Development of EMS-induced mutation population for amylose and resistant starch variation in bread wheat (Triticum aestivum) and identification of candidate genes responsible for amylose variation

Ankita Mishra, Anuradha Singh, Monica Sharma, Pankaj Kumar, Joy Roy

Starch is a major part of cereal grain. It comprises two glucose polymer fractions, amylose (AM) and amylopectin (AP), that make up about 25 and 75 % of total starch, respectively. The ratio of the two affects processing quality and digestibility of starch-based food products. Digestibility determines nutritional quality, as high amylose starch is considered a resistant or healthy starch (RS type 2) and is highly preferred for preventive measures against obesity and related health conditions. The topic of nutrition security is currently receiving much attention and consumer demand for food products with improved nutritional qualities has increased. In bread wheat (Triticum aestivum L.), variation in amylose content is narrow, hence its limited improvement. Therefore, it is necessary to produce wheat lines or populations showing wide variation in amylose/resistant starch content. In this study, a set of EMS-induced M4 mutant lines showing dynamic variation in amylose/resistant starch content were produced. Furthermore, two diverse mutant lines for amylose content were used to study quantitative expression patterns of 20 starch metabolic pathway genes and to identify candidate genes for amylose biosynthesis. A population comprising 101 EMS-induced mutation lines (M4 generation) was produced in a bread wheat (Triticum aestivum) variety. Two methods of amylose measurement in grain starch showed variation in amylose content ranging from ~3 to 76 % in the population. The method of in vitro digestion showed variation in resistant starch content from 1 to 41 %. One-way ANOVA analysis showed significant variation (p < 0.05) in amylose and resistant starch content within the population. A multiple comparison test (Dunnett's test) showed that significant variation in amylose and resistant starch content, with respect to the parent, was observed in about 89 and 38 % of the mutant lines, respectively. Expression pattern analysis of 20 starch metabolic pathway genes in two diverse mutant lines (low and high amylose mutants) showed higher expression of key genes of amylose biosynthesis (GBSSI and their isoforms) in the high amylose mutant line, in comparison to the parent. Higher expression of amylopectin biosynthesis (SBE) was observed in the low amylose mutant lines. An additional six candidate genes showed over-expression (BMY, SPA) and reduced-expression (SSIII, SBEI, SBEIII, ISA3) in the high amylose mutant line, indicating that other starch metabolic genes may also contribute to amylose biosynthesis. In this study a set of 101 EMS-induced mutant lines (M4 generation) showing variation in amylose and resistant starch content in seed were produced. This population serves as useful germplasm or pre-breeding material for genome-wide study and improvement of starch-based processing and nutrition quality in wheat. It is also useful for the study of the genetic and molecular basis of amylose/resistant starch variation in wheat. Furthermore, gene expression analysis of 20 starch metabolic genes in the two diverse mutant lines (low and high amylose mutants) indicates that in addition to key genes, several other genes (such as phosphorylases, isoamylases, and pullulanases) may also be involved in contributing to amylose/amylopectin biosynthesis.