high resolution melting analysis for the detection of ems induced mutations in wheat sbeiia genes高分辨率分析融化ems诱变小麦sbeiia基因的检测.pdfVIP

Botticella et al. BMC Plant Biology 2011, 11:156 /1471-2229/11/156 RESEARCH ARTICLE Open Access High resolution melting analysis for the detection of EMS induced mutations in wheat SbeIIa genes 1 1 2 2 1* Ermelinda Botticella , Francesco Sestili , Antonio Hernandez-Lopez , Andrew Phillips and Domenico Lafiandra 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