WO2015004638A2

WO2015004638A2 - Mutations in starch biosynthesis genes leading to high fiber and resistant starch expression in rice endosperm

Info

Publication number: WO2015004638A2
Application number: PCT/IB2014/063040
Authority: WO
Inventors: Bhikane Shankarrao NANDKISHOR
Original assignee: Texcity Bio-Sciences Pvt. Ltd
Priority date: 2013-07-12
Filing date: 2014-07-11
Publication date: 2015-01-15
Also published as: WO2015004638A3

Abstract

The present invention discloses the mutations in the starch synthase genes of Tx2 mutant rice that are associated with enhanced dietary fiber and resistant starch levels in the endosperm. The increased level of dietary fiber and resistant starch in rice results in low glycemic index with a value of value of 51.2+5.2 and hence these rice varieties are of great demand for diabetic population. The mutations in starch synthase genes target the structural modification of amylopectin instead of increasing the amylose content without affecting the cooking quality of the rice grains. The mutation in SSIIa gene is also associated with reduced chain elongation in L form of amylopectin such that amylopectin chain length can be manipulated in indica varieties of rice.

Description

Mutations in starch biosynthesis genes leading to high fiber and resistant starch expression in rice endosperm

[001] FIELD OF THE INVENTION

[002] The present invention relates to an endosperm of rice with increased dietary fiber and resistant starch expression. More particularly, the invention relates to five mutations in starch synthase genes of rice leading to increased dietary fiber and resistant starch expression by modifying the amylopectin structure of starch which reduces the glycemic index.

[003] BACKGROUND OF THE INVENTION

[004] Rapid urbanization and industrialization have produced advancement on the social and economic front in many countries that has resulted into dramatic lifestyle changes leading to lifestyle related diseases. The transition from a traditional to modern lifestyle, consumption of diets rich in fat and calories combined with a high level of mental stress has compounded the problem further. In today's world, more people are succumbed to various diseases owing to high lifestyle that includes eating pattern. Trend of eating food in restaurants and fast food joints is increasing day by day, making people more prone to diseases that develop due to unhealthy eating habits. One of such common diseases includes diabetes that happens due to increased blood sugar level in a person's body. There are several studies from various parts of the world that reveal a rising trend of diabetes in the urban areas.

[005] Diabetes mellitus (DM) generally known as Diabetes, is the most common endocrine disorder in both developing and developed nations. Diabetes is a chronic disease, which occurs when the pancreas fails to produce enough insulin, or when the body is not able to effectively use the insulin it produces. This leads to an increased concentration of glucose in the blood (hyperglycemia). Type 1 diabetes (previously known as insulin-dependent or childhood-onset diabetes), is characterized by lack of insulin production whereas, Type 2 diabetes (formerly called non-insulin-dependent or adult-onset diabetes) is caused by the body's inability to use insulin effectively. This happens due to excessive body weight and physical inactivity. Another type of diabetes, termed as gestational diabetes, is hyperglycemia which is first recognized during pregnancy.

[006] In urban countries, one in three and in rural population, one in five people are found to have latent (glucose intolerance) or frank DM. Though the exact reason for upsurge of diabetes is not known, changes in lifestyle, increased calorie intake, sedentary life, environmental factors and stress are among the potential contributors. Diabetes is usually treated by insulin and oral hypoglycemic agents. However, life style modification through practice of regular exercises, relaxation of body & mind and proper nutrition improves the condition to a greater extent and plays an important role in the long-term treatment of DM.

[007] Planning and achieving a proper diet for diabetic patients is the mainstay in clinical strategy of the diabetes management. As carbohydrates form the major fraction of food and an indispensible causal factor for glucose release, current dietary diabetes management strategies focus on altering the carbohydrate metabolism in humans to achieve slow release of glucose into the blood stream. This strategy warrants alterations in carbohydrate chemistry and composition in food stuffs to make them medically acceptable to manage diabetes.

[008] A large population of country eats rice everyday as rice is the most important cereal crop and staple food of over half the world's population. As a primary dietary source of carbohydrates in these populations, rice plays an important role in meeting energy requirements and nutrient intake. Cooked rice is readily digested because it contains a higher percentage of digestible starch (DS) and a lower percentage of resistant starch (RS).

