Harnessing particle disintegration of cooked rice grains for predicting glycaemic index.

This study investigated the particle disintegration of cooked milled rice during in vitro digestion to identify its potential for rapidly predicting glycaemic index (GI). Milled grains and flour of rice with varying GI were cooked, stirred and subjected to digestion followed by kinetics analyses. Despite variations in physicochemical parameters (typically amylose content), flours showed a single-phase-digestion rate (k, ∼0.12 min-1) which did not vary significantly between varieties. In contrast, intact grains were disintegrated into small/intermediate (d < 30 µm, 30 < d < 300 µm) and large (d > 300 µm) particles. The small/intermediate particles comprising 50-70 % starch were initially-digested (0-20 min) at a fast k-f (∼0.05-0.10 min-1), which enabled to differentiate rice digestibility; whereas the large was latter-digested (20-180 min) at a slow k-s (∼0.04 min-1). The sum-ratio of disintegrated-particle 0-300 µm (Q-300) correlated positively with clinical GI values, allowing for a digestibility prediction of intact milled rice grain.

The impact of the indica rice SSIIa allele on the apparent high amylose starch from rice grain with downregulated japonica SBEIIb.

KEY MESSAGE: Catalytically active indica SSIIa allele in high amylose rice with down-regulated japonica SBEIIb can increase starch content and modify the starch structure and properties without changing its amylose content. Rice (Oryza sativa) genotypes with inactive starch synthase IIa (SSIIa) with recessive variants of starch branching enzyme IIb (SBEIIb) exhibit a range of alterations in grain phenotype, starch granule morphology, starch granule bound proteins, starch structure, and functional properties. However, the interactions between the two enzymes have not been thoroughly investigated yet. We analysed recombinant rice lines having down-regulated SBEIIb expression (SBEIIbDR) with either indica or japonica type SSIIa (SSIIaind or SSIIajap). In SBEIIbDR rice starch granules, the increased abundance of two protein bands (SSI and SSIIa) was found with eight additional protein bands not generally associated with starch granules. The amount of SSIIa was higher in SSIIaindSBEIIbDR than SSIIajapSBEIIbDR, which indicated that indica type SSIIa, possibly in the monomer form, was extensively involved in starch biosynthesis in the SBEIIbDR endosperm. Furthermore, SSIIaindSBEIIbDR grains had higher total starch content and higher starch swelling power than SSIIajapSBEIIbDR lines, but the amylopectin gelatinization temperatures and enthalpy and the apparent amylose content remained similar. In summary, this work suggests that SSIIaind can partly compensate for the alteration of starch synthesis resulting from the SBEIIb down-regulation in japonica background without reducing its amylose content. The study provides insight into the starch structural and textural improvements of high amylose starch.

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

Balancing the double-edged sword effect of increased resistant starch content and its impact on rice texture: its genetics and molecular physiological mechanisms.

Resistant starch (RS) is the portion of starch that escapes gastrointestinal digestion and acts as a substrate for fermentation of probiotic bacteria in the gut. Aside from enhancing gut health, RS contributes to a lower glycemic index. A genome-wide association study coupled with targeted gene association studies was conducted utilizing a diverse panel of 281 resequenced Indica rice lines comprising of ~2.2 million single nucleotide polymorphisms. Low-to-intermediate RS phenotypic variations were identified in the rice diversity panel, resulting in novel associations of RS to several genes associated with amylopectin biosynthesis and degradation. Selected rice lines encoding superior alleles of SSIIa with medium RS and inferior alleles with low RS groups were subjected to detailed transcriptomic, metabolomic, non-starch dietary fibre (DF), starch structural and textural attributes. The gene regulatory networks highlighted the importance of a protein phosphatase alongside multiple genes of starch metabolism. Metabolomics analyses resulted in the identification of several metabolite hubs (carboxylic acid, sugars and polyamines) in the medium RS group. Among DF, mannose and galactose from the water-insoluble fraction were found to be highly associated with low and medium RS lines, respectively. Starch structural analyses revealed that a moderate increase in RS is also linked to an elevation of amylose 1 and amylose 2 fractions. Although rice lines with medium RS content negatively affected textural and viscosity properties in comparison to low RS, the textural property of medium RS lines was in the same acceptable range as IR64, a rice mega variety popular in Asia.

