Previous studies have shown the effects of null SBE and SS genes on starch composition in durum and hexaploid wheat backgrounds [6, 19, 20, 22, 23, 32, 35]. No full null ssiia mutants, however, have been developed in a single hexaploid wheat genotype. Yamamori et al. (2000) developed a full null ssiia individual by crossing different wheat varieties that contained null ssiia in each subgenome, respectively . The full null ssiia (named ‘sweet wheat’) genotype showed an increase in amylose content, as well as RS, however the seeds were highly shriveled .
In this study, we have developed a full null ssiia mutant, all within the genetic background of the elite cultivar ‘Jagger’. The full null mutant showed increases in RS and amylose content, similar to those reported for ssiia null mutants in durum wheat, as well as hexaploid wheat [21–23, 25]. Interestingly, our study showed variations within knock-out mutants of different genomes in relation to total starch and amylose content, suggesting a non-balanced homoeolog expression of SSIIa from the three genomes. It is known that ~ 30% of wheat genes show a non-balanced expression among the A, B and D genomes . The ssiia ∆ D mutant showed a decrease in amylose, in comparison with WT, and significantly more total starch than the ssiia ∆ ABD mutant. The trend in ssiia ∆ D is consistent with the expectation, as moderate negative correlations were observed between amylose content and total starch. It is important to note that amylose content and total starch quantification was conducted by two independent assays, so the results are consistent coming from two independent samplings of flour. In Yamamori et al. (2000), cv Turkey, which naturally lacked the ssiia gene in the D genome, was used to integrate a null D genome ssiia gene into a full null individual . No significant difference in amylose content between Turkey and Chinese Spring, and a slight increase between Turkey and Norin 61 was found, using a colorimetric as well as a titration assay, which were different than the protocols used in the present study. Chinese Spring and Norin 61 both contain functional SSIIa genes in all three genomes and were used as controls . Non-balanced homoeolog expression of the three SSIIa homoeoalleles in Jagger or background mutations in the D genome mutant could be the underlying factors for the reverse effect on amylose content and TS of the ssiia D genome knock-out mutant. Further studies will be needed to identify the exact reason for this unexpected trend with the D genome copy of ssiia.
In studies of ssiia null mutant durum wheat, which is generally used for pasta flour, studies have shown a decrease in cooking time, increased firmness, and a resistant to overcooking in pasta. This, in addition to a higher protein and fat content are indicators of beneficial properties of flour derived from ssiia mutant wheat . In addition, studies using high amylose bread wheat showed possibilities of using this flour as a replacement for both pasta flour as well as in steamed Chinese foods . However, as this study shows, there are negative effects on yield in ssiia null mutants. Additionally, some cooking properties have been shown to be negatively affected by high amylose content. Studies focusing on SBE genes in in durum and bread wheat have shown significant increases of 55.13% and 108.77% on average respectively in amylose content [5, 13, 32]. Alternatively, studies on SS genes in durum and bread wheat have shown an amylose content increase of 62.74% and 17.90% on average respectively [21, 22, 24]. Morita et al. (2002) showed that high amylose bread wheat flour was unsuitable for bread making, as it resulted in dense, small pocketed loaves . In an association study between 12 different soft wheat cultivars, Gaines et al. (2000) found a moderately negative correlation (P = -0.53) between amylose content and milling flour yield . However, it is important to note that the Jagger ssiia ∆ ABD mutant can be used as a specialty wheat for targeting weight management and other health benefits in humans.
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 [9–11, 40]. 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 [11, 15, 41]. 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, 42]. 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 [44, 45]. 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 .
The resistant starch content in our ssiia ∆ ABD mutant was 118.81% higher than WT, which is significantly lower than studies on SBE gene knockouts. On average, SBE null mutants had an RS increase of roughly 387.24% in durum wheat, and 1132.39% in bread wheat [32, 39]. This significant increase could be a result of the functionality of SBE genes. SBE genes are directly responsible for the branching of glucose polymers, whereas SS genes work on the linear portions of amylopectin [8, 16]. In durum wheat, a ssiia null individuals were developed by Botticella et al. (2016), and showed an increase in RS of 645% on average . It is important to note, however, that these null ssiia mutants were developed by crossing two varieties that had null ssiia genes in the A and B sub genomes, respectively.
This study marks the first development of a full null ssiia mutant in a single winter wheat background. It further exemplifies the power behind TILLING as a genetic tool. The benefits with working with a hexaploid wheat TILLING population are that a significantly smaller population of mutant individuals (< 2000 individuals) are required to get a knockout of any gene of interest. Tetraploid and diploid wheat species require significantly larger populations to have the same effect, ~ 3000 and ~ 5500 individuals, respectively [29, 30]. We used a previously established TILLING population, developed by Rawat et al.  and the full null mutants were all discovered in this population. Due to this individual being created via a TILLING approach, it circumvents the stigmas surrounding GM crops. ssiia null individuals harbor several background mutations, but these can be eliminated through just a few rounds of back-crossing.