High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes

Abstract

Background: Manipulation of the amylose-amylopectin ratio in cereal starch has been identified as a major target for the production of starches with novel functional properties. In wheat, silencing of starch branching enzyme genes by a transgenic approach reportedly caused an increase of amylose content up to 70% of total starch, exhibiting novel and interesting nutritional characteristics. In this work, the functionality of starch branching enzyme IIa (SBEIIa) has been targeted in bread wheat by TILLING. An EMS-mutagenised wheat population has been screened using High Resolution Melting of PCR products to identify functional SNPs in the three homoeologous genes encoding the target enzyme in the hexaploid genome.

Results: This analysis resulted in the identification of 56, 14 and 53 new allelic variants respectively for SBEIIa-A, SBEIIa-B and SBEIIa-D. The effects of the mutations on protein structure and functionality were evaluated by a bioinformatic approach. Two putative null alleles containing non-sense or splice site mutations were identified for each of the three homoeologous SBEIIa genes; qRT-PCR analysis showed a significant decrease of their gene expression and resulted in increased amylose content. Pyramiding of different single null homoeologous allowed to isolate double null mutants showing an increase of amylose content up to 21% compared to the control.

Conclusion: TILLING has successfully been used to generate novel alleles for SBEIIa genes known to control amylose content in wheat. Single and double null SBEIIa genotypes have been found to show a significant increase in amylose content.

Figure 1

Design and testing of primers for first round PCR. a) Electrophoretic profile of the PCR products obtained from Langdon (1), Langdon 2D(2A) (2), Langdon 2D(2B) (3) by using homoeoallele specific primer pairs. b) Graphical representation of the first round PCR amplicons. For SBEIIa-A the selected regions are: fragment from exon II to V (A^(II-V) ); from exon VI to IX (A^(VI-IX)); from exon X to XIII (A^(X-XIII)). For SBEIIa-B: from exon IV to IX (B^(IV-IX)). For SBEIIa-D: from exon II to VI (D^(II-VI)); from exon X to XIII (D^(X-XIII) ). Red, green and blue arrows represent PCR primers specific for genome A, B and D, respectively.

Figure 2

High Resolution Melting analysis of second round PCR products of 96 2-fold pooled samples. The figure shows the analysis of the amplicon correspondent to exon VI of the SBEIIa-B gene. a) Total fluorescence (F) vs temperature (T) curves; b) comparison of dF/T curves between normal and heteroduplex (indicated by arrows) DNA amplicons; c) normalized and temperature-shifted curves of fluorescence vs temperature showing wild types (grey) and mutants (red); d) ΔF/T difference curves with variants highlighted in red.

Figure 3

Representation of the allelic variants identified in SBEIIa genes by TILLING as obtained by PARSESNP. Red, black and violet triangles represent deleterious (non-sense and splicing junction), mis-sense and silent mutations, respectively.

Figure 4

3D Structures of normal and mutated SBEIIa-D protein. Secondary (above) and 3D (bottom) structures as elaborated by I-TASSER for wild type and mutant forms of SBEIIa-D protein (V398I and D462N). The ligand is depicted in magenta colored ball & stick, the predicted binding site residues interacting with the ligand are shown as transparent green spheres, while the N and C terminus in the model are marked by blue and red spheres respectively.

Figure 5

Semiquantitative and quantitative RT-PCR of SBEIIa transcripts. a) Semiquantitative RT-PCR of SBEIIa genes in SBEIIa homozygous single mutant genotypes: 1) SBEIIa-A^-1; 2) SBEIIa-A^-2; 3) SBEIIa-D^-1; 4) SBEIIa-B^-1; 5) SBEIIa-B^-2; 6) wild-type Cadenza. b) Relative expression of SBEIIa homoeologs in single null genotypes as determined by Real Time quantitative PCR analysis: W.T.= wild type Cadenza; A-(1)= SBEIIa-A^-1; A-(2)= SBEIIa-A^-2; B-(1)= SBEIIa-B^-1; B-(2)= SBEIIa-B^-2; D-(1)= SBEIIa-D^-1. Vertical bars indicate standard error.