title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Agriculture, Forests, Nature and Energy, University of Tuscia, 01100 Viterbo, Italy.
authors.forename: Ermelinda
authors.initials: E
authors.lastname: Botticella
authors.forename: Francesco
authors.initials: F
authors.lastname: Sestili
authors.forename: Antonio
authors.initials: A
authors.lastname: Hernandez-Lopez
authors.forename: Andrew
authors.initials: A
authors.lastname: Phillips
authors.forename: Domenico
authors.initials: D
authors.lastname: Lafiandra
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics*; D000483:Alleles; D000688:Amylose / Q000032:analysis*; D019295:Computational Biology; D018744:DNA, Plant / Q000235:genetics; D005020:Ethyl Methanesulfonate; D018506:Gene Expression Regulation, Plant; D018390:Gene Targeting; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D014908:Triticum / Q000201:enzymology / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D000688:Amylose; D005020:Ethyl Methanesulfonate; D015061:1,4-alpha-Glucan Branching Enzyme
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