Location: Crops Pathology and Genetics ResearchTitle: Identification of novel mutations in the rice starch branching enzyme I gene via TILLING by sequencing
|KIM, HYUNJUNG - University Of California|
|YOON, MI-RA - Rural Development Administration - Korea|
|CHUN, AREUM - Rural Development Administration - Korea|
Submitted to: Euphytica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/4/2018
Publication Date: 5/12/2018
Citation: Kim, H., Yoon, M., Chun, A., Tai, T. 2018. Identification of novel mutations in the rice starch branching enzyme I gene via TILLING by sequencing. Euphytica. 214:94. https://doi.org/10.1007/s10681-018-2174-7.
Interpretive Summary: Rice grains consist mainly of starch and the eating, cooking and processing quality of rice kernels is largely dependent on starch properties. Starch consists of branched amylopectin and straight-chain amylose molecules. Starch branching enzymes (SBEs) provide one of three major enzyme activities for starch biosynthesis. In rice, there are three isoforms of SBE (SBEI, IIa, and IIb). SBEI is the least characterized with regard to function as to date SBEI mutants that have been isolated show little to no effect on starch content or structure. Using a reverse genetics method called Targeting of Induced Local Lesions in Genomes (TILLING) by sequencing we have screened a library of about 4,000 rice mutants for mutations in the gene encoding SBEI, which is involved in starch biosynthesis. This resulted in identification of six novel mutations that are predicted to change single amino acids in the SBEI protein. We have isolated a mutant, NE-4936, harboring one of the six mutations and shown that the rice grains of this mutant have a number of altered grain quality properties compared to the wild-type variety. This mutant provides new insight into possible SBEI functions. Further characterization of this mutant and the remaining mutations will contribute to a better understanding of rice starch biosynthesis and grain quality. The germplasm identified in this study may also be useful for breeding novel, grain quality traits in rice.
Technical Abstract: Starch branching enzymes (SBEs) incorporate a-1,6-branched glucosidic linkages into a-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 to 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 = 4,096), 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% versus 19%) and protein content (9.2% versus 4.8%). Amylose chain length distribution analysis indicated that the mutant has a slight but significant decrease in B3 chains [degree of polymerization (DP) = 36, P<0.01] and a very slight but significant increase in B1 chains (DP 13-24; P<0.05). Identification of novel SBEI mutations will be useful in elucidating its function and may provide valuable germplasm for breeding novel quality traits.