Diabetes mellitus (DM) is a major global threat and its prevalence is increasing at an alarming rate worldwide, especially in Asian countries (Cummings and Englyst, 1991; Chan et al., 2009). About 415 million adults suffered with type 2 diabetes mellitus (T2DM) globally during 2015, which is predicted to increase to 642 million by 2040 (Herman, 2017). Apart from obesity and physical inactivity, another major cause of high T2DM prevalence is the consumption of rapidly digestible carbohydrate-rich foods capable of increasing the blood sugar concentration. “Rice is life” for human being especially in the Asian subcontinent, where 90% of world’s rice is grown and consumed by 60% of the population (Khush and Virk, 2000). Although rice is a major cereal globally, efforts to address or enhance the nutritional qualities of rice through crop improvement programs are rare. Rice grain lacks several micronutrients including vitamins and rapidly releases energy after consumption, therefore, has a relatively higher glycemic response compared to other starchy foods (Chassy et al., 2008).

Earlier dietary carbohydrates used to be derived from whole coarse grains loaded with sufficient dietary fibre however, they are currently replaced with refined carbohydrates devoid of any dietary fibre, e.g. modern day rice resulting from advanced milling technology (Chattopadhyay, 2005). It is known that refined carbohydrate-rich diet enhances the plasma glucose level, insulin, triglycerides (TAG), and non-esterified fatty acid (NEFA) thus play a significant role to insulin resistance (Wolever and Mehling, 2003). Due to lifestyle changes, cells of the body did not respond properly to the hormone insulin which precedes the development/onset of T2DM. In a recent systemic review based on four prospective cohort studies with a total of 13,284 incident cases of T2DM among 352,384 Asian participants, Hu et al. (2012) concluded that the higher consumption of white rice was associated with a significant increase T2DM risk. In contradiction, the scientific investigation from Miller et al. (1992) indicated that all processed forms of rice (white, brown, or parboiled) fell under the category of high-glycemic index (GI foods). This signifies the necessity to develop rice variety(ies) with reduced starch hydrolysis or glycemic index utilizing molecular breeding approaches.

Starch is a complex glucose polymer stored as distinct water insoluble granules in rice grains (∼90%) primarily composed of one-quarter amylose and three-quarters amylopectin (Ramadoss et al., 2019). Starch is divided into three fractions based on the enzymatic hydrolysis, readily digestible (RDS), slowly digestible (SDS), and resistant starch (RS) (Englyst and Hudson, 1996). Amylose, an important determinant of RS formation as it showed a positive correlation with amylose (Ramadoss et al., 2015), is more resistant to enzymatic hydrolysis compared to its counterpart amylopectin (Leeman et al., 2006; Lehmann and Robin, 2007) and therefore, is being widely used to enhance the RS content of processed foods. Therefore, amylose concentration in starch can be targeted to improve RS content in the rice grains. Increasing amylose and RS content in rice endosperm is envisaged as a potential target to enhance its starch quality and promote human health and nutrition (Fuentes-Zaragoza et al., 2010). RS has a significantly lower hydrolysis index compared to other fractions of starch and free glucose moieties thus assist to normalize blood sugar levels in diabetic people. In addition, RS is fermented by large intestinal microflora into short chain fatty acids promoting the growth of beneficial bacteria and lowering the pH of the intestinal microenvironment thus alleviating the risk of gastrointestinal (GI) disorders. Extensive studies also have shown that RS possess physiological functions like those of dietary fiber (Eerlingen and Delcour, 1995).

