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, 52 (4), 1754-1766

Induced Mutations in the starch Branching Enzyme II ( SBEII) Genes Increase Amylose and Resistant Starch Content in Durum Wheat

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Induced Mutations in the starch Branching Enzyme II ( SBEII) Genes Increase Amylose and Resistant Starch Content in Durum Wheat

Brittany Hazard et al. Crop Sci.

Abstract

Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat.

Figures

Figure 1
Figure 1
Structure of the SBEIIa and SBEIIb genes and location of the primers used to screen the tetraploid Kronos TILLING population (⇀ ↼) and the selected mutants. T4- numbers indicate the line ID and the numbers below the effect of the mutation on the protein. Mutation effects are described using a number for the position of the amino acid change, a letter on the left describing the original amino acid, and a letter on the right indicating the new amino acid (* indicates premature stop codon and # a deleted splicing site). Rectangles represent exons. Mutations on the A genome are indicated above the gene structure and mutations in the B genome below the gene structure.
Figure 2
Figure 2
Crossing scheme used to reduce the background mutations and to combine the different mutations. 2 BC indicates two backcross generations to the wild type Kronos line and ⊗ indicates self-pollination. Mutations were followed at each stage by sequencing the segregating lines.
Figure 3
Figure 3
Selected mutations for the B genome copy of the predicted SBEIIa protein. The E296# mutation results in the elimination of the splicing site between exons 8 (grey) and exon 9. The P303L mutation affects a highly conserved amino acid within the catalytic domain of the starch branching enzyme (line below sequences).
Figure 4
Figure 4
Characterization of durum sib lines with different combinations of SBEIIa alleles A) Percent amylose content in the grain. B) Resistant starch content in the grain C) Kernel weight. SBEIIa alleles: wild type (wt) alleles from Kronos in both A and B genomes (wt, white bar), mutations in SBEIIa-B alone (ΔB1= E296# and ΔB2=P303L, bar with horizontal lines), mutations in SBEIIa-A alone (ΔA= W220*, solid grey bar), and combinations of the mutations in both genomes (ΔAΔB1 and ΔAΔB2, black bars). Asterisks indicate probability values in Dunnett tests of mutant lines against the wild type sib line (*P< 0.05 and **P< 0.01).
Figure 5
Figure 5
Scanning electron micrographs of starch granules from wild type (A and B) and ΔAΔB1 SBEIIa double mutants (C and D). Bars represent 50 μm.

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