Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat.

Increased amylose in wheat (Triticum ssp.) starch is associated with increased resistant starch, a fermentable dietary fiber. Fermentation of resistant starch in the large intestine produces short‐chain fatty acids that are associated with human health benefits. Since wheat foods are an important component of the human diet, increases in amylose and resistant starch in wheat grains have the potential to deliver health benefits to a large number of people. In three replicated field trials we found that mutations in starch branching enzyme II genes (SBEIIa and SBEIIb) in both A and B genomes (SBEIIa/b‐AB) of durum wheat [T. turgidum L. subsp. durum (Desf.) Husn.] resulted in large increases of amylose and resistant starch content. The presence of these four mutations was also associated with an average 5% reduction in kernel weight (P = 0.0007) and 15% reduction in grain yield (P = 0.06) compared to the wild type. Complete milling and pasta quality analysis showed that the mutant lines have an acceptable quality with positive effects on pasta firmness and negative effects on semolina extraction and pasta color. Positive fermentation responses were detected in rats (Rattus spp.) fed with diets incorporating mutant wheat flour. This study quantifies benefits and limitations associated with the deployment of the SBEIIa/b‐AB mutations in durum wheat and provides the information required to develop realistic strategies to deploy durum wheat varieties with increased levels of amylose and resistant starch.

Starch present in the endosperm of common wheat (Triticum aestivum L.) grains is an important source of carbohydrates worldwide. Starches with a greater proportion of amylose have increased levels of resistant starch, a dietary fiber that can provide human health benefits. Induced mutations in STARCH BRANCHING ENZYME II (SBEII) genes in wheat are associated with increased amylose and resistant starch. Ethyl methane sulfonate mutations in SBEIIa and SBEIIb paralogs were combined in the hexaploid wheat cultivar Lassik. Four mutant combinations were generated: SBEIIa/b‐AB (Reg. No. GP‐997, PI 675644); SBEIIa/b‐A, SBEIIa‐D (Reg. No. GP‐998, PI 675645); SBEIIa/b‐B, SBEIIa‐D (Reg. No. GP‐999, PI 675646); and SBEIIa/b‐AB, SBEIIa‐D (Reg. No. GP‐1000, PI 675647). The SBEII mutant lines were compared with a wild‐type control in a greenhouse and field experiment. The quintuple mutant line (SBEIIa/b‐AB, SBEIIa‐D) presented significant increases in both amylose (51% greenhouse; 63% field) and resistant starch (947% greenhouse; 1057% field) relative to the control. A decrease in total starch content (7.8%) was observed in the field experiment. The quintuple mutant also differed in starch viscosity parameters. Registration of the hexaploid wheat SBEII‐mutant lines by University of California, Davis can help expedite the development of common wheat cultivars with increased amylose and resistant starch content.

Starch synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat ssIIIa mutants in the elite cultivar Cadenza using in silico TILLING in a mutagenized population. SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B and D). Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls (33.8% of starch in triple mutants, and 29.3% in double mutants vs. 25.5% in sibling controls) and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~ 11% less than the sibling control which was partially explained by loss in starch content. Interestingly, our study revealed gene dosage effects which could be useful for fine-tuning starch properties in wheat breeding applications while minimizing impact on grain weight and quality.