Abstract
BackgroundWheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes.ResultsUsing TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry.ConclusionsWe report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.
Highlights
Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement
Novel genetic variation was introduced into these TILLING populations using ethylmethane sulfonate (EMS), which primarily alkylates G residues resulting in G to A or C to T point mutations
The region containing exons 12–14 of the 22 exon starch branching enzyme IIa genes (SBEIIa) gene was targeted for TILLING because it contained 8 nucleotide positions that could be mutated to introduce a stop codon based on the action of EMS
Summary
Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. Wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. With the rise in human health concerns such as obesity and diabetes, there has been an increasing interest in altering starch composition in cereal grains to raise the proportion of resistant starch. Starch contains two major glucose polymers, amylose and amylopectin, which differ in the degree of polymerization (DP) of glucan chains and in the frequency of branches. Amylose is a predominantly linear molecule with glucan chains linked through alpha 1,4 linkages in the range of 1,000-2,000 DP that are produced mainly through the action of granule bound starch synthase (GBSSI). Amylopectin is produced through the combined action of many enzymes including multiple starch synthases that catalyse the formation of linear glucan chains, starch branching enzymes that cleave alpha 1,4 bonds and transfer glucan chains forming branches through alpha 1,6 linkages, and starch debranching enzymes that cleave alpha 1,6 linkages [14]
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