Cell wall degradation is a crucial process within the malting process of barley. Therefore the haplotype diversity of a gene for the cell wall degrading enzyme (1→3),(1→4)-β-D-Glucan-4-glucanohydrolase was investigated and associations to malting quality parameters were performed.
The gene contained several single base-pair insertions-deletions in the 3UTR and one 4bp-INDEL which could be converted into a marker. Six SNPs were discovered originally in both sequenced fragments of the gene. Only SNP3, 4, 5 were found to be highly polymorphic and were used for high throughput genotyping by pyrosequencing assays. While SNP4 (T/C) causes a silent mutation, the other two SNPs are leading to an amino acid exchange after translation. Each of them is coding for different amino acids. If in SNP3 (T/C) the allele T is present in the triplett GTC it will be translated into Valine while the allele C in GCC will cause Alanine, respectively. The opposite is true for SNP5 (C/T), where the allele C in the triplett GCG leads to an Alanine and the allele T in GTG is translated into Valine.
On protein level, the haplotypes glb_H1 and glb_H2 showed besides the two described amino acid exchanges the same deduced amino acid sequence in the considered gene region which only consists of the last exon before the 3'UTR.
By genotyping 448 cultivars two main haplotypes with three SNPs and the INDEL marker in complete linkage disequilibrium were detected. While haplotype glb_H1 is equally distributed in 2-rowed spring, 2- and 6-rowed winter cultivars, haplotype glb_H2 predominates in the spring subpopulation. The other two haplotypes occurred only rarely and are caused by break of the linkage disequilibrium of the INDEL to the other three SNPs. All four haplotypes could be confirmed by sequencing the varieties Barke (glb_H1), Orthega (glb_H2), Ares (glb_H3) and Valja (glb_H4).
Association studies were performed in the complete set of 141 varieties with and without regard of population structure and in the subsets of spring and winter varieties. Without regard of population structure, many spurious associations were discovered and no significant associations for both glb haplotypes were found when population structure was taken into account. When looking at the subsets, only for two important malting quality parameters significant associations either with the both main haplotypes or the three SNP- and one INDEL-marker were found in the subset of 41 (two-rowed) spring varieties. Malt extract is one of the most important properties for assessing brewing quality and describes the percentage of soluble ingredients, mostly starch and protein, in the wort. By comparing the average values for malt extract among the spring barleys a decrease of 1.2 % (82.7 to 81.7) from haplotype glb_H1 towards glb_H2 was observed, which is also reflected by each of the four markers. Malt extract content [%] and viscosity [mPas] were significant at p < 0.05 with F-values of 5.27 and 4.96, whereas 8.9 % and 9.5 % of the total variation of these traits is explained by the markers, respectively. All other traits regarded here were not significant.
No significant associations were detected in winter cultivars. This is explainable, because all 80 winter cultivars considered in the association study possessing sufficient phenotypic data are representing only the haplotype glb_H1.
The (1→3),(1→4)-β-D-Glucan-4-glucanohydrolase gene glb2 had two major haplotypes defined by three SNPs and one INDEL, which explained 8.9 % and 9.5 % of the total variation of malt extract content and viscosity in the spring barley gene pool, respectively.