Three genetic mechanisms have been described to increase amylose and RS content in cereals. Interestingly, the specific nature and impact of each mechanism can differ among cereals despite their close relatedness. The first mechanism includes overexpression of granule-bound starch synthase I (GBSSI) to increase amylose content (AC) (Flipse et al., 1996; Umemoto et al., 2004). The second mechanism to increase amylose accumulation in the developing grain is to down-regulate SSIIa or SSIIIa activity and decease amylopectin biosynthesis. Moreover, suppressed expression of SSIIIa has been associated with up-regulation of GBSSI and therefore, to increased amylose content (Umemoto et al., 2008; Sestili et al., 2012). The most prominent third mechanism, to increase apparent AC in cereals, targets suppression of major starch branching enzyme (SBEIIb) activity as structural changes in starch granule may have a great impact on RS content, particle size distribution and crystallinity. Mutation in starch synthases (SSIa, SSIIa, and SSIIIa) produce altered amylopectin chain length distribution in rice endosperm (Fujita et al., 2007). The suppression of starch branching enzyme SBEI alone has either no detectable effect (Regina et al., 2004), or only a minor effect on amylopectin chain length. Moreover, suppression of SBEIIb leads to elevated AC in maize and rice with altered amylopectin chain length distribution (Butardo et al., 2011) and suppression of both SBEIIb and SBEI in rice led to high AC (Zhu et al., 2012). Previous studies have been focused on the identification of mutations and characterization to understand the role of individual mutations in starch biosynthesis. This study was aimed to discover mutations responsible for increased RS content in the identified mutant line γ278 and to understand the role/contribution of individual mutations and/or combination of mutation towards trait improvement. Consequently, the present study characterized a high RS rice mutant line (mutant γ278), developed through gamma mutagenesis and biochemical analysis. The developed mutant was characterized for key candidate genes in the starch biosynthesis (GBSSI, SSI, SSIIa, SSIIIa, SBEIa, and SBEIIb) which is responsible for high RS content and to find out the sequence variants/mutations between the high RS mutant (γ278) and its wild type ADT43. Further, we analyzed the functional sequence variants/mutations for their contribution on the trait of interest through the development of segregating population (F₂) of ”wild type × mutant” cross and revealed the role of gene-specific mutations towards an increase in rice RS content.

The existing cereals contributing a major portion to the daily diet lack the appropriate concentration of RS to meet out the per day requirement, therefore, daily intake of RS is much lesser than the minimum recommended levels (6 g per meal) for health benefits (Dian et al., 2005). Currently, researchers and nutritionists have shown increased emphasis on research exploring the starches resistant to human digestive enzymes. Rice is one of the major staple cereals with low RS concentration utilized among the globe. Introducing an important trait such as RS in rice grains will assist to prepare food products rich in RS/dietary fibre, alleviating the burden of T2DM, and improve the socio-economic condition of the society. Earlier researchers postulated that RS concentration is positively correlated with amylose concentration (AC) in cereal crops (Benmoussa et al., 2007; Sang et al., 2008; Ramadoss et al., 2015) however, it would be disadvantageous to the cooking quality and palatability. Alternatively, other starch properties such as granule size, architecture, crystalline pattern, the degree of crystallinity, surface pores, or channels and degree of polymerization also influence starch digestibility (Tester et al., 2006; Noda et al., 2008). However, amylose concentration and amylopectin chain length variation influence the formation of RS concentration in rice endosperm (Fujita et al., 2007; Nakamura et al., 2010). Mutation/nucleotide changes in starch biosynthetic genes (GBSSI, SSI, SSIIa, SSIIIa, SBEIa, and SBEIIb) lead to variation in the starch quality/quantity in rice endosperm. Therefore, the present study was undertaken to study the effect of allelic variation in the target genes which contribute towards the enhancement of RS concentration in rice endosperm.

Exposing seeds to the radiation is one of the well-known approaches in mutation breeding. To know the effect of radiation on the change of gene structure and enzyme function, we characterized important candidate genes which are responsible for increasing the RS fraction of starch in the rice grains by adopting PCR and re-sequencing mutation/variant discovery method through in silico and in vivo approaches.

NGS sequencing results together with PARSESNP analysis revealed that the mutation C→T (2078) might cause an amino acid substitution of proline to serine at the 415 position in amino acid sequence of GBSSI gene. Mutations in GBSSI showed reduced activity by affecting starch-binding capacity (Liu et al., 2014), and its ADP–glucose-binding capability or its protein stability. Moreover, Delrue et al. (1992) reported that the Chlamydomonas mutant lacking GBSSI activity generated amylose free starch with the altered structure of amylopectin. Therefore, a deleterious mutation in GBSSI in the present study was expected to have lesser AC in γ278 mutant than wild type, but surprisingly the determined AC in γ278 was found to be more than the corresponding wild type. This could be explained by the mutations in the key candidate genes (SSIIa and SSIIIa) of amylopectin biosynthesis that might be responsible for the altered ratio of amylose and amylopectin which may lead to an incremental effect on AC than amylopectin in the total starch.