[009] The Glycemic index (GI) is a ranking of carbohydrates based on their immediate effect on blood glucose (blood sugar) levels. Foods that raise blood sugar content quickly, have high GI values. Conversely, foods that raise blood sugar content slowly have low GI values. As a result, the GI can be a useful indicator of starch digestion of food-based products. World health organization define GI as the incremental area under the blood glucose response curve of a 50 g available carbohydrate portion of a test food, expressed as a percent of the response to the same amount of carbohydrate from a standard food consumed by the same subject. The GI consists of a scale from 1 to 100, indicating the rate at which 50 grams of carbohydrate in a particular food is absorbed into the bloodstream as blood-sugar. Glucose itself is used as the main reference point and is rated 100. The GI values of foods are grouped into low GI (< 55), medium (55-70), and high (>70) (Miller et al, 1992). During digestion, carbohydrates that break down quickly have high GI (giving fast and high glucose responses). On the other hand, carbohydrates that break down slowly have low GI (releasing glucose gradually into the blood stream). Lowering postprandial blood glucose (by consuming low GI foods) has positive health outcomes for both healthy subjects and patients with insulin resistance.

[0010] Rice being a major cereal grown and eaten globally, is not the fittest food in the nutritional and medical terms, as it is known to have a relatively high Glycemic response compared with other starchy foods. The high starch content and low non-starch polysaccharide content of polished rice means that rice typically gives a high Glycemic response and contain low levels of dietary fiber and resistant starch. Jenkins et al. (1981) reported a GI of 83 for white rice.

[0011] Rice goes through a variety of processes before it is ready for cooking. After harvesting and drying, the seeds are run through a rice huller/husker for milling to remove the outer grain husks, which gives the brown rice. Most people are accustomed to white rice rather than brown rice and the steps to convert the brown rice into white rice leads to loss of almost 75 per cent of the dietary fiber content. Hence, decreasing the GI of rice grains by increasing its dietary fiber content is not feasible.

[0012] Hence, to address the problem of high GI of rice, the viable solution will be to increase the fraction of dietary fiber and resistant starch (RS) within the endosperm of rice grains. Dietary fiber and RS can elicit three major effects when included in the diet that is dilution of dietary metabolizable energy, a bulking effect, and fermentation to short-chain fatty acids and increase in expression of Peptide YY (PYY) and glucagon-like peptide (GLP)-l in the gut. RS that has physiologic effects similar to fiber is of utmost importance in rice based diet. Understanding the genetic control of dietary fiber and RS accumulation in rice is of utmost importance for enhancing its nutritional quality. Research on dietary fiber and RS contents in rice assumes considerable significance given the dramatic increase in the incidence of type II diabetes and colo-rectal cancer in South East Asian countries that are increasingly adopting western diets.

[0013] As it is well known that dietary fiber and resistant starch can lower Glycemic index because their interaction with a amylase, a key enzyme in the human gut responsible for release of glucose by breaking down the carbohydrates in food leading to slow release of glucose (Cummings and Englyst 1991 ; Englyst et ah, 1992). Increasing DF and RS in food will lower the insulin response by the body, thus helping people with diabetes to normalize their blood sugar. The lowered insulin response may also reduce the subsequent drop in blood sugar that triggers hunger for the next meal. This could result in a lower energy intake at the following meal and better body weight regulation. In addition, DF and RS also encourage the growth of beneficial bacteria in the large intestine and lower the pH of the intestinal contents, all of which may reduce the development of colon cancer. In this context, breeding rice with high DF and RS is of particular interest, as it will be easy to incorporate into the dietary-prevention strategy of diabetes. High DF and RS cereals are useful for various applications but the genetic and molecular basis of these desirable traits are not fully understood yet.

[0014] Proper understanding of the starch biosynthesis pathway, determination of key genes and discovery of natural and induced mutations involved in the enhancement of DF and RS followed by development and thorough validation of functional markers linked to this enhancement can help in genetic manipulation, transfer and introgression of these important nutritional attributes in the rice breeding programmes.

[0015] In the state of the art RNA silencing techniques have been deployed in order to down- regulate the expression of starch branching enzyme lib (SBEIIb) to produce rice lines exhibiting the amylose extender (ae) phenotype with the aim of increasing the levels of RS and attenuating glycemic response. The RNA silencing techniques as per the current genetically modified organism regulatory norms cannot be applied to food crops like rice. [0016] All the current strategies of enhancing the resistant starch in rice endosperm target the increase in amylose content, which has the drawback of severely affecting the cooking quality of rice grain. As rice is the only cereal cooked and eaten, augmenting the amylose content beyond 28% will lead to hardness in cooking of rice grains.

