Near-isogenic lines (NILs) of the eight haplotypes of starch synthase IIa (SSIIa) were used to analyze the effects of SSIIa gene dosage on branch chain length, gelatinization, pasting, retrogradation, and enzymatic hydrolysis of starches. Compared to wild-type, the amylopectin of lines missing one or more active SSIIa enzymes had increases in the proportion of short branch chains (DP6-10) and decreases in midlength chains (DP11-24), and the size of these differences depended on the dosage of active SSIIa enzymes. Of the three loci, SSIIa-A1 had the smallest contribution to amylopectin structure and SSIIa-B1 the largest. The different effects of the three SSIIa enzymes on starch properties were also seen in gelatinization, retrogradation, pasting, and enzymatic hydrolysis properties. Such differences in starch properties might be useful in influencing the texture and shelf life of food products.

Wheat (Triticum aestivum L.) starch synthase II, which is also known as starch granule protein 1 (SGP-1), plays a major role in endosperm starch synthesis. The three SGP-1 proteins, SGP-A1, B1 and D1, are produced by three homoeologous SSII genes, wSSII-A, B, and D. Lines carrying null alleles for each SGP-1 protein have previously been identified. In this report, the mutations occurring in each wSSII gene were characterized, and PCR-based DNA markers capable of detecting the mutations were developed. In the null wSSII-A allele, a 289 bp deletion accompanied by 8 bp of filler DNA was present near the initiation codon. A 175 bp insertion occurred in exon 8 of the null wSSII-B allele. The insertion represented a recently discovered miniature inverted-repeat transposable element (MITE) named Hikkoshi that was first found in a wheat waxy gene. A 63 bp deletion was found at the region surrounding the junction of the fifth exon and intron of the null wSSII-D allele. Based on this information, we designed primer sets to enable us to conduct allele-specific amplifications for each locus. The applicability of these primer sets for breeding programs was demonstrated by reconstructing a line lacking all three SGP-1 proteins using marker-assisted selection. These markers will also be useful in breeding programs aimed at obtaining partial mutants missing one or two SGP-1 proteins.

The major components of storage starch are amylose and amylopectin, and in wheat, both an amylose-free mutant lacking granule-bound starch synthase I and a high-amylose mutant lacking starch synthase IIa have been produced recently. Here, we report the production of an amylose-free/ high-amylose double mutant. This double mutant has kernel and carbohydrate characteristics that are remarkably different than those of either single mutant, including a dramatically shrunken seed shape. Surprisingly, the double mutant has maltose and sucrose levels that are high enough to make it worthy of being called "sweet wheat".

In hexaploid crops, such as bread wheat, it should be possible to fine-tune phenotypic traits by identifying wild-type and null genes from each of the three genomes and combining them in a calculated manner. Here, we demonstrate this with gene combinations for two starch synthesis genes, SSIIa and GBSSI. Lines with inactive copies of both enzymes show a very dramatic change in phenotype, so to create intermediate phenotypes, we used marker-assisted selection to develop near-isogenic lines (NILs) carrying homozygous combinations of null alleles. For both genes, gene dosage effects follow the order B > D ≥ A; therefore, we completed detailed analysis of starch characteristics for NIL 3-3, which is null for the B-genome copy of the SSIIa and GBSSI genes, and NIL 5-5, which has null mutations in the B- and D-genome-encoded copies of both of these genes. The effects of the combinations on phenotypic traits followed the order expected on the basis of genotype, with NIL 5-5 showing the largest differences from the wild type, while NIL 3-3 characteristics were intermediate between NIL 5-5 and the wild type. Differences among genotypes were significant for many starch characteristics, including percent amylose, chain length distribution, gelatinization temperature, retrogradation, and pasting properties, and these differences appeared to translate into improvements in end-product quality, since bread made from type 5-5 flour showed a 3 day lag in staling.

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition. Whole grain flour of SW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient.