Identification of novel mutations in the rice starch branching enzyme I gene via TILLING by sequencing

Starch branching enzymes (SBEs) incorporate α-1,6-branched glucosidic linkages into α-1,4-glucan chains. Rice has three SBE isoforms (SBEI, SBEIIa, and SBEIIb), which differ in the amylose chain lengths that they transfer to amylopectin. SBEI mutants characterized to date have had little or no effect on starch structure or content. In this study, TILLING by sequencing was employed to identify SBEI mutations in a population of chemically induced mutants of the cultivar Nipponbare (n = 4096), resulting in the detection of 37 putative mutations. After removing mutations in introns or predicted to be synonymous, Sanger sequencing of M2 and M3 generation mutants confirmed six mutations and homozygous lines for four of these mutations were isolated. Only one line, NM-4936, exhibited a visible grain phenotype and produced sufficient M4 seeds for morphological and physicochemical analyses. In contrast to Nipponbare, NM-4936 grains are opaque due to rounded, loosely packed starch granules, and consequently, grain hardness was significantly reduced. NM-4936 grains exhibited significantly reduced width and thickness, which is reflected in reduced grain weight. Physicochemical analysis revealed significant differences in apparent amylose content (15 vs. 19%) and protein content (9.2 vs. 4.8%). Amylopectin chain-length distribution analysis indicated that the mutant has a slight but significant decrease in B 3 chains [degree of polymerization (DP) ≥ 37, P < 0.01] and a very slight but significant increase in B 1 chains (DP 13–24; P < 0.05). Identification of novel SBEI mutations will be useful in elucidating its function and may provide useful germplasm for breeding novel quality traits.