low directions could have a potential market value. However, there are still few available details with regards to the molecular control of MLG content in barley grain and the regulatory genes controlling its biosynthesis other than the CslF6 gene. A better understanding of MLG biosynthesis would be valuable to researchers and industry alike and historically the use of mutants has been a powerful tool in dissecting complex processes. Three barley mutants with altered MLG profiles are thus used here to try and further our understanding of biosynthesis of this important cell wall polysaccharide. The work carried out in this project was designed to characterise and analyse three barley mutants showing reduced MLG content in mature grain known as 45(7), M-737 and Chalky Glen with their parents Flagship, Minerva and Glen, respectively. For the three MLG barley mutants, the reduction of grain MLG content was associated with perturbations in grain phenotypic traits, grain biochemical composition and/or internal grain morphology compared to their parents. MLG contents % (w/w) were significantly less in the three mutants compared to their parents during grain development. At a molecular level and throughout grain development, CslF6 transcript levels were outstandingly lower in the three mutants compared to abundant transcript levels in their parents. In addition, high transcript abundance of the Glb1 gene, especially in 45(7) and M-737 mutants, in mid and late grain development was also detected. Genetic variation within the CslF6 gene is rare since no differences were found in the putative promoter region, coding region and GT-motif region of the CslF6 gene that could explain the differences in grain MLG content, suggesting other regulatory sequences or proteins, acting independently or in combination are likely to be involved in the observed differences in the grain MLG content between the three mutants and their parents. Accordingly, a transcriptome analysis was performed for the two mutants 45(7) and M-737 and their parents Flagship and Minerva respectively, at 10, 14 and 18 days after pollination (DAP) of grain development to identify compromised molecular pathways, differentially expressed genes (DEGs), and sequence polymorphisms that might contribute to the low MLG phenotype in grain. In 45(7), the expression of the CslF6 gene was not significantly reduced, suggesting other factors could cause the phenotype. Sequence polymorphism analysis of 45(7) identified a point mutation in the soluble starch synthase III (SSIII) gene and the nuclear factor YB1 (NF-YB1) transcriptional regulator, both of which have previously been implicated in grain composition. Altered NF-YB1 activity might explain the reduced transcript levels of several starch biosynthesis genes including FLOURY ENDOSPERM6 (FLO6), consistent with a reduction in grain starch content such that the effect on grain MLG content is pleiotropic. A third mutation was detected in the myosin heavy chain gene that could affect MLG shuttling to the plasma membrane, accordingly reducing grain MLG content. In M-737, CslF6 expression was significantly reduced and was accompanied by a mutation in the aldose-1-epimerase gene. This enzyme is implicated in D-galactose uptake and reduced activity could potentially reduce the biosynthesis of the sugar donors (substrate availability) required for grain MLG biosynthesis. These findings could be used for designing crosses and planning marker selection strategies in mapping populations, and subsequently providing greater opportunity for quantitative trait locus (QTL) detection. The M-737 mutant was chosen for a micro-malting study as it displays good malting characteristics as judged by the low MLG content compared to the parent cultivar Minerva. This study quantified MLG content and the activity of the (1,3;1,4)-β-glucanase enzyme during the micro-malting experiment and a number of micro-malting quality traits of the final malt, including hot water extract, wort viscosity, wort MLG, free amino nitrogen and soluble protein were measured. During micro-malting stages, MLG content % (w/w) was significantly less in M-737 than Minerva, however the relative rate of hydrolysis was similar. M-737 and Minerva showed no significant differences in their (1,3;1,4)-β-glucanase enzyme activity during micro-malting stages, suggesting that a lower abundance of MLG does not translate into faster hydrolysis of MLG during germination. In terms of malt quality traits, M-737 exhibited better traits than Minerva, including significantly low wort viscosity and wort MLG, and significantly higher hot water extract, soluble protein and free amino nitrogen. The three low MLG mutants and their parents were used as a training set to develop a new screening method based on Fourier transform mid infrared (FTMIR) spectroscopy. Scans of non-embryo half grain were significantly (p-value <0.05) correlated with the Megazyme assay (r = 0.903). FTMIR spectroscopy was subsequently used to screen 300 M2 Compass mutant lines, resulting in the identification of 41 M3 mutants of interest via univariate and multivariate analysis. Megazyme assays confirmed that MLG levels were altered in these mutants. MLG quantification of M4 grain confirmed two mutants (182-5 and 1669-1) that showed significantly low MLG content in grain. This method also showed a strong correlation between absorbance and MLG content for six oat (Avena sativa) breeding lines that were tested. Thus, the FTMIR method appears to be a promising method for detecting variation in MLG content and with additional optimisation, this method could be used for research and breeding programs.