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.

Characterization of waxy proteins and waxy genes of Triticum timopheevii and T. zhukovskyi and implications for evolution of wheat

The granule-bound starch (GBSS I, waxy protein) in Triticum timopheevii (AtAtGG) and T. zhukovskyi (AtAtAzAzGG) and a diagnostic section of the genes encoding GBSS-I from the Wx-TtA and Wx-G loci of T. timopheevii and the Wx-TtA, Wx-G, and Wx-TzA loci of T. zhukovskyi were investigated in this study. The waxy proteins in these two polyploid wheats could not be separated into distinct bands, in contrast to those in the T. turgidum (AABB) – T. aestivum (AABBDD) lineage. Alignment of sequences of the section covering exon4–intron4–exon5 of the various waxy genes led to the identification of gene-specific sequences in intron 4. The sequences specific to the Wx-TtA and Wx-G genes of T. timopheevii were different from those of the Wx-A1 gene and Wx-B1 genes of T. turgidum and T. aestivum. A surprising observation was that the Wx-TzA of T. zhukovskyi did not match with the Wx-TmA of T. monococcum, a putative donor of the Az genome, but matched unexpectedly and perfectly with the Wx-B1 gene on chromosome 4A, which is proposed to have translocated from the chromosome 7B of T. aestivum. The possible genetic mechanism explaining these observations is discussed.Key words: waxy proteins, waxy genes, T. timopheevii, T. zhukovskyi, wheat evolution.