A High-Throughput In Vitro Assay for Screening Rice Starch Digestibility.

The development of rice that can produce slow and steady postprandial glucose in the bloodstream is a response to alarmingly high global rates of obesity and related chronic diseases. However, rice grain quality programs from all over the world currently do not have access to a high-throughput method to distinguish rice breeding materials that are digested slowly. The objective of this study was to develop a high-throughput in vitro assay to screen the digestibility of cooked white rice grains and to investigate its ability to differentiate rice genotypes with a low starch digestibility rate. The digestibility rate and extent of three commercial rice genotypes with diverse GI values (Doongara, Reiziq and Waxy) were successfully differentiated using the protocol. Further investigations with eight rice genotypes indicated the percentage of starch hydrolysed at a single time point of the assay (SH-60) successfully differentiated genotypes with a low digestibility rate (the SH-60 of Doongara and YRL127 was 50% and 59%, respectively) from genotypes with an intermediate or high digestibility rate (SH-60 values were between 64% and 93%). Application of this methodology in rice breeding programs may assist in the screening and development of new varieties with a desirable postprandial glycaemic response.

Dissecting the genome-wide genetic variants of milling and appearance quality traits in rice.

Higher head rice yield (HRY), which represents the proportion of intact grains that survive milling, and lower grain chalkiness (opacity) are key quality traits. We investigated the genetic basis of HRY and chalkiness in 320 diverse resequenced accessions of indica rice with integrated single- and multi-locus genome-wide association studies using 2.26 million single-nucleotide polymorphisms. We identified novel haplotypes that underly higher HRY on chromosomes 3, 6, 8, and 11, and that lower grain chalkiness in a fine-mapped region on chromosome 5. Whole-genome sequencing of 92 IRRI breeding lines was performed to identify the genetic variants of HRY and chalkiness. Rare and novel haplotypes were found for lowering chalkiness, but missing alleles hindered progress towards enhancing HRY in breeding material. The novel haplotypes that we identified have potential use in breeding programs aimed at improving these important traits in the rice crop.

Improving Head Rice Yield and Milling Quality: State-of-the-Art and Future Prospects.

Increasing paddy yield in rice does not directly translate to enhancing food security because significant decrease in grain yield can happen during postharvest processing of the rice paddy. In parallel with enhancing paddy yield, improving the milling quality of rice is essential in ensuring food security by mitigating the impact of significant losses during the postharvest processing of rice grains. From an industrial standpoint, maximizing the milling recovery of whole grain polished rice is crucial in fetching higher revenues to rice farmers. Significant advances in rice postharvest processing technology have been achieved which are geared toward reducing the incidence of fissures and chalkiness to increase head rice yield (HRY) in rice. The genetic bases of kernel development and grain dimension are also characterized. In addition to these advancements, an integrated phenotyping suite to simultaneously characterize phenotypes related to milling quality will help in screening for breeding lines with high HRY. Toward this goal, modern imaging tools and computer algorithms are currently being developed for high-throughput characterization of rice milling quality. With the availability of more sophisticated, affordable, automated, and nondestructive phenotyping methods of milling quality, it is envisioned that significant improvement in HRY will be made possible to ensure rice food security in the future.

Improving Rice Grain Quality: State-of-the-Art and Future Prospects.

Rice grain quality encompasses complex interrelated traits that cover biochemical composition, cooking, eating, nutritional, and sensory properties. Because rice endosperm is composed mainly of starch, rice grain quality is traditionally defined by characterizing starch structure and composition, which is then subsequently correlated with functional properties of the grain. The current proxy tests routinely used to describe rice grain quality preferences are rather limited to the estimation of apparent amylose content, gelatinization temperature, and gel consistency. Additional tests that characterize starch property, viscoelasticity, grain texture, and aroma are also employed in more advanced laboratories. However, these tests are not routinely applied in breeding programs to distinguish cooking quality classes to reflect evolving consumer preference and market demand. As consumer preferences in Asia and all over the world are diverse due to varied demographics and culture, defining uniform attributes to capture regional grain quality preferences becomes more challenging. Hence, novel and innovative proxy tests are needed to characterize rice grain quality to meet the demand for consumer preferences of commercially-released cultivars. In this chapter, the current methods employed in rice grain quality monitoring are succinctly reviewed. Future prospects for improvement are identified, introducing cutting edge technologies that can facilitate high-throughput screening of rice diversity panels and breeding lines. Aside from addressing the requirements for quality improvement in the traditional inbred rice breeding programs, we also tackled the need to enhance grain quality in the hybrid rice sector.

