Around 13% of the people in the world are obese and according to available data, obesity is one of the largest risk factors for human deaths globally . Diseases caused by poor diet, such as cardiovascular issues and type-2 diabetes, are directly related to obesity [1, 2]. In 2017, roughly 8% of deaths worldwide were a result of obesity related diseases . Obesity is an effect of a sedentary lifestyle and an overconsumption of cheap, high caloric foods . In order to get a better handle on an epidemic of this magnitude, developing affordable food sources with higher nutrition are imperative. Wheat, on average, is responsible for over 500 calories consumed daily worldwide, which equates to roughly 20% of the recommended daily calories [3, 4]. Enhancing the nutritional value of such an important food source can be a powerful tool in fighting obesity.
The calories and nutrition that are associated with wheat products come primarily from starch, which serves as the energy and carbon reservoir for the embryo. Roughly 70-90% of the dry weight of the endosperm is composed of starch [5, 6]. Starch is made up primarily of alternating sections of glucose polymers; linear amylose and highly branched amylopectin [6, 7]. Unbranched chains are organized by α-(1,4)-linked glucose, whereas branched sections are composed of α-(1,4)-linked glucose as well as branched α-(1,6)-linked glucose [8, 9]. Regions of highly branched amylopectin alternate with linear arrays of double helices that contribute to the semi-crystalline composition of starch granules . Starches, as a whole, are categorized based on their digestibility: Rapidly digestible starches (RDS) and slowly digestible starches (SDS). RDS are converted into their glucose constituents within the first 20 minutes of incubation with starch degrading enzymes. The SDS are completely digested in the small intestine. The resistant starches (RS) are the small fraction of starches that are resistant to α-amylase activity and are fermented by colonic bacteria [11, 12].
Studies on RS have shown incredible benefits to human health ranging from maintaining healthy bowel function to helping moderate glycemic indices, as well as the possibility to prevent colon cancer [11–14]. RS intake is lower in populations where obesity related diseases are prevelent  .Replacement of RDS with RS, has been shown to lower the rate of glucose entry into the blood stream, thus resulting in a reduction of insulin demand . In rats, fat tissue was reduced in individuals fed with a high RS diet . In an empirical study done by Regina et al. (2006), rats fed with a novel high RS transgenic wheat were found to have a roughly 100% increase in short chain fatty acid (SCFA) pools in large bowel digesta, as well as fecal excretion. Additionally, a lower pH was recorded in the bowels of these rats, indicating colonic fermentation . SCFAs have been shown to increase colonic blood flow, as well as lower the risk of malignant transformation. In addition, SCFAs play a role in acidifying digesta content, which has the ability to inactivate toxic compounds [14, 17, 18]. Furthermore, the obesity epidemic in the U.S., as well as world-wide, has a direct impact on type 2 diabetes and/or cardiovascular disease [2, 19]. In comparison with diets high in digestible starch (DS), diets consisting of higher RS showed a decrease in adipocyte cell size, as well as a reduced whole-body weight gain in rats. . In humans, Park et al. (2004) showed evidence that RS dietary supplementation showed a decrease in blood cholesterol concentrations ,. Moreover, studies have also shown longer times of satiety after eating foods containing high RS [21, 22]. In terms of helping those who have already developed type 2 diabetes, human study showed a significant decrease in insulin levels after subjects ingested bread with higher RS content . In order to develop wheat with higher RS, the ratio of amylose to amylopectin needs to be modified. Studies have shown that starches with higher amylose content are more resistant to digestion than those higher in amylopectin [18, 24].
Starch synthesis within the endosperm of wheat is accomplished by several enzymes, with differing roles in the elongation and branching of glucose polymers. Granule-bound starch synthase (GBSS) isoforms are the sole enzymes responsible for synthesis of amylose in starch formation . Amylose-free, also refered to as waxy wheats, have been created through mutagenesis as well as crosses of cultivars lacking functional copies of GBSS homoeologs [26, 27]. Amylopectin synthesis is more complicated due to the complex, yet organized structure of the branching pattern, and requires more enzymes in the biosynthesis pathway. Starch branching enzymes (SBEs) and debranching enzymes (DBEs) work together to create the α-(1,6) branching patterns that are characteristic of amylopectin [10, 25]. Mutations in SBE genes in cereals, including durum (Triticum durum) (genome AABB) and bread wheat (genome AABBDD), have shown an increase in amylose [7, 28, 29]. Starch synthase enzymes (SS) are responsible for the short chains of glucose polymers between branched clusters, thus are important for the organization of the higher structure of starch granules [10, 25, 30]. Individuals with full null SS genes in bread wheat have shown increases in amylose and RS content [31, 32]. Studies in durum and bread wheat have shown similar results, however the grains of these individuals were highly shriveled [7, 33–35].
In this study, an EMS (ethyl methanesulfonate)-mutagenized TILLING (Targeting Induced Local Lesions IN Genomes) population of hard red winter bread wheat variety ‘Jagger’ was used to study the impact of knock-out mutations of starch synthase genes on the RS and amylose content . Jagger was selected for this study due to its superior agronomic performance and excellent baking properties . TILLING is a powerful forward and reverse genetics tool where chemically or radiation induced SNPs are identified via high throughput screening methods [36, 38–42]. Several genes have been validated using TILLING, including genes that are responsible for starch synthesis [34, 35, 38, 42]. With the increasing amount of reference genomes available, as well the decreasing price for sequencing techniques, TILLING has become a popular functional genetics tool. In addition, development of cultivars with modified genes using TILLING techniques is not considered a GM approach and is not subject to the public and legal stigmas that surround GM crops.
With the use of our Jagger TILLING population, knockout mutants of SSIIa gene homoeologs were identified in “A”, “B”, and “D” sub genomes and combined by crossing to develop a full null ssiia mutant. With increased RS content in these individuals, we show the ability to develop increased nutritional value in an important cereal crop without the use of transgenics.