[0017] Hence, looking at the problems that exists in current state of the art it is desirable to make use of induced mutations in the key candidate genes that can modify the amylopectin structure, which in turn results in the enhancement of dietary fiber and resistant starch in the endosperm of rice. As this strategy does not involve the use of genetic manipulation technologies, it can be directly employed in the rice breeding programmes without any restrictions. It is desirable to target the structural modification of amylopectin for increasing DF and RS content instead of increasing the amylose content that destroys the cooking quality of the rice grains.

[0018] Summary of the invention

[0019] The present invention describes identification of the induced mutations in different starch synthase genes of rice. The mutation is associated with down-regulation of these genes and subsequently expression of enzymes encoded by these genes. The mutation targets the structural modification of amylopectin instead of increasing the amylose content, which does not offset the cooking quality of the rice grains and provides additional dietary fiber and resistant starch contents in the endosperm.

[0020] The present invention also discloses at increasing the fraction of dietary fiber and resistant starch (RS) within the endosperm of rice grains. The increased dietary fiber and resistant starch is associated with decreased glycemic index in rice.

[0021] In order to understand the association of the these mutations in starch synthase genes SSIIa and SSIIIa with the glycemic property of starch, the wild type ADT 43 is hybridized with a mutant Tx2. The first generation hybridization resulted in 27 true Fl plants, which were tagged based on morphological traits and selfed for the second generation. The second hybridization resulted in generation of 540 F2 plants. Out of this, 384 F2 plants were randomly selected for Single Nucleotide Polymorphism (SNP) genotyping. The Oryza sativa primers for the genes coding the enzymes Starch Synthase I (SSI), Starch Synthase Ila (SSIIa), Starch Synthase Ilia (SSIIIa), Granule Bound Starch Synthase (GBSSI), Starch Branching enzyme la (SBEIa), Starch Branching enzyme lib (SBEIIb) were manually designed using Gene Runner version 3.05. The PGR is carried out with initial denaturation at 95°C for 10 min followed by 40 cycles at 95°C for 30s, 60°C for lmin, 72 °C for 1 min. The results showed that there were no recombinants found for all the four SSIIIa alleles indicating that the gene is present in a recombination cold spot that does not allow free crossovers. The invention also describes the effect of combined mutations of SSIIa and SSIIIa genes in the modification of amylopectin structure. The endosperm of the Tx2 mutant showed the round shaped amyloplasts, which are relatively loosely packed into endosperm cell. The genes coding the enzymes SSIIa and SSIIIa showed reduced fold change in qPCR studies of the mRNA isolated from the developing grains of the mutants and wild type suggesting down regulation is resulting from higher functionality of the mutation in suppressing these genes.

[0022] The invention may be employed to enhance the dietary fiber and resistant starch (RS) levels in the endosperm of any rice variety of interest through marker assisted breeding strategies. Such an enhancement of dietary fiber and resistant starch results in the development of low Glycemic Index rice varieties, which are of great demand for diabetic population of the country. The fiber content of the mutant rice endosperm was found to be 8.20% and showed low glycemic index with a value of 51+5.2.

[0023] Brief description of the drawings

[0024] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings.

[0025] FIG 1 shows a table depicting resistant starch content in the seeds of the rice lines mutant Tx2 and wild type ADT 43, in accordance to one or more embodiments of the invention.

[0026] FIG 2 shows a table depicting the details of candidate genes explored for sequence variations, in accordance to one or more embodiments of the invention.

[0027] FIG 3 shows a table depicting primers and their fragments (PCR amplicon) size coverage, in accordance to one or more embodiments of the invention.

[0028] FIG 4 shows a PCR amplification of the seven candidate genes, in accordance to one or more embodiments of the invention.

[0029] FIG 5 shows position of mutations identified with respect to SSIIa (A) and gene SSIIIa (B), in accordance to one or more embodiments of the invention.

[0030] FIG 6 shows a table depicting the list of mutations identified in the key candidate genes of mutant Tx2, which are likely to increase the dietary fiber and resistant starch content in the endosperm, in accordance to one or more embodiments of the invention.

[0031] FIG 7 shows the sequence length and the size of the primers involved in starch biosynthesis, in accordance to one or more embodiments of the invention.

[0032] FIG 8 shows the delta ct values for the starch synthesis genes after qPCR with average ct values of target gene and average ct of UBQ5 gene, in accordance to one or more embodiments of the invention. [0033] FIG 9 shows the fold change values (DDCt) values for the starch synthesis genes, in accordance to one or more embodiments of the invention.

[0034] FIG 10 shows the schematic representation of hybridization of wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.

[0035] FIG 11 shows the results of genetic analysis, in accordance to one or more embodiments of the invention.

[0036] FIG 12 shows the results of scanning electron microscope, in accordance to one or more embodiments of the invention.

[0037] FIG 13 depicts a table for the results of particle size of starch granules with percentage volume distribution, in accordance to one or more embodiments of the invention.

[0038] FIG 14 illustrates the graph showing the distribution of particle size of starch granules, in accordance to one or more embodiments of the invention.

[0039] FIG 15 shows the microscopic view of the starch granules from wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention.

[0040] FIG 16 shows the values of fiber content and resistant starch of mutant Tx2, in accordance to one or more embodiments of the invention.

[0041] Detailed description of the invention

[0042] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0043] The term "Resistant starch", means portion of the starch, which is not broken down by human enzymes in the small intestine. It enters the large intestine where it is partially or wholly fermented, as context requires.

[0044] The term "DNA sequencing" , means the process of determining the precise order of nucleotides within a DNA molecule, as context requires.

[0045] The term "PCR", means polymerase chain reaction (PCR), which is a biochemical technology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence, as context requires.

[0046] The term "Mutation", means a permanent change in the DNA sequence of a gene that can alter the amino acid sequence of the protein encoded by the gene, as context requires. [0047] The term "Expressed sequence tags", means a short sub-sequence of a cDNA sequence. These tags may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination, as the context requires.

[0048] The term "Quantitative polymerase chain reaction", The term a laboratory technique based on the polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule, as the context requires.

[0049] The term "Glycemic index", we mean a numerical scale used to indicate how fast and how high a particular food can raise the blood glucose (blood sugar) level, as the context requires.

[0050] By the term "Hybridization", we mean a process of interbreeding between individuals of different species or genetically divergent individuals from different species, as context requires.

[0051] The present invention overcomes the drawback of the existing state of the art technologies by exhibiting mutations in the key target genes that are responsible for starch biosynthesis and modifies the amylopectin structure leading to increased dietary fiber (DF) and resistant starch (RS) content in rice. The above methodology is successful in achieving the desired increased level of dietary fiber and resistant starch content in rice without increasing the amylose content of starch, which may destroy the cooking quality of rice by making it hard after cooking.

[0052] Plant materials used for selection of suitable mutants were Txl and Tx2 rice lines with high resistant starch content (> 7 %). These rice lines were isolated after screening of more than 1200 rice mutant lines generated from the variety ADT 43 through a mutagenic treatment with ethyl methane sulfonate. Txl and Tx2 lines were tested for their stability of resistant starch expression over locations and seasons consistently. Txl was relatively less stable for resistant starch expression hence eliminated from further characterization. Tx2 was selected for further characterization.

[0053] FIG 1 shows a table depicting resistant starch content in the seeds of the rice lines including mutant Tx2 and wild type ADT 43, in accordance to one or more embodiments of the invention. The resistant starch content of the rice lines were estimated using the megazyme RS estimation kit. Tx2 was isolated through biochemical screening of a large number of mutant lines for RS content in the endosperm and showed high content of resistant starch. The highest RS expressing and stable line Tx2 was used for the identification of DNA sequence variants (mutations) leading to high RS and DF expression.

[0054] FIG 2 shows a table depicting the details of candidate genes explored for sequence variations, in accordance to one or more embodiments of the invention. The Expressed Sequenced Tag (ES s) sequences for six candidate genes governing the biosynthesis of starch in rice endosperm along with the amino acid sequences were retrieved from the gene bank database of NCBI. These EST sequences analyzed through BLAST in comparison with the rice whole genome sequence available in NCBI.

