High Amylose Wheat (SSIIa Mutant)
Screening for lines missing the SSIIa proteins was performed by Yamamori et al. (2000) using similar methodology as described above for waxy proteins. After SSIIa-A1, −B1 and -D1 null lines were identified, these lines were crossed and a fully null SSIIa mutant was selected. The amylose content in this line was increased by about one-third as compared to wild-type (Yamamori et al. 2000; Shimbata et al. 2005). Although no commercially available HA variety has been released yet, quality studies have shown dramatic changes in flour properties for this type of wheat (Yamamori et al. 2000; Shimbata et al. 2005). HA seed also contains a higher proportion of resistant starch (Yamamori et al. 2006), which is thought to have a beneficial influence on health. Although HA flour used alone does not appear to be suitable for baking or noodle products, combinations of HA and regular flour are giving promising results in ongoing quality tests.
However, our interest has focused more on the “partial null“ SSIIa genotypes. We were curious to know if there were significant effects due to the presence of one or two null genes, and if the contributions of the three SSIIa genes were different. Again, the use of MAS appeared to be the most efficient way to obtain the desired genotypes. We first characterized the mutations in the three SSIIa genes at the DNA sequence level, then used this information to develop PCR-based markers that were co-dominant and could be multiplexed (Shimbata et al. 2005). After reconstituting the HA line by MAS, we used MAS to develop near-isogenic lines of the eight possible homozygous genotypes for the SSIIa gene, using a leading variety from the southern area of Japan as the recurrent parent.
In less than three years, we obtained BC5F2 NIL lines for wild (type 1) and HA (type 8) lines, as well as all “partial“ lines (types 2–7). Of the eight lines, only the high amylose line showed significant differences in kernel weight and amylose content as compared to wild-type (Shimbata et al. 2012). However, differences in starch characteristics including chain length distribution, relative viscosity, retrogradation, pasting, and enzymatic hydrolysis properties were observed among partial null lines. Generally, larger effects were observed in lines carrying two null homoeologous genes than in lines carrying a single null gene. Significant differences were also observed among the three lines carrying two null SSIIa genes (Shimbata et al. 2012). These results largely concurred with results from similar studies using doubled haploid lines (Konik-Rose et al. 2007). We were also able to determine that the effect of SSIIa genes on amylopectin synthesis follows the order SSIIa-B1 > SSII-D1 ≥ SSIIa-A1 (Shimbata et al. 2012), which parallels the differential effects of the waxy genes on amylose synthesis (Wx-B1 > Wx-D1 ≥ Wx-A1).
Sweet Wheat (GBSSI and SSIIa Mutant)
With two fully null starch mutant lines available, creating a double mutant became possible. Since we had co-dominant markers for wild-type and null alleles of all six genes, selection of the fully null SSIIa/GBSSI double mutant was fairly straightforward. Immature seed of the double mutant showed a higher sugar content, with significant increases in sucrose, glucose, maltotriose and particularly maltose (Nakamura et al. 2006). The seeds of the double mutant appeared to develop normally until seed desiccation began, when the seeds became shrunken, resulting in a large decrease in seed weight, as is seen in some sweet corns. The wheat SSIIa/GBSSI double mutant also resembles sweet corn in terms of its increased sugar content, therefore we refer to it as “sweet wheat“ (SW). Mature seed of SW also showed a high sugar content, with increases in fructan and total dietary fiber levels (Shimbata et al. 2011).
The endosperm starch granules of SW are very small and appear degraded, even at earlier stages of seed development, when SW seeds still appear plump (Vrinten et al. 2012). Starch granules from the HA line also have a somewhat deflated appearance in comparison to the normal pancake-like shape found in wild-type granules, suggesting that the lack of functional SSIIa protein may play a major role in this phenotype.
The structure of starch from SW seeds also showed large changes in comparison to starch from wild-type or parental lines. When pasting properties of normal wheat starch are subjected to relative viscosity analysis, a strong peak indicating increased viscosity due to starch swelling is usually observed. However, in SW flour, this peak was essentially absent. As well, the amylopectin from SW seeds had a reduced molecular weight and an increased proportion of short branch chains (Vrinten et al. 2012).
Product tests conducted with SW have indicated that mixing SW flour with regular flour can impart a slightly sweet taste and a crispy texture to baked goods. However, SW flour does not seem to be suitable for use in pasta or noodles. Product tests are still ongoing, and other uses, such as using fresh immature seed as a topping or vegetable, are being considered.
The dramatic changes in starch and seed phenotypes seen in SW can predictably lead to certain problems in the field. For example, SW seed is very light and is therefore easily lost during harvesting, necessitating modification of harvesting machinery. SW also appears to be very susceptible to pre-harvest sprouting, probably due to the high levels of sugar in seeds.