High resolution melting analysis for the detection of EMS induced mutations in wheat Sbella genes
Open Access
- 10 November 2011
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Plant Biology
- Vol. 11 (1), 156
- https://doi.org/10.1186/1471-2229-11-156
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.Keywords
This publication has 59 references indexed in Scilit:
- Increasing the amylose content of durum wheat through silencing of the SBEIIagenesBMC Plant Biology, 2010
- Starch-based coatings for colon-specific delivery. Part II: Physicochemical properties and in vitro drug release from high amylose maize starch filmsEuropean Journal of Pharmaceutics and Biopharmaceutics, 2009
- A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheatBMC Plant Biology, 2009
- Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populationsPlant Methods, 2009
- Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®BMC Plant Biology, 2009
- I-TASSER server for protein 3D structure predictionBMC Bioinformatics, 2008
- Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamicsNature Reviews Molecular Cell Biology, 2004
- Multiple isoforms of starch branching enzyme-I in wheat: lack of the major SBE-I isoform does not alter starch phenotypeFunctional Plant Biology, 2004
- Waxy proteins in diploid, tetraploid and hexaploid wheatsPlant Breeding, 2002
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001