Quantifying Grain Digestibility of Starch Fractions in Milled Rice.

Rice is one of the staple foods which serves as the major source of carbohydrate in the human diet. A typical milled rice grain is mainly composed of starch of up to 80-90%, with an average of 6-8% proteins and some trace amounts of dietary fiber. Although cooked white rice can elicit variable glycemic response, a portion of rice starch may evade digestion in the human small intestine. The digested portion of rice can be estimated and characterized in vitro based on starch digestion extent and rate (kinetics). The indigestible portion of starch can also be quantified. This chapter will present micro-scale methods to quantify rice starch digestion rate and extent based on the sugar fractions released after treating the samples with digestive enzymes.

Analysis of Developing Rice Grain Transcriptome Using the Agilent Microarray Platform.

Transcriptome analysis reflects the status quo of transcribed genetic code present in the form of mRNA, which helps to infer biological processes and unravel metabolic status. Despite the increasing adoption of RNA-Seq technique in recent years, transcriptome analysis using the microarray platform remains the gold standard technique, which offers a simpler, more cost-effective, and efficient method for high-throughput gene expression profiling. In this chapter, we described a streamlined transcriptomic analyses pipeline employed to study developing rice grains that can also be applied to other tissue samples and species. We described a novel RNA extraction method that obviates the problem introduced by high-starch content during rice grain development that usually leads to reduction in RNA yield and quality. The detailed procedure of microarray analysis involved in cDNA synthesis, cRNA labeling, microarray hybridization, slide scanning, feature extraction to QC validation has been described. The description of a newly developed Indica- and Japonica-specific microarray slides developed from the genome information of subpopulation to study gene expression of 60,000 genes has been highlighted. The downstream bioinformatics analyses including expression QTL mapping and gene regulatory network analyses were mentioned.

Integrating a genome-wide association study with a large-scale transcriptome analysis to predict genetic regions influencing the glycaemic index and texture in rice.

Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome-wide association study (GWAS) with a transcriptome-wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re-sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate-to-high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate-to-high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis-acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wxa ) explained the intermediate-to-high GI variation. A SNP (C˃T) at exon-10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI. The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non-starch polysaccharide pathway in lowering GI.

Investigating glycemic potential of rice by unraveling compositional variations in mature grain and starch mobilization patterns during seed germination.

Rice lines with slower starch digestibility provide opportunities in mitigating the global rise in type II diabetes and related non-communicable diseases. However, screening for low glycemic index (GI) in rice breeding programs is not possible due to time and cost constraints. This study evaluated the feasibility of using in vitro cooked grain amylolysis, starch mobilization patterns during seed germination, and variation in starch structure and composition in the mature seed to differentiate patterns of starch digestibility. Mobilization patterns of total starch, resistant starch, amylose and amylopectin chains, and free sugars during seed germination revealed that the process is analogous to digestion in the human gastrointestinal tract. The combination of these biochemical markers can be used as an alternative measure to predict GI. Additionally, transcriptome analysis of stored mRNA transcripts in high and low GI lines detected differences in starch metabolism and confirmed the importance of seed storage pathways in influencing digestibility. Pathway analyses supported by metabolomics data revealed that resistant starch, cell wall non-starch polysaccharides and flavonoids potentially contribute to slower digestibility. These new insights can guide precision breeding programs to produce low GI rice with acceptable cooking quality to help mitigate the burden of diet-associated lifestyle diseases.

Systems Genetics Identifies a Novel Regulatory Domain of Amylose Synthesis.

