Creating Amylose-Free Barley Cultivars with High Soluble Sugar Content By Genome Editing

Background Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Waxy barley has extensive prospects for application in functional food development and the brewing industry; however, amylose-free waxy barleys are relatively scarce in nature. Results Here we created new alleles of the Waxy gene using CRISPR/Cas9 genome editing. Mutagenesis of single bases in these novel alleles caused absence of intact waxy protein in grain of the edited line. Consequently, B-type granules disappeared. The amylose and amylopectin contents of the edited line were zero and 31.73%, while those in the wild type (WT) were 33.50% and 39.00%, respectively. The absence of waxy protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the WT. Typical soluble sugars, sucrose and β-glucan, were 39.16% and 35.40% higher in the edited line than in the WT, respectively. Transcriptome analysis identified differences between the edited line and the WT that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and β-glucan contents. Conclusions The barley cultivar with novel alleles of the Waxy gene contained zero amylose, lower amylopectin, and higher soluble sugar, sucrose and β-glucan than the wild type. This new cultivar provides a good germplasm resource for improving the quality of barley.

THE STUDY OF TECHNOLOGICAL PROPERTIES OF WAXY WHEAT FLOUR AND ITS INFLUENCE ON REFINED SUGAR-FREE HARDTACK`S DOUGH

There was grounded the choice of ingredients for developing floury confectionary products without sugar and prospects of using amylose-free flour of waxy wheat and sunroot powder for their production. There were determined quality parameters of used flour, conditioned by the state of the protein-proteinase complex. Based on studying technological properties of flour of waxy wheat, there was demonstrated the expedience of its use in the technology of products of yeasty dough, especially hardtacks, at replacing sugar for sunroot powder. There was offered to introduce sunroot powder at producing hardtacks with the decreased sugar content in equal shares at the stage of kneading liquid fermented dough and ready one. There was determined the influence of using amylase-free flour of waxy-wheat and inulin-containing raw material on structural-mechanical and surface properties of dough for sugar-free hardtacks. It was demonstrated, that combined introduction of waxy wheat flour and inulin-containing raw material positively influences quality parameters of semi-products of yeasty dough at complete exclusion of sugar from their recipes. The replacement of bakery wheat flour by amylase-free one allows to get dough with sunroot powder for sugar-free hardtacks with less firmness, elasticity and tendency to adhesion.

Southeast Asian waxy maize (Zea mays L.), a resource for amylopectin starch quality types?

Amylose-free (waxy) maize has been a vegetable (cooked ears) and staple food in Southeast Asia for centuries, resulting in hundreds of landraces (LRs) across the region. The recessive waxy allele induces soft grains with preferred cooking and flavour properties. We hypothesized that eating preferences resulted in the additional selection for different starch properties, reflected in altered starch granule morphology or amylopectin structure. A total of 41 LRs were available as starting material that had been used by different ethnic groups in Vietnam and Thailand. Unluckily, some LR were not pure waxy, but we successfully regained the original pure waxy status for most. Twenty LR were chosen for analysis of starch traits according to their purity. Four different waxy mutations were identified, including two unknown alleles. This is a strong proof for parallel independent selection of waxy maize in the region. Starch granule morphology and size were similar among all LRs. Gelatinization properties differed only between waxy and wild-type LR, and all waxy LR were comparable to a commercial waxy hybrid. The fine structure of waxy amylopectin had fewer short chains compared with that in wild-type. So far, the differences observed in starch properties are likely associated exclusively with the waxy trait. Despite the strong selection for amylose-free starch, there was no evidence for additional region wide selection for other special starch properties in our collection. In conclusion, all analyses did not encourage further targeted research on allelic variation of other starch metabolism genes for future use in the food and feed industry.