Isolation of wheat starch pathway mutants using TILLING

Wheat starch pathway mutants with altered starch properties have a number of uses for functional food applications. High amylose starch is resistant to digestion in the gut and acts as a prebiotic in the gut, helps in prevention of colonic cancer, has hypoglycemic and hypocholesterolemic effects and enhances absorption of minerals. We have produced an EMS induced M₂ mutant population of 1296 individuals in a popular hard red winter bread wheat variety ‘Jagger’. We are using TILLING (Targeting Induced Local Lesions IN Genomes)  in 4x pools to screen for mutations in this population for important genes of starch synthesis pathway namely Starch branching enzyme (SBE), Starch synthase I (SSI), Starch synthase II (SSII), Waxy (Wx), Sucrose transporter (Sut), and ADP glucose pyrophosphorylase L & S subunit (ADPgpL/S). Genome specific primers have been designed for all the genes and genome specificity of primers was confirmed by using nulli-tetrasomic stocks. Standard TILLING method of heteroduplexing, Cel-I endonuclease digestion followed by visualization using agarose gel electrophoresis was used. This TILLING population has so far been screened for Sut, SBE and SSI genes for which 36, 37 and 10 mutants, respectively, were found. On an average we found 12 mutants for each of the homoeologous alleles of the genes. An average mutation rate of ~ 1/50kb was observed for the genes studied. Sequencing of the mutants to identify non-sense and functional mis-sense mutations is being done. Progress of the work will be presented.