A deeper understanding of the regulation of starch biosynthesis in rice (Oryza sativa) endosperm is crucial in tailoring digestibility without sacrificing grain quality. In this study, significant association peaks on chromosomes 6 and 7 were identified through a genomewide association study (GWAS) of debranched starch structure from grains of a 320 indica rice diversity panel using genotyping data from the high-density rice array. A systems genetics approach that interrelates starch structure data from GWAS to functional pathways from a gene regulatory network identified known genes with high correlation to the proportion of amylose and amylopectin. An SNP in the promoter region of Granule Bound Starch Synthase I was identified along with seven other SNPs to form haplotypes that discriminate samples into different phenotypic ranges of amylose. A GWAS peak on chromosome 7 between LOC_Os07g11020 and LOC_Os07g11520 indexed by a nonsynonymous SNP mutation on exon 5 of a bHLH transcription factor was found to elevate the proportion of amylose at the expense of reduced short-chain amylopectin. Linking starch structure with starch digestibility by determining the kinetics of cooked grain amylolysis of selected haplotypes revealed strong association of starch structure with estimated digestibility kinetics. Combining all results from grain quality genomics, systems genetics, and digestibility phenotyping, we propose target haplotypes for fine-tuning starch structure in rice through marker-assisted breeding that can be used to alter the digestibility of rice grain, thus offering rice consumers a new diet-based intervention to mitigate the impact of nutrition-related noncommunicable diseases.

Tailoring Grain Storage Reserves for a Healthier Rice Diet and its Comparative Status with Other Cereals.

A global rise of diet-related noncommunicable diseases calls for a focus on diet-based nutritional intervention across the entire socioeconomic consumer spectrum. We review recent reports in the area of healthier rice aimed at developing rice grains with improved dietary fiber compositions (increased amounts of nonstarch polysaccharides and resistant starch), and less digestible starch (higher amylose and phospholipid complex in the endosperm) resulting in reduced glycemic impact upon grain consumption. We furthermore elaborate on the interconnections of elevated amounts of protein and a balanced composition of essential amino acids. The importance of a nutritious aleurone layer and its role in lipid storage and micronutrient composition is discussed briefly in the context of brown rice benefits. We identify gene targets for precision breeding that will facilitate the production of rice grains and rice-based products to mitigate the impact of nutrition-related preventable diseases.

Influence of in situ progressive N-terminal is still controversial truncation of glycogen branching enzyme in Escherichia coli DH5α on glycogen structure, accumulation, and bacterial viability.

BACKGROUND: Glycogen average chain length (ACL) has been linked with bacterial durability, but this was on the basis of observations across different species. We therefore wished to investigate the relationship between bacterial durability and glycogen ACL by varying glycogen average chain length in a single species. It has been shown that progressive shortening of the N-terminus of glycogen branching enzyme (GBE) leads to a lengthening of oligosaccharide inter-α-1,6-glycosidic chain lengths, so we sought to harness this to create a set of Escherichia coli DH5α strains with a range of glycogen average chain lengths, and assess these strains for durability related attributes, such as starvation, cold and desiccation stress resistance, and biofilm formation. RESULTS: A series of Escherichia coli DH5α mutants were created with glgB genes that were in situ progressively N-terminus truncated. N-terminal truncation shifted the distribution of glycogen chain lengths from 5-11 DP toward 13-50 DP, but the relationship between glgB length and glycogen ACL was not linear. Surprisingly, removal of the first 270 nucleotides of glgB (glgBΔ270) resulted in comparatively high glycogen accumulation, with the glycogen having short ACL. Complete knockout of glgB led to the formation of amylose-like glycogen containing long, linear α1,4-glucan chains with significantly reduced branching frequency. Physiologically, the set of mutant strains had reduced bacterial starvation resistance, while minimally increasing bacterial desiccation resistance. Finally, although there were no obvious changes in cold stress resistance or biofilm forming ability, one strain (glgBΔ180) had significantly increased biofilm formation in favourable media. CONCLUSIONS: Despite glgB being the first gene of an operon, it is clear that in situ mutation is a viable means to create more biologically relevant mutant strains. Secondly, there was the suggestion in the data that impairments of starvation, cold and desiccation resistance were worse for the strain lacking glgB, though the first of these was not statistically significant. The results provide prima facie evidence linking abiotic stress tolerance with shorter glycogen ACL. However, further work needs to be done, perhaps in a less labile species. Further work is also required to tease out the complex relationship between glycogen abundance and glycogen structure.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

KEY MESSAGE: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

Designing climate-resilient rice with ideal grain quality suited for high-temperature stress.