[0055] FIG 3 shows a table depicting primers and their fragments (PCR amplicon) size coverage, in accordance to one or more embodiments of the invention Forward primers and reverse primers were designed for all the six candidate genes using the software Primer 3. The table also shows the length of the primers.

[0056] FIG 4 shows a PCR amplification of the seven candidate genes, in accordance to one or more embodiments of the invention. Mutations in these genes were identified through DNA sequencing of the PCR amplicons of the seven key candidate genes that are involved in the starch biosynthesis. Genomic DNA was isolated from ADT43 control and high RS expressing line Tx2 using the DNA isolation kit. Long Amp Taq DNA polymerase (Cat#M0323) was used for the PCR amplification of the genes. Next Generation Sequencing platform Ion Torrent was used for sequencing with the output of 1GB data on a 216 chip adopting suitable bar coding strategies. The shot gun sequences of ~ 100 base lengths were aligned over control sequence from ADT 43 and the mutations were confirmed using bioinformatics pipelines. Many mutations were identified as SNP changes between the wild and mutant.

[0057] FIG 5 shows position of mutations identified with respect to SSIIa and gene SSIIIa, in accordance to one or more embodiments of the invention. One mutation position C978G with respect to the exon position in SSIIa is furnished in FIG 5A and four mutation positions namely C1615T, C1834T, T3559A and C10761T with respect to the exon positions in SSIIIa are furnished in FIG 5B.

[0058] FIG 6 shows a table depicting the list of mutations identified in the key candidate genes of mutant Tx2, which are likely to increase the dietary fiber and resistant starch content in the endosperm, in accordance to one or more embodiments of the invention. The five mutations were identified from a high resistant starch expressing mutant line Tx2. Exon mutations were taken into account for functional analysis with PARSESNP, an online bioinformatics tool to know whether these mutations are capable of causing amino acid changes or not. Furthermore, PARSESNP analysis also gives details about introduction and disappearance of restriction sites. The efficacy of these mutations in inducing an amino acid change in the key enzymes SSIIa and SSIIIa were confirmed through the bio-informatic pipeline SIFT (Sorting Intolerant from Tolerant). SIFT is a sequence homology-based tool that identifies intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will have a phenotypic effect. SIFT is based on the concept that protein evolution is correlated with protein function. The amino acid positions important for protein function will be conserved in an alignment of the protein family, whereas unimportant positions will appear diverse in an alignment. The SIFT scores for all the five mutations of SSIIa and SSIIIa genes showed zero indicating that all these mutations are capable of altering an amino acid in the protein and these changes in the protein are intolerable and will either lead to disruption or down regulation of the concerned enzymes. The mutations performed in the candidate genes are capable of increasing the resistant starch and dietary fiber in the endosperm to a level more than 8 %. All cultivated rice varieties normally has a level of < 2-3 % of resistant starch in their endosperm thus this mutation has the capability to enhance resistant starch by two to three times as compared to other rice varieties.

[0059] FIG 7 shows the sequence length and the size of the primers involved in starch biosynthesis, in accordance to one or more embodiments of the invention. The starch synthase genes are subjected to qPCR amplification. The Oryza sativa primers SSI, SSIIa, SSIIIa, Granule Bound Starch Synthase (GBSSI), Starch Branching enzyme la (BEIa), and SBEIIb and reference 18S rRNA and UBQ5 were manually designed using Gene Runner version 3.05. PCR amplification curves were monitored for 40 cycles. Melting curves were analyzed for single product formation. Amplicons were run on the Bioanalyzer 2100 to confirm the expected sizes of the genes. Relative quantification by qPCR was done using Brilliant II SYBR Green qPCR Master mix (Lot # 1105284). Each sample was run in duplicates for each gene using 50ng input per reaction. The experiment was conducted using Stratagene Mx3005P (Agilent technologies) platform. The relative expression levels of the genes were determined after normalizing with 18s rRNA and UBQ5 as the reference genes by using Delta Ct method. The PCR is carried out with initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 30 s, 60°C for 1 min, 72°C for 1 min. A melt curve was also performed after the assay to check for specificity of the reaction. The down regulation of the concerned enzymes is clearly evident in the quantitative real time polymerase chain reaction (qPCR) study conducted from the RNA isolated from the endosperm of the wild type ADT 43 and mutant Tx2.