Mutation G→A (3797) in SSIIa caused an amino acid substitution of Glycine to Serine at the position of 604 in the mutant γ278. The same amino acid substitution (G604S) was reported to result in amylopectin chain length variation between japonica (Serine) and indica (Glycine) types (Nakamura et al., 2005). Among the four exon residing mutations identified in SSIIIa gene, the variant C→T (1615) cause amino acid substitution of Alanine to Valine at the position of 195 with deleterious in nature. Raja et al. (2017a) also reported the same mutation in rice found to have increased RS concentration. Previous reports also indicated that the loss of SSIIIa function leads to accumulation of white-core floury endosperm in rice (Fujita et al., 2007). Further, it was found that the accumulation of white core floury endosperm is due to enrichment of amylose and the loose packing of starch granules in the mutants (Fujita et al., 2007). Similar results were also reported by Yoshii (2000) and Nagato and Ebata (1958).

Upon successive mutant detection, SNP genotyping and trait association analysis was carried out to reveal the role of each mutation with respective to RS enhancement in the wild type × mutant derived F₂ population. Highest RS concentration of 8.68% was recorded in the marker combination of GBSSI: ssIIa: ssIIIa. Here we discovered wild type GBSSI and defective ssIIa and ssIIIa enhanced the RS level up to 8.68%. Zhou et al. (2016) reported active GBSSI and defective SSIIIa leads to high RS formation in rice endosperm. Marker combination GBSSI : SSIIa:ssIIIa was recorded RS value of 7.79%. Mutation free GBSSI resulted in enhanced AC, conversely mutations in SSIIa and SSIIIa leads to the formation of more short chains in amylopectin led to enhanced RS concentration. Similar results were also reported in rice by Sasaki et al. (2009). On the other hand, in spite of its lower AC, the marker combination of gbssI:ssIIa:ssIIIa recorded with 7.03% RS, than the marker combinations of GBSSI:ssIIa : SSIIIa (RS — 6.19%) and gbssI: SSIIa: ssIIIa (RS — 5.74%) with high AC. The marker combination gbssI:ssIIa:ssIIIa having mutant SNP in all the three genes lead to decreased AC and in-turn increasing the significant structural modifications by increasing the α -1-6 linkages which are less prone to α-amylose (Regina et al., 2015). Similar kind of findings were reported by Zihua et al. (2007) who reported that the concentrated α-1,6 linkages in amylopectin lead to the decrease in overall enzyme digestion rate. Because of mutant gbssI, lowest recorded AC (20.89%) and RS concentration (2.27%) were analyzed in marker combinations of gbssI:SSIIa : SSIIIa.

Among the analyzed marker combinations in the present study, wild type GBSSI with mutant gene alleles of ssIIa and ssIIIa will be the promising combinations to screen a large number of mutant/germplasm lines for enhanced RS in rice. This marker combination can also be used to develop a cultivar with increased RS concentration through marker-assisted breeding. High RS line developed in this study can be used to manage the global burden of T2DM lines. The outcomes of the present study may lead to propose a novel insight into the development of high yield rice varieties and hybrids with value-added properties in the near future.

All datasets generated for this study are included in the article/ Supplementary Material .

SG performed all the experiments and data analysis. BR helped in execution of all the experiments. VM, CS, and NK helped in execution of bioinformatic and structural analysis. JB and SG together conceptualized this study. SG, BR, CM, AA, AH, and EA drafted the manuscript with the input from JB. All authors read and approved the final manuscript.

Financial support for this study was provided by the pioneer international hi-bred student fellowship of the pioneer international USA to the first author to carry out this work as a part of doctoral program.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.