To ensure rice food security, the target outputs of future rice breeding programmes should focus on developing climate-resilient rice varieties with emphasis on increased head rice yield coupled with superior grain quality. This challenge is made greater by a world that is increasingly becoming warmer. Such environmental changes dramatically impact head rice and milling yield as well as increasing chalkiness because of impairment in starch accumulation and other storage biosynthetic pathways in the grain. This review highlights the knowledge gained through gene discovery via quantitative trait locus (QTL) cloning and structural-functional genomic strategies to reduce chalk, increase head rice yield, and develop stable lines with optimum grain quality in challenging environments. The newly discovered genes and the knowledge gained on the influence of specific alleles related to stability of grain quality attributes provide a robust platform for marker-assisted selection in breeding to design heat-tolerant rice varieties with superior grain quality. Using the chalkiness trait in rice as a case study, we demonstrate here that the emerging field of systems genetics can help fast-track the identification of novel alleles and gene targets that can be pyramided for the development of environmentally robust rice varieties that possess improved grain quality.

Rice starch granule amylolysis--differentiating effects of particle size, morphology, thermal properties and crystalline polymorph.

The underlying mechanism of amylolysis of rice starch granules was investigated using isolated starch granules from wild-type, as well as SBEIIb mutant and down-regulated lines. Fused granule agglomerates isolated from mutant and transgenic lines were hydrolysed at similar rates by amylases, and had similar crystalline patterns and thermal properties as individual granules. Surface pores, a feature previously only reported for A-polymorphic starch granules, were also observed in B- and C-polymorphic rice starch granules. Although the microscopic patterns of hydrolysis among granules with different crystalline polymorphs were qualitatively similar, the extent and the rate of amylolysis were different, suggesting that B-type crystalline polymorphs are intrinsically more resistant to enzymatic hydrolysis than A-type in rice starch granules. It is proposed that the slightly longer branch lengths of amylopectin which leads to the formation of more stable B-type double helical structures compared to their A-type counterparts is the major parameter, with other factors such as granule size, surface pores and interior channels having secondary roles, in determining the rate of enzymatic hydrolysis of rice starch granules.

Production of high oleic rice grains by suppressing the expression of the OsFAD2-1 gene.

The composition of rice (Oryza sativa L.) grain fatty acids (18% palmitic acid, 36% oleic acid and 37% linoleic acid) is suboptimal for rice storage and utilisation of rice bran oil as food grade oil or a source of biodiesel. Genetic manipulation of fatty acid composition in rice bran oil to increase oleic acid levels at the expense of linoleic acid and palmitic acid would not only add extra value to the rice, but also enhance health benefits for consumers. Four putative rice microsomal Δ12-fatty acid desaturase (OsFAD2) genes were identified as potentially important target genes to achieve this improvement. Reverse transcription-PCR analysis indicated that OsFAD2-1 was the most highly expressed gene in rice grains. RNA interference (RNAi) suppression of the expression of OsFAD2-1 resulted in an increase of oleic acid and a reduction of linoleic and palmitic acids in T3 grains. The research here showed that in the rice grains, the OsFAD2-1 enzyme was an effective target for raising oleic acid levels at the expense of the oxidatively unstable linoleic acid and the cholesterol-raising palmitic acid.

Biomolecular analyses of starch and starch granule proteins in the high-amylose rice mutant Goami 2.

Elevated proportions of amylose in cereals are commonly associated with either the loss of starch branching or starch synthase activity. Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound proteins revealed that SSI and SSIIa accumulate inside the mature starch granules of Goami 2, which is similar to the amylose extender mutant IR36ae. However, unlike the amylose extender mutants, SBEIIb was still detectable inside the starch granules of Goami 2. Detection of SBEIIb after protein fractionation revealed that most of the SBEIIb in Goami 2 accumulates inside the starch granules, whereas most of it accumulates at the granule surface in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound proteins failed to detect any peptide sequence mutation or major post-translational modifications in Goami 2. Moreover, the signal peptide was found to be cleaved normally from the precursor protein, and there is no apparent N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between exon and intron 11, which could potentially introduce a premature stop codon and produce a truncated form of SBEIIb. It is suggested that the mutation responsible for producing high amylose in Goami 2 is not due to a defect in SBEIIb gene as was observed in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic basis of this mutation will be important in identifying novel targets for increasing amylose and resistant starch contents in rice and other cereals.