[0060] FIG 8 shows the delta ct values for the starch synthesis genes after qPCR with respect to ct of target sample with ct of control, in accordance to one or more embodiments of the invention. The delta ct values are the difference of threshold cycles ct of the starch synthesis genes with ct of the control gene i.e. UBQ 5, which are determined to analyze the activity of the mutation. The delta ct values for all the starch synthesis genes like SSI, SSIIa, SSIIIa, GBSS I, SBE la and SBE lib are increased in the Tx2 mutant compared to ADT 43 wild type. The increased delta ct values indicate that the enzymes of starch biosynthesis are down regulated in the mutant Tx2 compared to the wild type ADT 43.

[0061] FIG 9 shows the fold change values (DDct) values for the starch synthesis genes, in accordance to one or more embodiments of the invention. The computed fold change values with a high degree of fold change of 0.46 and 0.68 in SSIIa and SSIIIa enzyme expression respectively confirms that the mutations are functional and effective in down regulating these genes. The lower reduction of fold change values in other genes including SSI, GBSS I, BE I, and SBE II B indicates that mechanisms like feedback inhibition of the enzymes has occurred in these enzymes.

[0062] FIG 10 shows the schematic representation of hybridization of wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention. In order to prove the association of the identified mutations in starch synthase genes SSIIa and SSIIIa with the glycemic property of starch, the wild type ADT 43 is hybridized with the mutant Tx2. The first generation hybridization resulted in 27 true Fl plants, which were tagged based on morphological traits and selfed for the second generation. This resulted in generation of 27 F2 families constituted by 20 plants each accounting to a population of 540 F2 plants. 384 F2 plants were randomly selected for Sequence Nucleotide Polymorphism (SNP) genotyping. The results showed that there were no recombinants found for all the five SSIIIa alleles indicating that the gene is present in a recombination cold spot that does not allow free cross overs and hence the possibility of analyzing the individual effects of the mutations is remote. As it is evident that all the five mutations in these genes are inherited as a unit, hence in plant breeding programs all the five SNP variants can be transferred together with the assistance of one or a couple of SNP based markers. In case of SSIIa gene, free recombination of alleles for one SNP is noticed indicating that the gene is in a recombination hot spot.

[0063] FIG 11 shows the results of genetic analysis, in accordance to one or more embodiments of the invention. The genetic analysis depicts the association of wild type and mutant alleles with various starch fractions released from a amylase digestion. The matured grains from 384 F2 plants were harvested, milled and grinded and subjected to biochemical analysis for determination of amylase digestion and estimating starch digestion fractions such as Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS) and Resistant Starch (RS). The results showed that among the 6 types of recombinants observed, the double mutant (SSIIa and SSIIIa) segregants alone showed the desired effect of low RDS and high levels of SDS and RS. Hence, the double mutant combination is uniquely responsible for reduced RDS level thereby lowering GI in rice. The present invention is a unique attempt to study the effect of combined mutations of these two key genes in the modification of amylopectin structure. These two genes are found to be the key enzymes involved in the amylopectin chain elongation in rice endosperm hence mutations in these genes will down regulate these key enzymes resulting in short chain amylopectin with more proportion of 1-6 linkages. As 1-6 linkages are not prone for a amylase digestion, this result in slower release of glucose finally resulting in low GI.

[0064] The L form of amylopectin is predominant in indica varities of rice and S form is predominant in japonica. The induced mutation is also effective in altering functional mutation in SSIIa gene, which gains significance with respect to manipulating amylopectin chain length in indica varieties cultivated in India. [0065] FIG 12 shows the results of scanning electron microscope, in accordance to one or more embodiments of the invention. The scanning electron microscopic studies of the cross section of the rice seed of wild type ADT 43 and mutant Tx2 were carried out to test the effects of SSIIa and SSIIIa activity on the granular structure of starch in the endosperm. In wild type ADT 43, the endosperm starch granules formed homogenous polygonal granules with sharp edges and the granules were tightly packed into the amyloplasts, which were very abundant in the endosperm cells as shown in Fig 12A. In contrast, the amyloplasts of the mutant Tx2 were round shaped and relatively loosely packed into the endosperm cell as shown in Fig 12B.

[0066] FIG 13 depicts a table for the results of particle size of starch granules with percentage volume distribution, in accordance to one or more embodiments of the invention. The particle size of the starch granules determines resistant starch and dietary fiber contents in natural and cooked foods. Hence, the particle size of the starch granules of wild type ADT43 and mutant Tx2 are determined in the present invention, lg of finely ground rice flour is mixed with 5ml of 0.01M sodium hydroxide and ΙΟΟμΙ of 1% protease overnight at 37°C with moderate shaking. The samples are neutralized with 1M hydrochloric acid and centrifuged at 3000g. After centrifugation, the precipitate is suspended with water, layered over 80% (w/v) caesium chloride solution and centrifuged at 13000g for 20 minutes. After centrifugation, again the pellet is suspended with water and filtered through ΙΟΟμπι pore size nylon filter. Supernatant is discarded and dark tailing layer is removed. The starch pellet is washed thrice with water, followed by acetone and finally overnight air dried in the fume hood. The dried starch pellet is subjected to particle size analysis using laser diffraction particle size analyzer. The table shows the results of percentage volume proportion in wild type ADT 43 and mutant Tx2 with reference to particle size of the starch granule.

[0067] FIG 14 illustrates the graph showing the distribution of particle size of starch granules, in accordance to one or more embodiments of the invention. The graph represents the association of the particle size of the starch granules with percentage volume proportion in wild type ADT 43 and mutant Tx2. It interprets that the mutant Tx2 showed higher dispersion of particle size than the wild type ADT 43.

[0068] FIG 15 shows the microscopic view of the starch granules from wild type ADT 43 and mutant Tx2, in accordance to one or more embodiments of the invention. The starch granules isolated from wild type ADT 43 showed higher dispersion of particles and smaller particles of less than 10 micron occupied more volume proportion. However, the starch granules from Tx2 showed reduced dispersion of particles and particles with 10-20 mcirons size occupied more than 90 % volume. Overall, the particle size in the Tx2 mutant is more homogenous with a shift in dispersion towards higher size as compared to wild type ADT 43.

[0069] Fig 16 shows the fiber content and the glycemic index of the mutant Tx2. The fiber content was found to be 8.20%. The rice is also subjected to glycemic analysis to determine the glycemic index. The rice showed the low glycemic index 51.2+5.2. This value falls under the low glycemic category.

[0070] The mutation in starch synthase genes of rice is associated with increased dietary fiber and resistant starch. This is also associated with modified amylopectin structure of starch, which reduces the glycemic index. The rice showed increased fiber content of 8.20% and low glycemic index with a value of 51.2+5.2, which is of high demand in the market. The induced mutation is also effective in altering functional mutation in SSIIa gene, which gains significance with respect to manipulation of amylopectin chain length in indica varieties cultivated in India.

Claims

[0071] Claims We claim:

1. An endosperm of rice grain which carries one or more mutated starch synthase genes such that dietary fiber and resistant starch contents are increased by modified structure of amylopectin thus reducing the glycemic index.

2. The endosperm of rice grain as claimed claim 1 wherein the starch synthase genes is one or more from a group consisting of Starch Synthase I (SSI), Starch Synthase Ila (SSIIa), Starch Synthase Ilia (SSIIIa), Granule Bound Starch Synthase I (GBSS I), Starch Branching enzyme la (SBE la) and Starch Branching enzyme lib (SBE lib).

3. The endosperm of rice grain as claimed in claim 1, wherein the mutation in SSIIa gene is associated with reduced chain elongation in L form of amylopectin such that amylopectin chain length can be manipulated in indica varieties of rice.

4. The endosperm of rice grain of as claimed in claim 1, wherein the combined mutations in SSIIa and SSIIIa genes are associated with altered amylopectin structure.

5. The endosperm of rice grain as claimed in claim 1, wherein the fiber content of rice is enhanced to 8.20%.

6. The endosperm of rice grain of claim 1, wherein the mutation in said starch synthase genes results in reduced level of Rapidly Digestible Starch (RDS) and increased levels of Slowly Digestible Starch (SDS) and Resistant Starch (RS) thereby reducing the glycemic index.

7. The endosperm of rice grain of claim 1, wherein the rice shows low glycemic index with a value of 51.2+5.2.

8. The endosperm of rice grain of claim 1, wherein one or more SNP markers are associated with the mutations of SSIIa and SSIIIa.